Two-stage Ultrafiltration Process Research Articles

A two-stage ultrafiltration process for separating multiple components of Tetraselmis suecica after cell disruption

A two-stage ultrafiltration process was applied to the aqueous phase of Tetraselmis suecica after breaking its cell wall by high-pressure homogenization. Microscopic observation revealed that the cells were completely disrupted from 600 bar and cell fragmentation of the cells was also noticeable after 800 bar. In addition, the highest concentration of all the molecules of interest in the aqueous phase was observed at 1,000 bar and a temperature of 46 °C while preserving the integrity of the molecules of interest in the downstream process. After centrifugation, the aqueous phase was submitted to ultrafiltration through two consecutive membranes of different molecular weight cutoffs. Complete retention of starch was possible with a 100-kDa membrane and separation of sugars from proteins with a 10-kDa membrane on the remaining mixture. After testing the process with model solutions, the transmembrane pressure selected was 2.07 bar, which succeeded in retaining starch and pigments during the first part of the process, and proteins during the second part. A linear correlation between the permeate flux rate and the pressure was observed in both parts of the process.

Optimization of conditions in ultrafiltration treatment of produced water by polymeric membrane using Taguchi approach

ABSTRACTIn this study, the ultrafiltration of produced water was studied using a two-stage ultrafiltration process. In the first stage, the influences of operating parameters, including transmembrane pressure, temperature, and cross-flow velocity on the amount of flux decline caused by membrane fouling, were investigated using a polymeric membrane. In order to design the experiments and optimize the experimental results, the Taguchi method was applied. L9 (33) orthogonal array for experimental planning and the smaller-the-better response category was selected to obtain optimum conditions because the lowest flux decline was our aim. Analysis of variance was used to determine the most important parameters affecting the flux decline caused by membrane fouling. The optimum conditions were found at the first level of transmembrane pressure (1.5 bar), second level of temperature (40°C), and third level of cross-flow velocity (1 m/s). In the second stage, performance of ultrafiltration system by the polymeric me...

Ultrafiltration of Coagulation-Pretreated Serratia marcescens Fermentation Broth: Flux Characteristics and Prodigiosin Recovery

The dead-end ultrafiltration (UF) of coagulation-pretreated fermentation broth of Serratia marcescens SMΔR for prodigiosin recovery was studied. Experiments were performed using different types (regenerated cellulose, YM; polyethersulfone, PES) and molecular weight cut-offs (MWCOs, 1–10 kDa) of the membranes, feed concentrations of prodigiosin (300–1000 mg/L), applied pressures (68.9–206.8 kPa), and stirring speeds (200–400 rpm). With the same MWCO, the YM membrane had a higher retention of prodigiosin and a lower flux than the PES membrane. A two-fold concentration of prodigiosin was observed in the retentate using a 1-kDa YM membrane compared to the concentration in the permeate using a 10-kDa YM membrane. In addition, the extent of membrane fouling was quantitatively analyzed in terms of the modified fouling index. Flux decline in the present batch UF process was mainly due to cake layer formation and partly due to pore blocking. A two-stage UF process was proposed for this purpose, with 81% recovery yield and four-fold concentration.

A novel membrane-based process to isolate peroxidase from horseradish roots: optimization of operating parameters

The optimization of operating parameters for the isolation of peroxidase from horseradish (Armoracia rusticana) roots with ultrafiltration (UF) technology was systemically studied. The effects of UF operating conditions on the transmission of proteins were quantified using the parameter scanning UF. These conditions included solution pH, ionic strength, stirring speed and permeate flux. Under optimized conditions, the purity of horseradish peroxidase (HRP) obtained was greater than 84 % after a two-stage UF process and the recovery of HRP from the feedstock was close to 90 %. The resulting peroxidase product was then analysed by isoelectric focusing, SDS-PAGE and circular dichroism, to confirm its isoelectric point, molecular weight and molecular secondary structure. The effects of calcium ion on HRP specific activities were also experimentally determined.

