An analysis of the growth of Gluconobacter oxydans in chemostat cultures

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

Kinetics of product formation in batch and continuous culture of Clostridium barkeri

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

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.     

The effect of vitamins and amino acids on glucose uptake in aerobic chemostat cultures of three Saccharomyces cerevisiae strains

In the respiro-fermentative region of aerobic chemostat cultures at steady state, Saccharomyces cerevisiae CBS 8066 produced high concentrations of ethanol with concomitant low levels of residual glucose which followed Monod kinetics. By contrast, very high residual glucose concentrations were observed in cultures of S. cerevisiae strains ATCC 4126 and NRRL Y132 at dilution rates above 60% of the washout dilution rate, resulting in much lower ethanol concentrations, even though clearly glucose-limited at lower dilution rates in the respiratory region. The addition of a vitamin mixture resulted in decreased residual glucose concentrations in respiro-fermentative cultures of all three strains, but the effect was much more pronounced with strains ATCC 4126 and NRRL Y132. Meso-inositol was mainly responsible for this effect, although with strain ATCC 4126 other vitamins as well as an amino acid mixture were also required to minimise the steady-state residual glucose levels. The residual glucose concentration in continuous culture was, therefore, greatly dependent on the growth factor requirements of the particular yeast strain, which apparently increased on increasing the dilution rate into the respiro-fermentative region. The strain differences with respect to growth factor requirements at high dilution rates, which were not evident at low dilution rates, had a profound effect on the kinetics of glucose assimilation in aerobic chemostat culture.     

Effects of glucose supply on myeloma growth and metabolism in chemostat culture

The effects of the glucose supply on growth and metabolism of an SP2/0 derived recombinant myeloma cell line were studied in chemostat culture during growth on IMDM medium at a fixed dilution rate of 0.032 h^?1. Lowering of the feed medium glucose concentration from 25.0 to 1.4 mmol/L resulted in a decrease of steady-state viable cell concentration from 1.9 × 10⁹ L^?1, whereas viability remained above 90%. Mass balances indicated that only a minor amount of glucose was utilized via the TCA cycle irrespective of the glucose concentration in the feed medium. The apparent biosynthetic yield of cells from ATP was independent of the ratio between the specific glucose and glutamine consumption rate. It is concluded that the primary role of glucose is the provision of intermediates for anabolic reactions. In addition, glucose may play an indirect catabolic role in the process of glutaminolysis by providing the pyruvate for the transamination of glutamate to alanine and α-ketoglutarate. At low glucose concentrations in the feed medium, glutamine is probably the sole energy source for this myeloma in chemostat culture. © 1995 Wiley-Liss, Inc.     

Influence of Environmental Factors and Medium Composition on Vibrio gazogenes Growth and Prodigiosin Production

Vibrio gazogenes ATCC 29988 growth and prodigiosin synthesis were studied in batch culture on complex and defined media and in chemostat cultures on defined medium. In batch culture on complex medium, a maximum growth rate of 0.75 h⁻¹ and a maximum prodigiosin concentration of 80 ng of prodigiosin · mg of cell protein⁻¹ were observed. In batch culture on defined medium, maximum growth rates were lower (maximum growth rate, 0.40 h⁻¹), and maximum prodigiosin concentrations were higher (1,500 ng · mg of protein⁻¹). In batch culture on either complex or defined medium, growth was characterized by a period of logarithmic growth followed by a period of linear growth; on either medium, prodigiosin biosynthesis was maximum during linear growth. In batch culture on defined medium, the initial concentration of glucose optimal for growth and pigment production was 3.0%; higher levels of glucose suppressed synthesis of the pigment. V. gazogenes had an absolute requirement for Na⁺; optimal growth occurred in the presence of 100 mM NaCl. Increases in the concentration of Na⁺ up to 600 mM resulted in further increases in the concentration of pigment in the broth. Prodigiosin was synthesized at a maximum level in the presence of inorganic phosphate concentrations suboptimal for growth. Concentrations of KH₂PO₄ above 0.4 mM caused decreased pigment synthesis, whereas maximum cell growth occurred at 1.0 mM. Optimal growth and pigment production occurred in the presence of 8 to 16 mg of ferric ion · liter⁻¹, with higher concentrations proving inhibitory to both growth and pigment production. Both growth and pigment production were found to decrease with increased concentrations of p-aminobenzoic acid. The highest specific concentration of prodigiosin (3,480 ng · mg protein⁻¹) was observed in chemostat cultures at a dilution rate of 0.057 h⁻¹. The specific rate of prodigiosin production at this dilution rate was approximately 80% greater than that observed in batch culture on defined medium. At dilution rates greater than 0.057 h⁻¹, the concentration of cells decreased with increasing dilution rate, resulting in a profile comparable to that expected for linear growth kinetics. No explanation could be found for the linear growth profiles obtained for both batch and chemostat cultures.     

