A novel nanolayer biosensor principle

A method for eliminating the mass transport limitation on biosensor surfaces is introduced. The measurement of macromolecular binding kinetics on plane surfaces is the key objective of many evanescent wave (e.g. total internal reflection fluorescence (TIRF)), and surface plasmon resonance (SPR) based biosensor systems, allowing the determination of binding constants within minutes or hours. However, these methods are limited in not being rigorously applicable to large macromolecules like proteins or DNA, since the on-rates are transport limited due to a Nernst diffusion layer of 5-10 microm thickness. Thus, for the binding of fibrinogen (340 kDa) to a surface current SPR biosensors will show a mass transport coefficient of ca. 2 x 10(-6) m/s. In a novel approach with an immiscible fluid vesicle (e.g. air bubble), it has been possible to generate nanoscopic fluid films of ca. 200 nm thickness on the sensor surface of an interfacial TIRF rheometer system. The thickness of the liquid film can be can be easily probed and measured by evanescent wave technology. This nanofilm technique increases the mass transport coefficient for fibrinogen to ca. 1 x 10(-4) m/s eliminating the mass transport limitation, making the binding rates reaction-rate limited. From the resulting exponential kinetic functions, lasting only 20-30s, the kinetic constants for the binding reaction can easily be extracted and the binding constants calculated. As a possible mechanism for the air bubble effect it is suggested that the aqueous fluid flow in the rheometer cell is separated by the air bubble below the level of the Nernst boundary layer into two independent laminar fluid flows of differing velocity: (i) a slow to stationary nanostream ca. 200 nm thick strongly adhering to the surface; and (ii) the bulk fluid streaming over it at a much higher rate in the wake of the air bubble. Surprising properties of the nanofluidic film are: (i) its long persistence for at least 30-60s after the air bubble has passed (2.5s); and (ii) the absence of solute depletion. It is suggested that a new liquid-liquid interface (i.e. a "vortex sheet") between the two fluid flows plays a decisive role, lending metastability to the nanofluidic film and replenishing its protein concentration via the vortices-thus upholding exponential binding kinetics. Finally, the system relaxes via turbulent reattachment of the two fluid flows to the original velocity profile. It is concluded that this technique opens a fundamentally novel approach to the construction of macromolecular biosensors.     

Effects of bubble–liquid two‐phase turbulent hydrodynamics on cell damage in sparged bioreactor

According to recent experimental studies on sparged bioreactors, significant cell damage may occur at the gas inlet region near the sparger. Although shear stress was proposed to be one of the potential causes for cell damage, detailed hydrodynamic studies at the gas inlet region of gas–liquid bioreactors have not been performed to date. In this work, a second‐order moment (SOM) bubble–liquid two‐phase turbulent model based on the two‐fluid continuum approach is used to investigate the gas–liquid hydrodynamics in the bubble column reactor and their potential impacts on cell viability, especially at the gas inlet region. By establishing fluctuation velocity and bubble–liquid two‐phase fluctuation velocities correlation transport equations, the anisotropy of two‐phase stresses and the bubble–liquid interactions are fully considered. Simulation results from the SOM model indicate that shear and normal stresses, turbulent energy dissipation rate, and the turbulent kinetic energy are generally smaller at the gas inlet region when compared with those in the fully developed region. In comparison, a newly proposed correlation expression, stress‐induced turbulent energy production (STEP), is found to correlate well with the unusually high cell death rate at the gas inlet region. Therefore, STEP, which represents turbulent energy transfer to a controlled volume induced by a combination of shear and normal stresses, has the potential to provide better explanation for increased cell death at the sparger region. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:48–58, 2014     

Quantitative studies of cell-bubble interactions and cell damage at different pluronic F-68 and cell concentrations

