- Email: [email protected]
Coupling of an electrodialysis unit to a hollow fiber bioreactor for separation of gluconic acid from sorbitol produced by Zymomonas mobilis permeabilized cells
Journal of Membrane Science 191 (2001) 43–51
Coupling of an electrodialysis unit to a hollow fiber bioreactor for separation of gluconic acid from sorbitol produced by Zymomonas mobilis permeabilized cells H.C. Ferraz, T.L.M. Alves, C.P. Borges∗ Chemical Engineering Program, COPPE, Federal University of Rio de Janeiro, P.O. Box 68502, 21945-970 Rio de Janeiro, Brazil Received 29 August 2000; received in revised form 11 April 2001; accepted 11 April 2001
Abstract The objective of this study was to evaluate the coupling of an electrodialysis unit to a reactor in which gluconic acid and sorbitol were produced by permeabilized and immobilized Zymomonas mobilis cells. The experimental results have shown that electrodialysis unit coupled to the bioreactor allowed an efficient removal of gluconic acid from the reaction medium, as well as an improvement in the stability of the enzyme. It was not observed any reduction in the reaction rate, even after 60 h of operation. On the other hand, when NaOH was applied to neutralize the gluconic acid produced, it was verified a reduction of 80% in the reaction rate in the same period. These results lead to the hypothesis that the enzyme stability is associated to gluconate accumulation in the reaction medium. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Zymomonas mobilis; Sorbitol; Gluconic acid; Hollow fibers; Electrodialysis
1. Introduction Sorbitol and gluconic acid production by permeabilized cells of Zymomonas mobilis has been extensively studied over the years [1–10]. Such efforts can be justified by relatively mild operation conditions of the biosynthesis when compared to the chemical route and by the enzyme specificity, which minimizes by-product formation, besides the importance of both products. Glucose–fructose oxidoreductase (GFOR) present in cells of Z. mobilis promotes simultaneous glucose oxidation to glucono-␦-lactone and fructose reduction to sorbitol. In a subsequent step, glucono-␦-lactone is hydrolyzed to gluconic acid, both spontaneously and ∗ Corresponding author. E-mail address: [email protected] (C.P. Borges).
under the action of the enzyme glucono-␦-lactonase. As shown in Fig. 1, in intact cells of Z. mobilis, sorbitol accumulates in the medium while gluconic acid is converted to ethanol, via Entner Doudoroff pathway. When cells are permeabilized, small compounds, such as metallic ions and cofactors can diffuse out of the cells, so the pathway from gluconate to ethanol is not functional anymore [3]. While this procedure is important to avoid ethanol formation, it has no effect on the activity of the enzyme, once the NADP(H) cofactor for GFOR remains bound to it. Fig. 2 shows the effect of permeabilization on cells of Z. mobilis [11]. The cytoplasmatic contents are very distinct due to intracellular compounds leakage. A better operational stability and an easier continuous operation can be achieved by immobilizing the permeabilized cells. Several methods have been applied [12,13] and most of them employed supports
0376-7388/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 6 - 7 3 8 8 ( 0 1 ) 0 0 4 4 7 - 1
44
H.C. Ferraz et al. / Journal of Membrane Science 191 (2001) 43–51
Fig. 1. Mechanism of sorbitol and gluconic acid production by permeabilized cells of Z. mobilis. When cells are fully permeabilized, the pathway from gluconate to ethanol, delimited by the broken line, becomes non functional [3].
based on k-carrageenan or alginate. The confinement of cells in the bore of hollow fiber membranes [14–16] has been used as an alternative and promising immobilization technique. Obviously, membrane pores have to be smaller than the cells and, furthermore, offer a low resistance to the substrates and products transport. As advantage, this type of system can be easily operated and regenerated. Down stream operations to concentrate and purify the products have not been taken into account in the previous studies, despite its importance to the process feasibility. In this context, techniques, such as electrodialysis (ED) and ion exchange could be useful. ED has the advantage of being easy to scale up and, in addition, it does not add any external compound to the medium. The dissociation constant K for gluconic acid is 1.99 × 10−4 corresponding to a pK of 3.7 [17]. If an aqueous solution of gluconic acid and sorbitol is pumped through an electrodialysis cell and an electrical potential is established between the anode and cathode, the gluconate ions will migrate across the anion exchange membrane toward the anode compartment. Likewise, the hydrogen ions will migrate through the cation exchange membrane to the cathode compartment. This permits the pH control without use of neutralizer [18]. Sorbitol, an uncharged molecule,
Fig. 2. Electron transmission photomicrograph of Z. mobilis cells: (A) before permeabilization; (B) after permeabilization. [11].
