Citric acid production from glucose. I. Growth and excretion kinetics in a stirred fermentor

The growth and citric acid production kinetics of Saccharomycopsis lipolytica on glucose are investigated in an aerated stirred fermentor. Cellular growth first proceeds exponentially until exhaustion of ammonia in the fermentation medium. Cells then continue to grow at a reduced rate with a concomitant decrease in intracellular nitrogen content. Citric and isocitric acid production starts at the end of the growth phase. During about 80 hr excretion proceeds at a constant rate of 0.7 g/liter/hr for citric acid and 0.1 g/liter/hr for isocitric acid. The final citric and isocitric acid concentrations are 95 and 10g/liter, respectively. During acid excretion cellular respiration accounts for 60 and 35% of consumed oxygen and glucose. Both acid and CO₂ production rates follow a Michaelis–Menten-type dependence on oxygen concentration with Michaelis–Menten constants of 0.9 and 0.15 mg/liter for acid and CO₂ productions, respectively.     

Microcalorimetric investigations of the metabolism of yeasts VII. Flow-calorimetry of aerobic batch cultures

Summary The heat evolution of aerobic batch cultures of growing yeast (Saccharomyces cerevisiae) in glucose media was investigated by a combination of a flow-microcalorimeter with a fermentor vessel. The course of heat production, cell production and the rate of oxygen consumption were qualitatively the same for all glucose concentrations between 10 mM and 100 mM. Under optimal aerobic conditions a triphasic growth was observed due to the fermentation of glucose to ethanol, respiration of ethanol to CO₂ and acetate, and respiration of acetate to C0₂. Energy and carbon were found to be in balance for all glucose concentrations.     

Constant dissolved oxygen concentrations in cephalosporin C fermentation: Applicability of different controllers and effect on fermentation parameters

Summary The behaviour and applicability of several controllers for maintaining a constant dissolved oxygen concentration (DO) during the cephalosporin C production with Cephalosporium acremonium in a laboratory fermentor is described. The process controllers were realized on a MC 68000 based process computer using the real-time language PEARL. The discrete signum integral controller showed the best control action. In addition some derived fermentation data were calculated on-line by the process computer.The results obtained by comparison of fermentations carried out at DO between 10% and 40% saturation during ideophase indicate that high DO leads to a high specific production rate for cephalosporin C and a low specific production rate for penicillin N and vice versa. In the range of DO investigated the production of deacetyl and deacetoxy cephalosporin C is not affected by DO. A direct correlation between DO and the yield coefficients Y _P/S and Y _P/X could be established. The yield coefficient Y _P/O for cephalosporin C is constant in the DO range from 10%–40%.Dedicated to Prof. Dr. H. J. Rehm on the occasion of his 60th birthday     

Microbial production of meso-2,3-butanediol by metabolically engineered Escherichia coli under low oxygen condition

A metabolically engineered Escherichia coli has been constructed for the production of meso-2,3-butanediol (2,3-BD) under low oxygen condition. Genes responsible for 2,3-BD formation from pyruvate were assembled together to generate a high-copy plasmid pEnBD, in which each gene was transcribed with a constitutive promoter. To eliminate by-product formation under low oxygen condition, genes including ldhA, pta, adhE, and poxB which functioned for the mixed acid fermentation pathways were deleted in E. coli JM109. Compared with the wild type, the quadruple gene deletion mutant produced smaller amounts of acetate, succinate, and ethanol from glucose when cultivated in LB medium in shake flasks under low-aeration. When 2,3-BD producing pathway was introduced via pEnBD into the mutant, higher glucose consumption and faster 2,3-BD production rate compared with that of the wild-type control were observed under aerobic condition in shake flasks. In a 6-L fermentor supplied with only 3% dissolved oxygen (DO), the mutant harboring pEnBD converted glucose to 2,3-BD much faster than the control did. When DO supply was further lowered to 1% DO, the recombinant mutant grew much slower but produced 2,3-BD as a major fermentation metabolic product. In addition, the 2,3-BD yield showed an increase from 0.20 g BD/g glucose for the control to 0.43 g BD/g glucose for the mixed acid pathway deleted mutant grown in fermentors under 1% DO. These results reveals the potential of production of enantiomerically pure 2,3-BD isomer by recombinant E. coli under low oxygen condition.     

