Fluidized bed ion exchange for improving purification of lactic acid from fermentation

A strong anionic exchange resin was used to recover lactic acid directly from fermentation in an upflow fluidized bed column, resulting in 0.18 g lactic acid/g resin bound with a subsequent elution of 94%. When the culture broth was heated and adjusted pH to 8.0, 0.4 g lactic acid was bound per g resin, with a subsequent elution of 90%. L(+) and D(–) lactic acid isomers distribution was analyzed in the elution product resulting in an increase of L(+) isomer concentration. The resin did not alter its binding capacity after 23 cycles.     

Lactate from cultures of Lactobacillus casei recovered in a fluidized bed column using ion exchange resin

Summary Lactic acid produced by continuous culture of L.casei in an upflow packed bed reactor, was recovered with Amberlite IRA 400 in a fluidized bed column. Bed expansions of 1.25 and 2.25 were applied. Reutilization did not alter the capability of net recovery of 0.048 ± 0.01 g lactic acid/g resin. When 2200 cm/h of ascensional velocity was used, (bed expansion of 2.25), the resin adsorbed 39.3% of the initial lactic acid and 63.5% was eluted. This resin supported the highest exchange capacity of 0.126 g lactic acid/g resin. Applying high flow rates, the process has potential industrial applications due to the short time employed.     

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.     

Effects of adsorbent properties on zone spreading in expanded bed chromatography

The mixing performance as well as the adsorption performance in expanded bed chromatography (EBC) was investigated by using various types of adsorption media (average particle size = 100–700 m, density = 1100–1700 kg/m³, base matrix = hydroxyapatite, styrene-divinylbenzene, cross-linked agarose). The scale down study with 0.8 cm diameter columns was also attempted. Pulse response curves were measured with vitamin B₁₂ as a tracer [Residence time distribution RTD experiments], and the HETP (height equivalent to a theoretical plate or plate height) values were calculated from the peak variance and the peak retention time. The HETP values for different types of packing media tested showed very similar values (0.5–1.0 cm), which did not depend on the flow-rate or the column diameter (0.8–2.6 cm). Dynamic binding capacity (DBC) values of lactic acid on a Dowex anion-exchange resin were determined from breakthrough curve (BTC) measurements for both EBC and fixed bed chromatography (FBC). The DBC values for EBC were similar to those for FBC. When the liquid feed contained insoluble particles (yeast cells) the degree of mixing increased. However, the contribution of the mixing to the total spreading of BTCs for EBC was usually small so that this increase in the mixing did not affect the adsorption performance or the DBC values significantly.     

Transport and binding characterization of a novel hybrid particle impregnated membrane material for bioseparations

The transport and binding properties of a novel hybrid particle-nonwoven membrane medium are described. In this construct, a polymeric chromatographic resin is entrapped between two layers of a nonwoven polypropylene membrane. The membrane-supported resin medium offers the advantage of increased interstitial pore diameter to allow passage of cells and other debris in the feed, while providing sufficiently high surface area for product capture within the resin particles. Columns packed with PIM displayed excellent flow distribution and had interstitial porosities of 0.48 ± 0.01, 25-60% larger than those typical of a packed bed. These columns were able to pass over 95% of E. coli cells and human red blood cell concentrate in 30 column volumes while maintaining a pressure drop significantly lower than that of a packed bed with a similar amount of resin. The dynamic binding capacity of bovine serum albumin (BSA) to the chromatographic resin entrapped in the PIM packed column was essentially the same as that observed with the same volume of resin in a packed bed. The General Rate (GR) model of chromatography was used to analyze experiments indicating the breakthrough behavior of the PIM columns is predictable, and very similar to those of a normal packed bed. These results suggest that PIM constructs can be designed to process viscous mobile phases containing particulates while retaining the desirable binding characteristics of the embedded chromatographic resin and could find uses in adsorption separation processes from complex feed streams such as whole blood, cell culture, and food processing.     