Fermentation characteristics of Clostridium pasteurianum LMG 3285 grown on glucose and mannitol

Clostridium pasteurianum fermented glucose to acetate, butyrate, CO₂ and H₂. In batch cultures the fermentation pattern was only slightly affected by culture pH over the range 8·0 to 5·5. The acetate/butyrate ratio was always higher than or equal to one. Between 2·14 and 2·33 mol H₂ was produced per mol glucose fermented. At unregulated pH, more butanol and less butyrate was formed. In a carbon-limited chemostat, the steady-state acetate/butyrate ratio was always lower than one. H₂ production was approximately 1·70 mol per mol glucose consumed. Substantial amounts of extracellular protein were formed. With decreasing pH, acetate and formate production decreased, while H₂ production was highest at pH 6.0. With increasing dilution rate ( D ), the product spectrum hardly changed, but more biomass was formed. Y _glucose^max and Y _ATP^max were 55·97 and 31·48 g dry weight per mol glucose or ATP respectively. With increasing glucose input the formation of fatty acids and H₂ slightly decreased.
Continuous cultures fermented mannitol to acetate, butyrate, butanol, CO₂ and H₂. With acetate as co-substrate, butanol production and molar growth yields, Y _mannitol and Y _ATP, markedly decreased, while the butyrate and H₂ production increased. The latter reached a value of 2·21 mol H₂ per mol mannitol consumed.     

Butyric fermentation: metabolic behaviour and production performance of Clostridium tyrobutyricum in a continuous culture with cell recycle

Summary Two physiological characteristics of butyric fermentation, inhibition by the acids produced, butyrate and acetate, and dependence on the growth rate of the distribution of these acids, prompted a study of butyrate production in a continuous fermentation system with cell recycle by microfiltration. The influence of the main operating parameters, glucose input (feed concentration and dilution rate) and bleed dilution rate on production of acids and biomass was studied. The performance of the system greatly exceeded the results obtained in batch and simple continuous fermentations as a high productivity for butyrate (9.5 g l^–1 h^–1) was achieved whilst retaining a satisfactory concentration of butyrate (29.7 g l^–1) and low acetate production (0.6 g l^–1) at a cell biomass concentration of 35 g l^–1. Cell growth rate was found to be a critical parameter for performance stability as oscillations in metabolic activity due to inhibition by acids were observed at bleed dilution rates below 0.016 h^–1.Offprint requests to: J. P. Vandecasteele     

Dynamic analysis of a microbial process: A systems engineering approach

A general mathematical model of the chemostat system is developed in order to define an experimental program of dynamic testing. A glucose-limited culture ofSaccharomyces cerevisiae was grown in a chemostat using chemically defined medium. The chemostat was perturbed from an initial steady state by changes in input glucose concentration, dilution rate, pH, and temperature. Dynamic responses of cell mass, glucose, cell number, RNA, and protein concentrations were measured. A number of simulation techniques were used in developing a dynamic mathematical model and in comparing the developed model with experimental data as well as the Monod model. The resulting model was found to be quantitatively accurate and superior to the Monod model. The developed model was interpreted in the light of cell physiology. Adjustment of intracellular RNA fraction was found to be rate limiting in acceleration of cell specific growth rate.     

