Calculations of intracapillary oxygen tension distributions in muscle

Characterizing the resistances to O(2) transport from the erythrocyte to the mitochondrion is important to understanding potential transport limitations. A mathematical model is developed to accurately determine the effects of erythrocyte spacing (hematocrit), velocity, and capillary radius on the mass transfer coefficient. Parameters of the hamster cheek pouch retractor muscle are used in the calculations, since significant amounts of experimental physiological data and mathematical modeling are available for this muscle. Capillary hematocrit was found to have a large effect on the PO(2) distribution and the intracapillary mass transfer coefficient per unit capillary area, k(cap), increased by a factor of 3.7 from the lowest (H=0.25) to the highest (H=0.55) capillary hematocrits considered. Erythrocyte velocity had a relatively minor effect, with only a 2.7% increase in the mass transfer coefficient as the velocity was increased from 5 to 25 times the observed velocity in resting muscle. The capillary radius is varied by up to two standard deviations of the experimental measurements, resulting in variations in k(cap) that are <15% at the reference case. The magnitude of these changes increases with hematocrit. An equation to approximate the dependence of the mass transfer coefficient on hematocrit is developed for use in simulations of O(2) transport from a capillary network.     

Combined dissolved oxygen tension and online viscosity measurements in shake flask cultivations via infrared fluorescent oxygen-sensitive nanoparticles

Oxygen supply is one of the most critical process parameters in aerobic cultivations. To assure sufficient oxygen supply, shake flasks are usually used in combination with orbital shaking machines. In this study, a measurement technique for the dissolved oxygen tension (DOT) in shake flask cultures with viscosity changes is presented. The movement of the shaker table is monitored by means of a Hall effect sensor. For DOT measurements, infrared fluorescent oxygen-sensitive nanoparticles are added to the culture broth. The position of the rotating bulk liquid needs to be determined to assure measurements inside the liquid. The leading edge of the bulk liquid is detected based on the fluorescence signal intensity of the oxygen-sensitive nanoparticles. Furthermore, online information about the viscosity of the culture broth is acquired due to the detection of the position of the leading edge of the bulk liquid relative to the direction of the centrifugal force, as described by Sieben et al. (2019. Sci. Rep., 9, 8335). The DOT measurement is combined with a respiration activity monitoring system which allows for the determination of the oxygen transfer rate (OTR) in eight parallel shake flasks. Based on DOT and OTR, the volumetric oxygen transfer coefficient (k_La) is calculated during cultivation. The new system was successfully applied in cultivations of Escherichia coli, Bacillus licheniformis, and Xanthomonas campestris.     

A method for the control of the dissolved oxygen tension in microbial cultures

A method for the control of dissolved oxygen tension in growing microbial cultures is described. The apparatus consists of a motor-driven air sparge pipe which may be lowered or raised to give a variable point of entry of the air stream into the culture liquid and hence a variable gas dispersion and gas–liquid contact time. Control of the sparge pipe position is by means of a feedback control loop consisting of a dissolved oxygen probe, an on/off controller, and a reversing electric motor which drives the sparge pipe. The difficulty presented by the relatively slow response of the oxygen probe has been overcome by incorporating an adjustable rate of control action.     

Protection of bioreactor from microbial contamination by high dissolved oxygen tension

Summary Repeated batch hydrolysis of casein using immobilized protease were carried out at various do tensions and pH. At any of the pH tested, microbial contamination could be satisfactorily suppressed by maintaining high do. In continuous reaction, contamination could be suppressed throughout 40 days under the do of 190 ppm, whereas contamination was observed after 24 hours with the do of 2.5 ppm.     

The effect of dissolved oxygen tension on the energy metabolism of yeasts]

The specific ATP generation rate in yeasts was examined on the glycolytic pathway and on the respiratory chain as a function of the dissolved oxygen tension of the culture medium. Two different strains were used: Saccharomyces cerevisiae sensitive to the glucose effect and Kluyveromyces fragilis insensitive to the catabolite respression when growing on lactose. The oxidative ATP generation rate followed by these two strains a Michaelis Menten kinetics against the dissolved oxygen concentration. Dissolved oxygen tension only influenced the glycolytic ATP generation rate in Kluyveromyces fragilis. Thus glucose and Pasteur effects are two mutually exclusive regulatory mechanisms of the energy yielding metabolism of the yeasts.     

