Fabrication and Use of Alginate-Based Cryogel Delivery Beads

Jun 21, 2016 - ABSTRACT: Alginate-based cryogel beads loaded with urea and phosphates were fabricated by the microinjecting and cryo-cross-...
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Fabrication and use of alginate-based cryogel delivery beads loaded with urea and phosphates as potential carriers for bioremediation Lishen Shan, Yunling Gao, Yuanchang Zhang, Wu-Bin Yu, Yujun Yang, Shaochuan Shen, Songhong Zhang, Lingyu Zhu, Linhong Xu, Bing Tian, and Junxian Yun Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.6b01256 • Publication Date (Web): 21 Jun 2016 Downloaded from http://pubs.acs.org on June 23, 2016

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Fabrication and use of alginate-based cryogel delivery beads loaded with urea and phosphates as potential carriers for bioremediation Lishen Shan†, Yunling Gao†, Yuanchang Zhang†, Wubin Yu†, Yujun Yang‡, Shaochuan Shen†, Songhong Zhang†, Lingyu Zhu†, Linhong Xu§, Bing Tian║, Junxian Yun†* †

State Key Laboratory Breeding Base of Green Chemistry Synthesis Technology, College of Chemical Engineering, Zhejiang University of Technology, Hangzhou 310032, China



Institute of Process Equipment and Control Engineering, Zhejiang University of Technology, Hangzhou 310032, China

§

Faculty of Mechanical & Electronic Information, China University of Geosciences (Wuhan), Wuhan 430074, China ║

Key Laboratory for Nuclear-Agricultural Sciences of Chinese Ministry of Agriculture and Zhejiang Province, Zhejiang University, Hangzhou 310029, China

* Corresponding author: Prof. Junxian Yun State Key Laboratory Breeding Base of Green Chemistry Synthesis Technology College of Chemical Engineering Zhejiang University of Technology Chaowang Road 18, Hangzhou 310032 China

Tel.: +86-571-88320951; Fax: +86-571-88320951. E-mail address: [email protected] (J.X. Yun)

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ABSTRACT: Alginate-based cryogel beads loaded with urea and phosphates were fabricated by the micro-injecting and cryo-cross-linking method, coated with ethyl cellulose films and their properties were investigated. The cryogel beads had narrow diameter distributions and fracture-like supermacropores with the width of 1 to 3 µm and length up to more than 30 µm. Their porosities were from 74.9% to 92.2% and the diameters were influenced by the micro-tube diameter, the injection flow velocity and the freezing temperature. The cryogel beads had higher loading capacities of 13.4 mg phosphates and 33.2 mg urea/g wet bead and the release rate of phosphates was much lower than that of urea. The beads with coating-films with the thickness of 3 to 8 µm had a lower release rate for phosphates than those without films. The inherent characteristic of alginate beads to be fully biodegradable makes them an interesting candidate for the bioremediation of coastal sediments.

Keywords: cryogel; cry-cross-linking; micro-tube; salt delivery; bioremediation; alginate.

1. INTRODUCTION The environmental pollution of hydrocarbons due to the petroleum industry in coastal sediments, riverside soils and muddy wetlands is a serious worldwide problem, which has received an increasing attention in recent years.1–5 The spreading of oil components into the marine offshore lines always causes the dynamic complex bacterial communities response to the oil pollutants and thus the threatening of the marine ecosystems.1,4 Bioremediation, including the bioaugmentation by adding oil2

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degrading microorganisms and the biostimulation by adding growth-limiting nutrients to stimulate the indigenous microorganisms for the degradation, is one of effective approaches for the eventual removal of hydrocarbons from the coastal environment and numerous investigations in lab-scale and field trials have been carried out in the past years.3,5 Biostimulation using key nutrients like salts and nitrogen resources to accelerate the biodegradation process is economical for the degradation of hydrocarbons in oil-contaminated sediments.5 However, the supplementation of ordinary salts always suffers from the rapid release and even remarkable loss of these water-soluble nutrients to the open wide seawater environment without utilization by the microorganisms and thus causes the low bioremediation efficiency in field trials. The slow release fertilizers have been suggested as an alternative resource for the delivery of nutrients in bioremediation process.6–8 However, the matrices of slow release fertilizers are usually oleophilic polymers, slow-release inorganics or synthetic materials using monomers like acrylic acid and acrylamide. The drawbacks of these fertilizers include the dense structure with poor permeability, the difficulty to be degraded in natural environments and sometimes the use of toxic chemicals in the preparation processes. Natural polymers like chitin, chitosan and alginate have been suggested as the fertilizer matrices.6 These materials are derived from marine resource and have excellent properties regarding the safety and natural

biodegradability.

