Ind. Eng. Chem. Res. 2002, 41, 3163-3168
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Poly(N-vinyl pyrrolidone)-Calcium Alginate (PVP-Ca-alg) Composite Hydrogels: Physical Properties and Activated Sludge Immobilization for Wastewater Treatment M. C. Doria-Serrano, G. Riva-Palacio, F. A. Ruiz-Trevin ˜ o,* and M. Herna´ ndez-Esparza Departamento de Ingenierı´as y Departamento de Ciencias, Universidad Iberoamericana, A.C., Me´ xico, D.F. 01210, Me´ xico
An alternative composite hydrogel material for cell immobilization is presented, and some of its important physical properties, such as weight swelling ratio (WSR) and cell viability, are evaluated. The hydrogel is prepared with cross-linked poly(vinyl pyrrolidone), PVP, and calcium alginate as the encapsulating rigid polymer. Composite hydrogels prepared with different proportions of sodium alginate lead to PVP/calcium alginate hydrogels with weight swelling ratios close to 11 (water content ≈ 90 wt %), higher than the WSR of PVP by a factor of 2. Low-vacuum SEM images reveal that the composite is made of PVP randomly distributed in a solid continuous cage formed by the rigid calcium alginate polymer. When cell viability of these composite hydrogels is evaluated from observations carried out using low-vacuum SEM, the images show that a concentrated bacterial population is attached to and growing on the surface of calcium alginate and entrapped PVP particles. In addition, when the composite hydrogels are used to entrap activated sludge and their efficiency to remove glucose from synthetic wastewater is evaluated and compared to the efficiency of a suspended activated sludge through kinetics studies, it is observed that the glucose depletion rate is practically the same for both systems, indicating that the composite materials have the right structure to allow the diffusion of substrate, byproducts, and oxygen for cell metabolism and may compete favorably with suspended activated sludge. Introduction During the past decade, the entrapment and immobilization of microorganisms in hydrogels has been the subject of ample research based on their potential application in the removal of biodegradable contaminants present in wastewater sources,1-5 in the removal of sour gases,6 and in the syntheses of bioproducts by fermentation.7 The key for the generalized use of these systems depends on developing hydrogels with an adequate structure in terms of porosity and physical properties, such as weight swelling ratio, water content, and, even more important, their viability for cell immobilization. For example, in the case of immobilization of microorganisms coming from wastewater treatment activated sludge, excellent hydrogels are those formed by polymers that swell in water, have a high water content, that is, above 90 wt %, and can be easily processed to give a material with pore-size distributions between 2 and 30 µm. These properties are required to allow for the entrapment and growth of living cells, the proper diffusion of the substrate, and exit of byproducts, and the oxygen required for cell metabolism. In addition to high mechanical resistance and because the polymer has to interact with water, hydrogels should have a molecular structure containing organic functional groups such as -OH, -COOH, -CONH2, -CONH, and -SO3H, which may favor the attachment and entrapment of the microorganisms.8 In recent years, it was reported that a hydrogel based on a mixture of poly(vinyl alcohol), PVA, and calcium * To whom correspondence should be addressed. E-mail:
[email protected]. Telephone: (52) 55 5267 4074. Fax: (52) 55 5267 4279.
alginate, Ca-alg, may result in a good candidate to immobilize microorganisms from activated sludge.9-11 This type of hydrogel is prepared by a method that involves the formation of spheres using a starting aqueous solution of PVA, sodium alginate, and microorganisms. The method also requires the physical crosslinking of PVA via freezing and thawing cycles once the hydrogel spheres are formed with entrapped microorganisms. During the cross-linking procedure, an elastic, non-water-soluble hydrogel is produced. Even though this method seems to be a reasonable procedure to entrap and immobilize microorganisms, it has some disadvantages because it requires long preparation times, it is difficult to process because of the high viscosity of the solution, and a large proportion of the viable population of microorganisms is lost during the freezing/thawing cycles (after three cycles almost 45% of the initial population is lost).11 To continue the search for different polymer/Ca-alg systems for cell entrapment that can overcome the drastic loss in microorganism population, as well as the preparation time, this work addresses the use of an already cross-linked polymer, such as poly(vinyl pyrrolidone), PVP, as the synthetic polymer part of the sphere. PVP is a component of hydrogels used for biomedical applications;12 the monomer VP has been copolymerized with acrylic acid, with methacrylates, and with other vinyl monomers for their application in contact lenses,13 for the controlled delivery of drugs,14 and for the immobilization of the enzyme lipase.15 PVP was selected because it has many of the mentioned properties required for a good hydrogel. It has a weight swelling ratio of 3-6, is biocompatible, and has
