Development of a process for large-scale purification of C-phycocyanin from Synechocystis aquatilis using expanded bed adsorption chromatography

In this paper a large and scaleable method for purification of C-phycocyanin (C-PC) from the cyanobacteria Synechocystis aquatilis has been developed. Phycobiliproteins are extracted from the cells by osmotic shock and separated by passing the centrifuged cell suspension through an expanded bed adsorption chromatography (EBAC) column using Streamline-DEAE as adsorbent. The eluted C-PC rich solution is finally purified by packed-bed chromatography using DEAE-cellulose. Optimal extraction is achieved using phosphate 0.05 M buffer pH 7.0 twice. The operation of EBAC is optimized on a small scale using a column of 15 mm internal diameter (I.D.). The optimal conditions are a sample load of 4.9 mg C-PC/mL adsorbent, an expanded bed volume twice the settled bed volume and a sample viscosity of 1.020 mP. The EBAC process is then scaled up by increasing the column I.D. (15, 25, 40, 60 and 90 mm) and the success of the scale-up process is verified by determining the protein breakthrough capacity and product recovery. The yield of the EBAC step is in the range of 90-93% for every column diameter. To obtain pure C-PC, conventional ion-exchange chromatography with DEAE-cellulose is utilized and a yield of 74% is obtained. The overall yield of the process, comprising all steps, is 69%. The purification steps are monitored using SDS-PAGE and the purity of recovered C-PC is confirmed by absorption and emission spectroscopy and RP-HPLC. Results show that EBAC method is a scalable technology that allows large quantities of C-PC to be obtained without product loss, maintaining a high protein recovery while reducing both processing cost and time.     

Evaluation of the effect of in-bed sampling on expanded bed adsorption

An expanded bed adsorption (EBA) column (5 cm diameter) has been modified to allow the abstraction of liquid samples from various positions along the height of an expanded bed. As the adsorbent particles were fluidized, in-bed monitoring of key component concentrations during feedstock application, washing and elution was achieved by the withdrawal of liquid samples from the voids within the expanded bed through ports along the wall of the column. Component levels in the withdrawn streams can be assayed using on-line analytical chromatography or samples can be collected and assayed off-line.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 and as a tool to provide information about the hydrodynamic and adsorption/desorption processes that occur during expanded bed adsorption. Studies of residence time distributions indicated that the modifications to the column do not significantly affect liquid dispersion. Using the adsorption of glucose-6-phosphate dehydrogenase from yeast homogenate on Streamline DEAE as a model system, comparison of breakthrough curves for runs when in-bed monitoring was and was not performed also suggested that separation efficiency is not appreciably affected by in-bed sampling.     

Hydrophobic interaction expanded bed adsorption chromatography (HI-EBAC) based facile purification of recombinant streptokinase from E. coli inclusion bodies

The downstream processing of recombinant streptokinase (rSK), a protein used for dissolution of blood clots has been investigated employing Escherichia coli inclusion bodies obtained after direct chemical extraction followed by expanded bed adsorption chromatography (EBAC). Streptokinase was over-expressed using high cell density (final OD(600)=40) culture of recombinant E. coli, and an SK protein concentration of 1080 mg l(-1) was achieved. The wet cell pellet after centrifugation was re-suspended in 8M urea containing buffer resulting in direct extraction of almost 97% of cellular proteins into solution. Compared to mechanical disruption using sonication, the direct extraction helped in simultaneous cell lysis and inclusion body (IB) solubilization in a single integrated step. The post-extraction solution containing cell debris and cellular proteins was diluted and directly loaded on to an EBAC column containing Streamline phenyl, without clarification. By passing the solution four times through the column and using 1M NaCl during loading, 82.7% rSK activity could be recovered in the 10mM sodium phosphate buffer used for elution. A 3-fold increase in specific activity of rSK, from 0.18 x 10(5) in cell lysate to 0.53 x 10(5)IU mg(-1) resulted after this step. rSK was further purified to near-homogeneity (specific activity=0.96 x 10(5)IU mg(-1)) by a subsequent ion-exchange step operated in packed bed mode. An overall downstream recovery of 63% rSK was achieved after EBAC and ion exchange chromatography. The paper thus describes the purification of rSK using a three-step regime involving simple, efficient and highly facile steps.     