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Cryogels are novel polymer materials with sponge-like porous microstructures prepared via cryo-polymerization of gel-forming monomers under frozen conditions. This class of materials can be used as the chromatography supports, tissue engineering scaffolds and immobilization matrices due to their interesting properties like the hydrophilic gel-skeleton, high porosity regarding the supermacropores, good elasticity, high permeability and efficient in-pore mass transfer of solutes.9–18 We have successfully prepared different monolithic, bead-form and disk-form cryogels by cryo-polymerization method using various polymeric systems.19–22 Recently, the preparation of biodegradable cryogel beads and their possible applications in bioremediation of oil contaminants have been explored in our group. Cryogels can also be prepared using biodegradable materials like alginate and some examples regarding alginate-based cryogels have been reported in recent years.23–26 In this work, novel alginate-based cryogel beads loaded with urea and phosphates will be fabricated via the micro-injection and cryo-cross-linking method under various conditions. The cryogels will be coated with ethyl cellulose film. The optimum concentration of alginate, the morphology, the microstructure, the diameter distribution and the porosity of the beads prepared under different conditions regarding the micro-injection, freezing and gelling factors, the loading capacity of urea and phosphorus as well as their release performance, will be investigated experimentally in detailed.

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2. MATERIALS AND METHODS 2.1. Materials. Sodium alginate (98%) was obtained from Sangon Biotech Co., Ltd (Shanghai, China). Ethyl cellulose, ammonium metavanadate (NH4VO3, 99%), pdimethylaminobenzaldehyde

(C9H11NO,

99%)

and

ammonium

molybdate

tetrahydrate ((NH4)6Mo7O24·4H2O, 99%) were purchased from Aladdin (Shanghai, China). Urea (CH4N2O, 99%), K2HPO4·3H2O (99.5%), KH2PO4 (99.5%) and other chemicals were of analytical grade from local sources. 2.2. Micro-injection and cryo-cross-linking fabrication of nutrients-loaded cryogel beads. The preparation of nutrients-loaded alginate cryogel beads was carried out via the micro-injection and cryo-cross-linking method. The formation of nutrients-loaded alginate cryogel beads by this method is a process including several stages, i.e., the generation of the aqueous drops by dripping with the micro-tube, the solvent crystallization within the drops suspended in the bulk CaCl2 solution under the freezing condition, the cross-linking process between alginate and the gelling ions Ca2+ to form the bead matrix and the melt of ice crystals to form the supermacropores during the thawing step, as schematically shown in Figure 1. This formation process is similar in some aspects as that occurred by the microchannel liquid-flow focusing and cryo-polymerization method,20 but more complex. The generation of aqueous drops is crucial to the final diameters and the perfect spherical morphology of the beads, while the cryo-cross-linking controls the microstructures of the supermacropores and gel matrices. Moreover, the simultaneous reaction between Ca2+ and HPO42‒ or H2PO4‒ caused the formation of tiny solid precipitates,

