Multistage affinity cross-flow filtration: mathematical modelling and analysis

A multistage affinity cross-flow filtration (mACFF) process for protein purification is proposed. The process is mathematically modelled taking into account a case of rapid equilibrium binding of a target protein to its macroligand. The process performance, i.e., dimensionless breakthrough volume (Q _b ⁺ )and recovery yield (REC) to obtain a desired purity is analysed by computer simulations. The results indicate that Q _b ⁺ increases with the increase of stage number (n) due to the increase of affinity binding efficiency. In addition, REC also increases with the increase of n, especially for lower affinity systems, even though the feed loading is the same as the corresponding breakthrough volume that increases with n. Thus both feed loading and recovery yield can be enhanced by raising the stage number. Incompletely permeable membranes reject the target and contaminant proteins. So they delay the appearance of the breakthrough point and compromise the contaminant washing efficiency. Hence although Q _b ⁺ increases with the increase of membrane rejection coefficient (R), REC decreases when the feed loading equals that of Q _b ⁺ . However, when the feed loading is kept unchanged and equals Q _b ⁺ at R=0, REC does not decrease, but slightly increases with the increase of R. This result indicates that incompletely permeable membranes may also be employed for the mACFF process. In general, the model gives a predictive evaluation of the mACFF process successfully.     

Simultaneous organic carbon removal-nitrification by an activated sludge process with cross-flow filtration

Simultaneous organic carbon removal-nitrification was carried out by an activated sludge process with cross-flow filtration while retaining a higher concentration of activated sludge. Operating the unit at ca. 0.10 gBOD·gVSS⁻¹·d⁻¹ made the sludge retention time very long and simultaneous carbon removal-nitrification was achieved quite well. Compared with conventional activated sludge processes, the present process made possible nearly twice the organic volumetric loading by increasing the sludge concentration five-fold. A nitrification rate of ca. 0.30 gTKN·l⁻¹·d⁻¹ was attained. More than 27% of the oxidized nitrogen in the reactor was denitrified at the same time in most cases.     

Affinity cross-flow filtration: purification of IgG with a novel protein a affinity matrix prepared from two-dimensional protein crystals

In this article, we describe the use of 1- to 2-mum sized affinity microparticles for the isolation and purification of IgG from artificial IgG-human serum albumin mixtures and clarified hybridoma cell culture supernatants by affinity cross-flow filtration. Affinity microparticles were prepared from cell wall fragments of Clostridium thermohydrosulfuricum L111-69, in which the peptidoglycan-containing layer was completely covered with a hexagonally ordered S-layer lattice. After crosslinking the S-layer protein with glutaraldehyde, carboxyl groups from acidic amino acids were activated with carbodiimide and used for immobilization of Protein. A. Quantitative determination confirmed that Protein A molecules formed a monomolecular layer on the outermost surface of the S-layer lattice. Affinity microparticles were found to withstand high centrifugal and shear forces and revealed no Protein A leakage or S-layer protein release under cross-flow conditions between pH 2 to 12. The IgG-binding capacity of affinity microparticles was investigated under crossflow conditions and compared with that obtained in batch adsorption processes. (c) 1994 John Wiley & Sons, Inc.     

Fractionation of dextran solutions by cross-flow filtration

The use of ultrafiltration to fractionate dextran solutions in order to obtain fractions for the synthesis of dextran derivatives was investigated. Several experiments were carried out in two available commercial ultrafilters. The operation was evaluated by the recovery yield and process time. Dextran solutions can be fractionated being concentrated up to 9 fold in a PM30, but no more than double in a PM5 hollow fibre membrane cartridge.     

Characteristics of cross-flow filtration of pullulan broth

Cross-flow filtration of culture broth from Aureobasidium pullulans, which elaborates pullulan, was done with a thin channel-type module and microfiltration membranes made of different materials and with different pore sizes. Various factors affecting the results of the filtration were studied. The specific resistance of the microbial cake was found to be higher than that of bakers yeast, the cells of which are about the same size as an A. pullulans cell, and resistance increased with cultivation time. The flux and transmission of pullulan through the membrane decreased with cultivation time as the specific resistance increased. The flux and transmission ] of pullulan depended on the structure and pore size of the membrane and also on the pH of the broth. With a polysulphone membrane with a nominal pore size of 2.0 m, transmission was nearly 100% with negligible leakage of cells and the flux was high when the pH of the broth was adjusted to 2.0.On leave from Hayashibara Co., Ltd., Amase-minamimachi, Okayama 700 Japan Correspondence to: K. Nakanishi     

