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A strategy of combining prefiltration and chromatography using composite cryogels for large-scale separation of biotransformation compounds from crude high-cell-density broth Junxian Yun, Hao Wu, Jie Liu, Shaochuan Shen, Songhong Zhang, Linhong Xu, Kejian Yao, and Shan-Jing Yao Ind. Eng. Chem. Res., Just Accepted Manuscript • Publication Date (Web): 18 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015
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Graphic Abstract 196x109mm (300 x 300 DPI)
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A strategy of combining prefiltration and chromatography using
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composite cryogels for large-scale separation of biotransformation
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compounds from crude high-cell-density broth
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Junxian Yun†, Hao Wu†, Jie Liu†, Shaochuan Shen†, Songhong Zhang*†, Linhong Xu‡, Kejian
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Yao†, Shan-jing Yao**§
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†
State Key Laboratory Breeding Base of Green Chemistry Synthesis Technology, College of Chemical Engineering,
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Zhejiang University of Technology, Hangzhou 310032, China ‡
Faculty of Mechanical & Electronic Information, China University of Geosciences (Wuhan), Wuhan 430074,
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China §
College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China
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* Corresponding author 1:
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E-mail address:
[email protected] (S.H. Zhang)
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** Corresponding author 2:
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E-mail address:
[email protected] (S.-J. Yao).
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ABSTRACT: The separation of interesting biomolecules or compounds from high-cell-density
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suspensions has received intensive attentions in biotechnology industry. In recent years, monolithic
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cryogels have been suggested as a novel class of chromatographic adsorbents for the direct
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separation of biomolecules from such crude microbial feedstocks. However, the preparation of
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large-scale monolithic cryogels for industrial applications and the direct separation of compounds
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from high-cell-density broths in industrial scale are challenge tasks. In this work, a strategy for the
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separation of biotransformation compounds from high-cell-density broths was developed by
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combination of the prefiltration of microbial cells using large-scale cryogels as the filter medium
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and the chromatography followed using large-scale ion-exchange cryogels as the adsorbents.
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Composite cryogel disks (diameter of 143 mm) of poly(2-hydroxyethyl methacrylate) embedded
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with SiO2 nanoparticles (pHEMA−SiO2) and their anion-exchange supports grafted with
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N,N-dimethylaminoethyl methacrylate (pHEMA−SiO2−DMAEMA) were prepared successfully. As
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an example, the separation of cytidine triphosphate (CTP) from a crude high-cell-density broth
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containing 233 g/L Saccharomyces cerevisiae cells was carried out by the prefiltration of cells using
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the cryogel bed packed with the large-scale pHEMA−SiO2 disks followed by the anion-exchange
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chromatography using the packed bed of the large-scale pHEMA−SiO2−DMAEMA cryogel disks.
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The results showed that more than half amount of the yeast cells have been removed successfully
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from the crude broth by the prefiltration using pHEMA−SiO2 cryogel disks, and the CTP with a
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high purity of 98.2% and the recovery of 98.3% were achieved by the anion-exchange
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chromatography using pHEMA−SiO2−DMAEMA cryogel disks from the dissolved feedstock
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filtrated thereafter, indicating that the present strategy is effective and these large-scale composite
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cryogels could be potentially applied as the interesting filter media and chromatography adsorbents
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in industrial biotechnology and downstream processes.
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Keywords: cryogel; bioseparation; filtration; scale-up; cytidine triphosphate.
