Expanded Bed Adsorption Column Research Articles

In situ recovery of taxadiene using solid adsorption in cultivations with Saccharomyces cerevisiae

In this study, an engineered strain of Saccharomyces cerevisiae was used to produce taxadiene, a precursor in the biosynthetic pathway of the anticancer drug paclitaxel. Taxadiene was recovered in situ with the polymeric adsorbent Diaion © HP-20. Here we tested two bioreactor configurations and adsorbent concentrations to maximize the production and recovery of taxadiene. An external recovery configuration (ERC) was performed with the integration of an expanded bed adsorption column, whereas the internal recovery configuration (IRC) consisted in dispersed beads inside the bioreactor vessel. Taxadiene titers recovered in IRC were higher to ERC by 3.4 and 3.5 fold by using 3% and 12% (w/v) adsorbent concentration respectively. On the other hand, cell growth kinetics were faster in ERC which represents an advantage in productivity (mg of taxadiene/L*h). High resin bead concentration (12% w/v) improved the partition of taxadiene onto the beads up to 98%. This result represents an advantage over previous studies using a 3% resin concentration where the partition of taxadiene on the beads was around 50%. This work highlights the potential of in situ product recovery to improve product partition, reduce processing steps and promote cell growth. Nevertheless, a careful design of bioreactor configuration and process conditions is critical.

Treatment of nano-oil polluted wastewater in an expanded bed adsorption column based on carboxymethyl cellulose-cellulose-nickel composite beads

In the present work, spherical carboxymethyl cellulose-cellulose-nickel (CMC-C-Ni) composite beads as novel adsorbent was synthesized to make a stable expanded bed adsorption (EBA) column for the treatment of the oily wastewater collected from the downstream of rapeseed industry. The morphology and structure of the CMC-C-Ni composite beads were studied by scanning electron microscopy (SEM) and optical microscope. The SEM images revealed that the synthesized composite beads were spherical with porous structure. The pore size of the beads was in the range of 90–200 nm. The physical characteristics of the CMC-C-Ni composite beads including wet density, porosity, and water content were respectively in the ranges of 1.23–1.63 g/cm3, 82.29–90.75%, and 52–76%. The factor of bed expansion in the range of 2–3 was corresponded with Richardson-Zaki equation. The results showed that by increasing the fluid viscosity, the terminal settling velocity (Ut) was reduced. The expansion index values were between 2.77 and 3.14 that were close to 4.8 (commonly utilized index in the laminar flow regimes). CMC-C-Ni composite beads were tested when the velocity of fluid was ˂ 700 cm/h, and the Daxl was found to be ˂ 1 × 10−5 m2/s (steady state).

Fabrication of novel agarose–nickel bilayer composite for purification of protein nanoparticles in expanded bed adsorption column

In the present work, a novel agarose–nickel bilayer composite (ANBC) adsorbents modified with Cibacron Blue 3GA (CB3) dye-ligand were fabricated via the three-phase emulsion method. The surface morphology of the novel adsorbents was evaluated using an optical microscope, scanning electron microscopy, and atomic force microscopy. Also, the commercially purchased adsorbents were modified with the same ligand and compared to novel composite (ANBC-CB3). Also, Lactoferrin (Lf) nanoparticles synthesized and employed as a protein model. Residence time distribution experiments were performed to consider the effect of particle density and fluid viscosity. The results established that the ANBC-CB3 bilayer composite follows a typical size range of 60–300μm and a mean diameter of 125μm with a spherical appearance. In batch adsorption, ANBC-CB3 revealed a higher dynamic binding capacity (DBC) of Lf versus Streamline™-CB3. Furthermore, the experimental data were consistent with the Langmuir isotherm model. Finally, The DBC at 10% breakthrough and the two-fold flow rate was more than 70% for the novel adsorbent. It was proved that ANBC-CB3 as high selective adsorbents for protein nanoparticle purification is suitable for high flow rate operations in an expanded bed column.

Improved Production and In Situ Recovery of Sesquiterpene (+)-Zizaene from Metabolically-Engineered E. coli.