The effect of dissolved oxygen tension and the utility of oxygen uptake rate in insect cell culture

Dissolved oxygen tension and oxygen uptake rate are critical parameters in animal cell culture. However, only scarce information of such variables is available for insect cell culture. In this work, the effect of dissolved oxygen tension (DOT) and the utility of on-line oxygen uptake rate (OUR) measurements in monitoring Spodoptera frugiperda (Sf9) cultures were determined. Sf9 cells were grown at constant dissolved oxygen tensions in the range of 0 to 30%. Sf9 metabolism was affected only at DOT below 10%, as no significant differences on specific growth rate, cell concentration, amino acid consumption/production nor carbohydrates consumption rates were found at DOT between 10 and 30%. The specific growth rate and specific oxygen uptake rate followed typical Monod kinetics with respect to DOT. The calculated _max and max were 0.033 h^-1 and 3.82×10^-10 mole cell^-1h^-1, respectively, and the corresponding saturation constants were 1.91 and 1.57%, respectively. In all aerated cultures, lactate was consumed only after glucose and fructose had been exhausted. The yield of lactate increased with decreasing DOT. It is proposed, that an apparent DOT in non-instrumented cultures can be inferred from the lactate yield of bioreactors as a function of DOT. Such a concept, can be a useful and important tool for determining the average dissolved oxygen tension in non-instrumented cultures. It was shown that the dynamic behavior of OUR can be correlated with monosaccharide (fructose and glucose) depletion and viable cell concentration. Accordingly, OUR can have two important applications in insect cell culture: for on-line estimation of viable cells, and as a possible feed-back control variable in automatic strategies of nutrient addition.Abbreviations DOT Dissolved oxygen tension - OUR Oxygen uptake rate - specific oxygen uptake rate - specific growth rate - X_v viable cell concentration - C_L, C^*, and oxygen concentrations in liquid phase, in equilibrium with gas phase, and medium molar concentration, respectively - H Henry's constant - K_La volumetric oxygen transfer coefficient - P_T total pressure - oxygen partial pressure - oxygen molar fraction - i discrete element     

Serum-independent modulation of hemicyst formation by dissolved oxygen in postconfluent epithelial monolayers

Summary Hemicyst formation is considered a manifestation of either transepithelial solute and fluid movement or secretory activity in culture. This study shows that hemicyst formation in postconfluent monolayers of rhesus monkey kidney (LLC-MK₂) cells is modulated by the dissolved oxygen concentration (P_O2) of the culture medium. Either daily replacement of serum-free medium or displacement of the gas phase with 18% vol/vol O₂ (initial medium P_O2=125 to 135 mm Hg) enhances formation of hemicysts. Use of 30% O₂ (medium P_O2≊175 mm Hg) does not further increase the incidence, but neither 10% O₂ (medium P_O2=90 to 95 mm Hg) nor 1% O₂ (medium P_O2=35 to 50 mm Hg), the approximate range of dissolved oxygen values in blood, supports hemicyst formation unless cultures are gently rocked to disrupt diffusion gradients. Phase photomicrography of living cultures shows that the surface of a turgid hemicyst is furrowed, and cinephoto-micrography reveals that the walls vibrate subtly. When hypoxic conditions (0 to 1% O₂) are introduced this vibration ceases within 2 to 3 h, whereas collapse and disappearance of turgid hemicysts requires 18 to 20 h, seems virtually synchronous, and is reversible. Hemicysts form in a broad osmotic range, and increased electrolyte concentration increases the incidence. Hemicysts persist in localyy dense areas when cell-free strips are etched in the postconfluent monolayer; no DNA synthesis is detected under these conditions, but two-dimensional cell spreading into the denuded area is seen along the edge of the wound. We conclude that the dissolved oxygen supply in the cellular microenvironment modulates functional expression by differentiated kidney epithelial cells in culture and that increased electrolyte concentration also enhances expression of this phenotypic marker.     

