Computational fluid dynamics modeling of mass transfer behavior in a bioreactor for hairy root culture. I. Model development and experimental validation

A two-dimensional axisymmetric computational fluid dynamics (CFD) model based on a porous media model and a discrete population balance model was established to investigate the hydrodynamics and mass transfer behavior in an airlift bioreactor for hairy root culture.During the hairy root culture of Echinacea purpurea, liquid and gas velocity, gas holdup, mass transfer rate, as well as oxygen concentration distribution in the airlift bioreactor were simulated by this CFD model. Simulative results indicated that liquid flow and turbulence played a dominant role in oxygen mass transfer in the growth domain of the hairy root culture. The dissolved oxygen concentration in the hairy root clump increased from the bottom to the top of the bioreactor cultured with the hairy roots, which was verified by the experimental detection of dissolved oxygen concentration in the hairy root clump. This methodology provided insight understanding on the complex system of hairy root culture and will help to eventually guide the bioreactor design and process intensification of large-scale hairy root culture.     

Production of catalase and glucose oxidase by Aspergillus niger using unconventional oxygenation of culture

A novel method for increasing dissolved oxygen concentration in culture media has been developed. It involves adding hydrogen peroxide (H2O2) to the medium which is then decomposed to oxygen and water by catalase. Some factors affecting oxygenation of culture were investigated. Maximal oxygen concentration occurred in 50 ml of the medium containing 0-2 g wet mycelium and 0.2% glucose at pH 5.0. A new apparatus for automated addition of H2O2 to the bioreactor to keep the dissolved oxygen concentration constant over the range 1-100% +/- 2% was tested. A significant increase (over sixfold) of intracellular catalase activity was obtained while the dissolved oxygen concentration remained stable (30% +/- 2%).     

Mathematical model for a three-phase fluidized bed biofilm reactor in wastewater treatment

A mathematical model for a three phase fluidized bed bioreactor (TFBBR) was proposed to describe oxygen utilization rate, biomass concentration and the removal efficiency of Chemical Oxygen Demand (COD) in wastewater treatment. The model consisted of the biofilm model to describe the oxygen uptake rate and the hydraulic model to describe flow characteristics to cause the oxygen distribution in the reactor. The biofilm model represented the oxygen uptake rate by individual bioparticle and the hydrodynamics of fluids presented an axial dispersion flow with back mixing in the liquid phase and a plug flow in the gas phase. The difference of settling velocity along the column height due to the distributions of size and number of bioparticle was considered. The proposed model was able to predict the biomass concentration and the dissolved oxygen concentration along the column height. The removal efficiency of COD was calculated based on the oxygen consumption amounts that were obtained from the dissolved oxygen concentration. The predicted oxygen concentration by the proposed model agreed reasonably well with experimental measurement in a TFBBR. The effects of various operating parameters on the oxygen concentration were simulated based on the proposed model. The media size and media density affected the performance of a TFBBR. The dissolved oxygen concentration was significantly affected by the superficial liquid velocity but the removal efficiency of COD was significantly affected by the superficial gas velocity. An erratum to this article can be found online at .     

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.     

NMR studies of water-gas interactions

Analysis of published data concerning the solubility of different gases in water as dependent on temperature was carried out. These dependences could be described by mono-or bi-exponential functions. Solubilities of nitrogen and oxygen are additive and depend on their percentage in the atmosphere over the liquid. The temperature dependence of oxygen and nitrogen dissolved in water corresponds to that in the atmospheric air. Measurements of water spin-lattice relaxation times, changing with the concentration of dissolved paramagnetic oxygen, showed that oxygen could be substantially but not completely eliminated by saturation with any gas. The best method is the contact with a water-immiscible liquid of gas capacity higher than water. However, all this leads to an unstable state of a gas-water system, converging to equilibrium.     

Comparison of polarographic and chemical measurements of oxygen uptake in complex media: the example of lipoxygenase reaction.

A polarographic method using a Clark oxygen electrode was used to assess oxygen concentrations in model and complex media before and during the lipoxygenase-catalyzed oxidation of linoleic acid to hydroperoxides. The results were in good agreement with those obtained with the chemical determination of dissolved oxygen. The electrode correctly responded when the dissolved oxygen concentration was decreased by the addition of water activity depressors (sorbitol, sucrose, glucose). The influence of the medium components on the gas solubility was discussed. The linear relationship between partial pressure of oxygen (determined by polarographic method) and the oxygen concentration (determined by chemical method) indicated that the Clark oxygen electrode can be used to study enzyme reactions consuming or evolving oxygen in non-Newtonian media.     