is not affected and will remain in the feed stream. This way, ED will promote the desired separation of gluconate from unreacted substrates. In this work, the reaction unit, consisting of a module of hollow fibers containing permeabilized cells of Z. mobilis, was coupled to the electrodialysis unit allowing the simultaneous extraction of the gluconic acid produced. A performance comparison of the systems with and without acid separation by electrodialysis is presented in terms of initial reaction rate and operational stability.
H.C. Ferraz et al. / Journal of Membrane Science 191 (2001) 43–51
2. Experimental 2.1. Microorganism and culture conditions All experiments were performed with the bacteria Z. mobilis CP4 (ATCC 31821), cultivated in a medium containing 100 g glucose/l as carbon source and 5 g yeast extract/l under controlled temperature of 30◦ C.
45
The pore size in the outer surface of the fiber is smaller than 0.1 m, while in the inner surface is smaller than 1 m. Considering cell dimensions, the range of pore size in the fiber is small enough to avoid any loss of cells during the experiment. The water permeability of these fibers was determined as 912 l/h m2 bar. 2.4. Assays and analytical methods
2.2. Preparation of permeabilized cells Z. mobilis was cultivated until the late exponential phase after about 20 h growth. After this period cetyltrimethylamonium bromide (CTAB) was added directly to the broth to give a final proportion of 0.04 g CTAB per gram of cells [12]. After gently stirring for 30 min, cells were separated by centrifugation at 5000 rpm and washed twice with distilled water, pH 5.5. 2.3. Immobilization method 2.3.1. Entrapment in alginate For the immobilization in calcium alginate, the concentrated suspension of cells was mixed with a solution of sodium alginate (8% w/v) [19]. Spherical beads were then produced by dropping the mixture into a 20 g/l calcium chloride solution through a syringe needle of 0.5 mm diameter. The mean diameter of obtained beads was ca. 2 mm. The beads were suspended in a 0.5% (v/v) glutaraldheyde solution with stirring for 30 min. After washing with water, the beads were stored at 4◦ C until further use. 2.3.2. Confinement in hollow fiber Permeabilized cells suspension was confined in the bore of microporous polycarbonate hollow fibers or in the shell side of the module. The fibers were prepared by phase inversion technique using spinning conditions reported in [20]. These fibers, about 20 cm long, were assembled in a longitudinal module of PVC as presented in Fig. 3(A). Fig. 3(B) shows a photomicrograph of a fiber cross-section, which has outer and inner diameters of 0.68 and 0.33 mm, respectively. The set up of the membrane reactor was done by filling either the bore side of the fibers or the shell side of the module with a cells suspension.
Cells concentration was determined by optical density at a wavelength of 600 nm. Protein assays were done according to the binding method of Bradford [21], slightly modified [22] using bovine serum albumin (BSA) as protein standard. Glucose, fructose, sorbitol and gluconic acid concentrations were analyzed by using a HPLC (Waters, Model 510) with refractive index detector and a Polyspher CH CA column. The column temperature was kept at 80◦ C and water was used as eluent at a flow rate of 0.5 ml/min. 2.5. Electrodialysis unit The ED stack contained three compartments made of acrylic, separated by two ion exchange membranes, 3.5 mm distant one of the other. Each membrane was ca. 6 cm of diameter, corresponding to a total effective area of 56 cm2 . Commercially available membranes designate by CR 67 HMR-412 (cationic exchange membrane) and AR 204 SZRA-412 (anionic exchange membrane), from the Ionics Inc. were used. Following the supplier, these membranes have a high water content (46%), which results in a water transport of 0.149 l/farad in 0.6N NaCl at 16 mA/cm2 . A centrifugal pump (Greycor Co model PQ-12 DC) with flow control by means of a power supply (Labo FR 3015) was used for continuously circulate the reaction medium through the feed chamber (ca. 20 ml), at flow rate of 12–24 l/h. A 20 g/l NaCl solution was used in the other two compartments of 800 ml each, for the nickel electrodes rinse. The voltage and the current across the ED stack were controlled by a dc power supply (Kepco model SM 325-2). The measured current density was always lower than 5 mA/cm2 . The temperature of the reaction medium and, consequently the temperature of the electrodialysis system, were
46
H.C. Ferraz et al. / Journal of Membrane Science 191 (2001) 43–51
Fig. 3. Hollow fiber module used to confine the permeabilized cells: (A) scheme of the hollow fiber bundle inside a module; (B) photomicrograph of the cross-section of a hollow fiber.