The sesquiterpene (+)-zizaene is the direct precursor of khusimol, the main fragrant compound of the vetiver essential oil from Chrysopogon zizanioides and used in nearly 20% of men’s fine perfumery. The biotechnological production of such fragrant sesquiterpenes is a promising alternative towards sustainability; nevertheless, product recovery from fermentation is one of the main constraints. In an effort to improve the (+)-zizaene recovery from a metabolically-engineered Escherichia coli, we developed an integrated bioprocess by coupling fermentation and (+)-zizaene recovery using adsorber extractants. Initially, (+)-zizaene volatilization was confirmed from cultivations with no extractants but application of liquid–liquid phase partitioning cultivation (LLPPC) improved (+)-zizaene recovery nearly 4-fold. Furthermore, solid–liquid phase partitioning cultivation (SLPPC) was evaluated by screening polymeric adsorbers, where Diaion HP20 reached the highest recovery. Bioprocess was scaled up to 2 L bioreactors and in situ recovery configurations integrated to fermentation were evaluated. External recovery configuration was performed with an expanded bed adsorption column and improved (+)-zizaene titers 2.5-fold higher than LLPPC. Moreover, internal recovery configuration (IRC) further enhanced the (+)-zizaene titers 2.2-fold, whereas adsorption velocity was determined as critical parameter for recovery efficiency. Consequently, IRC improved the (+)-zizaene titer 8.4-fold and productivity 3-fold from our last report, achieving a (+)-zizaene titer of 211.13 mg L−1 and productivity of 3.2 mg L−1 h−1. This study provides further knowledge for integration of terpene bioprocesses by in situ product recovery, which could be applied for many terpene studies towards the industrialization of fragrant molecules.

Hydrodynamic Characteristics and Adsorption Particularity of Nanobiological Feedstock Along the Bed Height in a Novel Chromatography Column

Expanded bed adsorption (EBA) is a practical method for the separation of nanoparticulates. In order to analysis the local hydrodynamic and adsorption behavior of nanoparticle (NP)-based biological feedstock, a modified Nano Biotechnology Group EBA column with a 26-mm inner diameter was used to withdraw liquid from different axial positions of the column. Fabricated egg albumin (EA) NPs with an average size of 70 nm were employed as a model system and viral size/charge mimic to assess the relationship between hydrodynamic and adsorption performance of NPs at the different column regions. The effects of influential factors, including flow velocity and initial concentration of NPs, on NP hydrodynamic behavior and adsorption kinetics along the bed height were investigated. NP hydrodynamic studies confirmed that non-uniform behavior dominated the system and a decreasing trend of liquid mixing/dispersion with increase of bed height was observed in this column. The results demonstrated an increase in the mixing/dispersion at certain bed heights with the increase in both the velocity and feed initial concentration. Breakthrough curves were measured at various column points to determine the adsorption performance [dynamic binding capacity (DBC) and yield] in different bed positions/zones. Yield and DBC of NPs were improved along the bed height, whereas liquid velocity had the opposite effect. Increasing the initial concentration of NPs enhanced only the DBC. Separation of EA NPs under optimal conditions was 87 %, which is an excellent result for a one-pass frontal chromatography method.

Evaluation of hydrodynamic parameters of fluidized bed adsorption on purification of nano‐bioproducts

AbstractThe biotechnology industry has recently been demanding nanoparticulate products (20‐200 nm) such as viruses, plasmids, virus‐like particles and drug delivery assemblies. Fluidized bed/expanded bed adsorption (EBA) is a technique that can be employed for nanoparticulate purification providing practical advantages such as the potential for direct process integration as has been seen with protein products. In the present work, the hydrodynamic behaviour of an expanded bed adsorption column for obtaining the optimal conditions of bovine serum albumin (BSA) nanoparticle recovery on streamline DEAE was studied. Residence time distribution (RTD) analysis was used to acquire hydrodynamic information from changes bed expansion, column diameter and settled bed height. Fluidization behaviour was predicted from the Richardson–Zaki correlation, with experimentally determined values of the expansion index. RTD showed axial dispersion increases with bed height as well as linear velocity. However, the effect of expansion degree of the bed on bovine serum albumin (BSA) nanoparticle purification was studied and the yield of recovery of BSA nanoparticle in optimal condition was 80.71%. Hydrodynamic behaviour of expanded bed adsorption and its potential for the purification and recovery of nanoparticle bioproducts is strongly discussed. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

A study of ion-exchange chromatography in an expanded bed for bovine albumin recovery

In the present work, the effect of bed expansion on BSA adsorption on Amberlite IRA 410 ion-exchange resin was studied. The hydrodynamic behavior of an expanded bed adsorption column on effects of the biomolecules and salt addition and temperature were studied to optimize the conditions for BSA recovery on ion-exchange resin. Residence time distribution showed that HEPT, axial dispersion and the Pecletl number increased with temperature and bed height, bed voidage and linear velocity. The binding capacity of the resin increased with bed height. The Amberlite IRA 410 ion-exchange showed an affinity for BSA with a recovery yield of 78.36 % of total protein.