10.1021/ie0109399 CCC: $22.00 © 2002 American Chemical Society Published on Web 05/25/2002
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amide groups that may be points for the adhesion of microorganisms. Thus, the present work reports a method to prepare PVP/Ca-alg composite hydrogel and evaluates some of its physical properties, such as the weight swelling ratio (WSR) and water content, as well as its viability for cell entrapment. Finally, the capacities to remove glucose from a synthetic wastewater by both the suspended activated sludge and the microorganisms entrapped in the PVP/Ca-alg hydrogels are evaluated in batch reactors and compared in terms of their efficiency to remove glucose. Materials and Methods Materials. Cross-linked poly(vinyl pyrrolidone), PVP, Kollidon CL from BASF Chemical Company, was kindly supplied by Abasto-Quim Me´xico and used as the already cross-linked polymer. It has a reported weight swelling ratio of 3-6, 1.5 wt % of soluble compounds, and a broad range of particle-size distribution 10 < Dp < 200 µm. Sodium alginate with a 99 wt % purity was used as purchased. The activated sludge seed was collected from the aerobic-submerged biofilm reactor of the Universidad Iberoamericana wastewater treatment plant. Hydrogel Preparation. Prior to the preparation of the composite hydrogels, the PVP was washed with warm water for 1 h to dissolve impurities that the commercial sample may have. Afterward, an aqueous suspension of the washed PVP was prepared to narrow the particle-size distribution using a combination of sedimentation and centrifugation techniques. To do this, 2 g of PVP was suspended in 50 mL of water and poured into a 50 mL graduated cylinder, and the well-stirred suspension was allowed to settle quiescent for 10 min. After this period, the supernatant dilute suspension was separated from the settled portion at the bottom and then centrifuged and dried overnight at 80 °C in a convection oven. The particle-size distribution for the fraction in the supernatant was in the range of 10 < Dp < 50 µm as determined using an optical and a highvacuum SEM. A typical aqueous suspension for hydrogel formation was then prepared by stirring a total mass made up of 5 wt % of the polymers (PVP of 10 < Dp < 50 µm and sodium alginate) and 95 wt % of distilled water. To study the effect of the amount of sodium alginate on the physical properties of the composite, the proportion of the mass of the two polymers, PVP/sodium alginate was varied within the 5 wt % of total polymer present. Similarly, a typical microorganism aqueous suspension to produce microorganism-composite hydrogels was made with 4 wt % of PVP, 1 wt % of Na-alg, and 5 wt % of settled activated sludge. The activated sludge was collected from the aerobic-submerged biofilm reactor and had an average concentration after settling of 3 × 107 colony-forming units/mL (CFU/mL) and an average concentration of volatile suspended solids (VSS) of 4 × 103 mg/L. Composite-hydrogel spheres with and without microorganisms were formed by dispensing the suspension in a dropwise manner with a 10 mL graduated pipet into a 0.4 M calcium chloride solution. The compositehydrogel spheres formed by the reaction of the Na-alg to form insoluble Ca-alg were then washed several times with deionized water. Characterization of Composite Hydrogels. The weight swelling ratio, WSR, relative to the dry compos-
ite hydrogel was estimated by the weight ratio of wet and dried composite hydrogels, whereas the water content was estimated by the difference in the weight of the wet and the dry composite hydrogels. The WSR and water content values reported here are average values of three determinations, each one including 30 hydrogel spheres. In this procedure, the dry weight of all hydrogels was determined after the spheres were dried for 4 days at room temperature, followed by vacuum-drying at 40 °C, until a constant weight was reached (approximately 4 h). The weight of the wet hydrogels was measured immediately after filtration to remove water excess. PVP/Ca-alg hydrogel spheres, with and without microorganisms, were cut along the equator using a razor blade to observe their morphology and to evaluate the viability of these materials to immobilize microorganisms (mainly composed of bacteria). The surface and cross section of the composite hydrogels were characterized by low-vacuum SEM (JEOL JMS-5900LV). Vacuum was applied to reach a pressure of 30 Pa. Composite hydrogels were not dehydrated or quenched in liquid nitrogen to avoid the crystallization of water. The amount of bacteria entrapped in the hydrogel, measured as CFU (colony-forming units), was determined by smashing spheres in 10 mL of 0.9 wt % NaCl solution, preparing serial dilutions, and spreading 0.1 mL samples in nutrient agar. The cultures were incubated for 48 h at room temperature and counted. Data reported are averages of three determinations with 30 spheres each. Kinetics of Glucose Removal. To compare the capacity of the immobilized and the suspended activated sludge in the removal of glucose from a synthetic wastewater (NH4Cl 0.56 g/L; MgSO4 0.029 g/L; K2HPO4 0.022 g/L, NaH2PO4 0.138 g/L; yeast extract 0.5 g/L), kinetics studies at room temperature were carried out in laboratory batch reactors using different initial