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which deposited on the surface of the cryogel beads and also gave a contribution both to the enhancement of the bead mechanical strength and prevention of the bead shrinkage during the cross-linking stage. Figure 1. The preparation was carried out using micro-tubes with inner diameters of 0.5, 1.1, 1.6, 1.9 and 2.2 mm, respectively. Typically, the alginate aqueous solution 2% (w/w) containing 2.18% K2HPO4, 2.18% KH2PO4, and 1.64% urea, i.e., the total concentration of solutes of 6% was used as an example here, was pre-cooled to 2-5 ºC and pumped through a given micro-tube to generate nutrients-loaded drops at a constant flow rate. The effluents drops were introduced into the bulk solution of CaCl2 containing 1.64% urea at a given freezing temperature and stirred mechanically at about 160 rpm for cryo-cross-linking. After the cross-linking for about 30 min, the frozen beads were filtered and thawed at room temperature for further use. The preparation was also carried out at different flow rates and CaCl2 concentrations under different temperature conditions. To prepare the beads with coating film, 5% (w/w) ethyl cellulose ethanol solution was sprayed onto the surface of the beads and ethanol was removed roughly by hot air and the obtained beads were used for the further test. 2.3. Characterization and release performance of the nutrients-loaded cryogel beads. The microstructures of the nutrients-loaded cryogel beads were characterized by scanning electron microscope (SEM) (Hitachi TM-1000, Japan). The water contents and porosities were measured by the weight method, as in our previous

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work.19,21 Morphology images of the beads were taken by a digital camera and used for the test of the diameter distributions and the mean diameters. The diameters of the beads were determined statistically by counting about 190 beads based on these images. The release tests were carried out by immersing 10 g beads samples in 200 mL water and maintained at room temperature. The contents of phosphorus and urea released in the water were analyzed. The solution was removed and the same volume of fresh water was supplied every 12 hours. Phosphorus content was measured by ammonium vanadate molybdate coloration method.27 Typically, 1 mL water sample was mixed with 10 mL colorant solution and 14 mL deionized water and the obtained mixture was kept for 30 min. The content was determined by measuring the optical density and using the calibration at given concentrations at 360 nm by UV spectrometer (Ultrospec 3300 Pro, GE Healthcare, UK). Similarly, the urea content was determined by p-dimethylaminobenzaldehyde coloration method at 400 nm.28

3. RESULTS AND DISCUSSION 3.1. Optimum concentration of alginate. In our experiment, the suitable concentration of alginate for the generation of perfect cryogel beads was observed in the range from 1% to 3% (w/w). The beads prepared at lower concentration, i.e., 3%, were not perfect in the sphericity due to the high viscosity and thus in this work the concentration of alginate was maintained at 2%. In order to

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increase the loading capacity of urea within the final beads, the same concentration of urea as that used in the aqueous drops was maintained within the CaCl2 solutions. 3.2. Morphology and microstructure of the beads. The preparation of nutrientsloaded alginate cryogel beads was conducted by the micro-tube with the inner diameter of 0.5, 1.1, 1.4, or 2.2 mm at the flow rate of 1, 2, 3 or 4 mL/min. The freezing temperature was maintained at 0, -5, -10, or -15 ºC and the concentration of CaCl2 was kept at 5%, 10%, 15%, 20% or 25%. For comparison purpose, the preparation of nutrients-loaded alginate beads without freezing but at room temperature (about 15 ºC) was conducted by the micro-tube with the inner diameter of 2.2 mm at the flow rate of 2 mL/min in 25% CaCl2. These beads and the cryogel beads prepared under the freezing temperature of -15 ºC by the micro-tube with the inner diameter of 2.2 mm at the flow rate of 2 mL/min in 25% CaCl2 were coated by spraying 5% ethyl cellulose in ethanol. Figure 2 shows examples of the morphology (a) and SEM (c) images of the nutrients-loaded alginate cryogel beads obtained by the micro-tube with the inner diameter of 2.2 mm at the flow rate of 2 mL/min in 25% CaCl2 under the freezing temperature of -15 ºC. Similarly, the morphology (b) and SEM (d) images of cryogel beads coated with ethyl cellulose films were also shown in this figure. As can be seen, both the beads with and without films have good sphericity. The cryogel beads were covered with white calcium phosphates due to the precipitation reaction between CaCl2 and phosphates and had fracture-like supermacropores with the width of about 1 to 3 µm and length of about several to more than 30 µm, which were much

