Effect of gas flow rate and oxygen concentration on the damping (filtering) action of fermenter head space

Summary The damping (filtering) action of head space and tubing of a fermenter was estimated. Dissolved oxygen tension in the culture and oxygen concentration in the outlet gas were measured during oscillatory condition and were used to estimate the damping action using fast Fourier transformation. The damping characteristics were found to be equal to a second order low pass filter with a cut off frequency of 1/500 Hz. The damping was found to be dependent on gas flow rate and oxygen concentration in the gas flow. Increasing the gas flow rate increased damping, however, increasing the oxygen content in the gas flow decreased damping. Data reconciliation of undamped oxygen concentrations in the outlet gas was performed using two different approaches.     

Optimising Cell Aggregate Expansion in a Perfused Hollow Fibre Bioreactor via Mathematical Modelling

The need for efficient and controlled expansion of cell populations is paramount in tissue engineering. Hollow fibre bioreactors (HFBs) have the potential to meet this need, but only with improved understanding of how operating conditions and cell seeding strategy affect cell proliferation in the bioreactor. This study is designed to assess the effects of two key operating parameters (the flow rate of culture medium into the fibre lumen and the fluid pressure imposed at the lumen outlet), together with the cell seeding distribution, on cell population growth in a single-fibre HFB. This is achieved using mathematical modelling and numerical methods to simulate the growth of cell aggregates along the outer surface of the fibre in response to the local oxygen concentration and fluid shear stress. The oxygen delivery to the cell aggregates and the fluid shear stress increase as the flow rate and pressure imposed at the lumen outlet are increased. Although the increased oxygen delivery promotes growth, the higher fluid shear stress can lead to cell death. For a given cell type and initial aggregate distribution, the operating parameters that give the most rapid overall growth can be identified from simulations. For example, when aggregates of rat cardiomyocytes that can tolerate shear stresses of up to are evenly distributed along the fibre, the inlet flow rate and outlet pressure that maximise the overall growth rate are predicted to be in the ranges to (equivalent to to ) and to (or 15.6 psi to 15.7 psi) respectively. The combined effects of the seeding distribution and flow on the growth are also investigated and the optimal conditions for growth found to depend on the shear tolerance and oxygen demands of the cells.     

Formation of steady-state oxygen gradients in vitro: application to liver zonation

We have developed a perfusion bioreactor system that allows the formation of steady state oxygen gradients in cell culture. In this study, gradients were formed in cultures of rat hepatocytes to study the role of oxygen in modulating cellular functions. A model of oxygen transport in our flat-plate reactor was developed to estimate oxygen distribution at the cell surface. Experimental measurements of outlet oxygen concentration from various flow conditions were used to validate model predictions. We showed that cell viability was maintained over a 24-h period when operating with a physiologic oxygen gradient at the cell surface from 76 to 5 mmHg O(2) at the outlet. Oxygen gradients have been implicated in the maintenance of regional compartmentalized metabolic and detoxification functions in the liver, termed zonation. In this system, physiologic oxygen gradients in reactor cultures contributed to a heterogeneous distribution of phosphoenolpyruvate carboxykinase (predominantly localized upstream) and cytochrome p450 2B (predominantly localized downstream) that correlates with the distribution of these enzymes in vivo. The oxygen gradient chamber provides a means of probing the oxygen effects in vitro over a continuous range of O(2) tensions. In addition, this system serves as an in vitro model of zonation that could be further extended to study the role of gradients in ischemia-reperfusion injury, toxicity, and bioartificial liver design.     

Monitoring the atmosphere in an anaerobic chamber

The Couloximeter, a fuel cell designed to measure trace amounts of oxygen, was used to monitor the atmosphere in an anaerobic chamber. The device, easy to operate and to maintain, allowed both major and minor fluctuations in oxygen concentration to be measured. Using a hose attached to the outlet within the box, defective (ruptured) gloves were consistently distinguishable from intact gloves.     

Long-term continuous monitoring of dissolved oxygen in cell culture medium for perfused bioreactors using optical oxygen sensors