Separation of yeast by cross-flow filtration with backwashing

The following results were obtained at the separation of yeast by cross-flow filtration from fermentation broth or yeast suspensions with or without backwashing by the filtrate, using micro-porous membranes: 1) the first stage of the filtration process was described by the standard blocking filtration model, 2) the mean filtration flux in one filtration cycle was kept almost constant for at least 3 h by backwashing, 3) the mean filtration flux with backwashing was roughly estimated from the results of filtration without backwashing, and 4) the mean filtration flux at certain yeast concentration in concentrating the yeast was not so much affected by the previous concentration path, and almost agreed that in the filtration of a constant concentration at the same concentration.     

Air slugs entrapped cross-flow filtration of bacterial suspensions

A novel cross-flow technique for membrane filtration of bacterial cell suspensions was established. This is an air slugs entrapped cross-flow method in which air slugs were generated by introducing air into the cross-flow stream. As air slugs moved along with cross-flow, the disturbance of cell sublayer formation on membrane surface was enhanced. As a consequence, filtration flux was improved and stabilized. The effect of air slugs on improving filtration flux was more pronounced in filtering gram-negative Escherichia coli cell than grampositive Brevibacterium flavum cell. Moreover, air slug was about 50% more effective on reducing filtration resistance using ultrafiltration (UF) membrane of 300,000 molecular weight cutoff (MWCO) than microfiltration (MF) membrane of 0.2 mum. (c)1993 John Wiley & Sons, Inc.     

Viability of an ectomycorrhizal fungus during cross-flow filtration

Factors affecting the viability and infectivity of an ectomycorrhizal fungus during moderate concentration by cross-flow filtration were determined. Mycelial suspensions were concentrated with three commercial membrane filters (Prostak Millipore Co., M14 Tech-Sep Co. and Ceraflo Norton Co.) under aseptic conditions. Medium components may reduce the filtration rate due to their low solubility. An antifoam agent did not reduce the average flux rates as much as did the malt extract. Clear unobstructed channels (I.D. 6mm) of the tubular modules (Tech-Sep) gave the best results both in terms of performance (filtration rate) and cell viability. Shear stresses caused by pumping and flow through narrow retentate channels were probably responsible for lowering viability and infectivity. There was no linear relationship between permeate fluxes and cell concentration. There is an optimum pore size both in terms of performance (filtration rate) and cell viability. Physical blockage of large pores by hyphae could explain lower permeate flux rates than those obtained with lower pore sizes membranes.     

On-line biofilm strength detection in cross-flow membrane filtration systems

A fluid dynamic gauging (FDG) technique was used for on-line and in situ measurements of Pseudomonas aeruginosa PAO1 biofilm thickness and strength on flat sheet polyethersulphone membranes. The measurements are the first to be successfully conducted in a membrane cross-flow filtration system under constant permeation. In addition, FDG was used to demonstrate the removal behaviour of biofilms through local biofilm strength and removal energy estimation, which other conventional measurements such as flux and TMP cannot provide. The findings suggest that FDG can provide valuable additional information related to biofilm properties that have not been measured by other monitoring methods.     

Extraction of laminarin from Saccharina latissima seaweed using cross-flow filtration