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different from the near circle-form supermacropores with diameters from several to 100 µm observed in most ordinary cryogels without loading salts.9–18. These fracturelike supermacropores were interconnected together to form the irregular pore network Precipitants and some solid crystals with sizes of about several to 10 µm deposited and filled in the pores. The reason is either the reaction between phosphates and calcium chloride or the crystallization of phosphates due to the supersaturation under the freezing conditions. For the bead coated with films, a very thin ethyl cellulose film with the thickness of about 3 to 8 µm was observed. The film thickness depended on the ethyl cellulose used. In our typical experimental runs, about 146 g 5% ethyl cellulose in ethanol was used for coating of 67 g cryogel beads and about 4.2 g ethyl cellulose was coated successfully, i.e., about 58% (w/w) of ethyl cellulose was available for the final coating. The core of the bead was dense and shrunk in some degree, as can be seen from Figure 2(d), due to the solvent evaporation during the spraying process. Figure 2. 3.3. Diameter distributions and porosities of the beads. Figure 3 shows the diameter distributions of the nutrients-loaded alginate croygel beads prepared under various preparation conditions. The mean diameters and the porosities are listed in

Table 1. Figure 3. Table 1.

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As can be seen, the porosities of the nutrients-loaded alginate beads were in the range from about 74.9% to 92.2% and varied randomly, indicating that these beads had large voids filled by the aqueous solution of nutrients and thus, could be benefit to the mass transfer of nutrients from the pores within the beads to the outside environment. These values were close to those monolithic or bead-form cryogels.9,18,20,22 It is also seen that the nutrients-loaded croygel beads have narrow diameter distributions, indicating that the beads are almost uniform in sizes and the microinjection and cryo-cross-linking method is effective in the control of beads sizes. Under the present conditions, the mean diameter increased with the increase of the micro-tube diameter. The relative errors in the calculation of the mean diameters were below 0.3% in this measurement. The diameters increased from 1.90 to 3.31 mm, i.e., the increment of about 74%, with the increase of the micro-tube diameter from 0.5 to 2.2 mm at the flow rate of 2 mL/min in 25% CaCl2 under the freezing temperature of -15 ºC. In fact, when the nutrients-loaded alginate solution was extruded through the micro-tube at a constant flow rate and reached the tube tip, the liquid was accumulated temporally without detaching due to the surface tension and tended to pendant to the tip and keep spherical shape. However, the gravitational force tended to pull the drop downward,29 which caused the elongation of the drops followed by the detachment from the micro-tube tip. The liquid volume accumulated at the tip before the detachment increased and consequently, the bigger drops were generated with the increase of micro-tube diameter. Increasing the liquid flow rate 10

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also caused the slight increase of the mean diameter of the beads due to the similar mechanisms. The mean diameter was also influenced by the freezing temperature. When the temperature was maintained at room temperature (about 15 ºC) or above -10 ºC, the crystallization of water within the nutrients-loaded alginate drops did not occur and thus, only the cross-linking reaction between alginate and the gelling ions without cryo-cross-linking happened, which caused the formation of the ordinary gel matrix with shrinkage. Therefore, smaller beads were obtained compared with those of cryogel beads at the temperature of -15 ºC. Moreover, after a long time storage, i.e., 30 days, there was a slightly decrease of the mean diameters of the nutrients-loaded alginate due to the fully gelling induced by ions Ca2+. The decrease percentages were of about 2.1% to 12.0% under the present conditions. Generally, the concentration of CaCl2 in the range from 1% to 3% is enough for the cross-linking of alginate beads and it had a less important effect on the mean diameter in the range from 5% to 25%. However, in the case that the concentration was below 10%, the density of the solution was low, which could cause the sedimentation of frozen beads during the cross-linking process. The solution could also be frozen under the present freezing conditions and thus the concentration of CaCl2 used in the formation of cryogel beads was maintained at 25%. The mean diameter of the cryogel beads coated with films was lower than that of the cryogel beads without film. The mean diameter of beads coated with films in Figure 1 (b) was about 2.56 mm, while the value that for beads without films in

