Foam fractionation of globular proteins

Foam fractionation of bovine serum albumin (BSA) was studied as a model system for potato wastewater. The effects of feed concentration, superficial gas velocity, feed flow rate, bubble size, pH, and ionic strength on the enrichment and recovery of BSA were investigated in a single-stage continuous foam fractionation column. Enrichments ranged from 1.5 to 6.0 and recoveries from 5 to 85%. The feed concentrations were varied from 0.01 to 0.2 wt %, and enrichments were found to increase with lower feed concentrations. Enrichments also increased with lower superficial gas velocities and larger bubble sizes. At sufficiently low feed flow rates, enrichment was found to increase with an increase in the flow rate, eventually becoming insensitive to the feed flow rate at higher values. The pH was varied from 3.5 to 7.0 and ionic strength from 0.001M to 0.2M. The effects of pH and ionic strength were found to be coupled with bubble size. A minimum bubble size was found at pH 4.8, the isoelectric point of BSA, resulting in a minimum in the enrichment. Bubble size, and thus enrichment, was found to increase as the ionic strength decreased from 0.2M to 0.01M. Previous models(1,2) for the hydrodynamics of foam column were extended for a singlestage continuous foam fractionation column for the prediction of enrichment and recovery. The model assumed adsorption equilibrium, infinite surface viscosity, and bubbles of the same size. Though coalescence was formally accounted for in the model by considering bubble size as a function of foam height, calculations for the experimental runs were performed only for the case of no coalescence. Quantitative predictions of enrichment and recovery could not be made with a single representative bubble size because of the broad inlet bubble size distribution as well as broadening of the distribution as a result of coalescence. The experimental enrichments were higher and recoveries were lower than the model predictions, the discrepancy being more pronounced at lower feed concentrations because of increased coalescence. The higher enrichments are due to the predominant effect of internal reflux as a result of coalescence whereas the lower recoveries are a result of detrimental effects of broadening bubble size distributions.     

Polarized light based scheme to monitor column performance in a continuous foam fractionation column

Background  

A polarized light scattering technique was used to monitor the performance of a continuously operated foam fractionation process. The S ₁₁ and S ₁₂ parameters, elements of the light scattering matrix, combined together (S ₁₁ +S ₁₂) have been correlated with the bubble size and liquid content for the case of a freely draining foam. The performance of a foam fractionation column is known to have a strong dependence on the bubble size distribution and liquid hold up in foam. In this study the enrichment is used as a metric, representative of foam properties and column performance, and correlated to the S ₁₁ +S ₁₂ parameter.     

Isolation and purification of protease from human placenta by foam fractionation

The effect of operating parameters like pH, protein concentration, column geometry, and gas flow rate on the separation efficiency of proteolytic enzymes from crude human placental homogenate has been studied in a batch foam column. Purification has been found to be optimum at pH 8.0, close to the isoelectric pH, at which the surface adsorption of the protein on the foam bubbles is maximum. Both purification and recovery varied significantly with total protein concentration. Stable bubble formation was hindered at lower protein concentrations, while extraneous proteins rather than the protease were preferentially adsorbed at higher protein concentrations, decreasing the purification efficiency. Column diameter and column height should be optimized for any specific feed protein concentration and gas flow rate. However, the enrichment ratio was found to decrease with the increase in flow rate. The results indicate that foam fractionation is an effective separation process for recovering valuable biochemicals from biological materials.     

On the denaturation of enzymes in the process of foam fractionation

Experimental study on the denaturation of enzyme during the separation by foaming was conducted with trypsin and catalase in aqueous medium as model system respectively. The effects of operating pH and sparging gas composition on the denaturation of an enzyme were examined respectively. The oxidative deactivation of enzyme at the gas-liquid interface was identified, which could be reduced by applying nitrogen or carbon dioxide as sparging gas. At suitable conditions, the loss of enzyme activity can be reduced to less than 10% in case of trypsin and to zero in case of catalase. With its proven mildness and effectiveness, foam fractionation in a loop bubble column is applicable for recovery and concentration of enzymes from aqueous solutions.     

Foam separation of microbial cells

Batch foam separation has been employed to separate Saccharomyces carlsbergensis cells from their broth without the use of any external surface-active agent. A model has been developed to predict the foamate cell concentration as well as the variation of cell concentration in the bulk liquid in the foam column as a function of time. The model assumes a linear equilibrium relation between the cell concentrations at the interface and the bulk. The foam has interface as well as interstitial liquid. The interface is assumed to be in equilibrium with the interstitial liquid, which in turn is assumed to have the same concentration as the bulk. The interfacial area is calculated by assuming the foam bubbles to be pentagonal dodecahedral in shape. The model has been found to explain the results of foam separation of cells quite well, particularly with respect to the effect of bubble size and aeration rate.     

