The effect of dissolved oxygen and aeration rate on antibiotic production of Streptomyces fradiae

Different dissolved oxygen concentrations and aeration rates were imposed on a stable mutant of Streptomyces fradiae during the antibiotic-producing phase. At high aeration rate (1 vvm), the tylosin yield in the fermentor broth with dissolved oxygen (DO) concentrations controlled close to 100% saturation (6-8 ppm) increased 10% as against uncontrolled. The rates of cellular growth, oil consumption, and tylosin production were severely reduced when DO concentration fell below 25% saturation, but all resumed to their initial rates when DO was raised to saturation level again. The DO concentration in combination with air flow rate affected the pattern of the antibiotics produced. At high DO levels, an additional macrolide antibiotic, macrocin, was synthesized to more than one-third the amount of tylosin at high aeration rate (1 vvm). On the other hand, tylosin production rate remained constant and no significant amount of macrocin was produced at low aeration rate (0.2 vvm).     

<Emphasis Type="Italic">Brettanomyces bruxellensis</Emphasis>: effect of oxygen on growth and acetic acid production

The influence of the oxygen supply on the growth, acetic acid and ethanol production by Brettanomyces bruxellensis in a glucose medium was investigated with different air flow rates in the range 0-300 l h(-1 ) x (0-0.5 vvm). This study shows that growth of this yeast is stimulated by moderate aeration. The optimal oxygen supply for cellular synthesis was an oxygen transfer rate (OTR) of 43 mg O(2) l(-1) x h(-1). In this case, there was an air flow rate of 60 l h(-1) (0.1 vvm). Above this value, the maximum biomass concentration decreased. Ethanol and acetic acid production was also dependent on the level of aeration: the higher the oxygen supply, the greater the acetic acid production and the lower the ethanol production. At the highest aeration rates, we observed a strong inhibition of the ethanol yield. Over 180 l h(-1) x (0.3 vvm, OTR =105 mg O(2) l(-1) x h(-1)), glucose consumption was inhibited and a high concentration of acetic acid (6.0 g x l(-1)) was produced. The ratio of "ethanol + acetic acid" produced per mole of consumed glucose using carbon balance calculations was analyzed. It was shown that this ratio remained constant in all cases. This makes it possible to establish a stoichiometric equation between oxygen supply and metabolite production.     

Enhancement of production of cloned glucoamylase under conditions of low aeration from recombinant yeast using a SUC2 promoter

The effect of aeration rate on the production of cloned glucoamylase in a recombinant yeast was investigated. This system consisted of Saccharomyces cerevisiae transformed with the 2 μ-based plasmid YEpSUCSTA which contains the SUC2 promoter, the STA signal sequence, and the STA structural gene. In contrast to typical yeast expression reports, high production of cloned glucoamylase was achieved at low aeration level (0·3 vvm). The recombinant yeast grown at 0·3 vvm aeration produced more glucoamylase (0·94 units/ml) than when grown at 0·0 vvm, 0·6 vvm, or 0·9 vvm (9·4, 1·4, and 3·1 times more, respectively). A high dissolved oxygen level early in the cultivation was important for cell growth and a low dissolved oxygen level during the production stage was important for glucoamylase production. In large scale processes for the production of recombinant proteins, the maintenance of aeration and dissolved oxygen at high levels is difficult and expensive. In this work, we have evaluated the coordination of oxygen level with growth and protein production and developed optimal conditions. Since a low aeration rate was optimal, our results demonstrate that the method described at the laboratory scale should be successfully applied at an industrial scale.     

Effects of pluronic F-68 on oxygen transport in an agitated,sparged bioreactor

The gassing out method was used to study the effect of Pluronic F-68 on oxygen transport in an agitated, sparged bioreactor. At a low air sparge rate (0.07 vvm), the addition of Pluronic F-68 had a minimal effect on K_La in both water and medium at low agitation rates. As the agitation rate was increased at this low sparge rate the relative inhibitory effect of Pluronic F-68 on K_La increased with increasing agitation rate. At an air sparge rate of 0.80 vvm, the addition of Pluronic F-68 resulted in a significant reduction in K_La at all agitation rates studied.     

