Physiological characterictics and energy balance ofKlebsiella aerogenes in a multistage tower fermentor

Biomass growth, consumption of carbon and energy source, specific rates of formation of metabolic byproducts, biomass yield referred to the C-source and to oxygen, respiration rate and the value of RQ were studied in Klebsiella aerogenes CCM 2318 (on a synthetic glucose medium) at different specific growth rates. Maintenance coefficients and the total energy balance of the cultivation process were evaluated for a multistage tower fermentor with a defined interstage mixing. The results pointed to changes in both glucose metabolism and the physiological state of the population, brought about by changes in specific growth rate. As compared with a chemostat, the culture was found to exhibit a different physiological character is stages 1 and 4 despite a considerable interstage mixing.     

Growth and metabolism of Saccharomyces cerevisiae in chemostat cultures under carbon-, nitrogen-, or carbon- and nitrogen-limiting conditions.

Aerobic chemostat cultures of Saccharomyces cerevisiae were performed under carbon-, nitrogen-, and dual carbon- and nitrogen-limiting conditions. The glucose concentration was kept constant, whereas the ammonium concentration was varied among different experiments and different dilution rates. It was found that both glucose and ammonium were consumed at the maximal possible rate, i.e., the feed rate, over a range of medium C/N ratios and dilution rates. To a small extent, this was due to a changing biomass composition, but much more important was the ability of uncoupling between anabolic biomass formation and catabolic energy substrate consumption. When ammonium started to limit the amount of biomass formed and hence the anabolic flow of glucose, this was totally or at least partly compensated for by an increased catabolic glucose consumption. The primary response when glucose was present in excess of the minimum requirements for biomass production was an increased rate of respiration. The calculated specific oxygen consumption rate, at D = 0.07 h-1, was more than doubled when an additional nitrogen limitation was imposed on the cells compared with that during single glucose limitation. However, the maximum respiratory capacity decreased with decreasing nitrogen concentration. The saturation level of the specific oxygen consumption rate decreased from 5.5 to 6.0 mmol/g/h under single glucose limitation to about 4.0 mmol/g/h at the lowest nitrogen concentration tested. The combined result of this was that the critical dilution rate, i.e., onset of fermentation, was as low as 0.10 h-1 during growth in a medium with a low nitrogen concentration compared with 0.20 h-1 obtained under single glucose limitation.     

Effect of interstage mixing in multistage culture systems on continuous biomass production

The significance of the interstage mixing on important process parameters of biomass production was studied. The experiments were performed in a multistage tower fermentor and in fermentors in series. The interstage mixing effect can be evaluated under conditions of geometrical similarity, identity of oxygen transfer rate, and identity of dilution rate per stage in the individual stages of both culture systems. Candida utilis was cultivated on a synthetic medium with ethanol as the sole carbon and energy source in the concentration range 10–100 g/liter. Dilution rate, temperature, and pH in each stage of both culture systems were kept constant. It was demonstrated that in the multistage tower fermentor the definite backflow which ensures the permanent reinoculation by adapted cells significantly decreases the inhibitory effect of higher ethanol concentrations on the cell growth and on the rate of ethanol utilization.     

Enzymic analysis of the crabtree effect in glucose-limited chemostat cultures of Saccharomyces cerevisiae