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.     

Influence of surface active agents on oxygen absorption to the free interface in a stirred fermentor

Liquid phase coefficients were measured for the absorption of oxygen from air to the free interface in stirred vessels. Coefficients for absorption into soft water were independent of the instantaneous dissolved oxygen concentration. Coefficients for absorption into soft water containing a surface active agent were strongly dependent on the instantaneous dissolved oxygen level. The degree of nonlinearity of the coefficients was a function of the rate of agitation of the liquid. The coefficients were independent of the amount of surface active agent added above a very low level. Absorption coefficients for bubble aeration in the same vessels were independent of dissolved oxygen concentration even when the surface active agent was present.     

A method for estimating oxygen availability of stationary plant cell cultures in liquid media

A method for estimating the oxygen availability in plant cell cultures grown in stationary liquid media (e.g. many protoplast cultures) was developed. The method is based on short-term measurements of respiration rate versus oxygen concentration on a sample of cells, suspended in liquid media. From such data it is possible to estimate the oxygen concentration at the bottom of a stagnant liquid culture, by calculating the amount of oxygen reaching the cells by diffusion. As an example, rape (Brassica napus L. cv. Omega) hypocotyl protoplasts were grown with different oxygen concentrations at the site of the cells, obtained by varying the cell density, the height of the liquid layer and the oxygen content of the gas phase. The number of surviving calli was positively correlated with the estimated oxygen availability in the range between 60 and 350 M O₂, below 60 M all cells died. This indicates that oxygen availability can be a limiting factor in the range usually encountered in protoplast cultures, and that the method can be useful when designing optimal growth conditions for stationary cultures of plant cells.Abbreviations C₁ bulk oxygen concentration in agitated medium - C_o oxygen concentration in medium at the gas-liquid interface, in equilibrium with the gas - C_x oxygen concentration at cell level - D diffusion constant of oxygen in water - K_La oxygen transfer rate - l height of liquid above cells - n number of cells per ml - R_x respiration rate per cell     

AN INVESTIGATION INTO THE CAUSE OF THE SPONTANEOUS AGGREGATION OF FLAGELLATES AND INTO THE REACTIONS OF FLAGELLATES TO DISSOLVED OXYGEN : PART II.

Spontaneous aggregations of flagellates are formed under the cover-glass because the organisms are attracted to and remain in regions where the concentration of dissolved oxygen is less than the saturation concentration under atmospheric partial pressure. These regions of lessened oxygen content arise towards the center of the liquid beneath the cover-glass, owing to the oxygen consumed by the flagellates in respiration not being replaced here by the solution of atmospheric oxygen, as it is along the edges of the liquid. The flagellates, however, are insensitive to the attraction of regions of lessened oxygen concentration when the oxygen concentration throughout the liquid is above a certain value. Therefore, for the aggregations to form, either the initial concentration of dissolved oxygen must be below this limiting value, or an interval of time must first elapse after the making of the preparation until the respiration of the organisms has reduced the oxygen concentration throughout the liquid down to this limiting value. The aggregations will then form because the flagellates have become positively chemotropic to the lower concentration of oxygen at the center of the liquid. Once established, such an aggregation of flagellates does not remain long in the same form. An area free from flagellates appears at the center of the aggregation so that the organisms lie in a circular band surrounding the clear area. The latter increases in size and its bordering band of flagellates in diameter, the band gradually becoming less circular and more square in shape, if the cover-glass is a square one. The clear central area is a region where the oxygen consumption of the flagellates has reduced the oxygen content to such a low value that the organisms are forced to leave the region. They collect in a band where the concentration of dissolved oxygen is an optimum for them. It is the equilibrium position between the oxygen consumed at the center and that diffusing in from the edges of the liquid. As the consumption at the center is more rapid than the replacement from the edge, the flagellate band moves outwards until it becomes stationary at a position where the rates of consumption and replacement of oxygen are equal. Although the flagellates collect in this manner in regions of optimum oxygen concentration, yet greater concentrations of dissolved oxygen have no injurious effect on them. Concentrations of dissolved oxygen lower than the optimum have the effect of inhibiting the movement of the flagellates. They recover their activity, however, immediately they are given access to dissolved oxygen again. Work done in the past on chemotropism of flagellates will have to be revised in the light of the above facts, since the oxygen content of solutions used has never been taken into account.     