Influence of dissolved oxygen tension on the production rate of 2,5-diketogluconic acid byGluconobacter melanogenum

Summary Increased constant dissolved oxygen tension (200% related to air saturation at 1 bar) has a positive influence on the direct oxidation of glucose byGluconobacter melanogenum. This effect is pronounced and apparent by the faster production of 2,5-diketogluconic acid, mediated through induction in activity of gluconic acid dehydrogenase.     

Effect of oscillating dissolved oxygen tension on the production of alginate by Azotobacter vinelandii.

The effect of oscillating dissolved oxygen tension (DOT) on the metabolism of an exopolysaccharide-producing bacteria (Azotobacter vinelandii) was investigated, particularly on the mean molecular weight (MMW) of the alginate produced. Sinusoidal DOT oscillations were attained by manipulating the oxygen and nitrogen partial pressures at the inlet of a 1.0 L working volume bioreactor. Periods of 1200, 2400, and 4000 s and average amplitudes between 1.0% and 2.2% DOT, with an oscillation axis fixed at 3% DOT, were tested. A culture carried out at constant 3% DOT was used as comparison. The average wave amplitude had an important effect on the maximum mean molecular weight (MMW(max)) of the alginate produced. The higher the amplitude, the lower the MMW(max). As the average wave amplitudes decreased from 2.2% to 1.0%, the MMW(max) increased from 64 to 240 KDa, respectively. Furthermore, at 3% constant DOT (0.0% of amplitude), a MMW(max) of 350 KDa was obtained. No important effect of the oscillating DOT on kinetics of biomass growth, alginate production, and sucrose consumption was observed, compared with constant DOT. The findings of this study point out that accurate DOT control is crucial if a particular molecular weight species of alginate needs to be produced, particularly in large fermentors, where bacteria are exposed to an oscillatory environment as a result of DOT gradients caused by the high viscosity of the broth and insufficient mixing.     

The influence of dissolved oxygen tension on the synthesis of the antibiotic difficidin by bacillus subtilis

The antibiotic, difficidin, and its hydroxylated derivative oxydifficidin, were synthesized by cultures of Bacillus subtilis grown on a complex medium. Maximum titers of about 200 and 130 mg/L, respectively, were obtained. In fermentations where the dissolved oxygen tension (DOT) was controlled, the maximum specific growth rate was only reduced below 5% air saturation. DOT had little effect on the volumetric rateof synthesis of oxydifficidin but greatly influenced the rate for difficidin, which was reduced at DOT values below 40% air saturation. (c) 1994 John Wiley & Sons, Inc.     

High oxygen tension constricts epineurial arterioles of the rat sciatic nerve via reactive oxygen species

Microcirculation of the sheath of the rat sciatic nerve fiber was investigated by using an intravital microscope, and changes in the diameter of the epineurial arterioles in response to highly oxygenated Krebs-bicarbonate solution were evaluated. Superfusion of low-oxygen (0%) Krebs-bicarbonate solution (LKS) onto rat sciatic nerves did not affect changes in the diameter of the arterioles. Nifedipine, a Ca(2+)-channel blocker, caused a dose-dependent dilation of the epineurial arterioles in LKS. In contrast, superfusion of high-oxygen (21%) Krebs-bicarbonate solution (HKS) onto rat sciatic nerves significantly constricted the epineurial arterioles in a time-dependent manner. The HKS-induced constriction of the epineurial arterioles was significantly reduced by treatment with 120 U/ml superoxide dismutase (SOD) alone or 5,000 U/ml catalase alone. In the presence of 120 U/ml SOD plus 5,000 U/ml catalase, 10(-4) M tempol, 10(-6) M diphenyleneiodium, 2 x 10(-4) M apocynin, or 10(-6) M allopurinol, the HKS-induced constriction of the epineurial arterioles completely disappeared. These results suggest that superfusion of highly oxygenated solution onto rat sciatic nerves constricts the epineurial arterioles through reactive oxygen species (ROS), including superoxide and hydrogen peroxide, and that production of superoxide involves a NADPH oxidase- or xanthine oxidase-dependent pathway. In conclusion, ROS play significant roles in the regulation of microcirculation of rat sciatic nerves in vivo.     