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1. INTRODUCTION
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The increasing demand for the isolation of interesting molecules from high-cell-density microbial
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fermentation broths and cultures has promoted the investigations of new separation methods and
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novel adsorbents. Monolithic cryogels are one class of such example materials used as the
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adsorbents for the chromatographic separation of compounds from unclarified feedstocks. These
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interesting sponge-like polymer supports have supermacropores with sizes from several to hundreds
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of microns and thus high permeability and porosity, which permit the convective mass transfer of
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compounds and free passage of microbial cells.1–10 Small or lab scale monolithic cryogels can be
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prepared conveniently via cryo-polymerization of aqueous gel-forming monomers or suspensions in
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small columns or molds under freezing conditions, as demonstrated by numerous researchers as
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examples reviewed in references. 1–10 However, the preparation of a monolithic cryogel with large
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diameter and height is difficult since it is needed to rapidly remove a great deal amount of heat from
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the relative big volume of gel-forming solutions or suspensions. Therefore, the direct scale-up of
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monolithic cryogels is a challenge work. Moreover, the large-scale separation of target compounds
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from high-cell-density culture suspensions using monolithic cryogels is also a difficult task.
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In recent years, pHEMA-based or polyacrylamide (pAAm)-based cryogel disks or discs have
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been prepared successfully in glass or metal molds with the diameters from several to more than
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one hundred of millimeters.4,11–14 These disk-form cryogels could be potentially employed in the
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scale-up of monolith cryogels because large-scale beds with an expected height can be prepared by
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packing these disks together in a column with a large diameter.
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CTP is known as an important nucleoside triphosphate and a precursor for RNA synthesis. This
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high-energy compound has received wide applications in biological and therapeutic areas. In
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industrial process, CTP can be produced by microbial synthesis using cytidine monophosphate
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(CMP) as the main substrate and Saccharomyces cerevisiae as the whole-cell catalysis and enzymes
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resource.15–18 In order to ensure the biosynthesis productivity and efficiency, a large amount of yeast
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cells is always used in industrial biotransformation process and thus the cell density in the crude
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broth is usually high. However, the separation of CTP from such unclarified broth with high density
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of cells is complex, and cost intensive. Anion-exchange chromatography using cryogels has been
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suggested as one of promising effective candidate methods for the direct isolation of nucleoside
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triphosphates from crude microbial feedstocks.14,17,19–21 For the high-cell-density suspensions, the
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entrapment or clogging of microbial cells always observed at the entrance of the cryogel beds,
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which could resulted in the decrease of flow rates and thus the complex chromatographic
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performance and unsatisfied separation efficiency.
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In order to perform the chromatography perfectly by cryogels, it is needed to remove some
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microbial cells from the broth to reduce the cell density to a suitable range before the loading.
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Traditionally, the separation of microbial cells from high-cell-density suspensions can be achieved
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by centrifugation or filtration.22–26 Centrifugation employs equipments of centrifuges or
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hydrocyclones to separate the microorganisms from the crude broth by utilizing the difference in
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densities of the cells and the liquid components, and thus usually gives a clarified supernatant. This
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approach is relatively mature in techniques, but suffers some disadvantages like the high cost and
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the requirements of complex equipments and a large amount of energy. Filtration offers an
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alternative effective way for the separation of cells from crude microbial suspensions. It usually
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employs permeable media as the filter material and several filtration methods like the dead-end
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filtration, cross-flow filtration as well as dynamic filtration with rotating disks or drums, and
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rotating or vibrating membranes, as examples in references, 23–26 have been applied in the removal
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of cells. Among them, the dead-end filtration is one of remaining useful tools for the separation of
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cells due to some advantages like the easy operation, low energy consumed and relatively low cost
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needed. In general, the dead-end filtration employs porous materials or membranes typically with
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pore sizes of sub-microns, which are always close to the sizes of microbial cells and thus, the
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filtration performance was influenced by the morphology, size and shape of the cells.23 The
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blockage by the cells and the filter supports themselves lead to a high filtration resistance, and the
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fouling problems in the filtration process. Therefore, it is of great importance to investigate new
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filter materials. Cryogels have porous structures and low flow resistance and thus could be a new
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choice as the novel dead-end filtration matrices. As yet, however, there is a lack of data regarding
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the cell filtration using cryogels and also the chromatographic separation of CTP in a large or
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industrial scale.