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Figure 1 (a) was 3.31 mm, i.e., the coating of films caused about 22% decrease of the diameter. The reason is that during the spray process in the film coating some amounts of water within the pores were removed and the matrices of these cryogel beads were shrunk, which caused the decrease of the diameters. 3.4. Urea and phosphorus loading capacity of the beads. The urea and phosphorus loading capacities of the nutrients-loaded alginate croygel beads with (marked as APN-A) and without ethyl cellulose films (marked as APN-B), and the beads prepared at room temperature and coated with (marked as APN-A-R) and without ethyl cellulose films (marked as APN-B-R), were listed in Table 2. These beads were prepared by the micro-tube with the inner diameter of 2.2 mm in the solution of 25% CaCl2 and 1.64% urea. The freezing temperature for cryogel beads was -15 ºC, while for the ordinary beads without cryo-cross-linking the preparation was achieved at room temperature. During the whole fabrication, some amounts of urea and phosphorus diffused into the bulk solution and thus a loss of nutrients existed. The encapsulation percentages of phosphorus and urea and the water contents in these beads were also listed in this table.

Table 2. As can be seen, these beads have high water contents in the range from about 59% to 86%. The beads without films have higher water content than those with coating films. The reason is that the spraying caused the evaporation of some amounts of water within the beads. The phosphorus loading capacities of the croygel beads with

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and without ethyl cellulose films were 12.4 and 13.4 mg/g bead, while those of the beads prepared at room temperature with and without films were 9.6 and 7.3 mg/g bead. The urea loading capacities of the croygel beads with and without films were 33.1 and 33.2 mg/g bead, while those of the beads prepared at room temperature with and without films were 26.9 and 28.7 mg/g bead, respectively. Therefore, the cryogel beads have higher nutrients-loading capacities than those of the beads prepared at room temperature. The encapsulation percentages of phosphorus and urea of cryogel beads were also higher than those of beads prepared at room temperature. In fact, during the cryo-cross-linking fabrication of the cryogel beads the crystallization of water within the drops caused the concentrated and supersaturation of phosphorus and urea, which resulted in the formation of nutrients crystals, as also observed from the SEM images. These solid precipitates were embedded in the bead matrices and thus decreased the loss to the bulk liquid outside the beads. However, during the cross-linking fabrication of beads at room temperature, no crystallization occurred but the shrinkage of bead volumes was observed, which could squeeze some amounts of the liquid containing phosphates and urea out the beads and thus decrease the loading capacity and the encapsulation percentage. 3.5. Release profiles of urea and phosphorus from the beads. Since several different phosphates, e.g., K2HPO4, KH2PO4, Ca(H2PO4)2 and CaHPO4, were generated during the preparation process, the total phosphorus content was employed as the parameter to describe the release of phosphates from the beads. The release profiles of urea and phosphorus from the nutrients-loaded alginate croygel beads

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with and without coating films are shown Figure 4. For comparison purpose, the release profiles from the beads prepared at room temperature and coated with and without films are also displayed in the figure, although the standard errors of the release data were not estimated in this work. These beads were fabricated under the same conditions of the micro-tube, the flow rate, the concentration of CaCl2 except the different temperature conditions and with or without coating films. It is seen that both the cryogel beads and those beads prepared at room temperature have similar slow release properties of phosphorus. The coating films enhanced the slow release properties of the beads as one can see that the release rates of those beads with coating films were lower than those without films at the same conditions. The reason is that the ethyl cellulose film is hydrophobic and occupies some sites on the bead surface, which could reduce the diffusion of phosphates from the beads into the bulk water and thus the decrease of the release rate. Some phosphates in the form of crystals or precipitates are needed to be dissolved in the water within pores and then transferred outside the beads, which could decrease the release rate. However, the release rate of urea was much higher than that of phosphorus. The release rates of urea in all these beads are almost the same, indicating that neither the coating films nor the cryo-cross-linking process contributed important effects on the diffusion of urea from the beads. The reason is that urea existed as the dissolution form in the water filled in the pores and can transfer freely even through the coating films. Figure 4.