Metabolic and energetic aspects of the growth of Clostridium butyricum on glucose in chemostat culture

The influence of a number of environmental parameters on the fermentation of glucose, and on the energetics of growth of Clostridium butyricum in chemostat culture, have been studied. With cultures that were continuously sparged with nitrogen gas, glucose was fermented primarily to acetate and butyrate with a fixed stoichiometry. Thus, irrespective of the growth rate, input glucose concentration specific nutrient limitation and, within limits, the culture pH value, the acetate/butyrate molar ratio in the culture extracellular fluids was uniformly 0.74±0.07. Thus, the efficiency with which ATP was generated from glucose catabolism also was constant at 3.27±0.02 mol ATP/mol glucose fermented. However, the rate of glucose fermentation at a fixed growth rate, and hence the rate of ATP generation, varied markedly under some conditions leading to changes in the Y _glucose and Y _ATP values. In general, glucose-sufficient cultures expressed lower yield values than a correponding glucose-limited culture, and this was particularly marked with a potassium-limited culture. However, with a glucose-limited culture increasing the input glucose concentration above 40g glucose·l^-1 also led to a significant decrease in the yield values that could be partially reversed by increasing the sparging rate of the nitrogen gas. Finally glucose-limited cultures immediately expressed an increased rate of glucose fermentation when relieved of their growth limitation. Since the rate of cell synthesis did not increase instantaneously, again the yield values with respect to glucose consumed and ATP generated transiently decreased.Two conditions were found to effect a change in the fermentation pattern with a lowering of the acetate/butyrate molar ratio. First, a significant decrease in this ratio was observed when a glucose-limited culture was not sparged with nitrogen gas; and second, a substantial (and progressive) decrease was observed to follow addition of increasing amounts of mannitol to a glucose-limited culture. In both cases, however, there was no apparent change in the Y _ATP value.These results are discussed with respect to two imponder-ables, namely the mechanism(s) by which C. butyricum might partially or totally dissociate catabolism from anabolism, and how it might dispose of the excess reductant [as NAD(P)H] that attends both the formation of acetate from glucose and the fermentation of mannitol. With regards to the latter, evidence is presented that supports the conclusion that the ferredoxin-mediated oxidation of NAD(P)H, generating H₂, is neither coupled to, nor driven by, an energy-yielding reaction.     

Protease and lipase production by a strain ofSerratia marcescens (532 S)

Summary Production of both exolipase and exoprotease activities bySerratia marcescens 532 S isolated from an aerobic fixed-biomass reactor were strongly influenced by nutritional factors which acted as inducers or repressors. In batch culture, protease and lipase activities were produced after the exponential phase. NH₄Cl, amino acids and simple carbon sources caused repression of protease activity. At a concentration of 1.5 g L^–1, the individual addition of maltose, mannitol, acetate, fructose or glucose, repressed exoprotease production, with the greatest effect by glucose. An inverse relationship existed between exoprotease synthesis and increasing glucose concentrations. Lipids activated lipase production, the most significant increase occurred when Tween 80 was added in the medium. Thus, glucidolytic, proteolytic and lipolytic activities could be efficiently expressed in batch cultures only successively.At low dilution rate of chemostat cultures with a constant glucose input concentration of 2 g L^–1, glucidolytic, proteolytic and lipolytic activities were produced, but did not have the same regulation: atD values <0.08 h^–1, the level of protease activity dropped while that of lipase showed a corresponding increase. Above these values, increasingD led to a decrease of the two hydrolase activities, at the level of the specific activities as well as in the specific rate of biosynthesis of each enzyme. Similar results were obtained in chemostat culture with a constant specific growth rate of 0.04 h^–1 with increasing glucose input concentrations, i.e. protease and lipase activities decreased when the specific glucose uptake rates were enhanced.     