Hypoxia tolerance of the mummichog: the role of access to the water surface

Low dissolved oxygen (DO) had a significant effect on specific growth rate (G_S), length increment (I_L) and haematocrit (Hct) of the mummichog Fundulus heteroclitus. Regardless of access to the water surface, F. heteroclitus maintained high growth rates (G_S and I_L) at DO concentrations as low as 3 mg O₂ l^?1. With access to the water surface, both G_S and I_L of F. heteroclitus decreased by c. 60% at 1·0 mg O₂ l^?1 compared to all higher DO treatments. When denied access to the water surface, a further decrease in G_S (c. 90%) and I_L (c. 75%) was observed at 1 mg O₂ l^?1. There was no effect of diel‐cycling DO (1–11 mg O₂ l^?1) with or without surface access on G_S, I_L or Hct of F. heteroclitus. Similar trends between G_S and faecal production across DO treatments suggest that decreased feeding contributed significantly to the observed decrease in growth rate. Haematocrit was significantly elevated at 1 mg O₂ l^?1 for fish with and without access to the water surface. Increased Hct, however, was not sufficient to maintain high G_S or I_L at severely low DO. When permitted to respire in the surface layer, however, F. heteroclitus was capable of maintaining moderate growth rates at DO concentrations of 1 mg O₂ l^?1 (c. 15% saturation). Although aquatic surface respiration (ASR) was not quantified in this study, F. heteroclitus routinely swam in contact with the water surface and performed ASR at DO concentrations ≤3 mg O₂ l^?1. No hypoxia‐related mortality was observed in any DO or surface access treatment for as long as 9 days. This study demonstrates that surface access, and thus potential for ASR, plays an important role in providing F. heteroclitus substantial independence of growth rate over a wide range of low DO conditions commonly encountered in shallow estuarine environments.     

Xanthan batch fermentations: compared performances of a bubble column and a stirred tank fermentor

Fermentations of Xanthomonas campestris, NRRL B-1459, were carried out in a bubble column fermentor (BCF) and in a stirred tank fermentor (STF) to allow comparison of representative variables measured during the microbial growth and the gum production. The microbial growth phase was described by a logistic rate equation where maximum cell concentration was provided by nitrogenous compounds balance. The average value of the maximum specific growth rate was higher in the bubble column (μ _M =0.5 h^?1) than in the stirred reactor (μ _M =0.4 h^?1). The upper values of xanthan yield (Y _g-x =0.65 kg xanthan/kg glucose; Y _{O 2?x} xanthan/kg oxygen) and specific production rate (q _x =0.26 kg xanthan/kg biomass · h) were measured when the oxygen transfer coefficient was kept up above 80 h^?1 in the STF fermentor. In the bubble column the fermentation achieved in the same culture medium lasts two times longer than in the stirred aerated tank; this was attributed to the low value of the oxygen transfer coefficient (K _L a =20 h^?1) at the beginning of the gum synthesis phase. The results obtained in the stirred tank were the basis to estimate the optimal biomass concentration which enables to achieve a culture in non-limiting oxygen transfer conditions. Nevertheless, the transfer characteristics were more homogeneous in the bubble column than in the stirred tank where dead stagnant zones were observed. This is of primary importance when establishing fermentation kinetics models.     