Recovery and partial purification of penicillin G acylase from E. coli homogenate and B. megaterium culture medium using an expanded bed adsorption column

The adsorption of penicillin G acylase (PGA) from B. megaterium and from Escherichia coli on a cationic resin, Streamline SP XL, was studied using both packed and expanded beds. Stability assays showed that penicillin acylases from the two sources presented high irreversible deactivation at pH 4.0 and 4.5, but remained stable at pH 4.8. Adsorption experiments performed in a packed bed (PB), in the pH range 4.8–5.8, showed highest adsorption yields at pH 4.8, for both enzymes. Using small expanded bed adsorption (EBA) columns, PGA was directly recovered and partially purified from E. coli crude extracts, E. coli homogenates, and from B. megaterium centrifuged broth in a single unit operation. Global recovery yields of 91.0, 55.0 and 7.4% and purification factors of 4.5-, 7.5- and 12.7-fold were achieved, respectively. The elution yields of penicillin acylase obtained with these cationic EBA processes when working with E. coli homogenate and B. megaterium centrifuged medium were of 100 and 52%, respectively. The comparison of adsorption capacities of E. coli penicillin acylase from crude extracts onto Streamline SP XL showed similar results for packed-bed and for expanded-bed modes. However, PGA adsorption yields for E. coli (homogenate) and B. megaterium (centrifuged medium) were substantially lower than the values obtained for E. coli crude extract, due to the competition of cell debris and other components present in the B. megaterium medium.

Intensified Process for the Purification of an Enzyme from Inclusion Bodies Using Integrated Expanded Bed Adsorption and Refolding

This work describes the integration of expanded bed adsorption (EBA) and adsorptive protein refolding operations in an intensified process used to recover purified and biologically active proteins from inclusion bodies expressed in E. coli. Delta(5)-3-Ketosteroid isomerase with a C-terminal hexahistidine tag was expressed as inclusion bodies in the cytoplasm of E. coli. Chemical extraction was used to disrupt the host cells and simultaneously solubilize the inclusion bodies, after which EBA utilizing immobilized metal affinity interactions was used to purify the polyhistidine-tagged protein. Adsorptive refolding was then initiated in the column by changing the denaturant concentration in the feed stream from 8 to 0 M urea. Three strategies were tested for performing the refolding step in the EBA column: (i) the denaturant was removed using a step change in feed-buffer composition, (ii) the denaturant was gradually removed using a gradient change in feed-buffer composition, and (iii) the liquid flow direction through the column was reversed and adsorptive refolding performed in the packed bed. Buoyancy-induced mixing disrupted the operation of the expanded bed when adsorptive refolding was performed using either a step change or a rapid gradient change in feed-buffer composition. A shallow gradient reduction in denaturant concentration of the feed stream over 30 min maintained the stability of the expanded bed during adsorptive refolding. In a separate experiment, buoyancy-induced mixing was completely avoided by performing refolding in a settled bed, which achieved comparable yields to refolding in an expanded bed but required a slightly more complex process. A total of 10% of the available KSI-(His(6)) was recovered as biologically active and purified protein using the described purification and refolding process, and the yield was further increased to 19% by performing a second iteration of the on-column refolding operation. This process should be applicable for other polyhistidine tagged proteins and is likely to have the greatest benefit for proteins that tend to aggregate when refolded by dilution.

A flow injection analysis system for on‐line monitoring of cutinase activity at outlet of an expanded bed adsorption column almost in real time

AbstractExpanded bed adsorption (EBA) was used to recover, concentrate and purify Fusarium solani pisi cutinase, secreted by a recombinant Saccharomyces cerevisiae strain, directly from a whole fermentation culture. A flow injection analysis (FIA) system for monitoring Fusarium solani pisi cutinase based on microencapsulation of p‐nitrophenylbutyrate (p‐NPB) in a micellar system (sodium cholate, tetrahydrofuran, phosphate buffer) was developed for monitoring this target enzyme at the outlet tube of the EBA column. Slight differences in yeast cultivation conditions during cutinase production may influence the fermentation performance, which affects directly the adsorption of cutinase during the loading step and consequently the efficiency of the EBA process. This effect can be especially relevant when it is necessary to stop the application of feedstock to the EBA column when the outlet concentration (A) of the desired product is lower than 5% of the feed concentration (Ao). Excellent correlations between the FIA system and the off‐line analytical method for monitoring cutinase activity during the different EBA steps were obtained. Additionally, the blocking/fouling of the sample injector and tubes of the FIA system initially observed were eliminated due to the excellent surfactant properties of the sodium cholate contained in the phosphate buffer and used to dilute the enzyme samples. This FIA system was shown to be a powerful analytical tool for monitoring cutinase activity almost in real time (45–60 s), maximizing enzyme adsorption while minimizing product loss and consequently maximizing the recovery yield of the product. Copyright © 2006 Society of Chemical Industry