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In this paper, a strategy of integrating the prefiltration of microbial cells and the chromatography
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followed using large-scale composite cryogels as the filter media and chromatographic adsorbents
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were proposed for the separation of CTP from a crude high-cell-density yeast broth. Large-scale
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pHEMA−SiO2 cryogel disks for the filtration and anion exchange pHEMA−SiO2−DMAEMA
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cryogel disks for the chromatography were prepared by the cryo-polymerization approach. A
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cryogel bed packed with pHEMA−SiO2 cryogel disks was employed to remove yeast cells from the
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crude feedstock, and another cryogel bed packed with pHEMA−SiO2−DMAEMA disks was used to
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isolate CTP by chromatography from the dissolved feedstock filtrate thereafter. Properties of these
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cryogel disks, the filtration performance as well as the chromatographic performance of the cryogel
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packed beds were investigated experimentally.
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2. MATERIALS AND METHODS
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2.1. Materials. Saccharomyces cerevisiae (CICC 1508) was from China Center of Industrial
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Culture Collection. Ammonium persulfate (APS, 98%), N,N,N’,N’–tetramethylethylenediamine
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(TEMED, 99%), 2-(dimethylamino) ethyl methacrylate (DMAEMA, 98%), poly(ethyleneglycol)
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diacrylate (PEGDA, 99%, Mn~258 g/mol), were from Sigma-Aldrich (Steinheim, Germany).
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Tetraethyl orthosilicate (TEOS, 99%), 2-hydroxyethyl methacrylate (HEMA, 97%) and other
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chemicals were analytical grade from local sources.
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2.2. Preparation of large-scale pHEMA composite cryogel disks embedded with SiO2
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nanoparticles. Monodispersed SiO2 nanoparticles with the mean size of about 364 nm were
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synthesized by two-step hydrolysis and condensation of TEOS in a mixture of ethanol, water and
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ammonia solution.19,27,28 Typically, 2 mL TEOS was added in 27.5 mL solution of ethanol and
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ammonia (10:1, v/v) in 5 min, stirred and maintained at 30℃ for 12 h. The obtained mixture was
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used as the seed and added into 100 mL solution of ethanol and ammonia. Then 7.5 mL TOES was
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added in 55 min, stirred and maintained at 30℃ for 12 h. Finally, SiO2 nanoparticles were obtained
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by centrifuge after washing with ethanol and deionized water.
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The large-scale composite cryogel disks were prepared by cryo-polymerization of the aqueous
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gel-forming mixture containing SiO2 nanoparticles under freezing conditions, as similar as that for
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the preparation of those small-scale monolith cryogels.17,19–21 The preparation of each disk was
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achieved in a stainless steel mold with the inner diameter of 143 mm, the height of 20 mm and the
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wall thickness of 2 mm, as same as that used in our previous work.13,14 The mold was assembled
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together with the top and bottom slabs (thickness of 2 mm). The inlet and outlet tubes with the inner
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diameter of 8 mm were connected to the mold for the injection and discharge of fluid. For the
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preparation of each cell-filtration cryogel disk, about 270 mL the aqueous suspension containing of
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90.8 g HEMA, 27.4 g PEGDA and 1.2 g SiO2 was stirred, pre-cooled to about 4℃, and then 33 mL
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aqueous solution containing 0.47 g APS and 33 mL aqueous solution containing 0.47 g TEMED
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were added. The obtained suspension was pumped into the mold for cryo-polymerized at -15℃ for 8
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h followed by maintaining at about -20℃ for one night. After thawed the cryogel disk was washed
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with enough water for further experiments. The large-scale composite cryogel disks for the
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chromatographic separation of CTP were similar but prepared with the suspensions containing
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gel-forming agents of low concentrations, i.e., the aqueous suspension of the same volume
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containing 38.1 g HEMA, 11.4 g PEGDA, 1 g SiO2, 0.396 g APS and 0.396 g TEMED was used in
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each preparation. The obtained cryogel disks of pHEMA−SiO2 were grafted by 0.5M DMAEMA
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with the initiator solution of 0.056 M K5[Cu(HIO6)2] and 1 M NaCl (2:1, v/v) at the temperature of
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53℃ for 2 h, as similar as those for other large-scale ion-exchange cryogel disks.17,29 The
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pHEMA−SiO2−DMAEMA cryogel disks were obtained by washing the cryogel with 0.1 M HCl
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and deionized water for further use.