Pluronic F-68 (PF-68) is routinely used as a shear-protection additive in mammalian cell cultures. However, most previous studies of its shear protection mechanisms have typically been qualitative in nature and have not covered a wide range of PF-68 and cell concentrations. In this study, interactions between air bubbles along with the associated cell damage were investigated using the novel adenovirus-producing cell line PER.C6, a human embryonic retinoblast transfected with the adenovirus type 5 E1 gene. A wide range of PF-68 and cell concentrations (approximately 3 orders of magnitude) were used in these studies. At low PF-68 concentrations (0.001 g/L), cells had a very high affinity for bubbles, indicated by a more than 10-fold increase in cell concentration in the foam layer liquid versus the bulk liquid. At high PF-68 concentrations ( approximately 3 g/L), however, the cell concentration in the foam layer liquid was only approximately 40% of that in the bulk cell suspension. The number of cells associated with each bubble decreased from approximately 1000 cells at 0.001 g/L PF-68 to approximately 120 cells at 3 g/L PF-68. Despite the lower cell affinity for bubbles at a high PF-68 concentration, at high cell concentrations (10(7) cells/mL and 1 g/L PF-68) significant cell entrapment occurred in the foam layer, on the order of 1000 cells/bubble. For the cells carried by the bubbles, quantitative cell damage data revealed that the probability of cell death from bubble rupture was independent of bulk cell concentration but was affected by PF-68 concentration. These quantitative studies further indicated that even at a low PF-68 concentration of 0.03 g/L, approximately 30% of the attached cells were killed during the bubble rupture process. At the same time, at low PF-68 concentration (<0.1 g/L), significant cell death occurred prior to bubble rupture. On average, a bubble disrupted more cells in the bulk liquid and/or foam layer than during rupture. For both mechanisms, the number of cells damaged by each bubble increased with decreasing PF-68 concentration and increasing bulk cell concentration.     

Surface properties of rat pulmonary surfactant studied with the captive bubble method: adsorption, hysteresis, stability.

Surface tension-area relations from pulmonary surfactant were obtained with a new apparatus that contains a leak free captive bubble of controllable size. Rat pulmonary surfactant was studied at phospholipid concentrations of 50, 200 and 400 micrograms/ml. At the highest concentration, adsorption was rapid, reaching surface tensions below 30 mN/m within 1 s, while at the lowest concentration, approximately 3 min were required. Upon a first quasi static or dynamic compression, stable surface tensions below 1 mN/m could be obtained by a film area reduction of approximately 50%. After three to four cycles the surface tension-area relations became stationary, and the tension fell from 25-30 to approximately 1 mN/m for a film area reduction of less than 20%. Hysteresis became negligible, provided the films were not collapsed by further area reduction. Under these conditions, the films could be cycled for more than 20 min without any noticeable loss in surface activity. After only three to four consecutive cycles, surfactant films exhibited the low surface tensions, collapse rates and compressibilities characteristic of alveolar surfaces in situ. Remarkably, surface tension and area are interrelated in the captive bubble which may promote low and stable surface tensions. If the surface tension of the captive bubble suddenly increases ('click') because of mechanical vibration or unstable surfactant, the bubble shape changes from flat to more spherical. The associated isovolumetric decrease in surface area prevents the surface tension from rising as much as it would have in a constant-area situation. This feedback mechanism may also have a favorable effect in stabilizing alveolar surface tension at low lung volumes.     

Palmitoylation of a pulmonary surfactant protein C analogue affects the surface associated lipid reservoir and film stability