Isolation and Purification of a Hyperthermostable Superoxide Dismutase from Thermus thermophilus HB27 Using Two-Stage Ultrafiltration

In the present work, a hyperthermostable superoxide dismutase (SOD) was isolated from Thermus thermophilus HB27 using a membrane-based process after being expressed in E. coli. It was found that a two-stage ultrafiltration process using a 100 kDa MWCO regenerated cellulose (RC) membrane and a 50 kDa MWCO polyethersulfone (PES) membrane could successfully employed to isolate SOD from the crude feedstock. The effects of solution pH, ionic strength and permeate flux on the transmission of proteins were quantified using parameter scanning ultrafiltration. Under optimized conditions, the purity of SOD obtained was 96% with a sepcific activity of 1412 U/mg protein and the recovery of SOD from the feedback was close to 87%. The enzyme was highly stable at 90°C and retained 63% activity after heat treatment at 100°C for 1 h.

Diafiltration and concentration of Reactive Brilliant Blue KN-R solution by two-stage ultrafiltration process at pilot scale: Technical and economic feasibility

In this study, a two-stage ultrafiltration (UF) membrane separation process on a pilot scale was introduced to resolve the shortcomings caused by the application of salt precipitation technique in the conventional process of Reactive Brilliant Blue KN-R production. The first stage was used for diafiltration and concentration of dye solution, and the second stage was applied to recover the dye from permeate of the first stage. Both the operating pressure and cross-flow velocity had significant and moderate effect on the permeate flux (24.9 to 129.7 L·m − 2 ·h − 1 ) and salt rejection (26.9%–55.7%), but insignificant effect on dye rejection since the rejections were always higher than 99.5%. The two-stage UF membrane process exhibited good performance as the dye and salt concentrations in concentrate of first stage were 203 and 5.4 g/L, and 67.1% of salt could be removed and the dye loss was negligible (0.1%) in the whole process. For a dye plant with a capacity of 1000 ton per year, the preliminary economic evaluation of an UF plant based on pilot scale showed that the cost of every ton of Reactive Brilliant Blue KN-R was US $154.

Purification and characterization of a hyperthermostable Mn-superoxide dismutase from Thermus thermophilus HB27

Thermostable Mn-dependent catalases are promising enzymes in biotechnological applications. In the present study, a Mn-containing superoxide dismutase of the hyperthermophilic Thermus thermophilus HB27 had been purified and characterized by a two-stage ultrafiltration process after being expressed in E. coli. The enzyme was highly stable at 90°C and retained 57% activity after heat treatment at 100°C for 1 h. The native form of the enzyme was determined as a homotetramer by analytical size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The final purified enzyme had an isoelectric point of 6.2 and a high α-helical content of 70%, consistent with the theoretical values. This showed that the purified SOD folded with a reasonable secondary structure.

Purification and characterization of superoxide dismutase from garlic

An efficient and easily scaled up method to isolate superoxide dismutase from garlic is proposed. The separation and purification procedure consists of phosphate buffer extraction, heat treatment and a two-stage ultrafiltration process. The enzyme was purified 139-fold with a specific activity of 2867 U/mg protein and a yield of 91%. The native molecular mass of superoxide dismutase estimated by fast protein liquid chromatography on a Superose 6 column was 28 kDa. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis showed a single band near 14 kDa, suggesting that native enzyme was homo-dimeric. The optimal pH for enzyme activity was found to be 7.0, and at this pH the enzyme exhibited maximum activity at 50 °C in 50 mM sodium phosphate buffer. Among various metal ions examined, Cu 2+ and Zn 2+ exerted a positive effect on superoxide dismutase activity, whereas Hg 2+ was found to be a strong inhibitor. The final purified enzyme had an isoelectric point of 5.1–5.4 and a sheet content of 46%, consistent with the literature values. This shows that the purified SOD folded with a reasonable secondary structure.

A new membrane based process to isolate immunoglobulin from chicken egg yolk

Although the importance of eggs as a source of specific antibodies has been well recognised, the generation of egg yolk immunoglobulins (IgY) is rarely chosen due to the peculiar composition of egg yolk and the lack of specific affinity ligands. In this work, we report a novel membrane based two-stage ultrafiltration process to isolate IgY from egg yolk. The effects of solution pH, ionic strength, stirring speed and permeate flux on the transmission of proteins were quantified using the pulsed sample injection technique and parameter scanning ultrafiltration. Under optimised conditions, the purity of immunoglobulin obtained was greater than 93% after the two-stage ultrafiltration process and the recovery of immunoglobulin from the feedstock was close to 87%. The resulting immunoglobulin product was then analysed by Isoelectric Focusing (IEF), SDS–PAGE and Circular Dichroism (CD), to confirm its isoelectric point, molecular weight and molecular secondary structure.

