The effects of cells on oxygen transfer in bioreactors

Cells may affect oxygen transfer rates by three mechanisms: respiration of cells accumulated at the gas/liquid interface, physical presence of cells as solid particles, and modification of the medium by cells. These effects were studied experimentally in bubble-aerated bioreactors using baker's yeast at different cell concentrations, agitation speeds, aeration rates, and specific oxygen uptake rates. The overall effect of cells was to enhance oxygen transfer rates. The physical presence of cells as solid particles was found to retard oxygen transfer, presumably due to the lower oxygen permeability in the cell layer accumulated near the bubble surfaces. Cell respiration and medium modification, on the other hand, enhanced oxygen transfer rates. The retardation by nonrespiring cells and the enhancement due to cell respiration were found stronger at higher agitation speeds and lower aeration rates employed. This was attributed to the higher interfacial cell accumulation associated with the smaller bubbles produced under these conditions in the systems studied.     

Effects of dissolved oxygen on hybridoma cell growth, metabolism, and antibody production kinetics in continuous culture.

The effects of dissolved oxygen concentration (DO) on hybridoma cell physiology were examined in a continuous stirred tank bioreactor with a murine hybridoma cell line (167.4G5.3). Dissolved oxygen concentration was varied between 0% and 100% air saturation. Cell growth and viability, carbohydrate, amino acid, and energy metabolism, oxygen uptake, and antibody production rates were investigated. Cell growth was inhibited at both high and low DO. Cells could grow at 0% DO and maintain viability under a nitrogen atmosphere. Cell viability was higher at low DO. Glucose, glutamine, and oxygen consumption rates changed little at DO above 1% air saturation. However, the metabolic uptake rates changed below 1% DO, where growth became oxygen limited, and a Km value of 0.6% DO was obtained for the specific oxygen uptake rate. The metabolic rates of glucose, glutamine, lactate, and ammonia increased 2-3-fold as the DO dropped from 1% to 0%. Amino acid metabolism followed the same general pattern as that of glutamine and glucose. Alanine was the only amino acid produced. The consumption rates of amino acids changed little above 1% DO, but under anaerobic conditions the consumption rates of all amino acids increased severalfold. Cells obtained most of their metabolic energy from glutamine oxidation except under oxygen limitation, when glucose provided most of the energy. The calculated ATP production rate was only slightly influenced by DO and rose at 0% DO. Antibody concentration was highest at 35% DO, while the specific antibody production rate was insensitive to DO.     

Fixed performance oxygen masks: an evaluation.

Fixed performance oxygen masks operate by supplying mixtures of oxygen and air at rates exceeding the inspiratory flow rate of the patient. In this study the oxygen concentration delivered by three fixed performance oxygen masks was determined non-invasively at various inspiratory flow rates. At low inspiratory flow rates all the masks studied acted as fixed performance devices. When the peak inspiratory rate increased the performance of all the masks showed some variability. The change from fixed to variable performance depended on the relation between inspiratory flow rate and the total gas flow delivered by the mask and was independent of the volume of the mask. Hence the use of low volume masks and high oxygen flow rates should produce more consistent results than high volume masks and lower flow rates.     

The steady-state transport of oxygen through hemoglobin solutions

The steady-state transport of oxygen through hemoglobin solutions was studied to identify the mechanism of the diffusion augmentation observed at low oxygen tensions. A novel technique employing a platinum-silver oxygen electrode was developed to measure the effective diffusion coefficient of oxygen in steady-state transport. The measurements were made over a wider range of hemoglobin and oxygen concentrations than previously reported. Values of the Brownian motion diffusion coefficient of oxygen in hemoglobin solution were obtained as well as measurements of facilitated transport at low oxygen tensions. Transport rates up to ten times greater than ordinary diffusion rates were found. Predictions of oxygen flux were made assuming that the oxyhemoglobin transport coefficient was equal to the Brownian motion diffusivity which was measured in a separate set of experiments. The close correlation between prediction and experiment indicates that the diffusion of oxyhemoglobin is the mechanism by which steady-state oxygen transport is facilitated.     