Recovery of lactic acid from simultaneous saccharification and fermentation media using anion exchange resins

The physicochemical properties (capacity, kinetics and selectivity) of the ion exchange resins Amberlite IRA900, IRA400, IRA96 and IRA67 were determined to evaluate their comparative suitability for lactic acid recovery. Both the kinetics of lactic acid sorption from aqueous solutions and the equilibrium were assessed using mathematical models, which provided a close interpretation of the experimental results. The best resins (Amberlite IRA96 and IRA67) were employed in further fixed-bed operation using aqueous lactic acid solutions as feed. In this set of experiments, parameters such as capacity, regenerant consumption, percentage of lactic acid recovery and product concentration were measured. Amberlite IRA67, a weak base resin, was selected for lactic acid recovery from SSF (simultaneous saccharification and fermentation) broths. Owing to the presence of nutrients and ions other than lactate, a slightly decreased capacity was determined when using SSF media instead aqueous lactic acid solutions, but quantitative lactic acid recoveries at constant capacities were obtained in four sequential load/regeneration cycles.     

Continuous separation of succinic acid and lactic acid by using a three-zone simulated moving bed process packed with Amberchrom-CG300C

The issue of separating succinic acid and lactic acid in a continuous mode has been a major concern in the biotechnological process for production of succinic acid. To address this issue, both the optimal design and the experimental validation of a three-zone simulated moving bed (SMB) process for such separation were attempted in this article using the Amberchrom-CG300C resin and a self-assembled SMB unit with three zones. First, the intrinsic parameters of the two organic acids on the Amberchrom-CG300 resin were estimated at 40 °C from a series of multiple frontal experiments. The resulting intrinsic parameters were then used in optimizing the experimental setting points for pump flow rates and switching time of the three-zone SMB equipment, which was assisted by an up-to-date genetic algorithm. Based on the optimized conditions, the relevant SMB experiment was conducted at 40 °C and all the resultant samples from the product ports and column outlets of the SMB unit were assayed. It was confirmed from the assay results that the continuous separation of succinic acid and lactic acid was performed successfully. The experimental data for the product concentration profiles and the internal concentration profiles were also in reasonable agreement with the model predictions.     

Separation of lactic acid from acetic acid using a four-zone SMB

A simulated moving bed (SMB) process has been developed to separate l-(+)-lactic acid from acetic acid, a major impurity in the fermentation broth of Lactobacillus rhamnosus. Poly(4-vinylpyridine) resin (PVP) was selected as the adsorbent. Adsorption isotherms and mass transfer parameters of the organic acids were estimated from single-column frontal tests. Experimental results show that the Langmuir isotherms obtained from the frontal tests can be used in the design of an SMB process to achieve 99.9% purity and over 93% yield of lactic acid. The column profiles and effluent histories, however, deviate from rate model predictions based on the Langmuir isotherms. They agree more closely with the predictions based on a modified Langmuir isotherm for lactic acid. The standing wave design method for systems with modified Langmuir isotherms is developed in this study. Rate model simulations show that the process based on the modified design method can achieve high purity (>99.9%) and high yield (>99.9%). For this nonlinear system, accurate isotherm model and model parameters are needed in the design, and the zone flow rates must be closely monitored and controlled in order to ensure high purity and high yield in the SMB process.     