The relationship between transport kinetics and glucose uptake by Saccharomyces cerevisiae in aerobic chemostat cultures

The steady-state residual glucose concentrations in aerobic chemostat cultures of Saccharomyces cerevisiae ATCC 4126, grown in a complex medium, increased sharply in the respiro-fermentative region, suggesting a large increase in the apparent k_s value. By contrast, strain CBS 8066 exhibited much lower steady-state residual glucose concentrations in this region. Glucose transport assays were conducted with these strains to determine the relationship between transport kinetics and sugar assimilation. With strain CBS 8066, a high-affinity glucose uptake system was evident up to a dilution rate of 0.41 h^–1, with a low-affinity uptake system and high residual glucose levels only evident at the higher dilution rates. With strain ATCC 4126, the high-affinity uptake system was present up to a dilution rate of about 0.38 h^–1, but a low-affinity uptake system was discerned already from a dilution rate of 0.27 h^–1, which coincided with the sharp increase in the residual glucose concentration. Neither of the above yeast strains had an absolute vitamin requirement for aerobic growth. Nevertheless, in the same medium supplemented with vitamins, no low-affinity uptake system was evident in cells of strain ATCC 4126 even at high dilution rates and the steady-state residual glucose concentration was much lower. The shift in the relative proportions of the high and low-affinity uptake systems of strain ATCC 4126, which might have been mediated by an inositol deficiency through its effect on the cell membrane, may offer an explanation for the unusually high steady-state residual glucose concentrations observed at dilution rates above 52% of the wash-out dilution rate.     

High production of 1,3-propanediol from industrial glycerol by a newly isolated Clostridium butyricum strain

Batch and continuous cultures of a newly isolated Clostridium butyricum strain were carried out on industrial glycerol, the major by-product of the bio-diesel production process. For both types of cultures, the conversion yield obtained was around 0.55 g of 1,3-propanediol formed per 1 g of glycerol consumed whereas the highest 1,3-propanediol concentration, achieved during the single-stage continuous cultures was 35-48 g l-1. Moreover, the strain presented a strong tolerance at the inhibitory effect of the 1,3-propanediol, even at high concentrations of this substance at the chemostat (e.g. 80 g l-1). 1,3-Propanediol was associated with cell growth whereas acetate and butyrate seemed non growth-associated products. At low and medium dilution rates (until 0.1 h-1), butyrate production was favoured, whereas at higher rates acetate production increased. The maximum 1,3-propanediol volumetric productivity obtained was 5.5 g l-1 h-1. A two-stage continuous fermentation was also carried out. The first stage presented high 1,3-propanediol volumetric productivity, whereas the second stage (with a lower dilution rate) served to further increase the final product concentration. High 1,3-propanediol concentrations were achieved (41-46 g l-1), with a maximum volumetric productivity of 3.4 g l-1 h-1. A cell concentration decrease was reported between the second and the first fermentor.     

Growth and anthocyanin production by carrot suspension cultures grown under chemostat conditions with phosphate as the limiting nutrient

Chemostat cultures of carrot suspension cultures, where growth was limited by the concentration of phosphate in the input medium, were achieved by replacing a fixed proportion of the culture with fresh medium at daily intervals. In the range 0.05–0.30mM phosphate in the input medium and at a specific growth rate of 0.357 days^?1, steady-state culture density but not anthocyanin in the cells was strictly proportional to the input phosphate concentration with no intercept. At a phosphate concentration of 0.10mM and growth rates from 0.105 to 0.430 days^?1, the steady-state culture density could not be described by Monod's model of chemostat cultures, but could be described by Nyholm's model. The steady-state levels of anthocyanin were not strictly proportional to the steady-state biomass under all conditions, showing that anthocyanin production is not completely growth associated.     