Effect of oxygen concentration on photosynthesis and respiration in two hypersaline microbial mats

The effects of oxygen concentration on photosynthesis and respiration in two hypersaline cyanobacterial mats were investigated. Experiments were carried out on mats from Eilat, Israel, with moderate photosynthetic activity, and mats from Mallorca, Spain, with high photosynthetic activity. The oxygen concentration in the overlying water above the mats was increased stepwise from 0% to 100% O2. Subsequent changes in oxygen concentration, gross photosynthetic rates, and pH values inside the mats were measured with microelectrodes. According to published reports on the regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), the key enzyme in the CO2-fixation pathway of phototrophs, we expected photosynthetic activity to decrease with increasing oxygen concentration. Gross photosynthetic and total respiration rates in both mats were highest when the O2 concentration was at 0% in the overlying water. Net oxygen production rates under these conditions were the same as under air saturation (21% O2), while gross photosynthetic and respiration rates were lowest at air saturation. In both mats, gross photosynthetic and respiration rates increased upon gradually increasing the oxygen concentration in the overlying water from 21% to 100%. These results contradict the expectation that photosynthesis decreases with increasing oxygen concentration. Increased photosynthetic rates at oxygen concentrations above 21% were probably caused by enhanced oxidation of organic matter and concomitant CO2 production due to the increased oxygen availability. The cause of the high respiration rates at 0% O2 in the overlying water was presumably the enhanced excretion of photosynthetic products during increased photosynthesis. We conclude that the effect of the O2/CO2 concentration ratio on the activity of Rubisco as demonstrated in vitro on enzyme extracts cannot be extrapolated to the situation in intact microbial mats, because the close coupling of the activity of primary producers and heterotrophic bacteria plays a major role in this ecosystem.     

Variations in tumor cell growth rates and metabolism with oxygen concentration, glucose concentration, and extracellular pH.

Tumors and multicellular tumor spheroids can develop gradients in oxygen concentration, glucose concentration, and extracellular pH as they grow. In order to calculate these gradients and assess their impact on tumor growth, it is necessary to quantify the effect of these variables on tumor cell metabolism and growth. In this work, the oxygen consumption rates, glucose consumption rates, and growth rates of EMT6/Ro mouse mammary tumor cells were measured at a variety of oxygen concentrations, glucose concentrations, and extracellular pH levels. At an extracellular pH of 7.25, the oxygen consumption rate of EMT6/Ro cells increased by nearly a factor of 2 as the glucose concentration was decreased from 5.5 mM to 0.4 mM. This effect of glucose concentration on oxygen consumption rate, however, was slight at an extracellular pH of 6.95 and disappeared completely at an extracellular pH of 6.60. The glucose consumption rate of EMT6/Ro cells increased by roughly 40% when the oxygen concentration was reduced from 0.21 mM to 0.023 mM and decreased by roughly 60% when the extracellular pH was decreased from 7.25 to 6.95. The growth rate of EMT6/Ro cells decreased with decreasing oxygen concentration and extracellular pH; however, severe conditions were required to stop cell growth (0.0082 mM oxygen and an extracellular pH of 6.60). Empirical correlations were developed from these data to express EMT6/Ro cell growth rates, oxygen consumption rates, and glucose consumption rates, as functions of oxygen concentration, glucose concentration, and extracellular pH. These empirical correlations make it possible to mathematically model the gradients in oxygen concentration, glucose concentration, and extracellular pH in EMT6/Ro multicellular spheroids by solution of the diffusion/reaction equations. Computations such as these, along with oxygen and pH microelectrode measurements in EMT6/Ro multicellular spheroids, indicated that nutrient concentration and pH levels in the inner regions of spheroids were low enough to cause significant changes in nutrient consumption rates and cell growth rates. However, pH and oxygen concentrations measured or calculated in EMT6/Ro spheroids where quiescent cells have been observed were not low enough to cause the cessation of cell growth, indicating that the observed quiescence must have been due to factors other than acidic pH, oxygen depletion, or glucose depletion.     