The physiology of Saccharomyces cerevisiae CBS 8066 was studied in glucose-limited chemostat cultures. Below a dilution rate of 0.30 h-1 glucose was completely respired, and biomass and CO2 were the only products formed. Above this dilution rate acetate and pyruvate appeared in the culture fluid, accompanied by disproportional increases in the rates of oxygen consumption and carbon dioxide production. This enhanced respiratory activity was accompanied by a drop in cell yield from 0.50 to 0.47 g (dry weight) g of glucose-1. At a dilution rate of 0.38 h-1 the culture reached its maximal oxidation capacity of 12 mmol of O2 g (dry weight)-1 h-1. A further increase in the dilution rate resulted in aerobic alcoholic fermentation in addition to respiration, accompanied by an additional decrease in cell yield from 0.47 to 0.16 g (dry weight) g of glucose-1. Since the high respiratory activity of the yeast at intermediary dilution rates would allow for full respiratory metabolism of glucose up to dilution rates close to mumax, we conclude that the occurrence of alcoholic fermentation is not primarily due to a limited respiratory capacity. Rather, organic acids produced by the organism may have an uncoupling effect on its respiration. As a result the respiratory activity is enhanced and reaches its maximum at a dilution rate of 0.38 h-1. An attempt was made to interpret the dilution rate-dependent formation of ethanol and acetate in glucose-limited chemostat cultures of S. cerevisiae CBS 8066 as an effect of overflow metabolism at the pyruvate level. Therefore, the activities of pyruvate decarboxylase, NAD+- and NADP+-dependent acetaldehyde dehydrogenases, acetyl coenzyme A (acetyl-CoA) synthetase, and alcohol dehydrogenase were determined in extracts of cells grown at various dilution rates. From the enzyme profiles, substrate affinities, and calculated intracellular pyruvate concentrations, the following conclusions were drawn with respect to product formation of cells growing under glucose limitation. (i) Pyruvate decarboxylase, the key enzyme of alcoholic fermentation, probably already is operative under conditions in which alcoholic fermentation is absent. The acetaldehyde produced by the enzyme is then oxidized via acetaldehyde dehydrogenases and acetyl-CoA synthetase. The acetyl-CoA thus formed is further oxidized in the mitochondria. (ii) Acetate formation results from insufficient activity of acetyl-CoA synthetase, required for the complete oxidation of acetate. Ethanol formation results from insufficient activity of acetaldehyde dehydrogenases.(ABSTRACT TRUNCATED AT 400 WORDS)     

Enzymic analysis of the crabtree effect in glucose-limited chemostat cultures of Saccharomyces cerevisiae.

The physiology of Saccharomyces cerevisiae CBS 8066 was studied in glucose-limited chemostat cultures. Below a dilution rate of 0.30 h-1 glucose was completely respired, and biomass and CO2 were the only products formed. Above this dilution rate acetate and pyruvate appeared in the culture fluid, accompanied by disproportional increases in the rates of oxygen consumption and carbon dioxide production. This enhanced respiratory activity was accompanied by a drop in cell yield from 0.50 to 0.47 g (dry weight) g of glucose-1. At a dilution rate of 0.38 h-1 the culture reached its maximal oxidation capacity of 12 mmol of O2 g (dry weight)-1 h-1. A further increase in the dilution rate resulted in aerobic alcoholic fermentation in addition to respiration, accompanied by an additional decrease in cell yield from 0.47 to 0.16 g (dry weight) g of glucose-1. Since the high respiratory activity of the yeast at intermediary dilution rates would allow for full respiratory metabolism of glucose up to dilution rates close to mumax, we conclude that the occurrence of alcoholic fermentation is not primarily due to a limited respiratory capacity. Rather, organic acids produced by the organism may have an uncoupling effect on its respiration. As a result the respiratory activity is enhanced and reaches its maximum at a dilution rate of 0.38 h-1. An attempt was made to interpret the dilution rate-dependent formation of ethanol and acetate in glucose-limited chemostat cultures of S. cerevisiae CBS 8066 as an effect of overflow metabolism at the pyruvate level. Therefore, the activities of pyruvate decarboxylase, NAD+- and NADP+-dependent acetaldehyde dehydrogenases, acetyl coenzyme A (acetyl-CoA) synthetase, and alcohol dehydrogenase were determined in extracts of cells grown at various dilution rates. From the enzyme profiles, substrate affinities, and calculated intracellular pyruvate concentrations, the following conclusions were drawn with respect to product formation of cells growing under glucose limitation. (i) Pyruvate decarboxylase, the key enzyme of alcoholic fermentation, probably already is operative under conditions in which alcoholic fermentation is absent. The acetaldehyde produced by the enzyme is then oxidized via acetaldehyde dehydrogenases and acetyl-CoA synthetase. The acetyl-CoA thus formed is further oxidized in the mitochondria. (ii) Acetate formation results from insufficient activity of acetyl-CoA synthetase, required for the complete oxidation of acetate. Ethanol formation results from insufficient activity of acetaldehyde dehydrogenases.(ABSTRACT TRUNCATED AT 400 WORDS)     