Characteristics of the chemostat culture of murine leukemia L 1210 cells

Mouse leukemia L 1210 cells were cultivated under glucose limitation in a chemostat. More than 20 steady-states were established over 9 different dilution rates ranging from 0.20 day⁻¹ (cell doubling time 83 h) to 2.0 days⁻¹ (cell doubling time 8.3 h). The steady-states were characterized by: a constant cell number, constant cell volume, constant concentrations of DNA, RNA, and L-lactate (in the culture supernatant), a constant percentage of cells labelled by autoradiography, and constant rate of incorporation of [³H]TdR, [³H]uridine, and ¹⁴C-labelled amino acids into cellular acid-precipitable material. Individual steady-states were maintained for periods up to 600 h continuous operation of the chemostat. A maximum output of 66.4 × 10⁶ cells/h was obtained at a dilution rate of 1.3 day⁻¹. The glucose substrate constant was determined as 0.0063 mg/ml. The relationships between dilution rate and the steady-state cell concentration, glucose concentration, and output of L 1210 cells from the chemostat, were in general agreement with the theoretical curves. It was found that the principles of continuous culture derived from the study of microorganisms are to a large extent applicable to the cultivation of animal cells.     

Amino acid consumption by Chloroflexus aurantiacus in batch and continuous cultures

Amino acid consumption was studied with batch and continuous chemostat cultures of Chloroflexus aurantiacus grown phototrophically in complex medium with casamino acids (Pierson and Castenholz 1974). Amino acids like Arg, Asx, Thr, Ala, Tyr, which were utilized during the early exponential phase by cells grown in batch cultures were consumed in chemostat cultures essentially at any of the dilution rates employed (0.018–0.104 h^-1). Those amino acids which were taken up during subsequent phases of growth were consumed in chemostat cultures preferentially at low dilution rates. For example, the consumption of Glx was enhanced during the late exponential phase and at low dilution rates. At high dilution rates Glx was not consumed at all. Since Glx utilization largely paralleled bacteriochlorophyll formation, it is discussed that formation of the photopigment depends on the intracellular availability of Glu as the exclusive precursor for tetrapyrrole synthesis.     

Production of butanol by Clostridium puniceum in batch and continuous culture

Summary The pink-pigmented, amylolytic and pectinolytic bacterium Clostridium puniceum in anaerobic batch culture at pH 5.5 and 25–30°C produced butan-1-ol as the major product of fermentation of glucose or starch. The alcohol was formed throughout the exponential phase of growth and surprisingly little acetone was simultaneously produced. Furthermore, acetic and butyric acids were only accumulated in low concentrations, and under optimal conditions were completely re-utilised before the fermentation ceased. Thus, in a minimal medium containing 4% w/v glucose as sole source of carbon and energy, after 65 h at 25°C, pH 5.5 all of the glucose had been consumed to yield (g product/100 g glucose utilised) butanol 32, acetone 3 and ethanol 2. Butanol was again the major product of glucose fermentation during phosphate-limited chemostat culture wherein, although the organism eventually lost its capacity to sporulate and to synthesize granulose, production of butanol continued for at least 100 volume changes. Under no growth condition was the organism capable of producing more than 13.3 g l^-1 of butanol. At pH 5.5, growth on pectin was slow and yielded a markedly lesser biomass concentration than when growth was on glucose or starch; acetic acid was the major fermentation product with lower concentrations of methanol, acetone, butanol and butyric acid. At pH 7, growth on all substrates produced virtually no solvents but high concentrations of both acetic and butyric acids.     

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.     

Energetics and product formation by Saccharomyces cerevisiae grown in anaerobic chemostats under nitrogen limitation

Anaerobic fermentation of glucose (20 g/l) by Saccharomyces cerevisiae CBS 8066 was studied in a chemostat (dilution rate = 0.05–0.25 h^–1) at different concentrations of the nitrogen source (5.00 g/l or 0.36 g/l ammonium sulphate). The ethanol yield (g ethanol produced/g glucose consumed) was found to be higher and the glycerol yield (g glycerol formed/g glucose consumed) lower during nitrogen limitation than under carbon limitation. The biomass yield on ATP (g dry weight biomass produced/mol ATP consumed) was consequently found to be lower during nitrogen-limited conditions.