Characterization of 3-chlorobenzoate degrading aerobic bacteria isolated under various environmental conditions

The rates of bacterial growth in nature are often restricted by low concentrations of oxygen or carbon substrates. In the present study the metabolic properties of 24 isolates that had been isolated using various concentrations of 3-chlorobenzoate, benzoate and oxygen as well as using continuous culture at high and low growth rates were determined to investigate the effects of these parameters on the metabolism of monoaromatic compounds. Bacteria were enriched from different sampling sites and subsequently isolated. In batch culture this was done both under low oxygen (2% O(2)) and air-saturated concentrations. Chemostat enrichments were performed under either oxygen or 3-chlorobenzoate limiting conditions. Bacteria metabolizing aromatics with gentisate or protocatechuate as intermediates (gp bacteria) as well as bacteria metabolizing aromatic compounds via catechols (cat bacteria) were isolated from batch cultures when either benzoate or 3CBA were used as C sources, regardless of the enrichment conditions applied. In contrast, enrichments performed in chemostats at low dilution rates resulted in gp-type organisms only, whereas at high dilution rates cat-type organisms were enriched, irrespective of the oxygen and 3-chlorobenzoate concentration used during enrichment. It is noteworthy that the gp-type of bacteria possessed relatively low μ(max) values on 3CBA and benzoate along with relatively high substrate and oxygen affinities for these compounds. This is in contrast with cat-type of bacteria, which seemed to be characterized by high maximum specific growth rates on the aromatic substrates and relatively high apparent half saturation constants. In contrast, bacteria degrading chlorobenzoate via gentisate or protocatechuate may possibly be better adapted to conditions leading to growth at reduced rates such as low oxygen and low substrate concentrations.     

A new method for studying microaerobic fermentations. II. An experimental investigation of xylose fermentation

A new experimental technique, called oxygen programmed fermentation (OPF), was used to study microbial cultures of the years Pichia stipitis and Candida utilis growing on xylose as carbon and energy source. In the oxygen programmed fermentation, the inlet oxygen mole fraction was continuously changed to scan through a wide range of oxygen uptake rates in a continuous culture. The largest ethanol yields and productivities of P. stipitis were found at oxygen transfer rates below 1.5 mmol L(-1) h(-1). It was found that the ratio between the culture fluorescence and near-IR absorbance increased at oxygen transfer rates lower than 1.5 mmol L(-1) h(-1). Small amounts of ethanol were produced also by C. utilis when the oxygen transfer rate was between 0 and 3 mmol L(-1) h(-1). It is suggested that OPF will form a nice complement to ordinary, microaerobic chemostat experiments, by making the identification of interesting regions of oxygen transfer rates possible in an efficient and time-saving initial experiment. (c) 1994 John Wiley & Sons, Inc.     

Measurement of low levels of oxygen and their effect on respiration in cell suspensions maintained in an open system

The presence of low levels of oxygen may have profound effects on the cytotoxic activity of radiation, radiosensitizers, and bioreductive alkylating agents. As others have shown, low oxygen tensions may significantly alter rates of cellular and chemical oxygen consumption. When experiments are performed at very low oxygen concentrations, the opposing effects of oxygen leakage into and cellular/chemical oxygen consumption from the system can lead to unpredictable results. Use of a newly designed, highly sensitive Clark-type oxygen sensor has permitted accurate and reproducible measurement of low levels of oxygen. Cellular depletion of oxygen at various cell densities has been monitored for a series of oxygen tensions in solution and the corresponding respiration rates have been calculated. Although oxygen depletion was found to be quite significant at low oxygen tensions, not all oxygen present could be removed by cellular respiration. Respiration rate decreased as oxygen tension decreased and approached zero at low oxygen tensions. This result was independent of cell density. A model is presented to account for the observed effect of oxygen tension on cellular oxygen utilization.     