Study of amylases recovery from maize malt by ion-exchange expanded bed chromatography

In the present work, the effect of bed expansion and buffer type on the adsorption of amylases from maize malt on Amberlite 410 ion-exchange resin was studied. The hydrodynamic behavior of an expanded bed adsorption column was studied to obtain the optimal conditions of amylases recovery on ion-exchange resin. Residence time distribution showed that HEPT, axial dispersion and the Prandt number increased with bed height, bed voidage and linear velocity. The adsorption capacity of amylases on the resin increased with bed height and the phosphate buffer shows the best recovering of amylases from maize malt on the Amberlite IRA 410 ion-exchange resin.

A novel system for continuous protein refolding and on‐line capture by expanded bed adsorption

A novel two-step protein refolding strategy has been developed, where continuous renaturation-bydilution is followed by direct capture on an expanded bed adsorption (EBA) column. The performance of the overall process was tested on a N-terminally tagged version of human beta2-microglobulin (HAT-hbeta2m) both at analytical, small, and preparative scale. In a single scalable operation, extracted and denatured inclusion body proteins from Escherichia coli were continuously diluted into refolding buffer, using a short pipe reactor, allowing for a defined retention and refolding time, and then fed directly to an EBA column, where the protein was captured, washed, and finally eluted as soluble folded protein. Not only was the eluted protein in a correctly folded state, the purity of the HAThbeta2m was increased from 34% to 94%, and the product was concentrated sevenfold. The yield of the overall process was 45%, and the product loss was primarily a consequence of the refolding reaction rather than the EBA step. Full biological activity of HAT-hbeta2m was demonstrated after removal of the HAT-tag. In contrast to batch refolding, a continuous refolding strategy allows the conditions to be controlled and maintained throughout the process, irrespective of the batch size; i.e., it is readily scalable. Furthermore, the procedure is fast and tolerant toward aggregate formation, a common complication of in vitro protein refolding. In conclusion, this system represents a novel approach to small and preparative scale protein refolding, which should be applicable to many other proteins.

The combined use of in-bed monitoring and an adsorption model to anticipate breakthrough during expanded bed adsorption

Tighter control of expanded bed adsorption (EBA) separations is shown to be possible through the combined use of on-line monitoring of samples abstracted from within a column and an adsorption model. A 5 cm diameter EBA column was modified to allow liquid sampling from within the fluidized bed during the adsorption of pure lysozyme and bovine serum albumin (BSA) on STREAMLINE SP and STREAMLINE DEAE, respectively. The developing breakthrough curve was measured at various positions along the bed. The adsorption model incorporated terms for liquid dispersion, film mass transfer, pore diffusion and adsorption. Parameters obtained by fitting the model to the experimental in-bed breakthrough curves were subsequently used to predict breakthrough at the bed exit. Comparison of the actual breakthrough curves at the bed exit ( 40 cm sample port) with those predicted based on the fit of the model to the experimental breakthrough data obtained from the 25 cm sample port, demonstrated that on-line, in-bed monitoring of the progress of the front of non-bound protein can provide information for tighter control of EBA separations. The accuracy of the model predictions was improved by employing information on the axial variations in the bed voidage, liquid phase axial dispersion and dynamic capacity for the top and bottom zones within the column. Ten per cent breakthrough at the 40 cm sample port was predicted to within 2% and 5% of the breakthrough time observed for the experimental runs for lysozyme and BSA, respectively.

The use of ion-selective electrodes for evaluating residence time distributions in expanded bed adsorption systems.

The suitability of ion-selective electrodes (ISE) for the determination of residence time distribution (RTD) in turbid, cell-containing fluids was examined. The electrodes were found to give reproducible signals in biomass-containing feedstock with up to 20% wet weight of solids. The enhanced feedstock compatibility of IES, when compared to other tracer sensing devices, allows the study of expanded bed system hydrodynamics under relevant operating conditions. Within the linear range of the corresponding ISE-tracer pair, both examined ISE (Li(+)- or Br(-)-selective) showed to be insensitive against the range of flow rate and pH normally employed during expanded bed adsorption (EBA) of proteins. Analyzing the RTD obtained after a perfect ion tracer pulse in terms of the PDE model (PDE, axially dispersed plug-flow exchanging mass with stagnant zones) gave a quantitative description of the underlying hydrodynamic situation during EBA processing. These data provided a powerful tool to make predictions on the adsorptive global process performance with a defined feedstock type and composition. The link between the hydrodynamic events during feedstock application and the actual process performance was shown when applying intact yeast cell suspensions at different biomass content (up to 7.5% wet weight) and buffer conductivity (5-12 mS) onto an EBA column filled with the adsorbent Streamline Q XL as fluidized phase. On the basis of our experimental results, a guideline for the successful application of the ISE/RTD method to EBA process design is presented.