Modeling of the mixed culture and periodic control for PHB production

The unstructured mathematical model was developed in the present investigation for the mixed culture, where the metabolites produced by one microorganism is assimilated by the other microorganism. For this, we specifically employed such model system in which sugars such as glucose were converted to lactate by Lactobacillus delbrueckii and the lactate was converted in turn to poly-β-hydroxybutyrate (PHB) by Ralstonia eutropha in one fermentor. Several batch and fed-batch culture experiments were conducted using each microorganism at different dissolved oxygen (DO) concentrations. Those experimental data were then fitted to the mathematical model, which can describe the dynamics of a mixed culture. Some of the model parameters were expressed as functions of DO concentrations, and some of the other model parameters were tuned based on the mixed culture experiments. The model developed describes the effects of such concentrations of glucose, lactate, DO, and NH₃ on the dynamic behavior of such concentrations as both microorganisms, glucose, lactate, and PHB. Optimal operating condition was then investigated using the model developed. It was found that the periodic change in DO concentration improved such performance as PHB yield, and it was verified by experiments. The optimal NH₃ concentration profile was also obtained for the efficient PHB production by the application of the maximum principle.     

Citric acid production from glucose. II. Growth and excretion kinetics in a trickleflow fermentor

The growth and citric acid production kinetics of Saccharomycopsis lipolytica on glucose is investigated in a trickle-flow fermentor. Liquid hold-up and oxygen-transfer coefficient in the reactor column filled with cylindrical wood chips have been determined and found in agreement with chemical engineering correlations. Citric acid production starts at the end of the growth phase and proceeds at a constant specific rate of 0.025 hr^?1for about 80 hr. The fermentor can then be regenerated by addition of ammonia, which induces new growth and excretion phases. Comparing the metabolic behavior of free and immobilized cells, two main kinetic differences are observed. First, the growth phase is linear with the bound cells instead of exponential in the stirred fermentor. Second, in the trickle-bed fermentor acid productivity and oxygen acid yield are reduced by 30%. Oxygen diffusional limitations, mainly in the biomass film, and alterations in bound cell metabolism are shown to be responsible of the kinetic modifications. Simple modelizations of oxygen diffusion effects are also presented to support the interpretation of the experimental data.     

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.     

Characteristics of sisomicin fermentation supplemented with MgSO4 in stirred and air lift fermentors

Summary Sisomicin fermentations were carried out in stirred and air lift fermentors, and various concentrations of MgSO₄ were supplemented to improve antibiotic yield. The highest antibiotic yield was obtained at 80 mM in an air lift fermentor due to effective liberation of intracellular sisomicin by MgSO₄. The presence of high concentrations of MgSO₄, along with shear stress due to strong agitation in the stirred fermentor, resulted in low values of cell activity, rheological parameter(n) and cell viability in the stirred fermentor, compared with the air lift fermentor. The better physiological states of cells grown in the air lift fermentor resulted in improved antibiotic yields.     

Pyranose 2-oxidase (P2O): Production from Trametes versicolor in Stirred Tank Reactor andits Partial Characterization

Abstract

Optimization of pyranose-2-oxidase (P2O) production conditions from Trametes versicolor was carried out in shaking cultures containing glucose, malt, and yeast extracts; the optimum concentration values were found to be 1.5% glucose, 1.0% yeast extract, and 1.0% malt extract, pH 5.0, temperature, 26°C, and agitation rate 150 rpm. For the first time, P2O production was also carried out in a stirred tank reactor (STR) with 2.2 L working volume in the optimized medium composition, and biomass, P2O activity, protein, nitrogen and glucose concentrations were also monitored besides pH and dissolved oxygen (DO). In the STR, P2O activity peaked on day 9. Partial enzyme characterization occurred and optimum pH and temperature were detected as 7.0 and 37°C, respectively. K _m value was found to be 1.009 mM.     

