Simultaneous measurements of oxygen diffusion coefficients and solubilities in fermentation media with polarographic oxygen electrodes

A membrane-covered oxygen electrode was used to measure oxygen diffusion coefficients and solubilities in aqueous glucose solutions and various fermentation media following a newly developed methodology. The fermentation media studied were tryptic soy broth and those for fermentations of Penicillium chrysogenum, Saccharomyces cerevisiae, and Micrococcus glutamicus. The experimental results of oxygen diffusion coefficients and solubilities in glucose solutions were in good accord with the literature data. As for the fermentation media, both oxygen diffusion coefficients and solubilities were found to decrease with an increased fractional composition of these media, and log-additive behaviors of the oxygen diffusion coefficients and solubilities in fermentation media were observed.     

Effects of microorganisms on effective oxygen diffusion coefficients and solubilities in fermentation media

Effective oxygen diffusion coefficients and solubilities were measured for submerged cultures of Saccharomyces cerevisiae, Escherichia coli, and Penicillium chrysogenum. Both effective oxygen diffusion coefficients and solubilities were found to decrease with increasing cell concentrations in the fermentation media. Comparison of the experimental results of effective oxygen diffusion coefficients in fermentation media with values theoretically predicted on the assumption of unpenetrable microbial cells indicates that oxygen molecules diffuse through the cells during the diffusion process. Within the cell concentration range of typical submerged fermentations, the effective oxygen diffusion coefficient of the fermentation media can be described as D(e) = A(1)f + A(2)f(2). In this equation, fis the cell volume fraction and both A(1) and A(2) are functions of the shape of the cells and the ratio of effective oxygen diffusion coefficient in microbial cells to that in the medium.     

Extraction of penicillin-G by aqueous two-phase partition

Summary Pure potassium penicillin-G solution and penicillin-G fermentation broth were extracted using the aqueous two-phase systems of poly-ethylene glycol and salts. The partition coefficients of penicillin-G were above 10 in both pure solution and broth systems. The partition coefficients of phenyl acetic acid were about 0.25. Cell mass and solid residue in the broth system were partly concentrated around the interfacial region and partly settled to the bottom when under 1000 × g centrifugal force. Accordingly, this technique is a promising alternative for the recovery of penicillins.     

The anomaly of oxygen diffusion in aqueous xanthan solutions

A membrane-covered polarographic oxygen electrode was used to measure oxygen diffusion coefficients in aqueous polyelectrolyte solutions of xanthan gum, sodium alginate, and sodium carboxymethylcellulose (CMC). In sodium alginate solutions, dilute xanthan solutions, and solutions containing more than 0.3 wt % CMC, oxygen diffusion coefficients decrease with increasing polymer concentrations. Interestingly, in dilute CMC solutions and concentrate xanthan solutions containing more than 0.5 wt % xanthan gum, oxygen diffusion coefficients increase with increasing polymer concentrations, and values exceeding that in pure water are generally observed.     

A comparison of three models for the diffusion of oxygen in electrolyte solutions

Diffusion of oxygen through aqueous solutions is of great importance in biological systems. In this work, three models for the diffusion of oxygen through aqueous salt solutions are compared. One model uses mole fraction as the driving force (Fick's Law) and another uses chemical potential. The third model uses the gradient in oxygen activity as the driving force. This new model was chosen because of the availability of oxygen electrodes which directly measure oxygen activity in aqueous solution. These models have been used to reevaluate the technique of measuring O(2) diffusivities. We show that Pick's Law diffusion coefficients do not vary strongly with salt concentration as was erroneously reported in the literature. In addition, we compare the predicted O(2) fluxes of the three models over a wide range in O(2) concentrations. For oxygen concentrations of biological interest, the three models give identical predictions of the flux.     

Oxygen solubilities in fermentation fluids

Summary Fermentation media consist of a large number of chemicals whose composition undergoes alteration during the course of fermentation. As a result of this, conventional methods and correlations for oxygen solubility measurement and prediction do not apply in these systems. Using a physical method, oxygen solubilities were measured in simulated chemical systems and in fermentation broths. Sugars, salts, and fermentation products were identified as major factors influencing oxygen solubility. Salt effect was correlated with electrical conductivity of the medium, which was easy to measure during fermentation. For mixtures and for fermentation medium, individual influences were found to be log-additive in accordance with Danckwerts (1970).     