Isolation and purification of superoxide dismutase from garlic using two-stage ultrafiltration

The objective of this work is to develop a membrane-based cost-effective process for the separation and purification of superoxide dismutase (SOD) from garlic. It was found that a two-stage ultrafiltration process using a 100 kDa MWCO regenerated cellulose membrane and a 50 kDa MWCO polyethersulfone membrane could successfully be employed to isolate SOD by removing the bigger macromolecules and smaller molecules from the crude feedstock. The effects of solution pH and ionic strength on the transmission of SOD and the presence of impurity proteins with molecular weights close to SOD were quantified using the pulsed sample injection ultrafiltration technique. The effect of other parameters including system hydrodynamics and mass transfer, in terms of stirring speed, and permeate flux on the separation of SOD was also studied using parameter scanning ultrafiltration. Under optimized conditions, the purity of SOD obtained was greater than 98.0% after two-stage ultrafiltration process and the recovery of SOD from the feedstock was close to 90%. The resulting SOD product was analyzed by isoelectric focusing (IEF), SDS-PAGE and circular dichroism (CD), to confirm its isoelectric point, molecular weight and molecular secondary structure of the garlic SOD. The purity and activity as well as effects of metal ions (copper and zinc ions) on SOD specific activities were experimentally determined.

Purification of surfactin in pretreated fermentation broths by adsorptive removal of impurities

An attempt was made to further improve the purity of surfactin in the fermentation broth of Bacillus subtilis ATCC 21332 culture by adsorption or ion exchange after the broth had been treated by a two-stage ultrafiltration process. The commonly used neutral adsorption resin (macroporous XAD-7) and charged ion-exchange resin (AG1-X4) were adopted. Under the conditions tested the impurities in the treated broth such as macromolecules (proteins, polysaccharides, peptides) or smaller molecules (glycine, serine, threonine, alanine) were adsorbed or exchanged more quickly and preferably within 5 h of operation, purification of surfactin was hence achieved by adsorption removal of the impurities. It was shown that XAD-7 resins gave higher purity of surfactin than AG1-X4 resins. The recovery of surfactin from the remaining solution after adsorption using XAD-7 resin exceeded 95% at pH 6.5 and the purity increased from 76% (the feed) to 88%; however, the purity of surfactin reached up to 80% only after ion exchange using AG1-X4 resin. Finally, the breakthrough phenomena of surfactin solutions in a XAD-7 column were tested and discussed.

A two-stage ultrafiltration process for fractionation of whey protein isolate

Recent studies have demonstrated that membrane ultrafiltration can be used to separate proteins with very similar size, but these investigations have been performed with model binary mixtures. This study examined the use of a two-stage tangential flow filtration system for the purification of both α-LA and β-LG from whey protein isolate. Separation was achieved using 100 and 30 kDa membranes in series, with the buffer concentration and filtration velocity for each stage adjusted to give optimal separation. Two purification strategies were examined: one using the 100 kDa membrane followed by the 30 kDa and one with the reverse order. In both cases, the α-LA purification was greater than 10-fold at 90% yield. The recovery of β-LG was more challenging since it was obtained in the permeate from one stage and the retentate from the other. The results provide important insights into the development of staged membrane systems for the purification of complex protein mixtures.

Quantitative Determination of Ca2+ Effects on Endotoxin Removal and Protein Yield in a Two-Stage Ultrafiltration Process

Endotoxin subunits normally aggregate into vesicles with a molecular mass around 1,000,000. Some proteins can bind and disaggregate endotoxin to form protein–endotoxin complexes, which makes endotoxin removal from protein solution more complicated. It is also known that divalent cations such as Ca2+ can act as “bridges” between lipopolysaccharide subunits, causing large vesicles to form. Such a property has potential utility in endotoxin removal from biological solutions. A two-stage ultrafiltration was used in this study during which Ca2+ reaggregated lipopolysaccharide subunits in protein solutions into large vesicles that hence were retained by a 300,000 nominal molecular weight cutoff ultrafiltration membrane. After the reaggregation of lipopolysaccharide subunits, the endotoxin removal efficiency was in the range of 54.78 to 73.10%, meanwhile protein yield was in the range of 71.57 to 89.54% at various protein/endotoxin concentration ratios.