Kinetics and stoichiometry of growth of plant cell cultures of Catharanthus roseus and Nicotiana tabacum in batch and continuous fermentors

Plant cell suspension cultures of Catharanthus roseus and Nicotiana tabacum were grown in stirred tank bioreactors operated in batch and continuous mode. The stoichiometry of growth of both species in steady-state glucose limited chemostats was studied at a range of different dilution rates. A linear relation was applied to describe specific glucose uptake, oxygen consumption, and carbon dioxide production as a function of the growth rate. Specific respiration deviated greatly from the linear relation. An unstructured mathematical model, based on the observed stoichiometry in the glucose limited chemostats, was applied to describe the growth in batch culture. From a comparison between the observed growth pattern in batch fermentors and computer simulations it appeared that the stoichiometry of growth of the C. roseus culture was different under steady-state and dynamic conditions. It was concluded that a mathematical model for the growth of suspension culture plant cells in which the biomass is considered to be a single compound with an average chemical composition is of limited value because large changes in the conmposition of the biomass may occur. (c) 1992 John Wiley & Sons, Inc.     

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.     

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.     

Calorimetric and stoichiometric analysis of growth of Kluyveromices fragilis in continuous culture: nitrogen limitation imposed upon carbon-limited growth

The calorimetric response of the yeast Kluyveromices fragilis was investigated for growth in continuous culture where nitrogen limitation was imposed on a carbon-limited culture. Calorimetric measurements were combined with off gas analysis, measurements of biomass, substrate and product concentrations, elemental biomass composition, and heat production to study the physiological response of K. fragilis. Regions where both carbon and nitrogen limited growth, were found over a broad range of dilution rates and feed carbon-to-nitrogen ratios. The principle mechanism by which K. fragilis accommodated regions of dual carbon and nitrogen limitation was by partial decoupling of the anabolic and catabolic pathways. When the culture was only nitrogen-limited, increased decoupling of the two pathways was observed. The principal effect of the decoupling was an increased catabolic consumption of glucose, generating an increased heat yield. The preferred way to process the excess glucose was through respiration but the cells were also capable of fermenting a small percentage of the excess glucose in specific cases where the dissolved oxygen partial pressure approached zero. In addition, these results were qualitatively compared to similar studies on Saccharomices cerevisiae. The two yeasts were similar in their ability to accommodate dual limitation by uncoupling anabolic biomass formation from substrate consumption. The two yeasts were dissimilar in how the catabolic substrate was processed. For S. cerevisiae the presence of a bottleneck in the respiration pathway dictated that the majority of the catabolic glucose consumption was by fermentation. For K. fragilis, the lack of a bottleneck in the respiration pathway dictated that the majority of catabolic glucose substrate consumption was by respiration.     

Kinetics of biphasic growth of yeast in continuous and fed-batch cultures

The influence of dilution rate on the production of biomass, ethanol, and invertase in an aerobic culture of Saccharomyces carlsbergensis was studied in a glucose-limited chemostat culture. A kinetic model was developed to analyze the biphasic growth of yeast on both the glucose remaining and the ethanol produced in the culture. The model assumes a double effect where glucose regulates the flux of glucose catabolism (respiration and aerobic fermentation) and the ethanol utilization in yeast cells. The model could successfully demonstrate the experimental results of a chemostat culture featuring the monotonic decrease of biomass concentration with an increase of dilution rate higher than 0.2 hr^?1 as well as the maximum ethanol concentration at a particular dilution rate around 0.5 hr^?1. Some supplementary data were collected from an ethanol-limited aerobic chemostat culture and a glucose-limited anaerobic chemostat culture to use in the model calculation. Some parametric constants of cell growth, ethanol production, and invertase formation were determined in batch cultures under aerobic and anaerobic states as summarized in a table in comparison with the chemostat data. Using the constants, a prediction of the optimal control of a glucose fed-batch yeast culture was conducted in connection with an experiment for harvesting a high yield of yeast cells with high invertase activity.     