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Properties of the pHEMA−SiO2 and pHEMA−SiO2−DMAEMA cryogel disks were characterized
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by measuring the microstructure, water permeability and porosity, according the methods reported
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in references.30–33
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2.3. Filtration of Saccharomyces cerevisiae cells from crude high-cell-density broth by
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large-scale pHEMA−SiO2 cryogel disks. Saccharomyces cerevisiae (CICC 1508) cells grown on
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yeast peptone dextrose (YPD) agar plates (1% yeast extract, 2% peptone, 2% dextrose and 2.0%
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agar) were transferred to YPD medium and the fermentation was achieved at 30℃. When the optical
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density (OD560) of the suspension was about 2.0, the cells were harvested from the broth by
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centrifugation at 6500 rpm for 6 min and washed with 0.9% NaCl solution. In order to enhance the
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passage of endoenzymes and CTP through the cell membrane, the yeast cells were treated by the
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freezing-thawing process, i.e., maintained at about -23℃ for one night and then thawed at room
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temperature. The freezing-thawing process was carried out three times. The obtained cells were
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employed in the biotransformation step.
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About 863 g treated yeast cells were transformed into 3L substrate solution containing 60 mM
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CMP, 100 mM KH2PO4, 12 mM MgSO4 and 150 mM dextrose, and the biotransformation was
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carried out at 35℃ for about 90 min. Then the suspension was heated to 90℃ and maintained for 20
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min to stop the reaction. The obtained suspension was used in the filtration test.
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The large-scale cryogel filtration system used in this work was shown in Figure 1. Five
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pHEMA−SiO2 cryogel disks were packed into a glass cylinder with the inner diameter of 140 mm
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and height of 200 mm. The glass cylinder was connected to a stainless steel vessel with the inner
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diameter of 164 mm and height of 250 mm filled with compressed low-pressure N2 for the supply
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of displacing gas at a constant pressure and a glass vessel for the supply of microbial suspension by
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a peristaltic pump. The filtration of cells was conducted under the constant-pressure mode by
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maintaining the gas pressure at about 0.015±0.005 MPa. About 3900 mL transformed yeast
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suspension was used in the filtration. Typically, the suspension was pumped into the glass cylinder
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and then the valve was switched to introduce N2 into the cylinder to displace the suspension passing
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through the pHEMA−SiO2 cryogel bed at a constant pressure. The effluent samples were collected
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at the time interval of about 1-2 min and the flow rates were measured by the sample volume and
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the time interval. The solid content in each sample was tested by the weight of cells and particulates
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like denatured proteins centrifuged from the sample. The effluent filtrate was collected and used in
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the following chromatographic experiments.
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Figure 1.
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2.4. Chromatographic isolation of CTP from the filtrate by the packed bed of large-scale
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pHEMA−SiO2−DMAEMA disks. The large-scale cryogel bed was prepared by packing
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pHEMA−SiO2−DMAEMA disks into a glass column with the inner diameter of 140 mm. The
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packing procedure was similar as that for polyacrylamide-based cryogel disks in our previous
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work.14 About 280 mL glass beads with diameter of 5 mm were packed on the top of the cryogel
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bed to enhance the uniform fluid distribution at the column inlet. The total bed height was about
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800 mm and the height of glass beads was 18 mm. Chromatographic isolation of CTP from the
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broth filtrate was carried out at a constant flow velocity of 0.80 cm/min for the loading process and
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0.68 cm/min at the wash and elution steps, respectively. The broth was diluted five folds with 0.01
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M HCl and used as the loading feedstock. The cryogel bed was equilibrated with 0.01 M HCl and
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then 9.98 L diluted filtrate broth was loaded onto the cryogel bed. The column was washed with
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0.01 M HCl and eluted with 0.02 M NaCl in 0.01 M HCl followed by 0.1 M NaCl in 0.01 M HCl,
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respectively. The column effluent was recorded over time and collected fractionally for further
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analysis. Column cleaning was performed with 1 M NaCl in 0.01 M HCl. The chromatography
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processes were monitored using a flow-through UV spectrometer at 254 nm by the flow branching
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from the main effluent stream.