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4. CONCLUSIONS Novel alginate-based cryogel beads loaded with urea and phosphates were prepared by the micro-injecting and cryo-cross-linking method. The cryogel beads have micrometer-scale supermacropores embedded with precipitates or crystals of phosphates within the skeleton matrices and pores. These pores have irregular fracture-like morphology with the width of about 1 to 3 µm and length of about several to more than 30 µm. The beads obtained under the present preparation conditions had the porosities of about 74.9% to 92.2%. The beads with narrow diameter distributions can be fabricated by adjusting the inner diameter of the injection tubes, the flow rate, the freezing temperature for cryo-cross-linking and the concentration of gelling ions. Among these factors, the inner diameter of micro-tube contributed remarkable influence on the mean diameter of the beads, while the concentration of gelling ions gave a less important effect on the bead sizes at a certain range of concentration. By coating the hydrophobic ethyl cellulose films with the thickness of about 3 to 8 µm over the beads, the release rate of phosphates can be decreased further than that without coating. The present cryogel beads have higher loading capacity of phosphates and urea, i.e., 13.4 mg phosphates/g wet bead and 33.2 mg urea/g wet bead, than those beads prepared under the same condition without cryo-cross-linking at room temperature, i.e., 7.3 mg phosphates/g wet bead and 28.4 mg urea/g wet bead, and similar release profiles. Slow-release properties of nutrients were observed and the release rate of phosphates was much lower than that of urea, indicating that the present naturally biodegradable cryogel beads could be

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interesting as the delivery carriers of urea and phosphates nutrients potentially in bioremediation areas.

ACKNOWLEDGMENTS The authors gratefully acknowledge the financial supports partially by the Zhejiang Provincial

Natural

Science

Foundation

of

China

(Nos.

LZ14B060001,

LY14B060005) and the National Natural Science Foundation of China (Nos. 21576240, 21036005).

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Mattiasson, B.; Kumar, A.; Galaev, I. Y. Macroporous polymers: Production Properties and Biotechnological/Biomedical Applications; CRC Press, Taylor & Francis Group: Boca Raton, USA, 2010.

(10) Gun'ko, V.M.; Savina, I.N.; Mikhalovsky, S.V. Cryogels: Morphological, structural and adsorption characterization. Adv. Colloid Interfac. 2013, 187−188, 1. (11) Kirsebom, H.; Mattiasson, B. Cryostructuration as a Tool for Preparing Highly Porous Polymer Materials. Polym. Chem. 2011, 2, 1059. (12) Plieva, F. M.; Kirsebom, H.; Mattiasson, B. Preparation of Macroporous Cryostructurated Gel Monoliths, Their Characterization and Main Applications. J. Sep. Sci. 2011, 34, 2164. (13) Plieva, F. M.; Galaev, I. Y.; Noppe, W.; Mattiasson, B. Cryogel Applications in Microbiology. Trends Microbiol. 2008, 16, 543. (14) Lozinsky, V. I. Polymeric Cryogels as a New Family of Macroporous and Supermacroporous Materials for Biotechnological Purposes. Russ. Chem. Bull. 2008, 57, 1015. (15) Plieva, F. M.; Galaev, I. Y.; Mattiasson, B. Macroporous Gels Prepared at Subzero

Temperatures

as

Novel

Materials

for

Chromatography

of

Particulate-containing Fluids and Cell Culture Applications. J. Sep. Sci. 2007, 30, 1657. (16) Lozinsky, V. I.; Galaev, I. Y.; Plieva, F. M.; Savina, I. N.; Jungvid, H.; Mattiasson, B. Polymeric Cryogels as Promising Materials of Biotechnological Interest. Trends Biotechnol. 2003, 21, 445. (17) Lozinsky, V. I. Cryogels On the Basis of Natural and Synthetic Polymers: Preparation, Properties and Applications. Russ. Chem. Rev. 2002, 71, 489.