Monoclonal antibody production using a new supermacroporous cryogel bioreactor

A supermacroporous cryogel bioreactor has been developed to culture hybridoma cells for long-term continuous production of monoclonal antibodies (mAb). Hybridoma clone M2139, secreting antibodies against J1 epitope (GERGAAGIAGPK; amino acids, 551-564) of collagen type II, are immobilized in the porous bed matrix of a cryogel column (10 mL bed volume). The cells got attached to the matrix within 48 h after inoculation and grew as a confluent sheet inside the cryogel matrix. Cells were in the lag phase for 15 days and secreted mAb into the circulation medium. Glucose consumption and lactic acid production were also monitored, and during the exponential phase (approximately 20 days), the hybridoma cell line consumed 0.75 mM day-1 glucose, produced 2.48 mM day-1 lactic acid, and produced 6.5 microg mL-1 day-1 mAb during the exponential phase. The mAb concentration reached 130 microg mL-1 after continuous run of the cryogel column for 36 days. The yield of the mAb after purification was 67.5 mg L-1, which was three times greater than the mAb yield obtained from T-flask batch cultivation. Even after the exchange of medium reservoir, cells in the cryogel column were still active and had relatively stable mAb production for an extended period of time. The bioreactor was operated continuously for 55 days without any contamination. The results from ELISA as well as arthritis experiments demonstrate that the antibodies secreted by cells grown on the cryogel column did not differ from antibodies purified from the cells grown in commercial CL-1000 culture flasks. Thus, supermacroporous cryogels can be useful as a supporting material for productive hybridoma cell culture. Cells were found to be viable inside the porous matrix of the cryogel during the study period and secreted antibodies continuously. The antibodies thus obtained from the cryogel reactor were found to be functionally active in vivo, as demonstrated by their capacity to induce arthritis in mice.     

三相流化床中固定化米根霉萃取发酵生产L-乳酸

以TRPO／磺化煤油为萃取剂,在2L三相流床反应器中进行了固定化米根霉原位萃取和异位萃取发酵生产L-乳酸的实验,结果表明,发酵液中的pH值能被控制在3．5左右．产酸速率高达每小时．每1L固定化颗粒产生11gL-乳酸。提出了一个数学模型用以描述萃取发酵中L-乳酸的积累及在各相的分配情况。模型计算曲线与实验值符合良好。     

变速上样在MabSelect填料上分离WLB303

随着抗体表达量的提升和生产规模的扩大,Protein A亲和层析不仅需要高载量填料,也需要提高工艺效率。变速上样的方法可以在满足载量要求的同时大大缩短工艺耗时。通过测定WLB303单克隆抗体在GE MabSelect填料多个保留时间的动态载量,建立一元三次方程拟合载量和保留时间的关系。以该方程计算获得填料在快、中、慢速下不同保留时间的动态载量表,并以此作为变速上样组合的参考依据。在2.7ml层析柱上使用精纯样品测试最快的变速上样组合的可行性;然后用常规纯化工艺在7ml层析柱上使用细胞培养澄清液,对最快的变速上样组合和恒速上样的两种工艺周期进行了比较,证实变速上样的方式能明显提升整体工艺效率。     

Extractive lactic acid fermentation using ion-exchange resin

Lactic acid fermentation is an end-product-inhibited reaction. The restriction imposed by lactic acid on its fermentation can be avoided by extractive fermentation techniques. Studies were performed by attaching an ion-exchange resin packed column with a 2-L fermentor for separation of lactic acid. The fermentation, in a conventional batch mode, resulted in a lactic acid yield of 0.828 g . g(-1) and a lactic acid productivity of 0.313 g . L(-1) . h(-1). However, these could be further enhanced to 0.929 g . g(-1) and 1.665 g . L(-1) . h(-1) by extractive fermentation techniques. The effect of temperature on extractive fermentation was remarkable and has been included in this work.     

Extractive fermentation of lactic acid by immobilizedLactobacillus casei using ion—exchange resin

Summary A novel method of lactic acid fermentation byLactobacillus casei immobilized in Ca—alginate gels is described, in which an ion—exchange resin packed column is attached to a fermentor for separation of lactic acid from fermentative broth. The technique successfully alleviated the restriction imposed by lactic acid on bacterial growth and product formation. As compared to the conventional batch fermentation, the new fermentation technique enhanced the lactic acid productivity and sugar conversion rate from 0.328g/L·h and 88. 2% to 0.482g/L·h and 98.6%, respectively.     