concentrations of glucose as carbon and energy source (0.5, 1, 1.5, and 3 g/L) and maintaining the reactors in a rotary shaker at 100 rpm. Samples were taken at different times, filtered through 0.45 µm pore diameter membranes and analyzed in HPLC (Hewlett-Packard, series 1050; column Aminex HPX-87H Biorad; column temperature ) 30 °C; mobile phase, H2SO4 0.05 mM; volumetric flow of the mobile phase 0.6 mL/min; detection by refraction index) so that the concentration of glucose in the reaction system could be evaluated at different times. The composite hydrogels for activated sludge growth and kinetics studies were prepared as follows: a suspension of 2.5 g of sodium alginate dissolved in 250 mL of distilled water, 10 g of PVP, and 250 mL of settled activated sludge (settled for 1 h) were thoroughly mixed and dispensed dropwise in the CaCl2 solution. The hydrogels for each batch reactor were prepared with 50 mL portions of the mixture, resulting in approximately 750 spheres. The microorganism-composite hydrogels were then washed with deionized water several times and incubated at 150 rpm at room temperature in 200 mL of synthetic wastewater supplemented with 3 g/L of glucose during 12 h so that microorganisms could adapt to these conditions before the kinetics experiments were run. After the adaptation period, spheres were thoroughly washed with deionized water before 200 mL of fresh medium was added to each reactor with the same
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Figure 1. Weight swelling ratio shown by PVP/calcium alginate composite hydrogels as a function of the amount of sodium alginate used to prepare the composite hydrogels.
composition as mentioned above but with different amounts of glucose to have different initial concentrations (0.5, 1, 1.5, and 3 g/L) equivalent to an initial organic loading as chemical oxygen demand (COD) of 1, 1.5, 2.0 and 3.5 kg/m3. Reactors were placed in the rotary shaker and the kinetics experiments were run simultaneously for the five reactors. Four samples of 50 mL of settled activated sludge were adapted to the synthetic wastewater mentioned above during the same 12 h period. Before the kinetics experiments, the activated sludge was centrifuged, washed thoroughly with saline solution, and centrifuged. To start the kinetics experiment, 200 mL of the same artificial medium used for immobilized biomass was added in each reactor, varying the initial concentration of glucose, as in the case of the hydrogel spheres. In this way, for each glucose concentration, there were two batch reactors: one with immobilized and another one with suspended microorganisms, both with similar concentrations of adapted biomass. Results and Discussion Hydrogels prepared according to the described procedure are very rigid and maintain the white color of their respective original powders. Even though the aqueous suspension is viscous, it can easily flow by gravity to the CaCl2 solution to form the white spheres. The capacity of the composite hydrogels to be swelled by water, measured as the WSR, is shown in Figure 1 as a function of the amount of sodium alginate in the total 5 wt % of polymer composition of the original aqueous suspension. The WSR of pure Ca-alg spheres (with water content ∼96 wt %) is higher than the WSR of the pure PVP (with water content ∼88 wt %) by a factor of 3.3. The dotted line in Figure 1 stands for the ideal additive behavior that the composite hydrogels would show after being formed. As seen from the experimental information, which includes the standard deviations, the composite hydrogels prepared with 10 and 20 wt % sodium alginate show positive deviations, whereas those hydrogels prepared with 30 and 40 wt % show negative deviations from the additive line. A
Figure 2. Low-vacuum SEM images of the interior and surface of (a) calcium alginate hydrogels and (b) PVP hydrogels. The particle-size distribution shown by of the PVP particles is after the use of a combination of sedimentation and centrifugation techniques to narrow the original distribution.
possible explanation for the positive deviation may be due to an additional amount of free water contained in some cavities left between the two polymers during the formation of the spheres. As the amount of sodium alginate increases, the number of cavities left diminishes and the WSR is closer to an additive behavior. The negative behavior shown by the 30 and 40 wt % data is hard to explain because it would suggest that a polymer is filling free spaces. In this regard, it is suggested that as the amount of sodium alginate increases, it penetrates the pores of the PVP particles and after precipitation in the CaCl2 solution, some pores of the PVP particles are blocked with calcium alginate. Figure 2 is a series of low-vacuum SEM images of spheres made with 1 wt % pure Ca-alg (part a) and pure PVP (part b). The left part of the image shows the interior of the pure Ca-alg hydrogel sphere, whereas the right part shows its surface. The surface of the Ca-alg hydrogel looks like a sponge, whereas the interior is a continuous solid phase containing pores in its structure. The PVP hydrogel particles, which were separated using a combination of sedimentation and centrifugation techniques, look like very rigid solids with particle sizes ranging from 10 to 50 µm.