controlled at 39◦ C. The absence of leaks in the system was confirmed by preliminary experiments with NaCl and sodium gluconate solutions. 2.6. Kinetic studies The experiments were operated in batch. When cells confined by hollow fibers were used, the system was operated by circulating an equimolar fructose/glucose solution through the hollow fiber module. The system
temperature was maintained constant at 39◦ C. In absence of the electrodialysis unit, the pH was also kept constant at 6.2 by automatic titration using 1 M NaOH, as shown in Fig. 4. The pH of the medium is an important parameter once if it is far from optimum for GFOR activity, the reaction stops [1]. The mass of the NaOH solution added to the system was measured during the reaction and used to monitor the gluconic acid production. In the coupled system, the setup is shown in Fig. 5.
H.C. Ferraz et al. / Journal of Membrane Science 191 (2001) 43–51
47
Fig. 4. Experimental set up for the experiments without electrodialysis: (1) digital balance; (2) NaOH tank; (3) pump; (4) pH controller; (5) magnetic stirrer; (6) substrates tank; (7) pump; (8) thermostatic bath; (9) hollow fiber module; (10) mixture vessel.
Fig. 5. Experimental set up for the experiments with electrodialysis: (1) thermostatic bath; (2) hollow fiber module; (3) pump; (4) pH controller; (5) magnetic stirrer; (6) substrates tank; (7) electrodialysis stack.
transfer. In this system, even applying same amount of protein, a comparison with the free cell system is not straight. In the gel some cells do not participate in the reaction and the mass transfer conditions are very different. Furthermore, during preparation of the beads may occur a reduction in enzyme activity. On the other hand, cells confined by hollow fibers also have a very different mass transfer condition when compared to free or immobilized cells. In this case, there is a stagnant layer of solution (including that inside of the membrane pores) which represents the main mass transfer resistance. Furthermore, the protein concentration in the layer of confined solution will be different of that used in the free cells system. A confined solution of the same concentration that is used in the free cells system would lead to different relation between protein and substrate. Table 1 shows the initial specific rate of sorbitol production and protein mass for each experiment. Ac-
3. Results In order to choose the most suitable procedure for cells immobilization, experiments using different supports were accomplished. Reactions with cells immobilized in calcium alginate or confined in hollow fibers membranes were carried out and their performances were compared to that using free cells. The alginate medium has been chosen to be the most favorable conditions of immobilization, i.e. the gel is soft and offers a minimum resistance to mass
Table 1 Performance of different cell immobilization methods without coupling ED unita Immobilization method
Initial specific reaction rate (gs /gp h)
Protein mass (mg)
Free cells Calcium alginate Bore of hollow fibers Shell side of hollow fibers module
32.8 3.8 27.9 16.5
64.5 145.7 8.6 17.4
a
A reaction volume of 100 ml was used in all experiments.