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(18) Yun, J.X.; Xu, L.H.; Lin, D.-Q.; Yao, K.J.; Yao, S.-J. Fabrication and Characterization of Cryogel Beads and Composite Monoliths, Chapter 4 in Supermacroporous cryogels: biomedical and biotechnological applications (ed. by Kumar, A.), CRC Press, Taylor & Francis Group: Boca Raton, USA, 2016. (19) Yun, J.X.; Wu, H.; Liu, J.; Shen, S.C.; Zhang, S.H.; Xu, L.H.; Yao, K.J.; Yao, S.J. Strategy of combining prefiltration and chromatography using composite cryogels for large-scale separation of biotransformation compounds from crude high-celldensity broth. Ind. & Eng. Chem. Res. 2015, 54, 2564. (20) Yun, J.X.; Dafoe, J.T.; Peterson, E.; Xu, L.H.; Yao, S.J.; Daugulis, A.J. Rapid freezing cryo-polymerization and microchannel liquid-flow focusing for cryogel beads: adsorbent preparation and characterization of supermacroporous bead-packed bed. J. Chromatogr. A 2013, 1284, 148. (21) Ye, J.L.; Yun, J.X.; Lin, D.-Q.; Xu, L.H.; Kirsebom, H.; Shen, S.C.; Yang, G.S.; Yao, K.J.; Guan, Y.-X.; Yao, S.J. Poly(hydroxyethyl methacrylate)-based composite cryogel with embedded macroporous cellulose beads for separation of human serum immunoglobulin and albumin. J. Sep. Sci. 2013, 36, 3813. (22) Yao, K.J.; Yun, J.X.; Shen, S.C.; Wang, L.H.; He, X.J.; Yu, X.M. Characterization of a novel continuous supermacroporous monolithic cryogel embedded with nanoparticles for protein chromatography. J. Chromatogr. A 2006, 1109, 103. (23) Zhao, Y.; Shen, W.; Chen, Z.G.; Wu, T. Freeze-thaw induced gelation of alginates. Carbohyd. Polym. 2016, 148, 45. (24) Singh, D.; Tripathi, A.; Zo, S.; Singh, D.; Han S.S. Synthesis of composite gelatinhyaluronic acid-alginate porous scaffold and evaluation for in vitro stem cell growth and in vivo tissue integration. Colloid Surface. B 2014, 116, 502.

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(25) Zain, N.A.M.; Suhaimi, M.S.; Idris, A. Development and modification of PVAalginate as a suitable immobilization matrix. Process Biochem. 2011, 46, 2122. (26) Chhatri, A.; Bajpai, J.; Bajpai, A.K; Sandhu, S.S.; Jain, N.; Biswas, J. Cryogenic fabrication of savlon loaded macroporous blends of alginate and polyvinyl alcohol (PVA). Swelling, deswelling and antibacterial behaviors. Carbohyd. Polym. 2011, 83, 876. (27) Kitson, R.E.; Mellon, M.G. Colorimetric determination of phosphorus as molybdivanadophosphoric acid. Ind. Eng. Chem. Anal. Ed. 1944, 16, 379. (28) Knorst, M.T.; Neubert, R.; Wohlrab, W. Analytical methods for measuring urea in pharmaceutical formulations. J. Parm. Biomed. Anal. 1997, 15, 1627. (29) Chan, E.-S.; Lee, B.-B.; Ravindra, P.; Poncelet, D. Prediction models for shape and size of ca-alginate macrobeads produced through extrusion-dripping method. J. Colloid Interf. Sci. 2009, 338, 63.

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Figure Captions

Figure 1. Schematic diagram of the Fabrication of alginate-based cryogel beads loaded with the nutrients of urea and phosphates by the micro-injection and cryocross-linking method under the freezing condition. Figure 2. Morphology (a, c) and SEM (b, d) images of the nutrients-loaded alginate cryogel beads. These beads were fabricated by the micro-tube with the inner diameter of 2.2 mm at the flow rate of 2 mL/min in 25% CaCl2 under the freezing temperature of -15 ºC, and coated without (a, b) or with ethyl cellulose films (c, d). Figure 3. Diameter distributions of the nutrients-loaded alginate beads prepared under various conditions. (a) The cryogel beads were prepared by the micro-tube with the inner diameter of 2.2 mm at the flow rate of 2 mL/min in 25% CaCl2 under the freezing temperature of -15 ºC and coated without or with ethyl cellulose films, (b) the cryogel beads were prepared in 25% CaCl2 under the freezing temperature of -15 ºC but different conditions of the micro-tube diameter and the flow rate, and (c) the beads were prepared by the micro-tube with the inner diameter of 2.2 mm at the flow rate of 2 mL/min but different conditions of the freezing temperature or room temperature and the concentration of CaCl2. Figure 4. Release performance of phosphates (a) and urea (b) from the nutrientsloaded alginate croygel beads coated with (APN-A) and without ethyl cellulose films (APN-B), and the beads prepared at room temperature and coated with (APN-A-R) and without ethyl cellulose films (APN-B-R). These beads were fabricated by the

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micro-tube with the inner diameter of 2.2 mm at the flow rate of 2 mL/min in 25% CaCl2 under the freezing temperature of -15 ºC for cryogels and room temperature for the ordinary beads. The release tests were carried out using 10g bead samples in 200 mL water at room temperature and the fresh water was supplied every 12 h.