Optimization of the aeration and agitation speed of Aeribacillus palidus 418 exopolysaccharide production and the emulsifying properties of the product

The specific properties of exopolysaccharides (EPS) from thermophilic microorganisms have attracted interest in their optimized production. In this study, the ability of Aeribacillus pallidus 418 to grow and produce polysaccharide in a 5-l stirred tank bioreactor was investigated. Agitation rates of 100, 200, 600, 900, and 1100 revolutions per minute (rpm), at an air flow rate of 0.5 gas volumes per unit medium volume per minute (vvm), and aeration rates of 0.25, 0.5, 1.0, and 1.5 vvm, at an agitation rate of 900 rpm, were examined. A maximum EPS yield of 170 μg/ml has been registered in a single impeller bioreactor equipped with an original Narcissus impeller at agitation speed of 900 rpm, with an aeration rate of 0.5 vvm. The bioprocess oxygen uptake rate (OUR) and oxygen mass transfer coefficient (K_La) were evaluated. The emulsifying properties of the specific EPS produced by A. pallidus 418 were determined. Stable oil-in-water emulsions, a low level of separated water phase and high dispersion stability were found, which together demonstrate the prospects for the industrial exploration of EPS production. Enhanced synergism between the A. pallidus 418 synthesized EPS and various commercially used hydrocolloids was observed; superior synergy was achieved in combination with xanthan gum.     

Effect of the dissolved oxygen on the bioproduction of glycerol and ethanol by Hansenula anomala growing under salt stress conditions

The effect of the dissolved oxygen on glycerol and ethanol productions by an osmotolerant yeast Hansenula anomala was examined during growth in media at low water activity resulting from the addition of 2M NaCl in the culture medium. High stirring rate, high culture medium aeration, as well as high mass transfer surface inhibited both glycerol and ethanol biosynthesis. In absence of oxygen, yeast used acetaldehyde as a hydrogen acceptor, leading to the stimulation of ethanol biosynthesis and accounting for the low biomass and glycerol production; the experimental ratio ethanol on glycerol produced was 5.1 when the available oxygen was lowered (low stirring rate, 500rpm) and increased to 10.2 in absence of aeration. Extracellular glycerol production was therefore optimal for a moderate stirring (1000rpm) and aeration (1.4vvm) rates. These optimal conditions resulted in an experimental ratio ethanol on glycerol produced of 4.1, namely close to the theoretical value of 4, illustrating the osmodependent channelling of carbon towards polyols production.     

Improved poly-ε-lysine biosynthesis using Streptomyces noursei NRRL 5126 by controlling dissolved oxygen during fermentation

The growth kinetics of Streptomyces noursei NRRL 5126 was investigated under different aeration and agitation combinations in a 5.0 l stirred tank fermenter. Poly-epsilon-lysine biosynthesis, cell mass formation, and glycerol utilization rates were affected markedly by both aeration and agitation. An agitation speed of 300 rpm and aeration rate at 2.0 vvm supported better yields of 1,622.81 mg/l with highest specific productivity of 15 mg/l.h. Fermentation kinetics performed under different aeration and agitation conditions showed poly- epsilon-lysine fermentation to be a growth-associated production. A constant DO at 40% in the growth phase and 20% in the production phase increased the poly-epsilon-lysine yield as well as cell mass to their maximum values of 1,992.35 mg/l and 20.73 g/l, respectively. The oxygen transfer rate (OTR), oxygen utilization rate (OUR), and specific oxygen uptake rates (qO2) in the fermentation broth increased in the growth phase and remained unchanged in the stationary phase.     

Systematic errors in data evaluation due to ethanol stripping and water vaporization

Systematic errors due to the neglect of water and/or ethanol partition between liquid and gaseous phases are discussed for bioreactors equipped with or without a condenser. Both water vapor and ethanol vapor are present in the off-gas leaving the condenser. Presence of residual water vapor largely influences the gas measurements by dilution. As a consequence, the oxygen consumption rate can be overestimated by a factor of 3 if calculations are not corrected for water vapor content or if no additional device is implemented after the condenser to completely dry the off-gases. The mass balance and partition equations predict that the condenser has only a small effect on reduction of the ethanol vapor content of the off-gas. The reason is the high ethanol concentration of the condensate droplets on the condenser wall in contact with the off-gases. Model predictions as well as experimental results show that ethanol evaporation represents a large fraction of the ethanol production rate and influences greatly the elemental recoveries. For a reactor working at 30 degrees C without condensation of the vapors and for a volumetric aeration rate of 0.63vvm, stripping of ethanol resulted in a gaseous dilution rate of 0.016 h-1 for ethanol. The dilution rate by stripping was reduced to 0.014 h-1 when a condenser at 12 degrees C was implemented. The fraction of ethanol that is stripped is mainly dependent on the ratio D/vvm (liquid to gaseous flow rates), and the effect is only slightly influenced by low condenser temperature. The evaporation of ethanol may account for more than 20% of the ethanol formation rate. Therefore, the condenser does not succeed to reflux all ethanol to the reactor broth. In terms of a unit operation, ethanol vapor can be efficiently reduced by absorption instead of condensation. To demonstrate the feasibility, a simple modification of the reactor was tested for continuous cultures: the feed port was changed from the top-plate to the top of the condenser, which was used as an absorption column. Ethanol stripping was reduced by a factor of 4 as compared to the condensation setup (at 12 degrees C): it accounted for 2% of the ethanol production rate as compared to 8.2% at D = 0.19 h-1 and 0.63vvm.     