Determination of oxygen profiles in agar-based gelled in vitro plant tissue culture media

Keeping account of the limited knowledge concerning the relevance of the oxygen status of the medium during in vitro culture, a technique was elaborated to systematically study the oxygen concentration in gelled media. In a first series of experiments, the Oxygen Diffusion Rate (ODR) technique was used to investigate the dissolved oxygen concentration as a function of time at different depths. The results obtained demonstrated that the oxygen concentration in agar media was reduced by 80% during the heating steps included in the preparation procedure. It took about one week to reach an oxygen concentration equal to 90% of the equilibrium value at a depth of 1 cm, irrespective of the brand of agar used. As the recovery of the oxygen concentration at various depths could be nicely modelled by Fick's law, it follows that this process is diffusion limited. In this respect, fluorescence recovery after photobleaching (FRAP) measurements revealed that the diffusion coefficient of oxygen in gelled media was only affected to a very small extent by the presence of up to 2% (w/v) agar. In a final experiment, explants of Ficus benjamina were cultured on a rooting medium. As the oxygen concentration in the gelled medium was not significantly affected by the presence of the biological material, it was concluded that the oxygen uptake by explants from gelled media is negligibly small and hence cannot be considered as being a growth-limiting factor during in vitro micropropagation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Measurement of oxygen solubility in fermentation media: a colorimetric method

Methods of measuring oxygen solubility in culture media are scarce, and those available are tedious to apply. A simple colorimetric assay was developed and applied to the analysis of oxygen solubility during alcoholic fermentation. The method was based on the consumption of oxygen by glucose oxidase activity and the production of the pink quinone of syringaldazine by coupled peroxidase activity. Color formation at 526 nm progressed through an optimum that was a linear function of the oxygen added to the assay. Sensitivity was maximized by operating at pH 7 and limiting the medium sample volume added. Each assay took 10-15 min to prepare and react. Reaction time was minimized by using abundant glucose and enzyme concentrations. Data obtained by the assay developed showed good agreement with published oxygen solubilities in water and selected media at various temperatures. Subsequent analyses of fermentation broths indicated falling sugar concentration to be primarily responsible for increases in oxygen solubility during fermentation. For example, during fermentations started with 230 g/L xylose or glucose, oxygen solubility could increase by 41% due to sugar consumption alone. This procedure can provide the solubility data needed to accurately calibrate in-line electronic probes for monitoring dissolved oxygen concentration during fermentation processes.     

Influence of glucose and dissolved oxygen concentrations on yields of Escherichia coli B in dialysis culture.

Yields of Escherichia coli B grown on glucose were determined in dialysis and non-dialysis culture. The molar growth yields were compared under conditions of excess glucose and oxygen as well as glucose- and oxygen-limiting conditions. The molar growth yields on glucose (YG) were determined for different periods during growth in non-dialysis cultures. A rapid decrease of YG was observed and growth ceased even in the presence of high concentrations of glucose and dissolved oxygen in the culture liquid. The decrease in YG was delayed in dialysis cultures where a high YG could be maintained at very high cell concentrations. The inhibition of growth depended on the accumulation of end-products of fermentative degradation of glucose. These products interfered with the oxidative phosphorylation. A large proportion of the glucose was fermented even in the presence of high concentrations of dissolved oxygen in the culture liquid. A decrease in the growth yield per g glucose was also observed.     

Apparent zero-order kinetics of phenol biodegradation by substrate-inhibited microbes at low substrate concentrations

The reaction kinetics for phenol biodegradation at low substrate concentrations can be estimated based on the analysis of changes in the dissolved oxygen concentration in the bulk liquid during biodegradation. The measured oxygen concentration changes with an interesting behavior as biodegradation proceeds. The oxygen concentration in the bulk liquid decreases rapidly in the early stages of degradation and subsequently decreases linearly and then rapidly recovers to the initial saturated level. Taking into account the oxygen transfer rate between gas and liquid phases and oxygen consumption rate by microbes, the change in the dissolved oxygen concentration can be simulated with an unsteady state mass balance equation and three kinetic models for the rate of phenol metabolism: a substrate-inhibited model; a zero-order model; and a combined model. In the combined model, it is assumed that, at phenol concentrations above 10 mg/L, the degradation rate is expressed by a substrate-inhibited model; whereas at concentrations below 10 mg/L the zero-order model is applied. It was found that the characteristics of the change in the dissolved oxygen concentration, especially the rapid increase at the end of degradation, can only be described by the combined kinetic model. This result suggests that conventional Haldane-type kinetics would be unsuitable for estimating the phenol consumption rate at low phenol concentrations, in particular, at concentrations less than 10 mg/L. (c) 1996 John Wiley & Sons, Inc.     