The effect of dissolved oxygen on PHB accumulation in activated sludge cultures

Nitrogen removal from wastewater is often limited by the availability of reducing power to perform denitrification, especially when treating wastewaters with a low carbon:nitrogen ratio. In the increasingly popular sequencing batch reactor (SBR), bacteria have the opportunity to preserve reducing power from incoming chemical oxygen demand (COD) as poly-beta-hydroxybutyrate (PHB). The current study uses laboratory experiments and mathematical modeling in an attempt to generate a better understanding of the effect of oxygen on microbial conversion of COD into PHB. Results from a laboratory SBR with acetate as the organic carbon source showed that the aerobic acetate uptake process was oxygen-dependent, producing higher uptake rates at higher dissolved oxygen (DO) supply rates. However, at the lower DO supply rates (k(L)a 6 to 16 h(-1), 0 mg L(-1) DO), a higher proportion of the substrate was preserved as PHB than at higher DO supply rates (k(L)a 30, 51 h(-1), DO >0.9 mg L(-1)). Up to 77% of the reducing equivalents available from acetate were converted to PHB under oxygen limitation (Y(PHB/Ac) 0.68 Cmol/Cmol), as opposed to only 54% under oxygen-excess conditions (Y(PHB/Ac) 0.48 Cmol/Cmol), where a higher fraction of acetate was used for biomass growth. It was calculated that, by oxygen management during the feast phase, the amount of PHB preserved (1.4 Cmmol L(-1) PHB) accounted for an additional denitrification potential of up to 18 mg L(-1) nitrate-nitrogen. The trends of the effect of oxygen (and hence ATP availability) on PHB accumulation could be reproduced by the simulation model, which was based on biochemical stoichiometry and maximum rates obtained from experiments. Simulated data showed that, at low DO concentrations, the limited availability of adenosine triphosphate (ATP) prevented significant biomass growth and most ATP was used for acetate transport into the cell. In contrast, high DO supply rates provided surplus ATP and hence higher growth rates, resulting in decreased PHB yields. The results suggest that oxygen management is crucial to conserving reducing power during the feast phase of SBR operation, as excessive aeration rates decrease the PHB yield and allow higher biomass growth.     

Growth of Trichosporon cutaneum under oxygen limitation: kinetics of oxygen uptake

The relationship between oxygen concentration and growth rate in the yeast Trichosporon cutaneum was studied. In order to establish the conditions for purely oxygen-limited growth, the cells were first grown in a carbon-limited chemostat, and kinetic parameters determined. The cells were then grown in an oxygen-limited chemostat at different dilution rates yielding different oxygen uptake rates. The steady-state dissolved oxygen tension was found at each dilution rate and the corresponding equilibrium dissolved oxygen tension was found at each dilution rate and the corresponding equilibrium dissolved oxygen concentration determined in the effluent medium. The relationship between oxygen concentration and growth rate followed Monod-type kinetics with an apparent K(O) of 4.38 x 10(-6)M.     

The responses of photosynthesis and oxygen consumption to short-term changes in temperature and irradiance in a cyanobacterial mat (Ebro Delta, Spain)

We have evaluated the effects of short-term changes in incident irradiance and temperature on oxygenic photosynthesis and oxygen consumption in a hypersaline cyanobacterial mat from the Ebro Delta, Spain, in which Microcoleus chthonoplastes was the dominant phototrophic organism. The mat was incubated in the laboratory at 15, 20, 25 and 30 degrees C at incident irradiances ranging from 0 to 1,000 micromol photons m(-2) s(-1). Oxygen microsensors were used to measure steady-state oxygen profiles and the rates of gross photosynthesis, which allowed the calculation of areal gross photosynthesis, areal net oxygen production, and oxygen consumption in the aphotic layer of the mat. The lowest surface irradiance that resulted in detectable rates of gross photosynthesis increased with increasing temperature from 50 micromol photons m(-2) s(-1) at 15 degrees C to 500 micromol photons m(-2) s(-1) at 30 degrees C. These threshold irradiances were also apparent from the areal rates of net oxygen production and point to the shift of M. chthonoplastes from anoxygenic to oxygenic photosynthesis and stimulation of sulphide production and oxidation rates at elevated temperatures. The rate of net oxygen production per unit area of mat at maximum irradiance, J0, did not change with temperature, whereas, JZphot, the flux of oxygen across the lower boundary of the euphotic zone increased linearly with temperature. The rate of oxygen consumption per volume of aphotic mat increased with temperature. This increase occurred in darkness, but was strongly enhanced at high irradiances, probably as a consequence of increased rates of photosynthate exudation, stimulating respiratory processes in the mat. The compensation irradiance (Ec) marking the change of the mat from a heterotrophic to an autotrophic community, increased exponentially in this range of temperatures.     