Optimization of culture condition for the production of D-amino acid oxidase in a recombinant Escherichia coli

The gene encoding D-amino acid oxidase (DAAO) from Trigonopsis variabilis CBS 4095 has been cloned and expressed in Escherichia coli BL21 (DE3). Unfortunately, it was observed that the host cell was negatively affected by the expressed DAAO, resulting in a remarkable decrease in cell growth. To overcome this problem, we investigated several factors that affect cell growth rate and DAAO production such as addition time of inducer and dissolved oxygen (DO) concentration. The addition time of lactose, which was used as an inducer, and DO concentration appeared to be critical for the cell growth of E. coli BL21 (DE3)/pET-DAAO. A two-stage DO control strategy was developed, in which the DO concentration was controlled above 50% until specific stage of bacterial growth (OD₆₀₀ 30–40) and then downshifted to 30% by changing the agitation speed and aeration rate, and they remained at these rates until the end of fermentation. With this strategy, the maximum DAAO activity and cell growth reached 18.5 U/mL and OD₆₀₀ 81, respectively. By reproducing these optimized conditions in a 12-m³ fermentor, we were able to produce DAAO at a productivity of 19 U/mL with a cell growth of OD₆₀₀ 80.     

Effects of culture conditions on the co-fermentation of a glucose and xylose mixture to ethanol by a mutant of Saccharomyces diastaticus associated with Pichia stipitis

Substrates that contain hexose as well as pentose sugars can form an interesting substrate for the production of ethanol. Pichia stipitis and a respiratory-deficient mutant of Saccharomyces diastaticus were used to convert such a substrate into ethanol under continuous culture conditions. With a sugar mixture (glucose 70%/xylose 30%) at 50 g/l, the xylose was entirely consumed when the dilution rate (D) did not exceed 0.006 h^–1 whereas the glucose was entirely consumed whatever the D. The study of influence of initial substrate concentration (S₀) was performed at D = 0.015 h^–1. Under these conditions the substrate was entirely consumed when its initial concentration did not exceed 20 g/l. With S₀ = 80 g/l the residual xylose concentration reached 20.5 g/l. At low D or at low S₀, P. stipitis was the dominant species in the fermentor. Increasing the D or S₀ resulted in the wash-out of P. stipitis mainly because of its low ethanol tolerance. Correspondence to: J. P. Delgenes     

溶氧对Blakeslea trispora分批发酵生产β-胡萝卜素的影响

在摇瓶和5 L发酵罐中研究了溶氧 (DO) 对Blakeslea trispora分批发酵生产β-胡萝卜素的影响,总结了5 L发酵罐中β-胡萝卜素发酵过程中溶氧的变化规律.结果表明,当500 mL摇瓶装液量为50 mL,转速为240 r/min条件下发酵生产β-胡萝卜素产量最大,达到3.416 g/L; 5 L发酵罐中,在搅拌转速为1 000 r/min,通气量为1.5 vvm的条件下,β-胡萝卜素的产量可达到3.712 g/L,略高于摇瓶,这可能是由于5 L发酵罐中的气液传递和混合状况好于摇瓶,促进了产物的合成.     

Metabolic changes during substrate shifts in continuous cultures of the yeast Candida boidinii variant 60