New method of immobilization of microbial cells by capture on the surface of insoluble pyridinium-type resin

A new method for the immobilization of microbial cells has been developed. Whole cells of Escherichia coli with aspartase activity were immobilized by capture on the surface of cross-linked poly(N-benzyl-4-vinylpyridinium bromide) containing styrene (BVPS resin), an insoluble pyridinium-type resin. When a suspension of the bacterial cells in buffer solution was passed through a glass column containing beads of BVPS resin, the cells were captured on the resin surface and formed an immobilized cell system. A fixed-bed column reactor containing 300 mg of the bacterial cells immobilized by capture on 10 g of BVPS resin beads was used for the preparation of L-aspartic acid from ammonium fumarate. Continuous operation of tne bioreactor produced L-aspartic acid in a quantitative yield when the influent substrate concentration was 0.1M and the flow rate was 0.41-0.83 bed volumes per hour at pH 7.4-7.7 at 30 degrees C.     

Continuous bind‐and‐elute protein A capture chromatography: Optimization under process scale column constraints and comparison to batch operation

Recently, continuous downstream processing has become a topic of discussion and analysis at conferences while no industrial applications of continuous downstream processing for biopharmaceutical manufacturing have been reported. There is significant potential to increase the productivity of a Protein A capture step by converting the operation to simulated moving bed (SMB) mode. In this mode, shorter columns are operated at higher process flow and corresponding short residence times. The ability to significantly shorten the product residence time during loading without appreciable capacity loss can dramatically increase productivity of the capture step and consequently reduce the amount of Protein A resin required in the process. Previous studies have not considered the physical limitations of how short columns can be packed and the flow rate limitations due to pressure drop of stacked columns. In this study, we are evaluating the process behavior of a continuous Protein A capture column cycling operation under the known pressure drop constraints of a compressible media. The results are compared to the same resin operated under traditional batch operating conditions. We analyze the optimum system design point for a range of feed concentrations, bed heights, and load residence times and determine achievable productivity for any feed concentration and any column bed height. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:938–948, 2016     

Expanded bed adsorption of human serum albumin from very dense Saccharomyces cerevesiae suspensions on fluoride-modified zirconia.

The adsorption of proteins from high cell density yeast suspensions on mixed-mode fluoride-modified zirconia (FmZr) particles (38 to 75 microm, surface area of 29 m(2)/g and density of 2.8 g/cm(3)) was investigated using human serum albumin (HSA) added to Saccharomyces cerevesiae as the model expression host. Because of the high density of the porous zirconia particles, HSA (4 mg/mL) can be adsorbed from a 100 g dry cell weight (DCW)/L yeast suspension in a threefold-expanded bed of FmZr. The expanded bed adsorption of any protein from a suspension containing >50 g DCW/L cells has not been previously reported. The FmZr bed expansion characteristics were well represented by the Richardson-Zaki correlation with a particle terminal velocity of 3.1 mm/s and a bed expansion index of 5.4. Expanded bed hydrodynamics were investigated as a function of bed expansion using residence time distribution studies with sodium nitrite as the tracer. The adsorption of HSA on FmZr exhibited features of multicomponent adsorption due to the presence of dimers. The protein binding capacity at 5% breakthrough decreased from 22 mg HSA/mL settled bed void volume for 20 g DCW/L yeast to 15 mg HSA/mL settled bed void volume for 40 g DCW/L yeast and remained unchanged for the higher yeast concentrations (60 to 100 g DCW/L). However, the batch (or equilibrium) binding capacity decreased monotonically as a function of yeast concentration (20 to 100 g DCW/L) and the binding capacity at 100 g DCW/L yeast was fivefold lower compared with that at 20 g DCW/L yeast. The lower batch binding capacity at high cell concentrations resulted from the adsorption of cells at the surface of the particles restricting access of HSA to the intraparticle surface area. Batch (or equilibrium) and column HSA adsorption results indicated that the adsorption of HSA on FmZr occurred at a time scale that may be much faster than that of yeast cells. The zirconia particles were cleaned of adsorbed HSA and yeast with a total of 1500 to 2000 column volumes (over many cycles) of 0. 25 M NaOH, without any significant effect on the chromatographic performance.     