Effects of dissolved oxygen tension and mechanical forces on fungal morphology in submerged fermentation

The effects of dissolved oxygen tension and mechanical forces on fungal morphology were both studied in the submerged fermentation of Aspergillus awamori. Pellet size, the hairy length of pellets, and the free filamentous mycelial fraction in the total biomass were found to be a function of the mechanical force intensity and to be independent of the dissolved oxygen tension provided that the dissolved oxygen tension was neither too low (5%) nor too high (330%). When the dissolved oxygen concentration was close to the saturation concentration corresponding to pure oxygen gas, A. awamori formed denser pellets and the free filamentous mycelial fraction was almost zero for a power input of about 1 W/kg. In the case of very low dissolved oxygen tension, the pellets were rather weak and fluffy so that they showed a very different appearance. The amount of biomass per pellet surface area appeared to be affected only by the dissolved oxygen tension and was proportional to the average dissolved oxygen tension to the power of 0.33. From this it was concluded that molecular diffusion was the dominant mechanism for oxygen transfer in the pellets and that convection and turbulent flow in the pellets were negligible in submerged fermentations. The biomass per wet pellet volume increased with the dissolved oxygen tension and decreased with the size of the pellets. This means that the smaller pellets formed under a higher dissolved oxygen tension had a higher intrinsic strength. Correspondingly, the porosity of the pellets was a function of the dissolved oxygen tension and the size of pellets. Within the studied range, the void fraction in the pellets was high and always much more than 50%.     

The effect of cycling dissolved oxygen tension on the synthesis of the antibiotic difficidin by bacillus subtilis

The antibiotic, difficidin, and its hydroxylated derivative, oxydifficidin, were synthesised by cultures of Bacillus subtilis grown on a complex medium in batch culture at dissolved oxygen tensions (DOT) of 15, 20 and 40% air saturation. During part of the growth phase the DOT was cycled about the control value and the effect on growth and antibiotic production observed. In fermentations with cycling at 15 and 20% DOT the growth yields were lower than for the fermentations done at constant DOT throughout. There appears to be a complex interaction between growth rate and difficidin production rate which led to a reduced specific production rate at 15% DOT as a result of cycling.UCL is the Biotechnology and Biological Sciences Research Council Interdisciplinary Research Centre for Biochemical Engineering and the Council's support is gratefully acknowledged. The authors wish to thank Merck & Co. for provision of the difficidin and oxydifficidin used to calibrate the HPLC assay.     

Control of the production of individual fusicoccins at different dissolved oxygen concentrations

The regulatory effect of different concentrations of dissolved oxygen on the production of fusicoccins by the fungus Fusicoccum amygdali Del. was studied. The maximum output of total fusicoccins was obtained by using a profiled dissolved oxygen tension (DOT) regime, in which the DOT was maintained at 15–20% during the biomass growth phase and at 5–8% during the fusicoccins production phase. In comparison with the profiled regime, the maintenance of DOT at 15–20% during the whole fermentation shortened the fusicoccins production phase. The fermentation performance at a low DOT (5–8%) inhibited both the accumulation of biomass and the production of fusicoccins. At high DOT (40–50%), an accelerated accumulation of the biomass with an expressed autolysis of mycelia took place, and the production of fusicoccins was lowered. The qualitative composition of individual fusicoccins varied substantially at different DOTs. Fusicoccins, A, C, D, J, H, 16-O-demethyl-J, detretpentenylfusicoccin and some minor fusicoccin metabolites were found in the fermentation broth using the method of liquid secondary ion mass spectrometry. It was established that the profiled DOT regime (15–20% to 5–8%) provided both the maximum concentration of fusicoccins and an enhanced accumulation of the main metabolite – fusicoccin A (FC A). The performance of the fermentation at a DOT of 15–20% decreased the content of FC A by 2–6% in comparison with the profiled DOT regime, and increased the content of fusicoccin C to 14–20% of the total fusicoccins. Fermentation at DOT of 5–8% was characterized by the highest content of the precursors of FC A, the less oxidized fusicoccins H and J, the contents of which were in range 7–12% and 16–17% of total fusicoccins, respectively.