Laminarin is a low-molecular-weight polysaccharide found in seaweed (kelp), often in equal concentrations to that in the commercially important hydrocolloid alginate. However, while alginate can be easily recovered by dissolution followed by acid precipitation, for laminarin, there is no such straightforward way of recovering it. Laminarin can be used as dietary fiber and, if efficiently extracted, it may be used for functional food/feed applications and as a component in plant defense stimulants for agriculture. One way of concentrating laminarin from dilute solutions is to press the solution through ultrafine membranes that the molecules cannot pass through. When alginate is extracted, an acid pretreatment step is used and the dilute acid residue from that process also contains laminarin. We used cross-flow filtration to concentrate laminarin from Saccharina latissima, retrieving it from the dilute acid solution of the acid pretreatment of an alginate extraction. Three ceramic membranes with 5, 15, and 50 kDa molecular weight cutoffs were used, and the pressure, temperature, and feed velocity were altered to reveal which parameters controlled the flow through the membrane and how efficiently laminarin was concentrated. The effects on laminarin extraction for fresh vs. frozen biomass were evaluated showing that frozen biomass releases more laminarin with a similar biomass homogenization technique. Thermal and microbial degradation of the feed components was studied during the course of the filtrations, showing that microbial degradation can affect the laminarin concentration, while the temperature of the process ~ 65 °C had little impact on laminarin. The techniques used to monitor the components in the feed and permeate during filtration were nuclear magnetic resonance, ¹H-NMR, and size exclusion chromatography. The filtrations were performed in a pilot-size filtration unit with ceramic membranes (ZrO₂/TiO₂, TiO₂-Al₂O support, 0.08 m²). To be able to operate without quick membrane fouling, the most important parameter was to have a high liquid velocity over the membrane, 4.7 m?s^?1. A good technique to concentrate laminarin was to prefilter it through a 50-kDa membrane using 2 bar liquid pressure and to concentrate it over a 5-kDa membrane using 5-bar liquid pressure. With these settings, the liquid flux through the filter became 60–80 and 30–40 L?m^?2?h^?1 over the 50-kDa and 5-kDa membrane.

     

Modeling shear damage to suspended CHO cells during cross-flow filtration

A mathematical model is presented for predicting the shear-induced decrease in live cells occurring over time during tangential flow filtration. The model uses a cell death rate constant (K) and considers the effects of flow rate, solution viability, and filtration system volumes and dimensions. Single pass and recycle capillary experiments with solutions of high (93%), medium (87%), and low (70%) viability were run, where the maximum laminar shear stress ranged from 10- 300 Pa, to validate the model and determine cell death rate constants. The K values for the suspended CHO cells used in this research ranged from 0.06 to 12.5 s-1. These K values increased with shear stress, as expected, and also as the solution viability decreased.     

Air-bubbling, hollow-fiber reactor with cell bleeding and cross-flow filtration

Continuous asymmetric reduction of dyhydrooxoisophorone (DOIP) to 4-hydroxy-2,2,6-trimethylcyclo-hexanone (4-HTMCH) was achieved by a thermophilic bacterium Bacillus stearothermophilus NK86-0151. Three reactors were used: an air-bubbling hollow-fiber reactor with cell bleeding and cross-flow filtration, an air-lift reactor, and a CSTR with PAA immobilized cells. The maximum cell concentration of 11.1 g dry wt L(-1) was obtained in an air-bubbling hollow-fiber reactor, while in the other reactors the cell densities were between 3.5 and 4.1 g dry wt L(-1) The optimum bleed ratio was 0.1 at the dilution rate 0.3 h(-1) in the hollow-fiber reactor. The highest viable cell concentration was maintained in the dilution range of 0.4-0.7 h(-1) by a combination of proper cell bleeding and cross-flow filtration. The maximum volumetric productivity of 4-HTMCH reached 826 mg L(-1) h(-1) at the dilution rate 0.54 h(-1). This value was 4 and 2 times higher than those in the air-lift reactor and CSTR, respectively. The increasing viable cell concentration increased the volumetric productivity of 4-HTMCH. A cell free product solution was continuously obtained by cross-flow filtration.     

Improvement of performance for cross-flow membrane filtration of pullulan broth

To improve the performance of cross-flow membrane filtration of pullulan broth from Aureobasidium pullulans, the effect of the cultivation conditions was examined. In particular, the sucrose concentration in the medium was changed over a wide range. By decreasing the sucrose concentration the distribution of morphology of the microbial cells in the broth changed; the yeast-like form became predominant and, as a result, the specific resistance of the microbial cake was lowered. When the broth was fermented with a sucrose concentration of 2.5% or lower, the filtration characteristics were greatly improved by periodic closure of permeation during cross-flow filtration.On leave from Hayashibara Co., Ltd., Amase-minamimachi, Okayama 700, Japan Correspondence to: K. Nakanishi     

High concentration cultivation of Bifidobacterium longum in fermenter with cross-flow filtration