48
H.C. Ferraz et al. / Journal of Membrane Science 191 (2001) 43–51
cording to these results, the lowest initial specific reaction rate was obtained in the system utilizing cells immobilized in calcium alginate. The results presented in Table 1 also indicate qualitatively that the use of confined cells can lead to reaction rates close to that obtained with free cells. Due to evident advantages of confined or immobilized cells, and considering that immobilization by gel gives lower reaction rates, the results with hollow fiber present a very interesting perspective, despite the fact that the systems are not optimized. When cells were confined in the shell side of the module it was observed a considerable cells deposition during the reaction. This fact can account for the 40% lower specific reaction rate in this experiment compared to that using cells inside the fibers. The main reason for these results was related to less efficient pH control due to cells deposition in the shell of the module [23]. Based on these preliminary results, the use of the calcium alginate as a support for cells was ruled out and the following experiments were carried out with cells confined either in the shell side of the hollow fiber module or in the bore of the fibers.
3.1. Coupled system 3.1.1. Cells confined in the shell side of the hollow fiber module In these experiments using cells confined in the shell side of the module, the reaction medium was continuously pumped through the electrodialysis unit at a flow rate of 24 l/h, corresponding to a Reynolds number of 1230. It is important to mention that the pumping speed through HF module was varied (12–84 l/h, corresponding to a Reynolds number of 185–1300) and no alteration in the reaction rate was observed. This result indicated that the limiting step to mass transfer is diffusion through the stagnant layer (membrane pores and confined solution). The gluconic acid was removed so that its concentration was always lower than 1 g/l, the reaction evolution was accompanied by the sorbitol production. However, despite the gluconic acid removal, the pH of the reaction medium was about 5.5, a little far from that ideal for GFOR activity [1]. At first, it was used a protein mass of 36 mg for 100 ml of reaction volume. In this case, during >25 h
Fig. 6. Cells confined in the shell side of the hollow fiber module. Effect of the protein concentration on the specific reaction rate: (䊏) 36.0 mg of protein; (䊉) 73.5 mg protein. Sorbitol specific concentration (—) and sorbitol mass (----) evolution. Initial concentration: glucose = 100 g/l; fructose = 100 g/l; reaction volume = 100 ml.
the specific reaction rate remained constant at about 2.6 gsorbitol /gprotein h (gs /gp h). In another experiment, in which more cells were confined in the module, corresponding to a protein mass of 73.5 mg for 100 ml of reaction volume, the specific reaction rate decreased. Both results are shown in Fig. 6. This fact was attributed to reduction of pH near the cells, which is more critical at high concentration of the cells suspension. The result present in Table 1 (rate of 16.5 gs /gp h) for the experiment at same configuration but without electrodialysis also reinforced this conclusion. Despite the fact of different protein masses (17.4 mg) were applied in that experiment, the huge reduction in the effective reaction rates only can be explained qualitatively by unfavorable conditions for the enzymatic reaction, i.e. the reduction in pH close to the cells due to its higher concentration. These results point out the necessity of module design optimization in order to obtain better conditions for product removal and pH control. A complete process optimization is not objective of this work. Therefore, to improve the product removal, the cells were confined in the bore of the fibers, where the stagnant layer has a lower thickness and the concentration near the cells is better controlled.
H.C. Ferraz et al. / Journal of Membrane Science 191 (2001) 43–51
Fig. 7. Cells confined in the bore of the hollow fiber module. Effect of the protein concentration on the reaction rate: (䊏) one module, initial protein mass of 2.2 mg; (䊉) two modules in series, initial protein mass of 4.4 mg. Sorbitol specific concentration (—) and sorbitol mass (----) evolution. Initial concentration: glucose = 100 g/l; fructose = 100 g/l; reaction volume = 100 ml.
3.1.2. Cells confined in the bore of the hollow fibers At this time, the flow was kept at 12 l/h (Reynolds number of 185) in order to minimize the water flux through ion exchange membranes. As a consequence of the reduced volume inside fibers, a lower cells mass was used, so a protein concentration of 16 mg/l was obtained. Fig. 7 shows the result of the experiments. A specific reaction rate of 17.5 gs /gp h was observed, which is four-times higher than that obtained in the system using cells confined in the shell side of the module, indicating a better pH control. Even when two identical modules were arranged in series, duplicating the total protein mass, the specific reaction rate did not differ significantly from that using only one module. Thus, it is possible to increase reaction rate proportionally to the protein concentration by distributing the total cells mass among distinct modules in order to avoid high cells concentration in the confined solution. The most important observation is, however, related to the stability of this system. The same hollow fiber module was used twice, for >64 h, with no noticeable decrease of the enzymatic activity. The same behavior was not observed in a system without gluconic acid removal by electrodialysis. In this case, it was verified a decrease of the enzyme activity in the early stages of the reaction what should
49
Fig. 8. Experiments without electrodialysis coupling. Specific reaction rate for the first experiment (—) and after substrate solution replacement (---). Sorbitol concentration ( ). Initial concentration: glucose = 100 g/l; fructose = 100 g/l.