Effect of oxygen transfer on glycerol biosynthesis by an osmophilic yeast Candida magnoliae I(2)B

The influence of oxygen on glycerol production by an osmophilic yeast, Candida magnoliae I(2)B, was studied in a bioreactor. Oxygen transfer rates (OTRs) and volumetric oxygen transfer coefficients (k(L)a) were determined at different aeration and agitation rates. Cell growth as well as glycerol production was strongly affected by oxygen supply. Improvement in OTRs resulted in increased cell growth and glycerol yield. However, at high OTRs, there was a reduction in glucose uptake rate, indicating Pasteur Effect, and glycerol accumulation was also reduced at k(L)a of 253 h(-1). The availability of oxygen per unit of cell mass was found to be the most important factor that controlled cell growth, glucose uptake, and glycerol yield. The overall productivity and yield of glycerol could be related with k(L)a. The biosynthesis of glycerol was found to both growth- and non-growth-associated, although glycerol was mainly produced in post-exponential phase.     

Growth, metabolic, and antibody production kinetics of hybridoma cell culture: 2. Effects of serum concentration, dissolved oxygen concentration, and medium pH in a batch reactor.

The effects of serum, dissolved oxygen (DO) concentration, and medium pH on hybridoma cell physiology were examined in a controlled batch bioreactor using a murine hybridoma cell line (167.4G5.3). The effect of serum was also studied for a second murine hybridoma cell line (S3H5/gamma 2bA). Cell growth, viability, cell density, carbohydrate and amino acid metabolism, respiration and energy production rates, and antibody production rates were studied. Cell growth was enhanced and cell death was decreased by increasing the serum level. The growth rates followed a Monod-type model with serum being the limiting component. Specific glucose, glutamine, and oxygen uptake rates and specific lactate and ammonia production rates did not change with serum concentrations. Amino acid metabolism was slightly influenced by the serum level. Cell growth rates were not influenced by DO between 20% and 80% air saturation, while the specific death rates were lowest at 20-50% air saturation. Glucose and glutamine uptake rates increased at DO above 10% and below 5% air saturation. Cell growth rate was optimal at pH 7.2. Glucose and glutamine uptake rates, as well as lactate and ammonia production rates, increased above pH 7.2. Metabolic rates for glutamine and ammonia were also higher below pH 7.2. The consumption or production rates of amino acids followed the glutamine consumption very closely. Cell-specific oxygen uptake rate was insensitive to the levels of serum, DO, and pH. Theoretical calculations based on experimentally determined uptake rates indicated that the ATP production rates did not change significantly with serum and DO while it increased continually with increasing pH. The oxidative phosphorylation accounted for about 60% of total energy production. This contribution, however, increased at low pH values to 76%. The specific antibody production rate was not growth associated and was independent of serum and DO concentrations and medium pH above 7.20. A 2-fold increase in specific antibody production rates was observed at pH values below 7.2. Higher concentrations of antibody were obtained at high serum levels, between 20% and 40% DO, and at pH 7.20 due to higher viable cell numbers obtained.     

Time course of O2-pulse during various tests of aerobic power.

The purpose of this study was to test the hypothesis that oxygen pulse typically reaches a maximum before maximal oxygen consumption by observing the time course of oxygen pulse throughout exercise to maximal stress and to discern those physiologic variables which might predispose an individual to reach a peak in oxygen pulse before achieving maximal oxygen consumption. Thirty male volunteers ranging in age from 18-25 (X = 20.5) years were recruited for this study. Maximal oxygen uptake was assessed on both bicycle ergometer and treadmill. Based upon the results of the exercise tests, subjects were classified into subgroups as a consequence of whether or not a maximal oxygen pulse or a plateau in oxygen pulse was demonstrated during submaximal exercise. The results indicate that submaximal peaking or at least the achieving of plateau values of oxygen pulse does in fact occur in some but not all indivuals. It was observed that this phenomenon occurs at a relatively high percentage of maximal heart rate and maximal oxygen consumption. It appeared that individuals who demonstrate low heart rates at low-work intensities, high maximal heart rates, and a disproportionate increase in R for a given ventilation are most likely to reach a submaximal peak in oxygen pulse. Oxygen pulse during submaximal exercise appears to provide a good indication of cardiorespiratory fitness.     