Performance of a continuous foam separation column as a function of process variables

Enrichment and recovery of bovine serum albumin has been examined in a continuous foam separation column. The effects of the operating factors, superficial air velocity, feed flow rate, feed concentration and pH on the above characteristics was investigated. The protein enrichment decreased with the increase in the value of each of these parameters. Protein recovery increased with increasing air velocity, decreased with increasing feed flow rate and did not change very much with increasing feed concentration. Maximum protein recovery was obtained at the isoelectric point (pH 4.8) of the protein. Maximum protein recovery was found to be a strong function of the air velocity in the range 0.05-0.15 cm/s. Further increase in air velocity did not have much effect on recovery because of very large bubbles formed as a result of coalescence. Bubble size was determined as a function of the above factors in the liquid and foam sections of the column. It was found to be dependent on protein concentration, feed flow rate and solution pH. The effect was more significant in the foam section of the column. The bubbles in the foam section were significantly larger (about 3-10 times) than those in the liquid, with a sharp change at the foam-liquid interface. The bubble size measurements were used to calculate the interfacial area and it was shown that the rate of protein removal increases with increasing interfacial area.     

Comparative evaluation of compact photobioreactors for large-scale monoculture of microalgae

Engineering analyses combined with experimental observations in horizontal tubular photobioreactors and vertical bubble columns are used to demonstrate the potential of pneumatically mixed vertical devices for large-scale outdoor culture of photosynthetic microorganisms. Whereas the horizontal tubular systems have been extensively investigated, their scalability is limited. Horizontal tubular photobioreactors and vertical bubble column type units differ substantially in many ways, particularly with respect to the surface–to–volume ratio, the amount of gas in dispersion, the gas–liquid mass transfer characteristics, the nature of the fluid movement and the internal irradiance levels. As illustrated for eicosapentaenoic acid production from the microalga Phaeodactylum tricornutum, a realistic commercial process cannot rely on horizontal tubular photobioreactor technology. In bubble columns, presence of gas bubbles generally enhances internal irradiance when the Sun is low on the horizon. Near solar noon, the bubbles diminish the internal column irradiance relative to the ungassed state. The optimal dimensions of vertical column photobioreactors are about 0.2 m diameter and 4 m column height. Parallel east–west oriented rows of such columns located at 36.8°N latitude need an optimal inter-row spacing of about 3.5 m. In vertical columns the biomass productivity varies substantially during the year: the peak productivity during summer may be several times greater than in the winter. This seasonal variation occurs also in horizontal tubular units, but is much less pronounced. Under identical conditions, the volumetric biomass productivity in a bubble column is 60% of that in a 0.06 m diameter horizontal tubular loop, but there is substantial scope for raising this value.     

Physical modeling of animal cell damage by hydrodynamic forces in suspension cultures

Physical damage of animal cells in suspension culture, due to stirring and sparging, is coupled with complex metabolic responses. Nylon microcapsules, therefore, were used as a physical model to study the mechanisms of damage in a stirred bioreactor and in a bubble column. Microcapsule breaskage folowed first-order kinetices in all experiments Entrainment of bubbles into the liquid phase in the stirred bioreactor gave more microcapsule breakage. In the bubble column, the bubble bursting zone at gas-liquid interface was primarilu responsible for microcapsule breakage. The forces on the microcapsules were equivalent to an external pressure of approximately 4 x 10(4) N . m(-2), based on the critical microcapsule diameter for survival of 190 mum. A stable foam layer, however, was found to be effective in protecting microcapsules from damage. The microcapsule transport to the gas-liquid interface and entrainment into the foam phase was consistent with flotation by air bubbles. This result implies that additives and operation of bioreactors should be selected to minimize flotation of cells. (c) 1992 John Wiley & Sons, Inc.     

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

Summary Electrical conductivity microprobes have been used to estimate the transverse variation of bubble size, local gas holdup and local specific gas/liquid interfacial area in bench scale bubble column bioreactors containing fermentation model media. Inserted O₂-electrodes and plane parallel windows alter the structure of the two phase flow. Even slight tilting of the column strongly influences the transverse profiles of the bubble size and local gas holdup. The larger bubbles are collected at the wall, where they can be redispersed. These observations open up new possibilities for the construction of bubble column bioreactors.     