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2.5 Analysis. Quantitative analysis of the purity of CTP was performed by HPLC in a 1260
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Infinity equipped with a photodiode array detector (1260 VL) and a ZORBAX SB-C18 column (5
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µm, 4.6 mm×150 mm).19 Mobile phase was the aqueous solution of methanol and 0.6% (v/v)
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phosphate (6:94 v/v, pH 6.6 titrated with triethylamine). The detection was at 271 nm with the flow
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rate of 0.5 mL/min, injection volume of 20 µL, and column temperature of 30℃.
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3. RESULTS AND DISCUSSION
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3.1. Properties of large-scale pHEMA−SiO2 and pHEMA−SiO2−DMAEMA composite
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cryogels. The formation of pHEMA−SiO2 composite cryogel disks in the large-scale stainless steel
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mold follows the same mechanisms as that for the preparation of monolithic cryogels in glass
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columns with small diameters.19 For the filtration application purpose, the pHEMA−SiO2 cryogel
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matrix was prepared by cryo-polymerization of the aqueous gel-forming mixture containing SiO2
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nanoparticles with the monomer and cross-linker weight concentration of 35%, while for the
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chromatographic application the pHEMA−SiO2−DMAEMA cryogel matrix was prepared with the
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aqueous gel-forming mixture with the weight concentration of 15%. In both disk preparations, the
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mass content of SiO2 nanoparticles to the gel-forming agents was maintained at about 2 %, which
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was preferred in the preparation of cryogels with satisfied mechanical strength.19
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Figure 2 shows scanning electron microscopy (SEM) images of a pHEMA−SiO2 cryogel disk for
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the filtration test, while the images of pHEMA−SiO2−DMAEMA disks were not shown here due to
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the same microstructures as those monolithic cryogels with the diameter of 10 mm prepared with
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the same concentration of gel-forming agents in a small-scale column.19 As can be seen, the
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microstructures within the pHEMA−SiO2 cryogel disk were also very similar as those in the
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small-scale pHEMA-based and polyacrylamide-based monolith cryogels.29–38 However, the
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gel-skeleton within the pHEMA−SiO2 cryogel disk prepared with the monomer and cross-linker of
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35% was much thicker than cryogels prepared with those of 15%,19 although the pore sizes seemed
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to be very close in both of these cryogels, indicating that the present disks have strong skeleton and
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thus could be expected to have relatively high mechanical strength for the filtration runs.
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Water permeabilities of pHEMA−SiO2 and pHEMA−SiO2−DMAEMA disks were determined by
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Darcy’s equation based on the data of flow rate vs. pressure drop measured on the beds packing
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with 2 to 4 disks, as similar as those in previous work.13,14 The permeabilities of pHEMA−SiO2 and
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pHEMA−SiO2−DMAEMA disks were 1.21×10-12 m2 and 1.68×10-12 m2, respectively. Porosities of
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these disks were also estimated by measuring the content of free water within a given cryogel disk
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sample at a certain volume. The obtained values of pHEMA−SiO2 and pHEMA−SiO2−DMAEMA
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disks were 52.4% and 73.9%, respectively. The porosity of pHEMA−SiO2−DMAEMA cryogels for
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chromatography was close to those of pHEMA cryogels without embedded nanoparticles prepared
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under similar conditions,13 while the porosity of pHEMA−SiO2 cryogels for filtration was lower
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than those of other pHEMA cryogels. The reason is that the total concentration of monomer and
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cross-linker (about 35%) for the present cryogel disks was higher that those usually used in the
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preparation of pHEMA cryogels (about 12-20%), which consequently resulted in the formation of
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smaller pores within the gels and thus the low porosity.