Gas chromatography and gateway sensors for on-line state estimation of complex fermentations (butanol-acetone fermentation)

A fermentation system has been designed to demonstrate the use of gas chromatography (GC) for on-line monitoring of the butanol-acetone and other complex saccharolytic fermentations. Tangential flow ultrafiltration was used to sterilely and continuously obtain a cell-free filtrate from the fermentation broth for on-line GC analysis of butanol, butyrate, acetate, acetone, ethanol, and acetoin. The liquid injection system consists of a phosphoric acid contactor, a slider-type injection valve, and a heater to address the difficulties (ghosting) encountered in the analysis of carboxylic acids. The fermentation headspace gas was also analyzed by on-line GC for nitrogen and carbon dioxide, while hydrogen was measured by difference. Raw chromatographic data were analyzed by a chromatography data system. Both raw and processed data were transmitted to a VAX 11/750 computer for further processing (using the fermentation equation) and archiving. The fermentation equation, which has recently been derived and tested on completed fermentation data, was also found to be valid during transient fermentations and thus useful as a gateway sensor for calculating various fermentation parameters on-line. Such parameters include glucose concentration and gas composition, as well as a number of unobservable parameters (such as Y(ATP), excess ATP, and NAD reduced by FdH(2)), which characterize the state of the fermentation.     

Hydrodynamics and performance in fluidized bed adsorption

The performance of fluidized bed adsorption is strongly influenced by the hydrodynamics of the fluidization process. Especially axial mixing in the liquid and solid phase may lead to reduced capacity and resolution. In this article axial mixing in the liquid phase of a classified fluidized bed based on porous glass granules is presented. Axial mixing was analyzed by measurements of residence time distributions in a fluidized bed, showing a reduction of mixing at increased ratio of bed height to diameter as well as at increased linear velocity of the liquid stream. These results were transferred to two real adsorption systems on two different scales: In a bench scale (up to 15 mL of adsorbent) the purification of monoclonal antibodies from hybridoma supernatant was performed with a cation exchanger, in a larger scale (up to 750 mL of matrix) the adsorption of bovine serum albumin (BSA) on the same matrix was investigated. The results showed an increase of capacity at increased bed height-to-diameter ratio; with regard to linear velocity a broad range of only slightly changed capacity was found. A shift from dispersion controlled to diffusion controlled adsorption at intermediate linear velocity was proposed by isolating the effect of pore diffusion from the effect of dispersion. (c) 1995 John Wiley & Sons, Inc.     

Cross-scale quality assessment of a mechanistic cation exchange chromatography model

Cation exchange chromatography (CEX) is an essential part of most monoclonal antibody (mAb) purification platforms. Process characterization and root cause investigation of chromatographic unit operations are performed using scale down models (SDM). SDM chromatography columns typically have the identical bed height as the respective manufacturing-scale, but a significantly reduced inner diameter. While SDMs enable process development demanding less material and time, their comparability to manufacturing-scale can be affected by variability in feed composition, mobile phase and resin properties, or dispersion effects depending on the chromatography system at hand. Mechanistic models can help to close gaps between scales and reduce experimental efforts compared to experimental SDM applications. In this study, a multicomponent steric mass-action (SMA) adsorption model was applied to the scale-up of a CEX polishing step. Based on chromatograms and elution pool data ranging from laboratory- to manufacturing-scale, the proposed modeling workflow enabled early identification of differences between scales, for example, system dispersion effects or ionic capacity variability. A multistage model qualification approach was introduced to measure the model quality and to understand the model's limitations across scales. The experimental SDM and the in silico model were qualified against large-scale data using the identical state of the art equivalence testing procedure. The mechanistic chromatography model avoided limitations of the SDM by capturing effects of bed height, loading density, feed composition, and mobile phase properties. The results demonstrate the applicability of mechanistic chromatography models as a possible alternative to conventional SDM approaches.     