Effect of invertase on the batch foam fractionation of bromelain

Foam fractionation can be used to enrich a hydrophobic protein such as bromelain from an aerated dilute protein solution because the protein foams. On the other hand, a protein such as invertase, which is hydrophilic, is not likely to foam under similar aerated conditions. While a foam fractionation process may not be approapriate for recovering a hydrophilic protein alone, it is of interest to see how that non-foaming protein affects the foaming protein when the two are together in a mixture. The bromelain enrichment, activity and mass recovery were observed as a function of the solution pH in order to explore how invertase can affect the recovery of bromelain in a foam fractionation process.     

Hydrodynamics and oxygen transfer in pneumatic bioreactor devices

Gas holdup and oxygen transfer studies in non-Newtonian suspensions of cellulose fibres conducted in two large (0.098 m(3) each) reactors are described. Both reactors-a bubble column and a similar internal loop airlift-were unusual in that they had rectangular cross-sections. In all cases gas holdups and k(L)a(L) declined with increasing solid concentration and, under identical conditions, the bubble column performed better than the airlift. The fluid systems used were carefully selected to represent mould fermentation broths.The behavior of true mass transfer coeffcient k(L) with changes in bubble size is discussed for these systems.     

Citric acid production by Aspergillus niger immobilized on polyurethane foam

Summary Citric acid was produced using Aspergillus niger immobilized on polyurethane foam in a bubble column reactor. Most of the adsorbed cells remained on the support and, as a result, high oxygen tension was maintained during the reactor operation. However, uncontrolled growth of the pellets made continuous reactor operation difficult. The citric acid productivity obtained from 15 vol.% foam particles containing immobilized cells was 0.135 g/l per hour. This productivity of immobilized cells was almost the same as that of free cells. The oxygen level dropped to half saturation in 5 days in the immobilized cell culture in contrast to 2 days in the free cell culture.     

Cell-microcarrier adhesion to gas-liquid interfaces and foam

The interaction of microcarriers, both with and without cells attached, with gas bubbles was studied. These studies consisted of qualitative microscopic observations of microcarriers with bubbles, quantitative measurements of microcarrier entrapment in foam, and quantitative measurements of the effect of bubble rupture at gas-medium interfaces. Ten different "protective additives" were evaluated for their ability to change the dynamic surface tension of the culture media and to prevent microcarrier adhesion to air bubbles during gas sparging and to prevent entrapment in the foam layer. These studies indicate that microcarriers, with and without cells, readily attach to gas-medium interfaces; yet unlike suspended cells, cells attached to microcarriers are not damaged by bubble ruptures at gas-medium interfaces. Only one surfactant was found to substantially prevent microcarrier entrapment in the foam layer; however, this surfactant was toxic to cells. No correlation was observed between surface tension and the prevention of microcarrier adhesion to gas-liquid interfaces. It is suggested that cell damage as a result of sparging in microcarrier cultures is the result of cells, attached to microcarriers, attaching to rising bubbles and then detaching from the microcarrier as this combination rises through the medium. It is further suggested that the hydrodynamic drag force of the rising microcarrier is sufficiently high to remove the bubble-attached cell from the microcarrier.     

Effects of kinetics of adsorption and coalescence on continuous foam concentration of proteins: Comparison of experimental results with model predictions

Protein enrichment and recovery were measured in a continuous foam concentration column for bovine serum albumin (BSA) for different pool heights, foam heights, superficial gas velocities, bubble sizes, feed flow rates, pH, and ionic strengths. Protein enrichment was found to decrease with an increase in pool height for low pool heights, reach a minimum at an intermediate pool height, and subsequently increase with pool height for sufficiently large pool heights eventually approaching an asymptotic value. Such a behavior was due to the combined effects of kinetics of adsorption of protein and coalescence. The increase in protein enrichment with pool height was due to the predominant effect of kinetics of adsorption of protein, whereas the opposite behavior at low pool heights was due to the predominant effect of coalescence in the foam. Protein enrichment was found to be higher for smaller feed concentrations, smaller gas velocities, larger bubble sizes, and larger foam heights. Enrichment at pH values different from the isoelectric point was found to be higher because of more coalescence. A model for foam concentration of proteins was employed to predict enrichment and recovery. The model predictions agreed well with the experimental data. (c) 1996 John Wiley & Sons, Inc.     