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Figure 2.
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3.2. Filtration performance of crude high-cell-density broth in the packed bed of large-scale
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pHEMA−SiO2 cryogel disks. The filtration of Saccharomyces cerevisiae from the crude broth was
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carried out in a batch manner. Due to the high cell density of the broth (233 mg/mL), some yeast
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cells were entrapped on the top surface of the cryogel bed to form the cell cake. However, other
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cells moved and passed through the bed into the filtrate bulk without being removed from the broth.
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In order to test if yeast cells in the filtrate could be further removed by the pHEMA−SiO2 cryogel
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bed with the cell cake, the refiltration was performed by employing the filtrate suspension collected
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from the former cycle as the loading broth in the next run. The total runs of three, i.e., Run 1, Run 2
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and Run 3, were conducted in this work. In each run, the filtration was operated in batch manner by
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displacing the suspension at a constant pressure using the gas until the interface of the gas and the
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suspension reached the positions about 6 to 40 mm above the cell cake surface to ensure no gas was
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injected through either the cell cake or the cryogel bed. Then the fresh suspension was supplied by
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the pump and the filtration continued. Three to four times of the suspension supply were used in
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each runs. Figure 3 shows examples of the filtration bed of pHEMA−SiO2 cryogel disks, the
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suspension broth, the cell cake, as well as the morphology of the cryogel disks and their
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cross-sections after the filtration test.
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Figure 3.
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Figure 4 shows the variations of cell content in the filtrate with the increase of the accumulated
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filtrate volume and the filtrate superficial flow velocity through the bed during the whole filtration
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process. As can be seen, the cell content decreased from 233 mg/mL in the crude broth to an
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averaged value of 129 mg/mL in the filtrate after Run 1. The results indicated that about half
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amount of the yeast cells in the crude broth (55%) were entrapped by the pHEMA−SiO2 cryogel
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bed only by one filtration. However, the averaged cell contents in the filtrates after Run 2 and Run 3
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were 122 and 133 g/L, respectively, indicating that there is no obvious enhancement of the cell
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removal efficiency by refiltration of the filtrates through the given cryogel bed. The reason is that
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the porous cell cake structures were formed during the filtration of Run 1. The flow channels or
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pores within the cell cake, similar as those supermacropores in the cryogel bed, were available and
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effective for the passage of most residual yeast cells with the flow of broth during the next two runs,
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and thus very small amounts of cells could be entrapped during Runs 2 and 3.
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From Figure 4, it is also seen that the filtration velocity during the refiltration runs was lower that
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that in Run 1 and decreased with the increase of refiltration cycles. The mean superficial velocities
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of filtrates observed in Run 1, Run 2 and Run 3 were 1.50, 0.96 and 0.58 cm/min, respectively,
271
indicating that the filtration productivity decreased. In fact, the suspension contained cells and
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particulates produced in the heat treatment procedure after the biotransformation. During the
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refiltration process, the migration of cells and these tiny solids within the pores of both the cell cake
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and the cryogel bed could further decrease the effective flow space and increase the filtration
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resistance probably due to the partial blocking or the redistribution of these particulates, and thus
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caused the decrease of the flow rates.
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Further increasing the filtration pressure (> 0.025 MPa) induced the collapse of the cell cake and
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the redistribution as well as the passage of those particulates through the cryogel bed. Therefore, a
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rather low linear velocity or low pressure should be used to avoid collapse of the filtrate cake. The
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suitable filtration pressure was below 0.02 MPa in the present work. The filtrate from the above
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filtration procedures was settled for about three hours and then sedimentation of additional amounts
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of cells occurred, which resulted in the cell content of about 20 mg/mL in the final filtrate. It should
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be noted that the agglomerates of those tiny solids were enhanced due to the interactions among the
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yeast cells and other particulates during the breakthrough of the suspensions through the cryogel
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bed. Therefore, the cell agglomeration gave further contributions to the cell separation from the
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suspension bulk following the gravitational settling mechanisms and thus reduced the final cell
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content in the filtrate bulk. It is also noted that the cell cake can be regenerated easily by the
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counter-current washing for the re-use purpose. Therefore, these cryogels are reusable and the
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regeneration procedure is easy.