Growth and final product formation by <Emphasis Type="Italic">Bifidobacterium infantis</Emphasis> in aerated fermentations

Fermentation conditions were developed to allow Bifidobacterium infantis to grow in the presence of air. Batch fermentations in TPYG medium, starting from anoxic conditions followed by the application of low airflow rates [0.02–0.1 air volume, per liquid media volume, per minute (vvm)], were analyzed for growth, oxygen uptake, and product formation by the bacterium. Under all aerated fermentations, B. infantis showed high aerotolerance, with a maximum oxygen-specific consumption rate of 0.34 mmol oxygen per gram dry cell weight per hour in the presence of 0.06 vvm. Similar growth yields were obtained under oxic and anoxic conditions (0.11–0.13 and 0.11 g dry cell weight per mmol glucose, respectively). Oxygen also influenced metabolite formation since lactate production and its molar relation to acetate increased and formate decreased with aeration rate. Under anoxic conditions, a maximum concentration of 8.1 mM lactate and an acetate/lactate ratio of 3.5:1 were obtained, while under oxic conditions the lactate concentration increased more than two-fold and the acetate/lactate molar ratio decreased to 1.5:1. The possibility of balancing acetate/lactate molar ratios for organoleptic purposes as well as for obtaining good growth under microaerated conditions was demonstrated.     

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.     

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.     

Investigation of liquid-solid hydrodynamic boundary layers and oxygen requirements in hairy root cultures.

Rates of oxygen uptake, growth and alkaloid production by hairy roots in submerged culture were investigated using a recirculation reactor allowing operation at high liquid velocities for removal of hydrodynamic boundary layers. Measurements were performed at dissolved oxygen tensions of 31-450% air saturation. Critical oxygen concentrations for Atropa belladonna hairy roots were above air saturation, viz. 100-125% air saturation for oxygen uptake and 150% air saturation for growth, demonstrating that these roots cultivated in reactors with air sparging are oxygen-limited. The critical oxygen tension for oxygen uptake by Solanum aviculare hairy roots was 75% air saturation. Both the specific oxygen uptake rate and specific growth rate of A. belladonna hairy roots were dependent on the mass (g dry weight) of roots present; even in the absence of boundary layers, growth did not remain exponential over the entire culture period. Cryo-scanning electron microscopy showed that hairy roots grown submerged in liquid medium were covered with thick layers of hydrated mucilage and root hairs, representing a significant additional barrier to oxygen transfer. Roots protruding out of the liquid medium showed no evidence of mucilage accumulation. The specific oxygen demand of A. belladonna root tips was 3.3-11.5 times higher than for the remainder of the roots, the ratio increasing as the dissolved oxygen tension was reduced. Specific growth rates, biomass yields from sugar, and atropine levels were maximum at around 150% air saturation, but decreased significantly with oxygen concentrations above ca. 200%.     

Influence of dissolved oxygen and shear conditions on clavulanic acid production by Streptomyces clavuligerus

Clavulanic acid (CA), a potent β-lactamase inhibitor, is produced by a filamentous bacterium. Here, the effect of DO and shear, expressed as impeller tip velocity, on CA production was examined. Cultivations were performed in a 4 L fermentor with speeds of 600, 800 and 1,000 rpm and a fixed air flow rate (0.5 vvm). Also, cultivation with automatic control of dissolved oxygen, at 50% air saturation, by varying stirrer speed and using a mixture of air and O₂ (10% v/v) in the inlet gas, and a cultivation with fixed stirrer speed of 800 rpm and air flow rate of 0.5 vvm, enriched with 10% v/v O₂, were performed. Significant variations in CA titer, CA production rate and O₂ uptake-rate were observed. It was also found that the DO level has no remarkable effect on CA production once a critical level is surpassed. The most significant improvement in CA production was related to high stirrer speeds.     