Characterization of cryogel monoliths for extraction of minor proteins from milk by cation exchange

Extraction and purification of high‐value minor proteins directly from milk without pre‐treatment is a challenge for the dairy industry. Pre‐treatment of milk before extraction of proteins by conventional packed‐bed chromatography is usually necessary to prevent column blockage but it requires several steps that result in significant loss of yield and activity for many minor proteins. In this paper, we demonstrate that it is possible to pass 40–50 column volumes of various milk samples (raw whole milk, homogenized milk, skim milk and acid whey) through a 5 mL cryogel chromatographic column at 550 cm/h without exceeding its pressure limits if the processing temperature is maintained above 35°C. The dynamic binding capacity obtained for the cryogel matrix (2.1 mg/mL) was similar to that of the binding capacity (2.01 mg/mL) at equilibrium with 0.1 mg/mL of lactoferrin in the feed samples. The cryogel column selectively binds lactoferrin and lactoperoxidase with only minor leakage in flowthrough fractions. Lactoferrin was recovered from elution fractions with a yield of over 85% and a purity of more than 90%. These results, together with the ease of manufacture, low cost and versatile surface chemistry of cryogels suggest that they may be a good alternative to packed‐bed chromatography for direct capture of proteins from milk. Biotechnol. Bioeng. 2009;103: 1155–1163. © 2009 Wiley Periodicals, Inc.     

固定化细胞利用离子交换树脂萃取发酵乳酸的研究

本文提出了利用海藻酸钙凝胶包埋固定化乳酸菌生产乳酸,用离子交换树脂从发酵液中分离出乳酸的新方法。该法成功地消除了产物乳酸对乳酸菌生长和产物乳酸形成的抑制作用,使发酵时间由１２０小时缩短到９６小时,乳酸的体积生产率由０．３２８ｇ／Ｌ·ｈ提高到０．４３２ｇ／Ｌ·ｈ。     

Human alpha-thrombin binding to nonpolymerized fibrin-Sepharose: evidence for an anionic binding region

In order to investigate ligand binding sites in alpha-thrombin that interact with nonpolymerized fibrin, fibrinogen was conjugated (with CNBr) to Sepharose 4B and converted to the nonpolymerized fibrin resin with alpha-thrombin. Human alpha-thrombin was bound to the resin at 22 degrees C and eluted with a linear NaCl gradient [50-300 mM in 50 mM tris(hydroxymethyl)aminomethane hydrochloride, pH 7.6] with midpeak elution occurring at an ionic strength that corresponds to 170 +/- 5 mM NaCl. Among various ligands examined, ATP and its analogues caused alpha-thrombin to elute with 125 mM or less salt. Apparent dissociation constants were estimated by the dependence of elution volume on ligand concentration. The most potent ligands for desorption from the column were anionic (e.g., adenine nucleotides), which also inhibit thrombin esterolytic/amidolytic and clotting activity [Conery, B. G., & Berliner, L. J. (1983) Biochemistry 22, 369-375]. The desorption series was at 10 mM concentrations: ATP = ADP greater than pyrophosphate greater than citrate greater than oxalate greater than PO4(3-). Contrastingly, serotonin and related apolar compounds did not cause dissociation of alpha-thrombin from the fibrin resin, even though several of these substances inhibit fibrinogen clotting and esterolytic/amidolytic activities of the enzyme. These data imply that independent sites for apolar and anionic binding in alpha-thrombin are required for converting fibrinogen into clottable fibrin and that alpha-thrombin-fibrin binding involves an anionic site.     

Rapid and simple gas chromatographic measurement of lactic acid in red blood cells, plasma, and tumor cells after hyperthermia.

The content of lactic acid in red blood cells, plasma, and Ehrlich ascites tumor cells were measured by a gas-liquid chromatography using a column with a terephtalic acid support coated with polyethylene glycol-6000. The lactic acid contents were directly determined in aqueous samples, because they were converted to a volatile derivative in the column. The method was rapid and simple, compared with previous methods which need time-consuming conversion of lactic acid to volatile derivatives. Our measurements showed the increase in the contents of intra- and extracellular lactic acid after hyperthermia.