Transfer free energies of peptide backbone unit from water to aqueous electrolyte solutions at 298.15 K

Transfer free energies (ΔG_tr) of amino acids from water to aqueous electrolyte solutions have been determined from the solubility measurements, as a function of salt concentration at 298.15 K under atmospheric pressure. The investigated aqueous systems contain amino acids of zwitterionic glycine peptides: glycine (Gly), diglycine (Gly₂), triglycine (Gly₃), and tetraglycine (Gly₄) and cyclic glycylglycine (c(GG)) with an electrolyte compound of potassium chloride (KCl), potassium bromide (KBr) or potassium acetate (KAc). The solubilities of glycine and diglycine in aqueous solution decrease with increasing the concentration of salts (salting-out effect), whereas those of triglycine and tetraglycine increase with increasing the concentration of salts (salting-in effect). Furthermore, salting-in effect was found in aqueous c(GG)/KBr system, while salting-out effect was observed in aqueous c(GG)/KCl or c(GG)/KAc system. The experimental results were used to estimate the transfer free energies (Δg_tr) of the peptide backbone unit (–CH₂CONH–) from water to the aqueous electrolyte solutions. We developed a new trail to determine the activity coefficients (γ) for aqueous and aqueous electrolyte solutions using an activity coefficient model, with which the total contribution of transfer free energy between solute and the solvent was calculated. We compared the difference between neglecting and using the activity coefficients term in predicting ΔG_tr. Since the transfer free energy contribution is negative, interactions between the ionic salts and the peptide backbone unit of zwitterionic glycine peptides are favorable and thus the ionic salts destabilize these amino acids. It was also found that KBr stabilizes c(GG), whereas KCl and KAc destabilize c(GG). These results provide evidence for the existence of interactions between the amide unit and ionic salts, in aqueous solution, which may be of importance in maintaining protein structure as well as in protein–solute and protein–solvent interactions.     

Effect of the interfacial surfactant layer on oxygen transfer through the oil/water phase boundary in perfluorocarbon emulsions

The effect of interfacial surfactant molecules on oxygen transfer through oil/water phase boundary has been studied in FlurO(2) (TM) emulsions, i.e., perfluorocarbon (PFC) emulsions developed as oxygen carriers in cell culture. Measurements of oxygen permeability were made with a polarographic oxygen electrode in pure PFCs and in emulsions with various PFC volume fractions. Comparison of the experimental results with the theoretically derived values of relative oxygen permeability clearly indicates that the mass transfer resistance caused by the interfacial surfactant layer in PFC emulsions is insignificant. Therefore, oxygen dissolved in the enclosed PFC phase is readily available to cells growing in the aqueous media and FlurO(2) emulsions with very fine emulsion particles (< 0.2 mum) can be used to effectively enhance gas/liquid interfacial oxygen transfer in bioreactors. The inadequacy in describing mass transfer in heterogeneous systems, such as the PFC emulsions, by conventional concentration-based oxygen diffusion coefficients has also been discussed.     

Hydrogen peroxide and superoxide radical formation in anaerobic broth media exposed to atmospheric oxygen.

Fourteen different broth media were autoclaved under anaerobic conditions and then exposed to atmospheric oxygen. The hydrogen peroxide and superoxide radical formation as well as the bactericidal effect of the media were studied. The rate of killing of Peptostreptococcus anaerobius VPI 4330-1 was high in media that rapidly autoxidized and accumulated hydrogen peroxide. In actinomyces broth (BBL), 50% of the cells were killed within 2 min, and in Brewer thioglycolate medium (Difco), 50% were killed within 11 min, whereas more than 50% of the cells survived for more than 2 h in Clausen medium (Oxoid), fluid thioglycolate medium (BBL), and thioglycolate medium without dextrose or indicator (Difco). Only media that contained phosphate and glucose had a tendency to accumulate hydrogen peroxide. A solution of phosphate and glucose autoxidized when it had been heated to 120 degrees C for at least 5 min and when the pH of the solution was higher than 6.5. Transitional metal ions catalyzed the autoxidation, but they were not necessary for the reaction to occur. Of the other substances heated in phosphate buffer, only alpha-hydroxycarbonyl compounds autoxidized with accumulation of hydrogen peroxide. Superoxide dismutase decreased the autoxidation rate of most of the broth media. This indicated that superoxide radicals were generated in these media.     

Hydrogen peroxide and superoxide radical formation in anaerobic broth media exposed to atmospheric oxygen.