On-line estimation of viable cells in a hybridoma culture at various DO levels using ATP balancing and redox potential measurement

Two on-line methods for the estimation of viable cell number in hybridoma cultivation were investigated. One used an empirical correlation between redox potential and animal cell density. The other was based on an ATP balance with ATP steady-state assumption. Oxygen uptake rate measurement provided the amount of ATP which was produced by oxidation of NADH. Oxygen uptake rate was measured either by stationary liquid phase balance with surface aeration or by gas balance during bubble aeration with headspace flushing with an inert gas. The amount of ATP produced through the glycolysis was estimated based on the amount of lactate produced. In cultures, in which pH was controlled via manipulation of the gas phase composition, the flow of CO(2) was linearly correlated with the lactate concentration. At constant dissolved oxygen levels, the viable cell density was proportional to the estimated ATP production rate, during exponential growth and during later phases. The estimated specific ATP production rate, however, varied from 2.2 pmol cell(-1) h(-1) at 10% air saturation to 4.5 pmol cell(-1) h(-1) at 100% air saturation. Specific rates of glutamine, glucose, and lactate followed the shape of the specific ATP production rate, whereas the specific oxygen uptake rate was minimal at around 50% air saturation. (c) 1996 John Wiley & Sons, Inc.     

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.     

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.     

Cell growth and oxygen transfer in Methylosinus trichosporium OB3b cultures

Summary The steady-state concentration of M. trichosporium OB3b increased about two-fold in the continuous culture when the feeding medium was supplemented by ferrous sulphate (50mg/L) and citric acid (100mg/L) at a steady state. In batch and continuous cultures, the cell growth was significantly inhibited by excess N-sources (NH₄OH, NH₄Cl, NH₄NO₃, HNO₃, and NaNO₃) and ammonium N-sources were more inhibitory. Both volumetric O₂ transfer coefficient and specific O₂ uptake rate increased monotonously in an extensive range of air flow rate (0.1–7 vvm) and the methane interfered with the O₂ transfer even at very low flow rates (0.01–0.1 vvm).     

Effect of bioreactor angle and aeration rate on growth and hydromechanics parameters in bioreactor culture of ginseng suspension cells

Suspension cells of Panax ginseng C.A. Meyer were cultivated in 3-L balloon-type bubble bioreactors and the bioreactor with the angle of 90° at the bottom side was optimized. The gaseous composition in plant cell and tissue cultures is regarded as an important factor affecting the plant growth. Gas hold-up was remarkably higher in the bioreactor with an angle of 90° than the other ones. Aeration rates impacted on the growth ratio, the specific O₂ uptake rate (SOUR) of ginseng cells were investigated. 0.4 vvm was selected as the optimal aeration rate with a dry weight of 6.45 g L^?1. The specific O₂ uptake rate in the culture time was detected and reached the top value at the maximum growth ratio.     

Oxygen uptake and citric acid production by Candida lipolytica Y 1095

The rates of oxygen uptake and oxygen transfer during cell growth and citric acid production by Candida lipolytica Y 1095 were determined. The maximum cell growth rate, 1.43 g cell/L . h, and volumetric oxygen uptake rate, 343 mg O(2)/L . h, occurred approximately 21 to 22 h after inoculation. At the time of maximum oxygen uptake, the biomass concentration was 1.3% w/v and the specific oxygen uptake rate was slightly greater than 26 mg O(2)/g cell . h. The specific oxygen uptake rate decreased to approximately 3 mg O(2)/g cell . h by the end of the growth phase.During citric acid production, as the concentration of dissolved oxygen was increased from 20% to 80% saturation, the specific oxygen uptake and specific citric acid productivity (mg citric acid/g cell . h) increased by 160% and 71%, respectively, at a biomass concentration of 3% w/v. At a biomass concentration of 5% w/v, the specific oxygen uptake and specific citric acid productivity increased by 230% and 82%, respectively, over the same range of dissolved oxygen concentrations.The effect of dissolved oxygen on citric acid yields and productivities was also determined. Citric acid yields appeared to be independent of dissolved oxygen concentration during the initial production phase; however, volumetric productivity (g citric acid/L . h) increased sharply with an increase in dissolved oxygen. During the second or subsequent production phase, citric acid yields increased by approximately 50%, but productivities decreased by roughly the same percentage due to a loss of cell viability under prolonged nitrogen-deficient conditions. (c) 1994 John Wiley & Sons, Inc.