Surfactant protein C (SP-C) is a lipopeptide that contains two thioester-linked palmitoyl groups and is considered to be important for formation of the alveolar surface active lipid film. Here, a non- or dipalmitoylated SP-C analogue (SP-C(Leu)), in which all helical Val residues were replaced with Leu and Cys-5 and Cys-6 were replaced with Ser, was tested for surface activity in a captive bubble system (CBS). SP-C(Leu), either palmitoylated at Ser-5 and Ser-6 or non-palmitoylated, was added to mixtures of 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/phosphatidyl glycerol (PG)/palmitic acid (PA), 68:22:9, (by mass) at a concentration of 2 and 5%. With 2% peptide, surface film formation was rapid, reaching a surface tension below 25 mN/m within 5 s, but the samples with 5% SP-C(Leu) required more than 20 s to reach values below 25 mN/m. Minimum surface tension for the samples with dipalmitoylated SP-C(Leu) was below 1.5 mN/m and very stable, as the surface tension increased by less than 0.5 mN/m within 10 min at constant bubble volume. Minimum surface tension for the non-palmitoylated SP-C(Leu) was approximately 2 and 5 mN/m for 2 and 5% peptide, respectively, but the films were less stable as seen by frequent bubble clicking at low surface tensions. Films with dipalmitoylated SP-C(Leu) that were dynamically cycled at 20-30 cycles/min were substantially less compressible at a surface tension of 20 mN/m (0.007 m/mN) than those that contained the non-palmitoylated peptide (0.02 m/mN). After subphase depletion, the incorporation of lipids into the surface active film during initial bubble expansion occurred at a relatively low surface tension (about 35 mN/m) for the samples with dipalmitoylated SP-C(Leu) compared to approximately 45 mN/m for those containing the non-palmitoylated peptide. Furthermore, for samples that contained non-palmitoylated SP-C(Leu), the ability to reach near zero stable surface tension was lost after a few adsorption steps, whereas with the dipalmitoylated peptide the film quality did not deteriorate even after more than 10 expansion steps and the incorporation of reservoir material equivalent to more than two monolayers. It appears that the covalently linked palmitoyl groups of the SP-C analogue studied are important for the mechanical stability of the lipid film, for the capacity to incorporate material from the reservoir into the surface active film upon area expansion, and for the low film compressibility of dynamically cycled films.     

Evaluation of the killing volume of gas bubbles in sparged animal cell culture bioreactors

The detrimental effect of direct gas sparging on insect cells was investigated in bubble columns with various gas flow rates and bubble sizes. The first-order cell death rate was shown to be directly proportional to the gas flow rate and inversely proportional to the bubble size. The specific killing volume of a bubble, killing volume per unit volume of bubble, was found to have a linear correlation with the specific interfacial area of a bubble. Based on these experimental results and the analysis of a bursting bubble at the liquid surface, it was concluded that the killing volume of a bubble is in the liquid layer surrounding the bubble before its rupture, and most important, in the liquid layer beneath the bubble cavity. Cell damage in the bubble film cap was relatively insignificant compared to that in the liquid layer underneath the bubble cavity, except for very large bubbles (i.e., bubble diameter over 5 mm).     

Overcoming shear stress of microalgae cultures in sparged photobioreactors

In the present work we identified and quantified the effect of hydrodynamic stress on two different microalgae strains, Dunaliella tertiolecta and D. salina, cultivated in bench-scale bubble columns. The cell death rate constant increased with increasing gas-entrance velocity at the sparger. Dunaliella salina was slightly more sensitive than D. tertiolecta. The critical gas-entrance velocities were approximately 50 and 30 m s(-1) for D. tertiolecta and D. salina, respectively. The effects of gas-flow rate, culture height, and nozzle diameter on the death rate constant were also studied. From these results it was concluded that bubble rising and bubble bursting are not responsible for cell death. Regarding nozzle diameter, small nozzles were more detrimental to cells. The bubble formation at the sparger was found to be the main event leading to cell death.     

Microscopic visualization of insect cell-bubble interactions. II: The bubble film and bubble rupture.

In this paper, the second in the series, the use of a microscopic, high-speed video system to study the interactions of two suspended insect cells strains, Trichoplusia ni (TN-368) and Spodoptera frugiperda (SF-9), with rupturing bubbles is reported. Events such as the adsorption of cells onto the bubble film and the mechanism of bubble rupture were observed. On the basis of these observations and the experimental and theoretical work of other researchers on bubble rupture and cell death as a result of sparging, it is proposed that cells are killed by the rapid acceleration of the bubble film after rupture and the high levels of shear stress in the boundary layer flow associated with bubble jet formation.     