Summary High concentration cultivation of Bifidobacterium longum in a fermenter with cross-flow filtration using a ceramic filter is described. Continuous cross-flow filtration allowed complete recycling of the cells to the fermenter and also continuous separation of inhibitory metabolites. The final cell concentration attained in the cultivation was 54.4 g dry wt./l; this was seven times as high as that without cross-flow filtration. The time course of the cultivation with cross-flow filtration was predicted, based on the assumption that the specific growth rate can be expressed only as a function of concentrations of metabolites (acetate and lactate) in a culture broth.Nomenclature D dilution rate (h^-1) - m maintenance coefficient (h^-1) - OD ₅₇₀ optimal density at 570 nm - P _A acetate concentration (g/l) - P _A0 initial acetate concentration (g/l) - P _L lactate concentration (g/l) - P _L0 initial lactate concentration (g/l) - S lactose (substrate) concentration (g/l) - S ₀ initial lactose (substrate) concentration (g/l) - t cultivation time (h) - Y _x/s growth yield (g/g) - X dry cell concentration (g/l) - X ₀ initial dry cell concentration (g/l) - constant - constant     

On-off regulation of gene expression from tryptophan promoter with cross-flow filtration

Summary Recombinant plasmid containing β-galactosidase gene fused to trp promoter (pMCT98) and that containing cloned trp repressor gene (pRLK13) were introduced into Escherichia coli C600. The bacterium was cultivated in a jar-fermetor equipped with a cross-flow filtration apparatus to attain the on-off regulation of the gene expression by controlling tryptophan concentration in the medium. In logarithmic growth phase, the cross-flow filtration was started. Tryptophan concentration dropped to a low level within 1 h and an efficient expression of β-galactosidase gene was started. By this twostage cultivation, very high biomass was achieved (final OD₅₇₀: 150) and the amount of produced β-galactosidase was about 10% of total cellular proteins.     

Extraction of rIL-2 inclusion bodies from Escherichia coli using cross-flow filtration

A cross-flow membrane filtration process was developed for the recovery of rIL-2 inclusion bodies from homogenized Escherichia coli. The membrane extraction process was comprised of a two-step diafiltration followed by an extraction with 7 M GuHCl and a 40-fold dilution of the solubilized inclusion bodies into 0.01 M Tris-HCl, 0.035 M NaCl, pH 7.9. The first diafiltration was with a 0.03 M Tris-HCl, 5 mM ethylenediaminetetraacetic acid (EDTA), pH 8, followed by a diafiltration with 1.75 M GuHCl. All of the insoluble rIL-2 was retained behind the membrane, whereas a GuHCl wash solubilized approximately 15% of the rIL-2. The membrane process increased the yield of rIL-2 in the diluted extract by threefold as compared to a similar centrifuge process with a significant increase in purity as determined by reverse-phase high-performance liquid chromatography (HPLC). (c) 1994 John Wiley & Sons, Inc.     

Purification of lysozyme by multistage affinity filtration

A multistage affinity filtration process was developed for the purification of proteins. An affinity adsorbent was prepared by immobilizing Cibacron Blue 3GA to TSK gel HW-65F. Adsorption equilibrium experiments showed that the blue TSK gel had a high affinity for lysozyme, while its binding to bovine serum albumin (BSA) was weaker. Using a three-stage affinity filtration system, lysozyme was purified from a model system (a mixture of lysozyme and BSA) and a natural source (chicken egg white). From the chicken egg white, the three-stage affinity filtration increased the recovery yield of lysozyme from 61 to 96%, compared with the one-stage process. A mathematical model taking into account the film and interior diffusions of protein and eluant was developed for the modeling and analysis of the experimental data. Both the experimental and modeling results indicate that the multistage affinity filtration technique can be employed for the selective recovery of proteins.     

Determination of maintenance coefficients of Saccharomyces cerevisiae cultures with cell recycle by cross-flow membrane filtration

The fermentation of glucose by a strain of Saccharomyces cerevisiae was studied in a continuous single-stage process with recycle of the cells via cross-flow micro-filtration membranes. Operating conditions were selected such that the culture was not carbon limited and inhibition by ethanol and cell death were minimized.Steady states were obtained for various biomass bleeding rates, i.e., various specific growth rates. From the experimental data, the stoichiometry of the simultaneous reactions, cell growth, ethanol production and maintenance were established using mass and degree of reduction balance relative to substrates (carbon source and oxygen) and products (biomass, ethanol, carbon dioxide etc.), and the growth parameters, yields, and maintenance cofficients were determined. It was shown that the oxygen consumption was not linked to the kinetics of the fermentation. The calculated growth constants were discussed and compared to the currently reported values.