Fourteen different broth media were autoclaved under anaerobic conditions and then exposed to atmospheric oxygen. The hydrogen peroxide and superoxide radical formation as well as the bactericidal effect of the media were studied. The rate of killing of Peptostreptococcus anaerobius VPI 4330-1 was high in media that rapidly autoxidized and accumulated hydrogen peroxide. In actinomyces broth (BBL), 50% of the cells were killed within 2 min, and in Brewer thioglycolate medium (Difco), 50% were killed within 11 min, whereas more than 50% of the cells survived for more than 2 h in Clausen medium (Oxoid), fluid thioglycolate medium (BBL), and thioglycolate medium without dextrose or indicator (Difco). Only media that contained phosphate and glucose had a tendency to accumulate hydrogen peroxide. A solution of phosphate and glucose autoxidized when it had been heated to 120 degrees C for at least 5 min and when the pH of the solution was higher than 6.5. Transitional metal ions catalyzed the autoxidation, but they were not necessary for the reaction to occur. Of the other substances heated in phosphate buffer, only alpha-hydroxycarbonyl compounds autoxidized with accumulation of hydrogen peroxide. Superoxide dismutase decreased the autoxidation rate of most of the broth media. This indicated that superoxide radicals were generated in these media.     

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

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

促溶剂对雷斯青霉发酵液流变特性的影响

目的:促溶剂通常用于甾体生物催化过程以提高底物溶解度,但在发酵液中添加促溶剂对菌体形态及发酵液特性的影响还少有报道。方法:利用旋转流变仪和顺磁分析仪分别对发酵液的流变特性及体积氧传递系数KLa进行监测。结果:无论是否添加促溶剂,发酵液都表现出非牛顿流体力学特性,但添加3%1,2丙二醇后同一时期发酵液稠度系数减小大约17%,而流动指数平均增加8%。结论:添加促溶剂使得发酵液表观黏度减小,体积氧传递系数增大,从而有利于甾体化合物的生物转化。     

Diffusion in bile and its implications on detergency.

Diffusion coefficients of bile salts, lecithin, and cholesterol above the critical micelle concentration have been measured with the diaphragm cell at varying concentrations of bile salts, lecithin, and added electrolyte. The diffusion of the bile salt can be five times faster than that of the solubilized lipids. This is shown not to be an artifact of multicomponent diffusion, but a result of a different transport mechanism of the bile salt. As a consequence, the concentration of bile salt and lipids at the surface of a cholesterol gallstone can differ from those in the bile solution. The effects of this upon growth and dissolution in detergent solutions are discussed.     

Pervaporative butanol fermentation by Clostridium acetobutylicum B18

Extractive acetone-butanol-ethanol (ABE) fermentation was carried out successfully using pervaporation and a low-acid-producing Clostridium acetobutylicum B18. A pervaporation module with 0.17 m(2) of surface area was made of silicone membrane of 240 mum thickness. Pervaporation experiments using make-up solutions showed that butanol and acetone fluxes increased linearly with their concentrations in the aqueous phase. Fickian diffusion coefficients were constants for fixed air flow rates, and increased at higher sweep air flow rates. During batch and fed-batch fermentations, pervaporation at an air flow rate of 8 L/min removed butanol and acetone efficiently. Butanol concentration was maintained below 4.5 g/L even though Clostridium acetobutylicum B18 produced butanol steadily. Pervaporation could not remove organic acids efficiently, but organic acids did not accumulate because strain B18 produced little organic acid and recycled added organic acids efficiently. With pervaporation, glucose consumption rate increased compared to without pervaporation, and up to 160 g/L of glucose was consumed during 80 h. Cell growth was not inhibited by possible salt accumulation or oxygen diffusion through the silicone tubing. The culture volume was maintained relatively constant during fed-batch operation because of an offsetting effect of water and product removal by pervaporation and addition of nutrient supplements. (c) 1994 John Wiley & Sons, Inc.     

On the methodology of dissolved oxygen saturation concentration measurements

Summary An experimental method for the determination of the concentration of dissolved oxygen saturation in real fermentation media is described. It is based on a joint analysis of gas and liquid phases, applying a mass spectrometer for gas analyses and an oxygen electrode for the liquid phase measurements. This method enables the experimental measurement of oxygen solubilities in real fermentation broth within the bioreactor during the process and its application seems to be of general validity.     

Comparison of measured and calculated oxygen solubilities in fermentation media

A fast physical method is used to measure oxygen solubilities in various solutions of sugars and salts, in nutrition media, and in fermentation broths of Penicillium chrysogenum. The findings were compared with the predictions of a solubility model which assumes that the solubility reduction is log-additive with respect to various compounds (mainly sugars and electrolytes). A striking agreement between experimental results and predictions was found.     