铜藻岩藻黄素提取及纯化工艺研究

为了对岩藻黄素的提取、纯化进行系统研究,进而为高纯度岩藻黄素的工业化生产提供研究基础,筛选了适用于提取铜藻（Sargassum horneri）鲜藻中岩藻黄素的有机溶剂,并通过单因素实验和正交实验确定了最佳的提取溶剂浓度、提取温度、提取时间、料液比等工艺参数。随后采用硅胶柱层析法进行纯化,并通过单因素实验确定了最佳的硅胶柱床高度、上样量和洗脱流速。最后采用制备液相法对经层析纯化的岩藻黄素进一步纯化。结果表明,有机溶剂萃取的最佳工艺条件为：甲醇浓度90%,提取温度50 ℃,提取时间1 h,料液比1∶10,此条件下岩藻黄素提取率达到(0.258 9±0.003 6) mg·g-1鲜重（FW）［(1.078 8±0.015 0) mg·g-1干重（DW）］。硅胶柱层析的最佳工艺条件为：硅胶柱床高度10 cm,上样量6 g,洗脱流速10 mL·min-1,此条件下岩藻黄素得率为0.176 5 mg·g-1FW（0.735 3 mg·g-1 DW）,纯度为87.01%±0.88%。经制备液相进一步纯化后,岩藻黄素得率为0.127 1 mg·g-1 FW（0.529 4 mg·g-1 DW）,纯度为99.27%±0.22%。研究所用工艺简单,岩藻黄素得率高,为高纯度岩藻黄素的制备提供了实验基础。     

Mathematical model for a three-phase fluidized bed biofilm reactor in wastewater treatment

A mathematical model for a three phase fluidized bed bioreactor (TFBBR) was proposed to describe oxygen utilization rate, biomass concentration and the removal efficiency of Chemical Oxygen Demand (COD) in wastewater treatment. The model consisted of the biofilm model to describe the oxygen uptake rate and the hydraulic model to describe flow characteristics to cause the oxygen distribution in the reactor. The biofilm model represented the oxygen uptake rate by individual bioparticle and the hydrodynamics of fluids presented an axial dispersion flow with back mixing in the liquid phase and a plug flow in the gas phase. The difference of settling velocity along the column height due to the distributions of size and number of bioparticle was considered. The proposed model was able to predict the biomass concentration and the dissolved oxygen concentration along the column height. The removal efficiency of COD was calculated based on the oxygen consumption amounts that were obtained from the dissolved oxygen concentration. The predicted oxygen concentration by the proposed model agreed reasonably well with experimental measurement in a TFBBR. The effects of various operating parameters on the oxygen concentration were simulated based on the proposed model. The media size and media density affected the performance of a TFBBR. The dissolved oxygen concentration was significantly affected by the superficial liquid velocity but the removal efficiency of COD was significantly affected by the superficial gas velocity. An erratum to this article can be found online at .     

Bed height monitoring and control for expanded bed chromatography

The height of a stable expanded bed depends upon the physical properties of the inlet liquid of which viscosity, density and flow rate have the most significant influence. A change in any of these parameters will subject the top of the bed to fluctuations and effective control would require monitoring the position of the top of the bed. In this work, the use of an LED based sensor configuration for providing information on the movement of the top of the bed in terms of its direction and rate of change is described. The information is incorporated in two different algorithms for pump control for appropriate adjustment of flow rate to counter the observed changes. The effectiveness of the sensor assembly and control algorithm was evaluated by testing the system on an expanded bed subject to a step change in the viscosity of the inlet irrigating liquid. The algorithm was further evaluated under scale-up and excellent control could be achieved using the protocol developed as a result of the high correlation of the expansion characteristics at the different scales.     

Fixed-Bed Studies on Removal of Arsenic from Simulated Aqueous Solutions Using Chitosan Nanoparticles