Quantification of damage to suspended insect cells as a result of bubble rupture

It is proposed that when cells are either attached to, or very near, a rupturing bubble, the hydrodynamic forces associated with the rupture are sufficient to kill the cells. Four types of experiments were conducted to quantify the number and location of these killed cells. We determined: (1) the number of cells killed as a result of a single, 3.5-mm bubble rupture; (2) the number and viability of cells in the upward jet that results when a bubble ruptures; (3) the number of cells on the bubble film; and (4) the fate of cells attached to the bubble film after film rupture. All experiments were conducted with Spodoptera frugiperda (SF-9) insect cells, in TNM-FH and SFML medium, with and without Pluronic F-68. Experiments indicate that approximately 1050 cells are killed per single, 3.5-mm bubble rupture in TNM-FH medium and approximately the same number of dead cells are present in the upward jet. It was also observed that the concentration of cells in this upward jet is higher than the cell suspension in TNM-FH medium without Pluronic F-68 by a factor of two. It is believed that this higher concentration is the result of cells adhering to the bubble interface. These cells are swept up into the upward jet during the bubble rupture process. Finally, it is suggested that a thin layer around the bubble containing these absorbed cells is the "hypothetical killing volume" presented by other researchers. (c) 1994 John Wiley & Sons, Inc.     

Investigation of the structure of two-phase flow model-media in bubble-column bioreactors

Summary By applying photographic, electrical conductivity, and electrooptical methods, the transverse variation of bubble size and velocity, the local gas holdup, and the local specific gas/liquid interfacial area were estimated in a bench scale bubble-column bioreactor containing distilled water. The liquid velocity profile, the transverse turbulence intensity variations, and the turbulence energy dissipation scale were also measured by a hot film turbulence probe and constant temperature anemometer technique.     

NS0 cell damage by high gas velocity sparging in protein-free and cholesterol-free cultures

Recent developments in high cell density and high productivity fed-batch animal cell cultures have placed a high demand on oxygenation and carbon dioxide removal in bioreactors. The high oxygen demand is often met by increasing agitation and sparging rates of air/O2 in the bioreactors. However, as we demonstrate in this study, an increase of gas sparging can result in cell damage at the sparger site due to high gas entrance velocities. Previous studies have showed that gas bubble breakup at the culture surface was primarily responsible for cell damage in sparged bioreactors. Such cell damage can be reduced by use of surfactants such as Pluronic F-68 in the culture. In our results, where NS0 cells were grown in a protein-free and cholesterol-free medium containing 0.5 g/L Pluronic F-68, high gas entrance velocity at the sparger site was observed as the second mechanism for cell damage. Experiments were performed in scaled-down spinners to model the effect of hydrodynamic force resulting from high gas velocities on antibody-producing NS0 cells. Cell growth and cell death were described by first-order kinetics. Cell death rate constant increased significantly from 0.04 to 0.18 day(-1) with increasing gas entrance velocity from 2.3 to 82.9 m/s at the sparger site. The critical gas entrance velocity for the NS0 cell line studied was found to be approximately 30 m/s; velocities greater than 30 m/s caused cell damage which resulted in reduced viability and consequently reduced antibody production. Observations from a second cholesterol-independent NS0 cell line confirmed the occurrence of cell damage due to high gas velocities. Increasing the concentration of Pluronic F-68 from 0.5 to 2 g/L had no additional protective effect on cell damage associated with high gas velocity at the sparger. The results of gas velocity analysis for cell damage have been applied in two case studies of large-scale antibody manufacturing. The first is a troubleshooting study for antibody production carried out in a 600 L bioreactor, and the second is the development of a gas sparger design for a large bioreactor scale (e.g., 10,000 L) for antibody manufacturing.     