Influence of the ethanol and glucose supply rate on the rate and enantioselectivity of 3-oxo ester reduction by baker's yeast

Baker's-yeast-mediated reductions of ketones hold great potential for the industrial production of enantiopure alcohols. In this article we describe the stoichiometry and kinetics of asymmetric ketone reduction by cell suspensions of bakers' yeast (Saccharomyces cerevisiae). A system for quantitative analysis of 3-oxo ester reduction was developed and allowed construction of full mass and redox balances as well as determination of the influence of different process parameters on aerobic ketone reduction. The nature of the electron donor (ethanol or glucose) and its specific consumption rate by the biomass (0-1 mol.kg dw(-1).h(-1)) affected the overall stoichiometry and rate of the process and the final enantiomeric excess of the product. Excess glucose as the electron donor, i.e. a very high consumption rate of glucose, resulted in a high rate of alcoholic fermentation, oxygen consumption, and biomass formation and therefore causing low efficiency of glucose utilization. Controlled supply of the electron donor at the highest rates applied prevented alcoholic fermentation but still resulted in biomass formation and a high oxygen requirement, while low rates resulted in a more efficient use of the electron donor. Low supply rates of ethanol resulted in biomass decrease while low supply rates of glucose provided the most efficient strategy for electron donor provision and yielded a high enantiomeric excess of ethyl (S)-3-hydroxybutanoate. In contrast to batchwise conversions with excess glucose as the electron donor, this strategy prevented by-product formation and biomass increase, and resulted in a low oxygen requirement.     

Growth of a Catharanthus roseus cell suspension culture in a modified chemostat under glucose-limiting conditions

Summary A system for the continuous cultivation of plant cells has been developed, based on a commercially available 3–1 turbine-stirred fermentor. A special device was constructed to provide for homogeneous effluent from the culture at low dilution rates. Two steady states with Catharanthus roseus cells growing under glucose limitation are described with respect to biomass yield on the carbon and energy source glucose, specific oxygen consumption, specific carbon dioxide production and (by)product formation. From a carbon balance for each steady state it is shown that the flow of carbon to the culture (as glucose) practically equalled the flow of carbon from the culture (as biomass, carbon dioxide and (by)product). Biomass yields on glucose were 0.31 g/g and 0.35 g/g at dilution rates of 0.0060 l/h and 0.0081 l/h respectively. The striking difference between the obtained yield coefficients and biomass yield commonly found for batch-cultured plant cells is discussed.     

Physiology of the yeast Kluyveromyces marxianus during batch and chemostat cultures with glucose as the sole carbon source

Growth, substrate consumption, metabolite formation, biomass composition and respiratory parameters of Kluyveromyces marxianus ATCC 26548 were determined during aerobic batch and chemostat cultivations, using mineral medium with glucose as the sole carbon source, at 30 degrees C and pH 5.0. Carbon balances closed within 95-101% in all experiments. A maximum specific growth rate of 0.56 h(-1), a biomass yield on glucose of 0.51 g g(-1), and a maximum specific consumption of oxygen of 11.1 mmol g(-1) h(-1) were obtained during batch cultures. The concentration of excreted metabolites was very low at the culture conditions applied, representing 6% of the consumed carbon at most. Acetate and pyruvate were excreted to a larger extent than ethanol under the batch conditions, and the protein content accounted for 54.6% of the biomass dry weight. Steady states were obtained during chemostats at dilution rates of 0.1, 0.25 and 0.5 h(-1). At the two former dilution rates, cells grew at carbon limitation and the biomass yield on glucose was similar to that obtained under the batch conditions. Metabolite formation was rather low, accounting for a total of 0.005 C-mol C-mol(-1) substrate. At 0.5 h(-1), although the biomass yield on glucose was similar to the value obtained under the above-mentioned conditions, the cultivation was not under carbon limitation. Under this condition, 2-oxoglutarate, acetate, pyruvate and ethanol were the prevalent metabolites excreted. Total metabolite formation only accounted to 0.056 C-mol C-mol(-1) of substrate. A very high protein and a low carbohydrate content (71.9% and 9.6% of biomass dry weight, respectively) were measured in cells under this condition. It is concluded that K. marxianus aligns with the so-called aerobic-respiring or Crabtree-negative yeasts. Furthermore, it has one of the highest growth rates among yeasts, and a high capacity of converting sugar into biomass, even when carbon is not the limiting nutrient. These results provide useful data regarding the future application of K. marxianus in processes aimed at the production of biomass-linked compounds, with high yields and productivities.     