Arsenic is a toxic element and may be found in natural as well as in industrial water; therefore, before using water for drinking purpose, its proper treatment is required. Thus, the aim of this work was to evaluate the potential of chitosan nanoparticles, in a continuous-flow method, for the removal of arsenic (III) and (V) from aqueous solutions. All experiments were conducted in fixed-bed columns. Experiments were carried out as a function of varying liquid flow rate (0.3–1.0 ml/min), initial metal concentration (0.5–1.5 mg/L), and bed height (3–9 cm) of adsorbent. The total adsorbed quantity, equilibrium uptake, and total percentage removal of arsenic ions were determined by evaluating the breakthrough curves obtained at different flow rates, initial concentrations, and bed heights. The results showed that the column performed well at the lowest flow rate. Also, column bed capacity and exhaustion time were found to increase with increasing bed height. When initial metal ion concentration was increased from 0.5 to 1.5 mg/L, the corresponding adsorption bed capacity decreased from 0.076 to 0.028 mg/g. The bed depth service time model (BDST) model was used to analyze the experimental data and the model parameters were evaluated. The calculated values of N _o and K _a were found to be 19.28 × 10^?2 mg/L and 0.662 L/mg·min, respectively. Good agreement was found between the experimental breakthrough curves and the model predictions.     

Separation of yeast cells from aqueous solutions using magnetically stabilized fluidized beds

AIMS: To separate Saccharomyces cerevisiae cells from aqueous solutions using magnetically stabilized fluidized beds (MSFB) that utilize a horizontal magnetic field, and to study the effect of some parameters, such as bed porosity and height, liquid flow rate and inlet concentration on cell removal efficiency and breakthrough curves. METHODS AND RESULTS: The separation process was conducted in an MSFB under the effect of horizontal magnetic field. The magnetic particles used consist of a ferromagnetic core of magnetite (Fe3O4) covered by a stable layer of activated carbon to adsorb the yeast cells from the suspension. The yeast cell concentration in the effluent was determined periodically by measuring the absorbance at 610 nm. The effect of the magnetic field intensity on the bed porosity and consequently the exit-normalized cell concentration from the bed was studied. It was found that bed porosity increased by 75%, and the normalized cell concentration in the bed effluent decreased by 30%, when the magnetic field intensity was increased from 0 to 110 mT. In addition, increasing the magnetic field intensity and bed height delayed the breakthrough point, and allowed efficient cell removal. These results demonstrate an improved method to separate cells of low concentration from cell suspension. CONCLUSIONS: This study allows the continuous separation of yeast cells from aqueous solutions in an MSFB. The removal efficiency is affected by different parameters including the bed height, flow rate and initial concentration. The removal efficiency reaches 82%, and could be improved by varying the operational parameters. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained in this investigation show that the MSFB using horizontal fields represents a potential tool for the continuous separation of cell suspension from aqueous solution. This study will contribute to a better understanding of the hydrodynamic parameters on the separation efficiencies of the cell.     

Comparison of non-catalytic and catalytic fast pyrolysis of corncob in a fluidized bed reactor

Fast pyrolysis of corncob with and without catalyst was investigated in a fluidized bed to determine the effects of pyrolysis parameters (temperature, gas flow rate, static bed height and particle size) and a HZSM-5 zeolite catalyst on the product yields and the qualities of the liquid products. The result showed that the optimal conditions for liquid yield (56.8%) were a pyrolysis temperature of 550 degrees C, gas flow rate of 3.4 L/min, static bed height of 10 cm and particle size of 1.0-2.0mm. The presence of the catalyst increased the yields of non-condensable gas, water and coke, while decreased the liquid and char yields. The elemental analysis showed that more than 25% decrease in oxygen content of the collected liquid in the second condenser with HZSM-5 was observed compared with that without catalyst. The H/C, O/C molar ratios and the higher heating value of the oil fraction in the collected liquid with the catalyst were 1.511, 0.149 and 34.6 MJ/kg, respectively. It was indicated that the collected liquid in the second condenser had high qualities and might be used as transport oil.     

The influence of particle size distribution and operating conditions on the adsorption performance in fluidized beds

The influence of matrix properties and operating conditions on the performance in fluidized-bed adsorption has been studied using Streamline diethyl-aminoethyl (DEAE), an ion exchange matrix based on quartz-weighted agarose, and bovine serum albumin (BSA) as a model protein. Three different particle size fractions (120-160 mum, 120-300 mum, and 250-300 mum) were investigated. Dispersion in the liquid phase was reduced when particles with a wide size distribution were fluidized compared to narrow particle size distributions. When the mean particle diameter was reduced, the breakthrough capacities during frontal adsorption were enlarged due to a shorter diffusion path length within the matrix. At small particle diameters the effect of film mass transfer became more relevant to the adsorption performance in comparison to larger particles. Therefore matrices designed for fluidized-bed adsorption should have small particle diameter and increased mean particle density to ensure small diffusion path length in the particle and a high interstitial velocity to improve film mass transfer. Studies on the influence of sedimented matrix height on axial mixing showed an increased Bodenstein number with increasing bed length. Higher breakthrough capacities were also found for longer adsorbent beds due to reduced dispersion and improved fluid and particle side mass transfer. With increasing bed height the influence of flow rate on breakthrough capacity was reduced. For a settled bed height of 50 cm breakthrough capacities of 80% of the equilibrium capacity for flow rates varying from 3 to 9 cm/min could be achieved. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 54-64, 1997.     