Hydrodynamic stress and lethal events in sparged microalgae cultures

The effect of high superficial gas velocities in continuous and batch cultures of the strains Dunaliella tertiolecta, Chlamydomonas reinhardtii wild-type and cell wall-lacking mutant was studied in bubble columns. No cell damage was found for D. tertiolecta and C. reinhardtii (wild-type) up to superficial gas velocities of 0.076 and 0.085 m s(-1), respectively, suggesting that high superficial gas velocities alone cannot be responsible for cell death and, consequently, bubble bursting cannot be the sole cause for cell injury. A death rate of 0.46 +/- 0.08 h(-1) was found for C. reinhardtii (cell wall-lacking mutant) at a superficial gas velocity of 0.076 m s(-1), and increased to 1.01 +/- 0.29 h(-1) on increasing superficial gas velocity to 0.085 m s(-1). Shear sensitivity is thus strain-dependent and to some extent the cell wall plays a role in the protection against hydrodynamic shear. When studying the effect of bubble formation at the sparger in batch cultures of D. tertiolecta by varying the number of nozzles, a death rate of 0.047 +/- 0.016 h(-1) was obtained at high gas entrance velocities. D. tertiolecta was cultivated in a pilot-plant reactor under different superficial gas velocities of up to 0.026 m s(-1), with relatively low gas entrance velocities and no cell damage was observed. There is some indication that the main parameter causing cell death and damage was the gas entrance velocity at the sparger.     

Analysis of cell-to-bubble attachment in sparged bioreactors in the presence of cell-protecting additives

To investigate the mechanisms of cell protection provided by medium additives against animal cell injury in sparged bioreactors, we have analyzed the effect of various additives on the cell-to-bubble attachment process using CHO cells in suspension. Cell-to-bubble attachment was examined using three experimental techniques: (1) cell-bubble induction time analysis (cell-to-bubble attachment times); (2) forming thin liquid films and observing the movement and location of cells in the thin films; and (3) foam flotation experiments. The induction times we measured for the various additives are as follows: no additive (50 to 500 ms), polyvinyl pyrrolidone (PVP: 20 to 500 ms), polyethylene glycol (PEG: 200 to 1000 ms), 3% serum (500 to 1000 ms), polyvinyl alcohol (PVA: 2 to 10 s), Pluronic F68 (5 to 20 s), and Methocel (20 to 60 s). In the thin film formation experiments, cells in medium with either F68, PVA, or Methocel quickly flowed out of draining thin liquid films and entered the plateau border. When using media with no additive or with serum, the flow of cells out of the thin liquid film and film drainage were slower than for media containing Pluronic F68. PVA, or Methocel. With PVP and PEG, the thin film drainage was much slower and cells remained trapped in the film. For the foam flotation experiments, a separation factor (ratio of cell concentration in the foam catch to that in the bubble column) was determined for the various additives. In the order of increasing separation factors (i.e., increasing cell attachment to bubbles), the additives are as follows: Methocel, PVA, Pluronic F68, 3% serum, serum-free medium with no additives, PEG, and PVP. Based on the results of these three different cell-to-bubble attachment experiments, we have classified the cell-protecting additives into three groups: (1) Pluronic F68, PVA, and Methocel (reduced cell-to-bubble attachment); (2) PEG and PVP (high or increased cell-to-bubble attachment); and (3) FBS (reduced cell attachment butslower drainage films compared with F68, PVA, and Methocel with some cell entrapment in those films). These phenomena are discussed in relation to the interfacial properties of the media reported in a companion Study (this issue). (c) 1995 John Wiley & Sons Inc.     

Steady propagation of a liquid plug in a two-dimensional channel

In this study, we investigate the steady propagation of a liquid plug within a two-dimensional channel lined by a uniform, thin liquid film. The Navier-Stokes equations with free-surface boundary conditions are solved using the finite volume numerical scheme. We examine the effect of varying plug propagation speed and plug length in both the Stokes flow limit and for finite Reynolds number (Re). For a fixed plug length, the trailing film thickness increases with plug propagation speed. If the plug length is greater than the channel width, the trailing film thickness agrees with previous theories for semi-infinite bubble propagation. As the plug length decreases below the channel width, the trailing film thickness decreases, and for finite Re there is significant interaction between the leading and trailing menisci and their local flow effects. A recirculation flow forms inside the plug core and is skewed towards the rear meniscus as Re increases. The recirculation velocity between both tips decreases with the plug length. The macroscopic pressure gradient, which is the pressure drop between the leading and trailing gas phases divided by the plug length, is a function of U and U2, where U is the plug propagation speed, when the fluid property and the channel geometry are fixed. The U2 term becomes dominant at small values of the plug length. A capillary wave develops at the front meniscus, with an amplitude that increases with Re, and this causes large local changes in wall shear stresses and pressures.     