Production of extracellular protease and glucose uptake in Bacillus clausii in steady-state and transient continuous cultures

The production of the extracellular alkaline protease Savinase (EC 3.4.21.62) and glucose uptake in a non-sporulating strain of Bacillus clausii were investigated by analysing steady-state and transients during continuous cultivations. The specific production rate was found to have an optimum at a dilution rate between 0.14 and 0.17 h(-1), whereas the yield of Savinase on glucose was found to increase with decreasing specific growth rate. A linear relationship between the ribosomal RNA content and the specific production rate was found, indicating that the translational capacity may be limiting for product formation. The dynamics of the production of Savinase were studied during step changes in the dilution rate. During a step down in the dilution rate the specific production rate decreased immediately until it reached a new steady value. During a step-up an initial cease in the production rate was observed, but when glucose stopped to accumulate the production rate was regained. The glucose uptake was further investigated when chemostat cultures growing at different dilution rates were exposed to glucose pulses. The maximal glucose uptake capacity was found to be dependent on the initial specific growth rate. Furthermore, the adaptation to high glucose concentrations was faster at high dilution rates than at low dilution rates.     

Effect of dilution rate on growth characteristics of Candida utilis in multistage culture systems with different interstage mixing

The effect of dilution rate on important process parameters of biomass production in two multistage culture systems with different interstage mixing has been examined. Experiments were performed in a multistage tower fermenter and in a cascade of fermenters. Measurements were made at steady-state of continuous culture under constant and identical values of ethanol concentration of 50 gl^?1 in the feed, temperature, OTR and pH in both culture systems used. The microorganism used was Candida utilis. Ethanol inhibition influenced cell growth rate due to the complete dissimilation of the restricted quantity of acetate to H₂O and CO₂, leading to insufficient energy generation. The value of ethanol concentration at which ethanol started to inhibit cell growth was a combined function of OTR, S_R and D. The presence of the interstage mixing resulted in more efficient ethanol conversion to biomass in the whole range of dilution rates and significantly lowered the risk of washing-out at high values of both S_R and D.     

Activities of plasma membrane-bound enzymes isolated from roots of spruce (Picea abies) grown in the presence of aluminium

In batch cultures of Petunia hybrida cv. Rosy Morn Fertile. one respiratory peak is usually observed shortly after subculturing. However, two types of peak respiration could be distinguished, one connected with the dilution process and one with sugar addition at low biomass concentrations. The dilution peak was observed when cells were diluted in medium without sugar, in the presence or absence of mannitol. The sugar peak occurred only after previous dilution of the cells and not when sugar is added at high biomass concentrations Apparently the existence of a dilute suspension is a prerequisite for the induction of the peak. The presence of sugar is not a prerequisite for the increased respiratory activity but it is necessary lor growth: however, growth is possible without the increase in respiration, as was shown by the addition of sugar to a culture with a high biomass concentration. The peak caused by dilution either in the presence or absence of sugar showed no significant differences in height. The height of the peak caused by sugar addition to a previously diluted cell suspension was correlated with the sugar concentration. The respiratory peak disappeared long before the end of the growth period; this decline of the respiratory rates was not connected to sugar or oxygen limitation. In a continuous culture of Petunia hybrida growing at low biomass concentration, the respiration was always at the high level as observed during the peak of batch culture. Growing at lower biomass concentrations might be more expensive for plant cell suspensions.     