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Figure 3. Low-vacuum SEM images of PVP/calcium alginate composite hydrogels made with a 4 wt % of PVP and 1 wt % of sodium alginate in the original solution. Image in part a represents the surface, whereas images in parts b and c represent the interior of the composite hydrogels.
The morphology as seen through a low-vacuum SEM of composite hydrogels made with a 4 wt % of PVP and 1wt % of Na-alg is shown in Figure 3 in which part a represents its surface and part b represents an image of the cross-section of the sphere. Figure 3 part c is a magnified image of a small area shown in part b. Specifically, this ratio was selected for further studies because it has the same polymer/alg ratio that is used to prepare the PVA/Ca-alg hydrogels reported in the literature9-11 and because this composite hydrogel still shows an excellent WSR, close to 11, and high water content, close to 92 wt % (see Figure 1). As seen in part a, PVP/Ca-alg hydrogels show a spongelike surface constituted by Ca-alg, which has trapped some of the PVP particles. The interior of the sphere (see Figure 3 parts b and c) shows a porous-lettuce structure with a well-defined material identification. The Ca-alg polymer is the solid semicontinuous phase and the small PVP particles are trapped and randomly distributed. The viability of this type of composite hydrogels to entrap living microorganisms is shown in Figure 4, in which part a represents an image of the surface and part b shows a cross-section of the microorganismloaded hydrogel. Figure 4 part c is a magnification of Figure 4 part b. The hydrogels shown in this figure were made with 4 wt % of PVP and 1 wt % of sodium alginate and an initial activated sludge containing a large population of bacteria as described before. As can be
seen in Figure 4 part a, the surface contains a large population of the activated sludge microorganisms and some PVP particles. In addition, the images in Figure 4 parts b and c show the proliferation of bacteria (white rods and cocci) entrapped inside the composite polymer matrix, growing on the surface of PVP particles and calcium alginate surface. It is well know that the typical size of bacteria is on the order of 1.5-3 µm, which corresponds to the size of the white rods shown in parts b and c. An additional observation is also related to the type of microorganisms that have been immobilized inside the composite hydrogel. The white rods observed in these images are mainly bacteria, even though the original activated sludge seed contained bacteria and fungi. To know whether the amount of PVP affects the concentration of microorganisms that can be immobilized and whether their growth is affected by the presence of the synthetic polymer, pure Ca-alg hydrogels were prepared from a 1 wt % aqueous Na-alg solution and another set of composite hydrogels were prepared from an aqueous solution containing PVP and calcium alginate in a 4/1 ratio, both with the same amount of initial settled activated sludge. Table 1 reports the amount of CFU determined just after the preparation of the hydrogels, as well as the amount determined after a 48 h incubation period in synthetic wastewater supplemented with glucose. Data show the average of
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Figure 4. Low-vacuum SEM images of PVP/calcium alginate hydrogels loaded with an original activated sludge that contained a high concentration of bacteria and fungi. The image in part a is a picture of the surface, while the images in parts b and c are pictures of the interior of the composite. The white rods, with sizes from 2 to 3 µm, are bacteria growing on the inside of the composite and apparently attached on top of the PVP hydrogel. Table 1: Amount of CFU Entrapped in Pure 1 wt % Calcium Alginate and in 4 wt % PVP/1 wt % Ca-alg Spheres Measured after Preparation and after a 48 Hour Incubation Perioda CFU × 10-8 CFU × 10 -8 (after preparation) (after incubation) 1 wt % Ca-alg 4 wt % PVP/1 wt % Ca-alg a
1.1 ( 0.47 1.1 ( 0.47
9.1 ( 11.7 13.8 ( 9.1
Data are the average of three samples of 30 spheres each.
six determinations obtained with three different samples. It can be seen that PVP/Ca-alg hydrogels show a higher increase in CFU after incubation; thus, the presence of the PVP not only does not inhibit the growth of the immobilized microorganisms but also seems to allow for a higher concentration of bacteria inside the spheres because of the increase in superficial area due to the random distribution of PVP particles. Figure 5 shows the kinetics of degradation of glucose by microorganisms in both the suspended and immobilized activated sludge reactors when different initial glucose concentrations are used (0.5,1, 1.5, 3 g/L). It can be seen that both the suspended and the immobilized activated sludge show a similar decrease in
the concentration of glucose for all of the initial concentrations studied. It is also observed that the rate at which glucose is being depleted is practically the same for both types of activated sludge, considering that both reactors started with the same concentration of microorganisms. The organic volumetric loadings tested in these experiments are high (1, 1.5, 2.0, and 3.5 kg COD/ m3), considering the typical loadings in activated sludge systems (