Water-to-Air Transfer and Enrichment of Bacteria in Drops from Bursting Bubbles

An electrostatic induction technique was used to determine both drop size distribution and concentration of bacteria in the film drops produced by bubbles bursting at the surface of a suspension of Serratia marcescens. Film drops are produced from the collapse of the thin film of water that just before bursting separates the air in the bubble from the atmosphere. Bubbles of 1.7-mm diameter produced from 10 to 20 film drops which ranged from <2 μm to over 30 μm in diameter. Half the drops were <10 μm. For bubbles rising a distance of less than 2 cm through the bacterial suspension, bacterial enrichment factors in the drops were between 10 and 20. Electrostatic methods can be used to determine the enrichment of bacteria in film drops as a function of bubble size and distance of rise through the bacterial suspension.     

Interactions between animal cells and gas bubbles: The influence of serum and pluronic F68 on the physical properties of the bubble surface

We describe a method by which the degree of bubble saturation can be determined by measuring the velocity of single bubbles at different heights from the bubble source in pure water containing increasing concentrations of surfactants. The highest rising velocities were measured in pure water. Addition of surfactants caused a concentration-dependent and height-dependent decrease in bubble velocity; thus, bubbles are covered with surfactants as they rise, and the distance traveled until saturation is reached decreases with increased concentration of surfactant. Pluronic F68 is a potent effector of bubble saturation, 500 times more active than serum. At Pluronic F68 concentrations of 0.1% (w/v), bubbles are saturated essentially at their source. The effect of bubble saturation on the interactions between animal cells and gas bubbles was investigated by using light microscopy and a micromanipulator. In the absence of surfactants, bubbles had a killing effect on cells; hybridoma cells and Chinese hamster ovary (CHO) cells were ruptured when coming into contact with a bubble. Bubbles only partially covered by surfactants adsorbed the cells. The adsorbed cells were not damaged and they also could survive subsequent detachment. Saturated bubbles, on the other hand, did not show any interactions with cells. It is concluded that the protective effect of serum and Pluronic F68 in sparged cultivation systems is based on covering the medium-bubble interface with surfaceactive components and that cell death occurs either after contact of cells with an uncovered bubble or by adsorption of cells through partially saturated bubbles and subsequent transport of cells into the foam region. (c) 1994 John Wiley & Sons, Inc.     

Phospholipases introduced into the hypophase affect the surfactant film outlining a bubble.

The hypophase exchanger is a recently developed device that makes it possible to replace the liquid in the sample chamber of a pulsating bubble surfactometer, after a bubble has been formed, without changing the size of the bubble. A surfactant film outlining the bubble will retain its surface properties, provided the liquid entering the sample chamber and replacing the hypophase is inert. If, on the other hand, the new hypophase consists of a phospholipase solution, the physical properties of the film are seriously affected. It was found that when phospholipase C, even at low concentration, entered the sample chamber, the physical properties were significantly changed. Phospholipase A2 had to be added at a higher concentration to exert a similar effect. It is postulated that the site of action of phospholipase A2 may be partly protected in the hydrophobic region of the tightly packed surfactant film.     