Hydrodynamics and adsorption behaviour within an expanded bed adsorption column studied using in-bed sampling

To study axial variations in particle size, bed voidage, liquid dispersion and dynamic capacity, a 5 cm diameter STREAMLINE expanded bed adsorption column was modified to allow liquid or particle sampling from various radial and axial positions within the fluidized bed. Particle size distributions of adsorbent samples withdrawn from within the expanded bed showed that there was no significant radial variation in particle size and confirmed that mean particle size was larger near the bottom of the column and smallest near the top. Residence time distribution (RTD) analysis was used to acquire hydrodynamic information from changes to the shape of a tracer pulse as it passed from the highly mixed region above the distributor to the calmer regions further up the expanded bed. Liquid jets issuing from the distributor bypassed a portion of the bottom 10 cm of the column. The velocity of liquid flow near the walls of the column was slower than at the centre, and the tracer pulse was more dispersed, resulting in a radial variation in the flow profile. The adsorption of pure lysozyme and bovine serum albumin (BSA) on STREAMLINE SP and STREAMLINE DEAE, respectively, were used to examine how adsorption behaviour was affected by the hydrodynamics of the flow through the different regions of the column. Dynamic capacity of the adsorbent was calculated for two different regions of the column: the bottom zone between the distributor and the height of 25 cm and the top zone between 25 and 40 cm . For both proteins, the dynamic capacity was more than 20% greater in the upper region than in the lower region. Although dynamic capacities were smaller in the lower region of the column, a higher total amount of adsorbate binding occurred here because of the greater adsorbent volume in this part of the bed that is due to the lower degree of bed expansion. Roughly twice the mass of adsorbate was bound (expressed as mg/ml column volume) to the adsorbent in the bottom zone compared to the top zone.

Monitoring of adsorbate breakthrough curves within an expanded bed adsorption column

An expanded bed adsorption column (5 cm diameter) has been modified to allow the ion of liquid samples from various positions along the height of an expanded bed. As the adsorbent particles are fluidized, in-bed monitoring of protein concentration during feedstock application, washing and elution is achieved by the withdrawal of liquid samples from the voids within the expanded bed at ports along the wall of the column. Protein levels in the withdrawn streams can be assayed using on-line analytical chromatography, or samples can be collected and assayed off-line. On-line monitoring can be used to control the duration of the loading stage, or as a tool to provide information about the hydrodynamic and adsorption/desorption processes that occur during expanded bed adsorption. Breakthrough, washing and elution profiles have been monitored for a range of different adsorbate/adsorbent systems, including the adsorption of pure lysozyme on STREAMLINE SP, the adsorption of glucose-6-phosphate dehydrogenase from yeast homogenate on STREAMLINE DEAE and the adsorption of glutathione S-transferase from Escherichia coli homogenate on STREAMLINE Chelating.

Simple two-step procedure for purification of cloned small sialidase from unclarified E. coli feedstocks

An efficient process for the purification of cloned small sialidase (pCPN-1, small nanH cloned from Clostridium perfringens) from unclarified E. coli feedstocks has been developed. In contrast to conventional purification processes, expanded bed adsorption (EBA) technique allows clarification, product recovery, and concentration to be combined into a single step operation. The cloned NanH was collected from both intra- and extracellular fractions, and purified by expanded bed ion-exchange chromatography followed by affinity chromatography. The EBA technique using STREAMLINE DEAE was shown to be successful in purifying NanH with a purification factor of 12 and yield of 93%. Successive purification of the concentrated NanH purified by an EBA column was achieved by affinity chromatography. The elution of adsorbed NanH was performed with 0.1 m NaHCO 3 buffer pH 9.0 and the pH of the eluted NanH was adjusted to 7.0. An overall yield of 88% was obtained after the affinity chromatographic step. The enzyme appeared to be homogeneous by polyacrylamide gel electrophoresis analysis. The molecular weight was measured as 43 KDa for cloned NanH. The procedure described is suitable for large-scale purification of cloned small NanH from unclarified E. coli feedstocks.