Effect of oxygen on steady-state product distribution in Bacillus polymyxa fermentations

Bacillus polymyxa ferments glucose to 1-2,3 butanediol, acetoin, ethanol, acetic acid, lactic acid, and formic acid. This research investigates product formation as a function of oxygen availability. A predictive model that simulates product distribution at known oxygen transfer rates is developed on the hypothesis that, in an energy-limited environment, B. polymyxa utilizes glucose and oxygen in the most efficient manner. The efficiency of utilization of glucose and oxygen is measured in terms of the ATP yields of each oxidative pathway. The identity of the products constituting the profile at the given oxygen transfer rate is determined by comparing the ATP production and consumption rates. While the ATP generated is calculated from a knowledge of the oxygen transfer rate and ATP yields of the oxidative pathways, the ATP consumption is estimated by the Pirt expression in terms of growth- and nongrowth-associated components. The product formation rates are obtained by solving ATP and NAD balance equations. They equate the production and consumption rates of these intermediates and are derived from the pseudo-steady-state hypothesis. The model is applied to continuous culture systems that are both open and closed with respect to biomass. At a given oxygen transfer rate, dilution rate, and inlet glucose concentration, the model predicts steady-state concentrations of two dominant fermentation endproducts with the help of four parameters that can be determined from independent experiments. In contrast with earlier approaches, the experimental studies are carried out in continuous culture. Product profiles are obtained at various oxygen transfer rates, fer rates, inlet glucose concentrations, and dilution rates. The effect of pH on the relative distribution of products is also demonstrated. Results indicate that the model is fairly successful in predicting product profiles as a function of oxygen availability. (c) 1992 John Wiley & Sons, Inc.     

Synthesis of staphylococcal enterotoxin A and nuclease under controlled fermentor conditions.

The production of enterotoxin A and nuclease by Staphylococcus aureus strain 100 was studied in a 1.0-liter fermentor. The effects of the gas flow rate, pH, and dissolved oxygen were evaluated. Toxin and nuclease secretion occurred under all conditions which permitted growth of the organism. Final yields of toxin and nuclease in cultures grown at constant air flow rates, ranging from 50 to 500 cm3 per min, were higher at successively higher flow rates. An optimum flow rate for either toxin or nuclease production was not observed. When the aeration rate alone or aeration rate and pH were held constant, the dissolved oxygen levels in the culture decreased from the initial 100% level to 0 to 5% 3 to 4 h after inoculation. The O2 demand of the culture then maintained this level for an additional 4 to 5 h. This low dissolved oxygen interval was characterized by rapid growth and extracellular protein production. Controlling the dissolved oxygen at a constant level throughout growth did not increase the final levels of toxin and nuclease above those achieved at the respective constant pH values. Growth under the influence of a constant aeration rate of 500 cm3 per min and a constant pH of 6.5 and 7.0 yielded the highest titers of nuclease (1,550 units/ml) and toxin (10.5 mug/ml) obtained in any of the fermentations conducted in this study. Sparging fermentor cultures with pure oxygen at a rate of 100 cm3 per min yielded growth and extracellular protein levels similar to those achieved at the sparge rate of 500 cm3 of air per min. Controlling the dissolved oxygen at 100% of pure oxygen saturation appeared to inhibit the culture, as the final cultural turbidity as well as the levels of toxin and nuclease were reduced. These data indicate that enterotoxin and nuclease secretions are closely associated with the growth of strain 100. Analyses of the production rates of these components indicated that early log phase was the most efficient production interval in the growth cycle and that this efficiency was increased by pH control at 6.7 to 6.8 and dissolved oxygen control at 10% of air saturation.     

Synthesis of staphylococcal enterotoxin A and nuclease under controlled fermentor conditions.