Cell death from bursting bubbles: Role of cell attachment to rising bubbles in sparged reactors

Bursting bubbles are thought to be the dominant cause of cell death in sparged animal or insect cell cultures. Cells that die during the bubble burst can come from three sources: cells suspended near the bubble; cells trapped in the bubble lamella; and cells that attached to the rising bubble. This article examines cell attachment to rising bubbles using a model in which cell attachment depends on cell radius, bubble radius, and cell–bubble attachment time. For bubble columns over 1 m in height and without protective additives, the model predicts significant attachment for 0.5‐ to 3‐mm radius bubbles, but no significant attachment in the presence of protective additives. For bubble columns over 10 cm in height, and without protective additives, the model predicts significant attachment for 50‐ to 100‐μm radius bubbles, but not all protective additives prevent attachment for these bubbles. The model is consistent with three sets of published data and with our experimental results. Using hybridoma cells, serum‐free medium with antifoam, and 1.60 ± 0.05 mm (standard error) radius bubbles, we measured death rates consistent with cell attachment to rising bubbles, as predicted by the model. With 1.40 ± 0.05 mm (SE) radius bubbles and either 0.1% w/v Pluronic‐F68 or 0.1% w/v methylcellulose added to the medium, we measured death rates consistent with no significant cell attachment to rising bubbles, as predicted by the model. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 468–478, 1999.     

Quantitative investigations of cell-bubble interactions using a foam fractionation technique

Previous work by the authors and others has shown that suspended animal cell damage in bioreactors is caused by cell-bubble interactions, regardless whether the bubbles are from bubble entrainment or direct gas sparging. As approach to measure the adsorptivity of animal cells to bubbles, a modified batch foam fractionation technique has been developed in this work and proven to be applicable. By using this technique, the number of cells adsorbed per unit bubble surface area and the adsorption coefficients have been measured to quantify hybridoma cell-bubble interactions, and the prevetive effects of serum and Pluronic F68 on these interactions. It was demonstrated quantitatively that the hybridoma cells adhere to bubbles spontaneously and significant numbers exist in the foam, and that both the serum and Pluronic F68 provide strong prevention to these cell-bubble interactions. The results obtained provide criteria for bioreactor operation and medium formulation to prevent cell-bubble interactions and cell damage in the culture processes.Abbreviations NBCS new born calf serum - SFM serum-free medium     

Improved production of phenylethanoid glycosides by Cistanche deserticola cells cultured in an internal loop airlift bioreactor with sifter riser

An internal loop airlift bioreactor with sifter riser (ILABSR) was composed of a bubble column and a draught-tube rolled with 40-mesh sifter that placed 5 cm above the bottom at the center of the column. A 2 L ILABSR was used for the suspension cultivation of Cistanche deserticola cells and its performance was compared with shake flask culture and a bubble column. Under the optimum culture conditions with the air flowrate of 0.075 m³/h and the inoculation size of 4.7%, about one-fifth cells were attached to the sifter draught-tube. PeG content in these cells was 16.3%, which was 104% higher than that of suspension cells. The production of phenylethanoid glycosides reached 0.85 g/L, which was 102 and 4% higher than those cultured in a 2 L bubble column and shake flasks respectively under their optimal culture conditions.     

Efficient large-scale purification of restriction fragments by solute-displacement ion-exchange HPLC.

Extreme overloading of HPLC columns with sample can create a condition of binding site saturation causing competition and displacement among solutes during column elution. This has been termed solute-displacement chromatography (SD-HPLC). We present an example of this phenomenon for the preparative fractionation and purification of restriction fragments of almost identical size (1337 and 1388 bp) which cannot be resolved by agarose gel electrophoresis. Standard analytical ion-exchange HPLC chromatography failed to separate these fragments from each other and from an unexpectedly early eluting pUC-derived vector fragment of 2.7 kbp. We demonstrate that by intentional overloading of the small (4.6 x 35 mm) non-porous TSK-DEAE HPLC column, hundreds of micrograms of DNA restriction fragments could be resolved and purified in a single HPLC run of less than 30 minutes.     

Lethal events during gas sparging in animal cell culture

The lethal effects of gas sparging on hybridoma cells obtained from a chemostat culture were examined in a bubble column. Experiments were performed to identify and quantify the main hazardous event: bubble formation, bubble rising, or bubble breakup. The results indicate that bubble breakup is the main cause of cell death. The protective activity of the surfactant Pluronic F68 against sparging seems to result from a direct interaction with the cells rather than influencing bubble-liquid interface properties.