Oxygen absorption in stirred tanks: A correlation for ionic strength effects

A new correlation is given for the prediction of the volumetric coefficient for mass transfer (K_La) in stirred tanks from dispersed gas bubbles to basal salt solutions of ionic strengths representative of fermentation media. The correlation includes the effects of both the operating parameters (agitation power per unit volume and gas superficial velocity) and the physicochemical properties of the system: interfacial tension, viscosity, density, diffusion, coefficient and, in particular, ionic strength. The effect of the latter was found to be most significant in the Newtonian systems of water-like viscosity investigated; no previous correlations have included the effect of ionic strength. K_La values were determined by using a dissolved oxygen probe to monitor the steady-state oxygen tension in continuous flow experiments, and/or the rate of change of oxygen tension in unsteady-state semibatch experiments. In the latter cases, results were computed by a nonlinear, least squares computer program which fitted the experimental data to a model of probe transient response characteristics. The general applicability of the model and the computational procedure was verified by comparing the results to those obtained with the same electrolyte solution in the steady-state mode. The experiments were run over a wide range of agitation power inputs, including those typical of both soluble- and insoluble-substrate fermentations. The correlation appears to be valid for both oxygen mass transfer with and without homogeneous chemical reaction in the liquid phase; in the former case, for example, sulfite oxidation, knowledge of the chemical reaction enhancement factor is required. In addition to predicting oxygen transfer capabilities, the correlation may be used for other sparingly soluble gases of interest in fermentation systems, such as methane, hydrogen, and carbon dioxide.     

Enhancement of oxygen transfer rates in fermentation using oxygen-vectors

Oxygen transfer is one of the bottlenecks in conventional fermentation technology and it has so far been almost totally overlooked with regards to high cell densities and immobilized cells. This review presents some new concepts to improve oxygen supply in aerobic fermentations, especially the use of oxygen-vectors. The oxygen-vectors generally used are liquids which are insoluble in the fermentation media. Their utilization in an emulsified form can significantly increase the oxygen transfer coefficient between gas and aqueous phases. It seems that the vector acts as an active intermediate in the oxygen transport from gas bubbles to aqueous phase, but the mechanisms involved in this unconventional technique of aeration are not yet known.     

Ethanol production by extractive fermentation

The ideal method to produce a terminal metabolite inhibitor of cell growth and production is to remove and recover it from the fermenting broth as it formed. Extractive fermentation is achieved in the case of ethanol production by coupling both fermentation and liquid-liquid extraction, The solvent of extraction is 1-dodecanol (or a mixture 1-dedecanol, 1-tetradecanol); study of the inhibitory effect of primary aliphatic alcohols of different chain lengths shows that no growth is observed in the presence of alcohols which have between 2 and 12 carbons. This effect is suppressed when the carbon number is 12 or higher. A new reactor has been used-1 pulsed packed column. Pulsation is performed pneumatically. Porous material used as a package adsorbs the cells. The fermentation broth is pulsed in order to (1) increase the interfacial area between the aqueous phase and the dodecanol, (2) decrease gas holdup. Alcoholic fermentation, performed at 35 degrees C on glucose syrup, permits the total utilization of glucose solution of 409 g/L with a yeast which cannot-in classical process- completely use solutions with 200 g/L of glucose. The feasibility of a new method of fermentation coupling both liquid-liquid extraction and fermentation is demonstrated. Extension of this method is possible to any microbial production inhibited by its metabolite excretion.     

Oxygen mass transfer into aerated CMC solutions in a bubble column

Differing findings on the volumetric mass transfer coefficients k(L)a in CMC solutions in bubble column bioreactors have been reported in the literature. Therefore, oxygen mass transfer was studied again in CMC solutions in a 14-cm-i.d. x 270-cm-height bubble column using different spargers. The k(L)a values were determined along with the dispersion coefficients by fitting the prediction of the axial dispersed plug model with the experimental oxygen concentration profiles in the liquid phase. Surprisingly, the obtained liquid phase dispersion coefficients for CMC solution are higher than one would expect from correlations. The k(L)a data depend largely on the flow regime. In general, they are lower than those reported in the literature. The data for developing slug and established slug flow are dependent on the gas velocity and the effective viscosity of the solution and can br correlated by a simple correlation. This correlation describes k(L)a values measured on fermentation broth of Penicillium chrysogenum with striking agreement.