not be attributed only to the reduction of substrate concentration in the reaction medium. To confirm the hypothesis of enzyme inactivation, after about 100 h of reaction the medium was replaced by a fresh sugar solution. The specific reaction rates presented in Figs. 8 and 9 were calculated from the sorbitol
Fig. 9. Specific reaction rate for the system with (---) and without (—) electrodialysis.
50
H.C. Ferraz et al. / Journal of Membrane Science 191 (2001) 43–51
concentration in the reaction medium. As shown in Fig. 8, the specific reaction rate for the new substrate solution was almost the same as that observed in the final stages of the previous reaction, which ratifies the enzyme inactivation hypothesis. Fig. 9 compares the specific reaction rate for this experiment and that obtained with electrodialysis coupling. It is clear the enzyme stability improvement when gluconic acid was removed by electrodialysis. It must be clarified that the absolute value of specific reaction rates is not very reliable when cells are confined in the bore of hollow fibers. This is due to the difficulty in determining the volume of cells suspension actually contained inside the fibers, for once it was not assured that fibers were totally filled with the suspension. Anyway, the removal of gluconate by electrodialysis seems to be responsible for the greater stability of the GFOR. The presence of gluconic acid causes an inhibition of the lactone hydrolysis and its accumulation in the reaction medium. According to Fürlinger et al. [10,24] the interaction with the lactone produced is responsible for the enzyme GFOR inactivation. In this way, the removal of gluconate ion by electrodialysis has a favorable effect once it prevents the lactone accumulation.
4. Conclusions During the reaction, the pH of the medium decreases quickly due to gluconic acid production. In order to prevent the enzyme inhibition, it is usually employed an alkali solution, such as NaOH or KOH. With electrodialysis, the acid produced was continuously removed and the pH was maintained constant, without alkali addition. It was found that a lower enzyme activity for cells confined in the shell side of hollow fiber module, which was attributed to local reduction of pH due to cell deposition. The experiments with different cells loading made evident the importance of pH control. The highest specific reaction rates were obtained by using low protein concentrations, which points out that the enzyme activity is affected by the pH near the cells, as it was indicated previously. The electrodialysis unit coupled to the reactor allowed an efficient removal of gluconic acid. Its concentration in the reaction medium was kept as low as 1 g/l. Furthermore, an improvement in the stability of
the enzyme was observed. No reduction in the reaction rate was found, even after 60 h of reaction. On the other hand, when NaOH was applied to neutralize the gluconic acid produced, a reduction of 80% in the reaction rate in the same period was observed. These results emphasize the hypothesis that the enzyme stability is associated to lactone concentration in the reaction medium. Although, the experimental condition were not optimized, the performance of different investigated systems leads to the conclusion that coupling of a separation unit has very good perspective to be applied in a technical scale.