Characterization of oxygen transfer in miniature and lab-scale bubble column bioreactors and comparison of microbial growth performance based on constant k(L)a

This work describes the engineering characterization of miniature (2 mL) and laboratory-scale (100 mL) bubble column bioreactors useful for the cultivation of microbial cells. These bioreactors were constructed of glass and used a range of sintered glass gas diffusers with differently sized pores to disperse humidified air within the liquid biomedium. The effect of the pressure of this supplied air on the breakthrough point for gas diffusers with different pore sizes was examined and could be predicted using the Laplace-Young equation. The influence of the superficial gas velocity (u(g)) on the volumetric mass transfer coefficient (k(L)a) was determined, and values of up to 0.09 s(-1) were observed in this work. Two modeling approaches were considered in order to predict and provide comparison criteria. The first related the volumetric power consumption (P/V) to the k(L)a and a good correlation was obtained for differently sized reactors with a given pore size, but this correlation was not satisfactory for bubble columns with different gas diffusers. Values for P/V ranged from about 10 to 400 W.m(-3). Second, a model was developed predicting bubble size (d(b)), bubble rising velocity (u(b)), gas hold-up (phi), liquid side mass transfer coefficient (k(L)), and thus the k(L)a using established theory and empirical correlations. Good agreement was found with our experimental data at different scales and pore sizes. Values for d(b) varied from 0.1 to 0.6 mm, and k(L) values between 1.7 and 9.8 x 10(-4) m.s(-1) were determined. Several E. coli cultivations were performed in the miniature bubble column at low and high k(L)a values, and the results were compared to those from a conventional stirred tank operated under identical k(L)a values. Results from the two systems were similar in terms of biomass growth rate and carbon source utilization.     

Outdoor helical tubular photobioreactors for microalgal production: modeling of fluid-dynamics and mass transfer and assessment of biomass productivity

The production of the microalga Phaeodactylum tricornutum in an outdoor helical reactor was analyzed. First, fluid dynamics, mass-transfer capability, and mixing of the reactor was evaluated at different superficial gas velocities. Performance of the reactor was controlled by power input per culture volume. A maximum liquid velocity of 0.32 m s(-1) and mass transfer coefficient of 0.006 s(-1) were measured at 3200 W m(-3). A model of the influence of superficial gas velocity on the following reactor parameters was proposed: gas hold-up, induced liquid velocity, and mass transfer coefficient, with the accuracy of the model being demonstrated. Second, the influence of superficial gas velocity on the yield of the culture was evaluated in discontinuous and continuous cultures. Mean daily values of culture parameters, including dissolved oxygen, biomass concentration, chlorophyll fluorescence (F(v)/F(m) ratio), growth rate, biomass productivity, and photosynthetic efficiency, were determined. Different growth curves were measured when the superficial gas velocity was modified-the higher the superficial gas velocity, the higher the yield of the system. In continuous mode, biomass productivity increased by 35%, from 1.02 to 1.38 g L(-1) d(-1), when the superficial gas velocity increased from 0.27 to 0.41 m s(-1). Maximal growth rates of 0.068 h(-1), biomass productivities up to 1.4 g L(-1) d(-1), and photosynthetic efficiency of up to 15% were obtained at the higher superficial gas velocity of 0.41 m s(-1). The fluorescence parameter, F(v)/F(m), which reflects the maximal efficiency of PSII photochemistry, showed that the cultures were stressed at average irradiances within the culture higher than 280 microE m(-2) s(-1) at every superficial gas velocity. For nonstressed cultures, the yield of the system was a function of average irradiance inside the culture, with the superficial gas velocity determining this relationship. When superficial gas velocity was increased, higher growth rates, biomass productivities, and photosynthetic efficiencies were obtained for similar average irradiance values. The higher the superficial gas velocity, the higher the liquid velocity, with this increase enhancing the movement of the cells inside the culture. In this way the efficiency of the cells increased and higher biomass concentrations and productivities were reached for the same solar irradiance.     

Reverse Enrichment in a Multi-stage Bubble Separator

An unexpected phenomenon named “reverse enrichment” was observed, in which, contrary to the normal behaviour, isoelectric hemoglobin coagula concentrated in the bottom stage of a multistage bubble separator. The phenomenon was observed when the hemoglobin particles were highly concentrated, the bubble diameter was larger than one third of the hole diameter of the partition plate, and the superficial air velocity at the hole was high. Transient and final behaviours of the multi-stage bubble separator with the reverse enrichment were analyzed by assuming the rejection of particles from upward thin liquid streams.