The production of enterotoxin A and nuclease by Staphylococcus aureus strain 100 was studied in a 1.0-liter fermentor. The effects of the gas flow rate, pH, and dissolved oxygen were evaluated. Toxin and nuclease secretion occurred under all conditions which permitted growth of the organism. Final yields of toxin and nuclease in cultures grown at constant air flow rates, ranging from 50 to 500 cm3 per min, were higher at successively higher flow rates. An optimum flow rate for either toxin or nuclease production was not observed. When the aeration rate alone or aeration rate and pH were held constant, the dissolved oxygen levels in the culture decreased from the initial 100% level to 0 to 5% 3 to 4 h after inoculation. The O2 demand of the culture then maintained this level for an additional 4 to 5 h. This low dissolved oxygen interval was characterized by rapid growth and extracellular protein production. Controlling the dissolved oxygen at a constant level throughout growth did not increase the final levels of toxin and nuclease above those achieved at the respective constant pH values. Growth under the influence of a constant aeration rate of 500 cm3 per min and a constant pH of 6.5 and 7.0 yielded the highest titers of nuclease (1,550 units/ml) and toxin (10.5 mug/ml) obtained in any of the fermentations conducted in this study. Sparging fermentor cultures with pure oxygen at a rate of 100 cm3 per min yielded growth and extracellular protein levels similar to those achieved at the sparge rate of 500 cm3 of air per min. Controlling the dissolved oxygen at 100% of pure oxygen saturation appeared to inhibit the culture, as the final cultural turbidity as well as the levels of toxin and nuclease were reduced. These data indicate that enterotoxin and nuclease secretions are closely associated with the growth of strain 100. Analyses of the production rates of these components indicated that early log phase was the most efficient production interval in the growth cycle and that this efficiency was increased by pH control at 6.7 to 6.8 and dissolved oxygen control at 10% of air saturation.     

Nitrogenase activity and regeneration of the cellular ATP pool in Azotobacter vinelandii adapted to different oxygen concentrations.

The in vivo activity of nitrogenase under aerobiosis was studied with diazotrophic chemostat cultures of Azotobacter vinelandii grown under glucose- or phosphate-limited conditions at different dilution rates (Ds, representing the growth rate mu) and different dissolved oxygen concentrations. Under steady-state conditions, the concentration as well as the cellular level of ATP increased in glucose-limited cultures when D was increased. Irrespective of the type of growth limitation or the dissolved oxygen concentration, the steady-state concentrations of ATP and of dinitrogen fixed by nitrogenase increased in direct proportion to each other. Specific rates of dinitrogen fixation as well as of the regeneration of the cellular ATP pool were compared with specific rates of cellular respiration. With glucose-limited cultures, the rate of regeneration of the ATP pool and the rate of respiration varied in direct proportion to each other. This relationship, however, was dependent on the dissolved oxygen concentration. As compared to the phosphate-sufficient control, phosphate-limited cultures exhibited the same nitrogenase activity but significantly increased respiratory activities. Rates of ATP regeneration and of cellular respiration of phosphate-limited cultures did not fit into the relationship characteristic of glucose-limited cultures. However, a linear relationship between the rates of dinitrogen fixation and ATP regeneration was identified irrespective of the type of growth limitation and the dissolved oxygen concentration. The results suggest that the ATP supply rather than cellular oxygen consumption is of primary importance in keeping nitrogenase activity in aerobic cultures of A. vinelandii.     

Oxygen transfer kinetics of riboflavin fermentation by Ashbya gossypii in agitated fermentors

Effects of agitation and aeration rates on volumetric oxygen transfer coefficient and oxygen uptake rate of a riboflavin broth containing Ashbya gossypii were investigated in three batch, sparged, and agitated fermentors having the working volumes of 0.42, 0.85, and 2.5 l. The change of oxygen uptake rate with time at 250 rev min⁻¹ stirring and vvm aeration rates was shown. The volumetric oxygen transfer coefficients and maximum oxygen uptake rates obtained have been correlated to mechanical power inputs per unit volume of the fermentation broth and the superficial air velocities.