Continuous culture of Candida boidinii variant 60 growing on methanol

Summary A new variant, Candida boidinii variant 60, which is less sensitive to methanol and formaldehyde shocks was grown in continuous cultures with methanol as sole carbon source. The substrate concentration in the feeding medium was either 1% methanol or 3% methanol. Biomass production, methanol consumption, the formation of formaldehyde and gas exchange were measured at different dilution rates. With low methanol feeding (10 g/l) maximal productivity of 0.44 g biomass/l·h is obtained at a dilution rate of 0.14 h^–1. Maximal specific growth rate is 0.18 h^–1. A yield of 0.32 g biomass/g methanol was obtained and the respiration quotient was determined as 0.55. Independently of initial substrate concentration, biomass decreases if methanol and formaldehyde are accumulating in the culture broth.In the culture with high methanol feeding (30 g/l) cell concentratioon increases up to 9 g/l at D=0.04 h^–1. At higher dilution rates methanol and form-aldehyde appear in the medium. Formaldehyde is then preferably oxidized without energy advantages for the cells. It seems that this enables the cells to overcome toxic effects caused by methanol and formaldehyde.     

Kinetics of growth and leukotoxin production by <Emphasis Type="Italic">Mannheimia haemolytica</Emphasis> in continuous culture

The growth and product formation kinetics of the bovine pathogen Mannheimia (Pasteurella) haemolytica strain OVI-1 in continuous culture were investigated. The leukotoxin (LKT) concentration and yield on biomass could substantially be enhanced by supplementation of a carbon-limited medium with an amino acid mixture or a mixture of cysteine and glutamine. Acetic acid was a major product, increasing to 1.66 g l(-1) in carbon-limited chemostat culture at intermediate dilution rates and accounting for more than 80% of the glucose carbon, whereas in amino acid-limited cultures high acetic acid concentrations were produced at low dilution rates, suggesting a carbon-overflow metabolism. The maintenance coefficients of carbon-limited and carbon-sufficient cultures were 0.07 and 0.88 mmol glucose g(-1) h(-1), respectively. LKT production was partially growth-associated and the LKT concentration was maximised to 0.15 g l(-1) and acetic acid production minimised by using a carbon-limited medium and a low dilution rate.     

Physiological, biochemical, and mathematical studies of micro-aerobic continuous ethanol fermentation by Saccharomyces cerevisiae. I: hysteresis, oscillations, and maximum specific ethanol productivities in chemostat culture

The growth and metabolism of Saccharomyces cerevisiae was studied in steady-state chemostat cultures under conditions of scarce oxygen and excess glucose. The specific ethanol productivity and specific glucose uptake rate were stimulated by 50% within a narrow range of air/nitrogen mixtures to the fermentor. Fermentation was inhibited at slightly higher and lower air/nitrogen ratios, confirming similar results by previous investigators. This stimulation could not be caused by obvious mechanisms, such as the Pasteur or Crabtree effects. Since this maximum in the fermentation rate occurred in a steady-state chemostat and at a constant dilution rate, the ATP yield of the culture necessarily attained a minimum. Thus, changes in the energetic efficiency of growth or the degree of wasting of ATP were surmised. The steady-state biomass concentration at various oxygenation rates exhibited hysteresis phenomena. Ignition and extinction of the biomass concentration occurred as critical oxygen feed rates were passed. The hysteresis was prevented by adding yeast extract to or reducing the antifoam concentration in the medium. These medium alterations had the simultaneous effect of stimulating the fermentation rate, suggesting that ATP has a critical role in dictating the biomass concentration in micro-aerobic culture. Silicone polymer antifoam was found to stimulate glycerol production at the expense of ethanol production, having consequences for the energy generation and the biomass concentration of the culture.