Tubular photobioreactor design for algal cultures.

Principles of fluid mechanics, gas-liquid mass transfer, and irradiance controlled algal growth are integrated into a method for designing tubular photobioreactors in which the culture is circulated by an airlift pump. A 0.2 m(3) photobioreactor designed using the proposed approach was proved in continuous outdoor culture of the microalga Phaeodactylum tricornutum. The culture performance was assessed under various conditions of irradiance, dilution rates and liquid velocities through the tubular solar collector. A biomass productivity of 1.90 g l(-1) d(-1) (or 32 g m(-2) d(-1)) could be obtained at a dilution rate of 0.04 h(-1). Photoinhibition was observed during hours of peak irradiance; the photosynthetic activity of the cells recovered a few hours later. Linear liquid velocities of 0.50 and 0.35 m s(-1) in the solar collector gave similar biomass productivities, but the culture collapsed at lower velocities. The effect of dissolved oxygen concentration on productivity was quantified in indoor conditions; dissolved oxygen levels higher or lower than air saturation values reduced productivity. Under outdoor conditions, for given levels of oxygen supersaturation, the productivity decline was greater outdoors than indoors, suggesting that under intense outdoor illumination photooxidation contributed to loss of productivity in comparison with productivity loss due to oxygen inhibition alone. Dissolved oxygen values at the outlet of solar collector tube were up to 400% of air saturation.     

Effect of fermentation conditions on N2 fixation by Methylococcus capsulatus

Nitrogen fixation has been investigated during chemostat fermentations with a culture of Methylococcus capsulatus with natural gas. It is demonstrated that nitrogen fixation occurs under conditions when either nitrate or ammonia as nitrogen source is insufficient for the growth on fixed supply of methane and oxygen. The fixation occurs contrary to expectations within a wide range of dilution rates and with variation of concentration of liquid source of nitrogen. An O₂ optimum is determined for the nitrogenase system of the culture in an assay. During fermentation a complete abolishment of nitrogenase reaction is attained at 15% air saturation (dissolved oxygen). Conditions for N₂ fixation is unaltered with change of pH from 6.8 to 5.7.     

Application of bioreactor design principles to plant micropropagation

Principles of oxygen consumption, oxygen transport, suspension, and mixing are discussed in the context of propagating aggregates of plant tissue in liquid suspension bioreactors. Although micropropagated plants have a relatively low biological oxygen demand (BOD), the relatively large tissue size and localization of BOD in meristematic regions will typically result in oxygen mass transfer limitations in liquid culture. In contrast to the typical focus of bioreactor design on gas–liquid mass transfer, it is shown that media-solid mass transfer limitations limit oxygen available for aerobic plant tissue respiration. Approaches to improve oxygen availability through gas supplementation and bioreactor pressurization are discussed. The influence of media components on oxygen availability are also quantified for plant culture media. Experimental studies of polystyrene beads in suspension in a 30-l air-lift and stirred bioreactors are used to illustrate design principles for circulation and mixing. Potential limitations to the use of liquid suspension culture due to plant physiological requirements are acknowledged.     

Microplates with integrated oxygen sensing for medium optimization in animal cell culture

A new approach using microtiter plate cultivation with on-line measurement of dissolved oxygen (DO) was applied for medium optimization of mammalian cell culture. Applying dynamic liquid phase balance, oxygen uptake rates were calculated from the DO level and used as an indicator for culture viability. The developed method was successfully applied to optimization of the concentration of glucose, glutamine and inorganic salts for cultivation of a Chinese Hamster Ovary (CHO) cell line. Using 2³ full factorial central composite design, the optimum medium composition could be identified in one single run. The concentration of inorganic salts had a significant influence on cultivation. The developed method exhibits high potential to improve procedures of medium optimization for animal cell cultivation by allowing the investigation of large sets of potentially important variables in short time and with reduced effort.     

酒精酵母在连续发酵中的振荡行为研究

初步分析酒精酵母在连续发酵中的振荡行为的产生条件及产生机理。通过改变稀释率、pH值、溶氧和进料葡萄糖浓度等条件 ,观察不同操作条件对酒精酵母菌生长和代谢行为的影响。在 10～ 15 g/L的较低葡萄糖浓度 ,0 .10～ 0 .2 0h-1的较低稀释率 ,以及 70 %左右的适度的溶氧浓度等发酵条件下 ,酒精酵母会出现同步的代谢振荡现象。一定条件下 ,菌体浓度处于振荡状态 ,残余葡萄糖浓度不可测或在很低水平振荡 ,这些发现预示着控制机制的新发展。