For long-term growth of mammalian cells in perfused bioreactors, it is essential to monitor the concentration of dissolved oxygen (DO) present in the culture medium to ascertain the health of the cells. An optical oxygen sensor based on dynamic fluorescent quenching was developed for long-term continuous measurement of DO for NASA-designed rotating perfused bioreactors. Tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) chloride is employed as the fluorescent dye indicator. A pulsed, blue LED was chosen as the excitation light source. The sensor can be sterilized using an autoclave. The sensors were tested in a perfused rotating bioreactor supporting a BHK-21 (baby hamster kidney) cell culture over one 28-day, one 43-day, and one 180-day cell runs. The sensors were initially calibrated in sterile phosphate-buffered saline (PBS) against a blood-gas analyzer (BGA), and then used continuously during the entire cell culture without recalibration. In the 180-day cell run, two oxygen sensors were employed; one interfaced at the outlet of the bioreactor and the other at the inlet of the bioreactor. The DO concentrations determined by both sensors were compared with those sampled and measured regularly with the BGA reference. The sensor outputs were found to correlate well with the BGA data throughout the experiment using a single calibration, where the DO of the culture medium varied between 25 and 60 mm Hg at the bioreactor outlet and 80-116 mm Hg at the bioreactor inlet. During all 180 days of culture, the precision and the bias were +/-5.1 mm Hg and -3.8 mm Hg at the bioreactor outlet, and +/- 19 mm Hg and -18 mm Hg at inlet. The sensor dynamic range is between 0 and 200 mm Hg and the response time is less than 1 minute. The resolution of the sensor is 0.1 mm Hg at 50 mm Hg, and 0.25 mm Hg at 130 mm Hg.     

Singlet oxygen production by chloroperoxidase-hydrogen peroxide-halide systems

Singlet oxygen production in the chloroperoxidase-hydrogen peroxide-halide system was studied using 1268 nm chemiluminescence. With chloride or bromide ions, singlet oxygen is produced by the mechanism (formula; see text) (formula; see text) where X- is chloride or bromide ion. Under conditions where there is high enzyme activity and when Reaction B is fast relative to Reaction A, singlet oxygen is produced in near stoichiometric amounts. In contrast, when Reaction A is fast relative to Reaction B, oxidized halogen species (chlorine and hypochlorous acid for chloride ion; bromide, tribromide ion, and hypobromous acid for bromide ion) are the principle reaction products. With iodide ion, no 1268 nm chemiluminescence was detected. Past studies have shown that iodine and iodate ion are the major end products of this system.     

Design of a system for the control of low dissolved oxygen concentrations: critical oxygen concentrations for Azotobacter vinelandii and Escherichia coli

The physiological activity of microorganisms in environments with low dissolved oxygen concentrations often differs from the metabolic activity of the same cells growing under fully aerobic or anaerobic conditions. This article describes a laboratory-scale system for the control of dissolved oxygen at low levels while maintaining other parameters, such as agitator speed, gas flowrate, position of sparger outlet, and temperature at fixed values. Thus, it is possible to attribute in dilute nonviscous fermentations all physiologic changes solely to changes in dissolved oxygen. Experiments were conducted with Azotobacter vinelandii and Escherichia coli. Critical oxygen concentrations for growth (that value of oxygen allowing growth at 97% of mu max) were measured as 0.35 +/- 0.03 mg/L for A. vinelandii and 0.12 +/- 0.03 mg/L for E. coli. These values are significantly different from the commonly quoted values for critical oxygen concentrations based on respiration rates. Because of the superior dissolved oxygen control system and an improved experimental protocol preventing CO2 limitation, we believe that the values reported in this work more closely represent reality.     

Estimation of human endurance time

A prediction formula has been evolved for estimation of human endurance time from aerobic and anaerobic fraction of the total oxygen utilization. The derivation of the formula is based on the assumption that fractional change in endurance time varies directly as the fractional change in aerobic fraction in the same direction and varies as the fractional change in anaerobic fraction in the opposite direction. The validity of the prediction formula has been tested on two sets of data. The first set is consisting of 31 observations on 13 Indian subjects and a second set of data is consisting of 7 observations on one subject collected from literature. The multiple correlations for these sets of data were 0.9650 and 0.9996, respectively. These multiple correlations were highly significant (p < 0.001). It has been concluded that aerobic and anaerobic fractions of total oxygen utilization are significant predictors of human endurance time.     

Scaling oxygen mass transfer in agitated fermentors

There are many scaling formulas that predict the oxygen mass transfer coefficient as k(L).a = constant.(Hp/V)(alpha)Vs(beta) Exponents alpha and beta frequently are scale dependent themselves. A general formula has been derived from the work of Calderbank,(1) Miller,(2) and Tilton,(3) resulting in k(L).a = C(1) phi + C(2) log (Pm/V) phi where phi equals the gas-holdup fraction and Pm/V equals the effective mechanical power input per unit of volume. This formula is consistent with the formula of Westerterp(4) modified by Miller.(2) Gas holdup can be predicted in several ways. Gas-sparged isothermal expansion power input, used for predicting phi, demonstrates that scaling can be done by using either superficial air velocity or volume per volume per minute for aeration.The importance of mixing in replenishing oxygen at the boundary layers of microorganisms will be assessed and compared with the k(L).a as the oxygen transfer ratelimiting step.     