290
Figure 4.
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291 292
3.3. Separation of CTP by the packed bed of large-scale pHEMA−SiO2−DMAEMA cryogel
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disks. By the above filtration and sedimentation, the filtrate contained 20 mg/mL cells, 6.94 mg/mL
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CTP, 1.57 mg/mL CDP, 0.61 mg/mL CMP and 0.59 mg/mL other impurities. In order to decrease
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the
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pHEMA−SiO2−DMAEMA cryogel, the filtrate broth was diluted five folds with 0.01 M HCl and
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then used as the feedstock. The loading broth had the electro conductivity of 7.88 mS/cm and pH
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2.6. The cell content of the loading broth was low (about 4 mg/mL) and during breakthrough and
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wash processes those yeast cells had passed freely through the chromatographic bed without
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blocking due to the supermacropores in the pHEMA−SiO2−DMAEMA disks. In our previous work,
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the elution of CTP from small scale anion exchange cryogels has been carried out successfully
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using the 0.02 M NaCl in 0.01 M HCl (elution 1) followed by either 0.5 M NaCl in 0.01 M HCl
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(elution 2) for the polyacrylamide-DMAEMA cryogel (bed diameter of 16 mm) or 0.1 M NaCl in
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0.01 M HCl (elution 2) for the pHEMA−SiO2−DMAEMA cryogel (bed diameter of 10 mm).17,19
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Therefore, for the present large scale chromatography the elution strategy used in the small scale
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pHEMA−SiO2−DMAEMA cryogel was employed directly.
concentration
of
ions
to
ensure
the
effective
adsorption
of
CTP
by
the
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In the present chromatography, 864 samples were collected from the loading to the elution 2, i.e.,
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47 samples at the loading (the volume from 0 to 9.98 L), 171 samples at the wash (9.98 to 35.18 L),
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266 samples at elution 1 (35.18 to 60.53 L) and 380 samples at elution 2 (60.53 to 105.89 L),
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respectively. Another 57 samples during the cleaning procedure were also collected. The
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concentrations and purities of AMP, ADP and CTP in these effluent samples were analyzed by
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HPLC and the results are shown in Figure 5. Some examples of the detailed HPLC profiles of the
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loading broth and the effluent samples during the loading, the wash and the elution stages are also
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shown in Figure 6, in which A271 denotes the recorded signals at 271 nm. From these figures, it is
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seen that even there are several compounds in the loading broth (Figure 6(a)), the main compound
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in the effluent during the loading stage was CMP (Figures 5(a, b) and 6(b)) and its concentration
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reached 0.03 mg/mL, while the maximal concentration of CDP was very low, i.e., 0.002 mg/mL
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(Figure 5(a)). It should be noted that during the loading stage the effluent contained 67.3% to 87.8%
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of CMP, 2.7% to 4.1% of CDP and 12.2% to 30% of other impurities, but no CTP (Figure 5(b)),
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indicating that most of CDP and CTP were adsorbed or remained within the bed. The reason is that
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the volume ratio of the loaded filtrate broth to the cryogel bed was low (0.81 in this work) and thus
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the main broth stream did not move through the whole bed. During the wash stage, the unbound
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CMP and CDP were washed out the bed and thus the breakthrough of CMP and CDP were
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observed. The maximal of the concentrations of CMP and CDP were 0.091 and 0.16 mg/mL
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(Figure 5(a)), respectively, which were higher than those in the loading procedure. It is also seen
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that most amounts of CMP passed through the cryogel bed during the loading and wash steps
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without being bound (Figures 5(a, b) and 6(b, c)) and the concentrations of CMP in the elution
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stages were very low (