Modeling of transient flow through a viscoelastic preparative chromatography packing

The common method for purification of macromolecular bioproducts is preparative packed‐bed chromatography using polymer‐based, compressible, viscoelastic resins. Because of a downstream processing bottleneck, the chromatography equipment is often operated at its hydrodynamic limit. In this case, the resins may exhibit a complex behavior which results in compression–relaxation hystereses. Up to now, no modeling approach of transient flow through a chromatography packing has been made considering the viscoelasticity of the resins. The aim of the present work was to develop a novel model and compare model calculations with experimental data of two agarose‐based resins. Fluid flow and bed permeability were modeled by Darcy's law and the Kozeny–Carman equation, respectively. Fluid flow was coupled to solid matrix stress via an axial force balance and a continuity equation of a deformable packing. Viscoelasticity was considered according to a Kelvin–Voigt material. The coupled equations were solved with a finite difference scheme using a deformable mesh. The model boundary conditions were preset transient pressure drop functions which resemble simulated load/elution/equilibration cycles. Calculations using a homogeneous model (assuming constant variables along the column height) gave a fair agreement with experimental data with regard to predicted flow rate, bed height, and compression–relaxation hysteresis for symmetric as well as asymmetric pressure drop functions. Calculations using an inhomogeneous model gave profiles of the bed porosity as a function of the bed height. In addition, the influence of medium wall support and intraparticle porosity was illustrated. The inhomogeneous model provides insights that so far are not easily experimentally accessible. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:958–967, 2013     

One-step isolation of adenosine triphosphate from crude fermentation broth of Saccharomyces cerevisiae by anion-exchange chromatography using supermacroporous cryogel

Adenosine triphosphate (ATP) is an important high-energy compound widely used in biological and therapeutic fields. It can be produced by phosphorylation of adenosine monophosphate (AMP) with microbial cells in industrial scale and the effective isolation of ATP from microbial fermentation broth is a challenging work. In this work, we develop a novel one-step method to directly separate ATP from fermentation broth of Saccharomyces cerevisiae by anion-exchange chromatography using supermacroporous cryogel. The cryogel bed with tertiary amine groups was prepared by grafting N,N-dimethylaminoethyl methacrylate (DMAEMA) monomer chains onto the matrix of a polyacrylamide-based cryogel in a glass column and its properties of liquid dispersion, water permeability, porosity as well as the ligand density were measured. Chromatographic separation of ATP from the fermentation broth by the cryogel was carried out using deionised water and 0.01 M HCl as running buffer, respectively. The breakthrough characteristics and elution performance in the cryogel bed were revealed and analyzed. The purities of the obtained ATP were analyzed quantitatively by high performance liquid chromatography (HPLC). The maximal purity of ATP by the one-step separation method was 95.5% using 0.01 M HCl as running buffer in this work. The corresponding chromatographic behaviors were investigated and analyzed.     

On-line monitoring of breakthrough curves within an expanded bed adsorber

By abstracting samples of the liquid phase from various positions along the height of an expanded bed, it has been possible to monitor the breakthrough profiles of adsorbing components during the application of feedstock. Similarly, the concentration profiles of the subsequent washing and elution procedures were also followed. The procedure involves the abstraction of liquid samples from the voids of the expanded bed using a specially modified column and assaying the levels of proteins in the withdrawn stream by on-line rapid chromatographic monitoring. Studies of the residence time distribution showed that the modifications to the expanded bed did not cause additional mixing and dispersion. Breakthrough profiles have been measured in a simple single component system and in a complex feedstock in which the adsorption of lysozyme from skimmed cows' milk was monitored. The system shows promise for the on-line control and monitoring of expanded bed adsorption separations, together with providing additional insight into the hydrodynamic and adsorption/desorption processes that occur during bioseparations using expanded bed adsorption.     