Summary Substrate shift experiments in chemostat cultures with either methanol or glucose as carbon source were performed with the yeast Candida boidinii variant 60. At low dilution rates of 0.064 h^–1 the culture may be easily shifted from methanol to glucose medium and back again to methanol. From these experiments it can be seen that glucose does not give rise to any catabolite inhibition of alcohol oxidase. Alcohol oxidase and formaldehyde dehydrogenase seem to be regulated by a repression-derepression mechanism, as small basal activities of both these enzymes can still be measured during growth on glucose. On the other hand, formate dehydrogenase activity is completely absent in the presence of glucose. This kind of regulation seems to favor the smooth switch from growth on glucose to methanol metabolism.With methanol or glucose, growth yields (Y_S) of 0.3 and 0.35, respectively may be obtained, and oxygen consumption (Q_{O 2}) is much higher in methanol cultures than in glucose-grown cells. Accordingly, the RQ values during growth on methanol decrease to about 0.5. Based on the yield coefficient of 0.3, it is possible to calculate that 38% of the methanol consumed must be incorporated into biomass, whereas 62% of the methanol is oxidized to CO₂. The corresponding RQ of 0.56 could not be experimentally ascertained.The activities of three mitochondrial enzymes were found to be higher in methanol-grown cells than in cells from glucose cultures. The low activites of enzymes for the phosphogluconate route in methanol-grown cells indicates that a cyclic oxidation of formaldehyde via hexose phosphate to CO₂ cannot be of great importance for methanol metabolism.List of Symbols D 1/h Dilution rate - 1/h Specific growth rate - Q_{CO 2} mmol/g·h Specific CO₂ production rate - Q_{O 2} mmol/g·h Specific O₂ comsumption rate - Q_S g/g·h Specific substrate consumption rate - RQ ./. Respiratory quotient (Q_{CO 2}/Q_{O 2}) - S_O g/l Substrate concentration in the feeding medium - $#x0073;$#x0304 g/l Substrate concentration in the fermentor - $#x0078;$#x0304 g/l Biomass in the fermentor - Y_{O 2} g/mmol O₂ Biomass yield on oxygen - Y_S g/g Biomass yield on carbon source     

Hydroxyethylation of Comenic Acid

A small jar fermentor was developed in order to investigate the effect of oxygen supply on hydrocarbon fermentation. Several indices to oxygen transfer were examined with this small jar fermentor. Conditions for suitable oxygen supply were examined in l-glutamic acid fermentation from hydrocarbon by use of shaking flasks and these small jar fermentors. The data indicated that the rate of oxygen transfer ought to be more than 14.3 × 10^?7 mole/ml·min in order to obtain satisfactory results. The coefficient of oxygen transfer rate (K_La/H) decreased as the fermentation went on, so the supply of oxygen enriched gas mixture was effective to increase the production of l-glutamic acid.     

Inhibitory effect of oxygen accumulation on the growth of Spirulina platensis

Summary The growth of Spirulina platensis was studied in a light-limited culture under various dissolved oxygen (DO) concentrations. At high DO concentration, e.g. at 1.25 mM DO, the growth rate was decreased up to 36 % compared with that of 0.063 mM DO. The retarded growth rate at high DO concentrations seemed to be coupled with the degeneration of photosynthetic activity in terms of O₂ evolution. Under higher DO concentrations, superoxide dismutase and ascorbate peroxidase activities tended to increase, while the contents of photosynthetic pigment, such phycocyanin, carotenoid and chlorophyll-a decreased distinctly.     

Effect of inoculum size on the aeration pattern of batch cultures of a fungal microorganism

Trichoderma reesei QM 9123 has been grown in batch culture in a 10 liter stirred fermentor, at a temperature of 30°C and pH 4.0. The fermentor was operated at a single stirrer speed of 400 rpm and air rate of 1 v/v/m. The effect of four inoculum sizes (0.5, 1.0, 3.0 and 5.0%) on the growth pattern and the aeration profiles was examined. Logarithmic growth of the fungus was observed. The aeration profile changed with inoculum size and at 5.0%, it was found that the oxygen uptake rate was controlled by the oxygen supply rate, during which the oxygen tension was zero.     

On-line continuous measurement of the oxygen consumption rate in mammalian cell culture

A method for on-line, continuous measurement of the oxygen consumption rate (Io₂) by mammalian cells on a microcarrier was developed and its reliability investigated. Utilizing the periodic dissolved oxygen (DO) fluctuation in the normal on-off control of DO, on-line, continuous measurement of Io₂ was carried out in which real-time estimation of the DO saturation concentration was made by measuring the gas-phase pressure and the gas-phase oxygen concentration. It was found that the continuously measured Io₂ value was quantitatively exact and could be applied commercially using the oxidation reaction of glucose by glucose oxidase.