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Figure 1. Schematic diagram of the Fabrication of alginate-based cryogel beads loaded with the nutrients of urea and phosphates by the micro-injection and cryocross-linking method under the freezing condition.

(a)

(b)

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(c)

(d)

Figure 2. Morphology (a, c) and SEM (b, d) images of the nutrients-loaded alginate cryogel beads. These beads were fabricated by the micro-tube with the inner diameter of 2.2 mm at the flow rate of 2 mL/min in 25% CaCl2 under the freezing temperature of -15 ºC, and coated without (a, b) or with ethyl cellulose films (c, d).

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(a)

(b)

(c) Figure 3. Diameter distributions of the nutrients-loaded alginate beads prepared under various conditions. (a) The cryogel beads were prepared by the micro-tube with the inner diameter of 2.2 mm at the flow rate of 2 mL/min in 25% CaCl2 under the freezing temperature of -15 ºC and coated without or with ethyl cellulose films, (b) the cryogel beads were prepared in 25% CaCl2 under the freezing temperature of -15 ºC but different conditions of the micro-tube diameter and the flow rate, and (c) the beads were prepared by the micro-tube with the inner diameter of 2.2 mm at the flow rate of 2 mL/min but different conditions of the freezing temperature or room temperature and the concentration of CaCl2.

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(a)

(b)

Figure 4. Release performance of phosphates (a) and urea (b) from the nutrientsloaded alginate croygel beads coated with (APN-A) and without ethyl cellulose films (APN-B), and the beads prepared at room temperature and coated with (APN-A-R) and without ethyl cellulose films (APN-B-R). These beads were fabricated by the micro-tube with the inner diameter of 2.2 mm at the flow rate of 2 mL/min in 25% CaCl2 under the freezing temperature of -15 ºC for cryogels and room temperature for the ordinary beads. The release tests were carried out using 10 g bead samples in 200 mL water at room temperature and the fresh water was supplied every 12 h.

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Table 1 The mean diameters of nutrients-loaded alginate croygel beads under various 505 conditions and the beads prepared at room temperature Micro-tube diameter (mm)

Flow rate

CaCl2

Temperature

Porosity

(mL/min)

(%)

(ºC)

(%)

Mean bead diameter (mm)

0.5

2

25

-15

74.9±2.9

1.90

1.1

2

25

-15

82.0±0.9

2.35

1.4

2

25

-15

76.7±1.1

2.50

2.2

2

25

-15

84.9±3.0

3.31

2.2

1

25

-15

89.5±0.9

3.51

2.2

3

25

-15

81.2±1.1

3.49

2.2

4

25

-15

91.6±3.4

3.66

2.2

2

5

15

83.7±2.2

3.25

2.2

2

10

15

92.2±1.9

3.16

2.2

2

15

15

91.7±2.5

3.16

2.2

2

20

15

81.1±0.8

2.96

2.2

2

25

0

83.7±2.7

3.21

2.2

2

25

-5

83.2±1.7

3.06

2.2

2

25

-10

76.2±1.7

3.01

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Table 2 The urea and phosphorus loading capacities of the croygel beads with (APN-A) or without ethyl cellulose films (APN-B), and the beads with (APN-A-R) and without ethyl cellulose films (APN-B-R) prepared at room temperature. Beads

Water content (%)

Phosphorus loading capacities (mg/g bead)

Urea loading capacities (mg/g bead)

Phosphorus encapsulation percentage (%)

Urea encapsulation percentage (%)

APN-A

66

12.4

33.1

47.7

68.8

APN-B

86

13.4

33.2

58.4

78.2

APN-A-R

59

9.6

26.9

32.1

49.0

APN-B-R

77

7.3

28.7

36.1

76.5

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