Pressure-flow relationships for packed beds of compressible chromatography media at laboratory and production scale

Pressure drop across chromatography beds employing soft or semirigid media can be a significant problem in the operation of large-scale preparative chromatography columns. The shape or aspect ratio (length/diameter) of a packed bed has a significant effect on column pressure drop due to wall effects, which can result in unexpectedly high pressures in manufacturing. Two types of agarose-based media were packed in chromatography columns at various column aspect ratios, during which pressure drop, bed height, and flow rate were carefully monitored. Compression of the packed beds with increasing flow velocities was observed. An empirical model was developed to correlate pressure drop with the aspect ratio of the packed beds and the superficial velocity. Modeling employed the Blake-Kozeny equation in which empirical relationships were used to predict bed porosity as a function of aspect ratio and flow velocity. Model predictions were in good agreement with observed pressure drops of industrial scale chromatography columns. A protocol was developed to predict compression in industrial chromatography applications by a few laboratory experiments. The protocol is shown to be useful in the development of chromatographic methods and sizing of preparative columns.     

Degradation of aromatic compounds by Pseudomonas putida

The influence of different process kinetics on the course of phenol degradation has been studied as well as the influence of axial dispersion in the liquid phase on the reactor height with relatively large biofilm thickness in a conventional fluidized bed and air-lift bioreactor. The object of this was to achieve a high conversion of substrate in a device of real size in real process time. For calculating the mathematical model, the method of orthogonal collocation with the STIFF integration routine has been used.     

Simulation of the dynamic packing behavior of preparative chromatography columns via discrete particle modeling

Preparative packed‐bed chromatography using polymer‐based, compressible, porous resins is a powerful method for purification of macromolecular bioproducts. During operation, a complex, hysteretic, thus, history‐dependent packed bed behavior is often observed but theoretical understanding of the causes is limited. Therefore, a rigorous modeling approach of the chromatography column on the particle scale has been made which takes into account interparticle micromechanics and fluid–particle interactions for the first time. A three‐dimensional deterministic model was created by applying Computational Fluid Dynamics (CFD) coupled with the Discrete Element Method (DEM). The column packing behavior during either flow or mechanical compression was investigated in‐silico and in laboratory experiments. A pronounced axial compression–relaxation profile was identified that differed for both compression strategies. Void spaces were clearly visible in the packed bed after compression. It was assumed that the observed bed inhomogeneity was because of a force‐chain network at the particle scale. The simulation satisfactorily reproduced the measured behavior regarding packing compression as well as pressure‐flow dependency. Furthermore, the particle Young's modulus and particle–wall friction as well as interparticle friction were identified as crucial parameters affecting packing dynamics. It was concluded that compaction of the chromatographic bed is rather because of particle rearrangement than particle deformation. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:363–371, 2016     

Modeling of direct recovery of lactic acid from whole broths by ion exchange adsorption

Lactic acid fermentation process with L. casei CRL 686 was performed. The static adsorption isotherm over a strong anionic exchange resin, Amberlite^TM IRA-400 was measured, and the static binding capacity parameters were quantified. Early recovery of lactic acid from this lactate producer from unclarified culture broth was performed in a liquid solid fluidized bed, with the resin as the solid adsorbent, and the dynamic adsorption capacity was calculated. Good agreement was found between static and dynamic binding capacity values. The fluidized bed height was twice the settled bed height and the overall process was controlled by the liquid solid mass transfer. This operation was also simulated by continuously well stirred tanks arranged in series and superficial solid deactivation as in a gas solid catalytic reactor. The deactivation process takes into account liquid channeling and agglomerations of solid induced by the viscosity of the broth and also by the cells during the adsorption. These patterns were also verified by experimental observations, and are in agreement with the results found in the literature. The breakthrough data together with others from previous works were satisfactorily fitted until the 90% dimensionless concentration was reached for both culture broths. The model could be used in future studies on predictions about the liquid solid fluidized bed behavior and other different operating conditions.