Uniform tissues engineered by seeding and culturing cells in 3D scaffolds under perfusion at defined oxygen tensions

In this work, we assessed whether culture of uniformly seeded chondrocytes under direct perfusion, which supplies the cells with normoxic oxygen levels, can maintain a uniform distribution of viable cells throughout porous scaffolds several milimeters in thickness, and support the development of uniform tissue grafts. An integrated bioreactor system was first developed to streamline the steps of perfusion cell seeding of porous scaffolds and perfusion culture of the cell-seeded scaffolds. Oxygen tensions in perfused constructs were monitored by in-line oxygen sensors incorporated at the construct inlet and outlet. Adult human articular chondrocytes were perfusion-seeded into 4.5 mm thick foam scaffolds at a rate of 1 mm/s. Cell-seeded foams were then either cultured statically in dishes or further cultured under perfusion at a rate of 100 microm/s for 2 weeks. Following perfusion seeding, viable cells were uniformly distributed throughout the foams. Constructs subsequently cultured statically were highly heterogeneous, with cells and matrix concentrated at the construct periphery. In contrast, constructs cultured under perfusion were highly homogeneous, with uniform distributions of cells and matrix. Oxygen tensions of the perfused medium were maintained near normoxic levels (inlet congruent with 20%, outlet > 15%) at all times of culture. We have demonstrated that perfusion culture of cells seeded uniformly within porous scaffolds, at a flow rate maintaining a homogeneous oxygen supply, supports the development of uniform engineering tissue grafts of clinically relevant thicknesses.     

A prototype of an electric-discharge gas flow oxygen−iodine laser: I. Modeling of the processes of singlet oxygen generation in a transverse cryogenic slab RF discharge

The existing kinetic model describing self-sustained and electroionization discharges in mixtures enriched with singlet oxygen has been modified to calculate the characteristics of a flow RF discharge in molecular oxygen and its mixtures with helium. The simulations were performed in the gas plug-flow approximation, i.e., the evolution of the plasma components during their motion along the channel was represented as their evolution in time. The calculations were carried out for the O₂: He = 1: 0, 1: 1, 1: 2, and 1: 3 mixtures at an oxygen partial pressure of 7.5 Torr. It is shown that, under these conditions, volumetric gas heating in a discharge in pure molecular oxygen prevails over gas cooling via heat conduction even at an electrode temperature as low as ~100 K. When molecular oxygen is diluted with helium, the behavior of the gas temperature changes substantially: heat removal begins to prevail over volumetric gas heating, and the gas temperature at the outlet of the discharge zone drops to ~220–230 K at room gas temperature at the inlet, which is very important in the context of achieving the generation threshold in an electric-discharge oxygen?iodine laser based on a slab cryogenic RF discharge.     

Growth kinetics and cellulase biosynthesis in the continuous culture of Trichoderma viride.

Continuous culture studies have been carried out growing Trichoderma viride QM 9123 in a 10 liter stirred fermentor on a medium containing commercial glucose as the carbon source. Experiments were carried out at 30 degrees C and at three controlled pH values of 2.5, 3.0, and 4.0 over a range of dilution rates from 0.01 to 0.11 hr-1. Steady-state values of cell, glucose, and cellulase concentration oxygen tension, and outlet gas oxygen partial pressure were recorded. Values of maximum specific growth rate, endogenous metabolism coefficient, Michaelis-Menten coefficient, yield and maintenance coefficient for glucose were derived and correlated the effect of the hydrogen ion concentration. Specific oxygen uptake rates were correlated with specific growth rates and absorption coefficients were shown to be a function of dilution rate independent of pH. Some data on cellulase biosynthesis were examined and correlated in terms of a maturation time model.     

A new method for breath-to-breath determination of oxygen flux across the alveolar membrane

A new method for breath-to-breath determination of the oxygen flux across the alveolar membrane is described. The principle of the method is to integrate the product of oxygen concentration and flow in the respiratory gas over an interval, which covers a complete respiratory cycle. The result is corrected for the change in oxygen content of the lungs through a formula, which, in contrast to those used in other methods, is independent of the residual capacity of the lungs. The method was evaluated with respect to repeatability by repetitive measurement of oxygen flux in twenty volunteer subjects, and with respect to accuracy by comparing the measured oxygen fluxes with those obtained by the gas collection method. The coefficient of variation was found to be 8% and the breath to breath determinations were, on an average, 6% lower than those of the gas collection method.     

Analysis of a continuous, aerobic, fixed-film bioreactor. II. Dynamic behavior

The dynamic analysis of a continuous, aerobic, fixed-film bioreactor has been performed. Rigorous mathematical models have been developed for a fluidized-bed fermentor with biofilm growth. The transient performance of the reactor is appraised in terms of outlet penicillin concentration for constant, as well as variable carbon substrate feed rates. The effect of the reactor oxygen transfer capacity is elucidated for those cases employing substrate feeding strategies. The results show that penicillin production in a continuous, fixed-film bioreactor reaches a maximum with processing time, but subsequently decreases as cell mass accumulates and substrate deficiencies occur. The maximum production level can be maintained for increased operating times if the substrate supply is continuously increased. The duration of this prolonged production is a direct function of the rate of increase and the operating time at which the increase is initiated. The oxygen transfer capacity of the reactor was found to be important to the effectiveness of a feeding strategy.     

Sinusoidal tide models: Design,construction and laboratory performance

The laboratory performance of sinusoidal tide models is discussed in relation to their construction and use. Constant-flow sinusoidal models are discussed in detail and a formula is derived to described their maximal rate of change of water-level, which is shown to occur at mid-tide. This maximum rate of change is directly proportional to the tidal amplitude and inversely proportional to the tidal period. It is shown that the maximal rate of change of water-level is limiting in the construction of tide models because it governs the rates of inflow and outflow of sea water. The inflow rate must be sufficiently great to keep up with changes in the level of the outflow pipe as it rotate. The outlet pipe, however, must be sufficiently large to remove water at an adequate rate during the ebb of the tide.     

Improved method for the dynamic measurement of mass transfer coefficient for application to solid–substrate fermentation

A new method has been developed for the measurement of overall volumetric mass transfer coefficient (K_L a) in gas-liquid-solid systems. This method is based on the examination of gas phase dynamics in a three-phase contactor and consists of measuring continuously the response of the outlet gas composition to a step input change of CO₂ in the inlet gas stream. The advantages and limitations of the new method are presented and its sensitivity is discussed on the basis of model predictions. Preliminary results on the implementation of the CO₂ method are also reported. Experimental data obtained in a nonviscous electrolyte solution show that the proposed method compares favorably with the conventional dissolved oxygen technique, provided that a correction is made to take account of the difference in diffusivity of oxygen and carbon dioxide.     

The use of resonance Raman spectroscopy to monitor catalytically important bonds during enzymic catalysis. Application to the hydrolysis of methyl thionohippurate by papain.

Resonance Raman spectra were obtained of the dithioacyl-enzyme intermediate produced during the papain-catalayzed hydrolysis of methyl thionohippurate. Intense resonance Raman features were observed in (formula; see text) and (formula; see text) stretching regions from the intermediate's (formula; see text) chromophore. These results demonstrate that by using a single atom replacement, i.e. sulfur for oxygen, the catalytically crucial bonds in the ester moiety can be monitored during enzymolysis via the resonance Raman spectrum. The method can be extended to other enzymes whose catalytic mechanisms involve the formation of a thiol-acyl intermediate.     

Estimation of heats of combustion of biomass from elemental analysis using available electron concepts

The method of Thornton and Dulong's formula for estimating heats of combustion are compared in this work. Heats of combustion predicted by Thornton's method for renewable resources such as wood, straw, and municipal solid wastes are considerably closer to experimentally measured values compared to values predicted by Dulong's formula. Thornton's method states that the heat of combustion is directly proportional to the quantity of oxygen consumed in the combustion process. A method which utilizes the weight fraction carbon on a dry basis and the reluctance degree to predict the heat of combustion of renewable resources is presented.     

Robust control of the activated sludge process

In this work, a robust control strategy is proposed for maintaining the oxygen concentration in the aerobic tank and the pollutant, i.e., ammonium, nitrate, nitrite, concentrations at acceptable levels in the effluent water at the outlet of the activated sludge process. To this end, the Activated Sludge Model no. 1 (ASM1) is first reduced using biological arguments and a singular perturbation method, and a simplified model of the secondary settler is included. In contrast with previous studies that make use of piecewise linear models, an average operating point is evaluated using available data (here data from the COST Action 624) and the reduced‐order model is linearized around it using standard techniques. Finally, a H₂ robust control strategy acting on the oxygen injection and the recirculated flow rate is designed and tested in simulation. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

E.s.r. study of the effect of oxygen on the irradiation of polyglycine

Radiation damage in polycrystalline polyglycine irradiated under different partial pressures of oxygen has been investigated by e.s.r. spectroscopy. Although the general form of the e.s.r. spectrum remained unaltered as compared to irradiation under vacuum, a decrease in the intensity of the e.s.r. signal was observed. The results were analysed in correlation with Alper's formula. A correspondence between free radical transformation into nonradical species in the presence of oxygen and the biological centres of damage is suggested.