Acknowledgements The authors would like to thank CNPq (Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnológico) for financial support. References [1] M. Zachariou, R.K. Scopes, Glucose–fructose oxidoreductase, a new enzyme isolated from Zymomonas mobilis that is responsible for sorbitol production, J. Bacteriol. 167 (1986) 863–869. [2] J. Hardman, R.K. Scopes, The kinetics of glucose–fructose oxidoreductase from Zymomonas mobilis, Bur. J. Biochem. 173 (1988) 203–209. [3] U.H. Chun, P.L. Rogers, The simultaneous production of sorbitol from fructose and gluconic acid from glucose using an oxidoreductase of Zymomonas mobilis, Appl. Microbiol. Biotechnol. 29 (1988) 19–24. [4] H.S. Ro, H.S. Kim, Continuous production of gluconic acid and sorbitol from sucrose using invertase and an oxidoreductase of Zymomonas mobilis, Enzyme Microbiol. Biotechnol. 13 (1991) 920–924. [5] B. Rehr, C. Wilhelm, H. Sahm, Production of sorbitol and gluconic acid by permeabilized cells of Zymomonas mobilis, Appl. Microbiol. Biotecnhol. 35 (1991) 144–148. [6] K.H. Jang, C.J. Park, U.H. Chun, Improvement of oxidoreductase stability of cethyltrimethylammoniumbromide permeabilized cells of Zymomonas mobilis through glutaraldheyde crosslinking, Biotechnol. Lett. 14 (1992) 311–316. [7] H. Loos, R. Krämer, H. Sahm, et al., Sorbitol promotes growth of Zymomonas mobilis in environments with high concentrations of sugar: evidence for a physiological function of glucose–fructose oxidoreductase in osmoprotection, J. Bacteriol. 176 (1994) 7688–7693. [8] D. Gollhofer, B Nidetzky, M. Furlinger, et al., Efficient protection of glucose–fructose oxidoreductase from
H.C. Ferraz et al. / Journal of Membrane Science 191 (2001) 43–51
[9]
[10]
[11]
[12] [13]
[14]
[15]
[16]
Zymomonas mobilis against irreversible inactivation during its catalytic action, Enzyme Microb. Technol. 17 (1995) 235–240. C. Parker, N. Peekhaus, X. Zhang, et al., Kinetics of sugar transport and phosphorylation influence glucose and fructose cometabolism by Zymomonas mobilis, Appl. Environ. Microbiol. 63 (1997) 3519–3525. M. Fürlinger, D. Haltrich, D. Kulbe, et al., A multistep process is responsible for product-induced inactivation of glucose–fructose oxidoreductase from Zymomonas mobilis, Eur. J. Biochem. 251 (1998) 955–963. K.Q. Wilberg, T.L.M. Alves, R. Nobrega, Enzymatic catalysis by permeabilized cells, Brazilian J. Chem. Eng. 14 (1997) 347–352. J. Woodward, Methods of immobilization of microbial cells, J. Microbiol. Methods 8 (1988) 91–102. H.W. Doelle, L. Kirk, R. Crittenden, et al., Zymomonas mobilis, science and industrial application, Critical. Rev. Biotechnol. 13 (1993) 57–98. A.W. Bunch, The uses and future potential of microbial hollow-fibre bioreactors, J. Microbiol. Methods 8 (1988) 103– 119. D.M.F. Prazeres, J.M.S. Cabral, Enzymatic membranes bioreactors and their applications, Enzyme Microb. Technol. 16 (1994) 738–750. S.L. Paterson, A.G. Fane, C.J.D. Fell, et al., Sorbitol and gluconate production in a hollow fibre membrane reactor by immobilized Zymomonas mobilis, Biocatalysis 1 (1988) 217–229.
51
[17] S. Novalic, T. Kongbangkerd, K.D. Kulbe, Separation of gluconate with conventional and bipolar electrodialysis, Desalination 114 (1997) 45–50. [18] H. Strathmann, Electrodialysis, in: W.S.W. Ho, K.K. Sirkar, (Eds), Membrane Handbook, Van Nostrand Reinhold, New York, NY, pp. 219–262. [19] J.E. Bailey, D.F. Ollis, Biochemical Engineering Fundamentals, McGraw-Hill, New York, NY, 1986. [20] M. Di Luccio, C.P. Borges, R. Nobrega, A.C. Habert, Spinning of polycarbonate hollow fibers, in: Proceedings of the 1996 International Congress on Membranes and Membrane Processes, ICOM’96, Yokohama, Japan, August 1996, pp. 972–973. [21] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [22] C. Cordeiro, A.P. Freire, Protein determination in permeabilized yeast cells using the coomassie brilliant blue dye binding assay, Anal. Biochem. 223 (1994) 321–323. [23] H.C. Ferraz, Produção de Sorbitol e Ácido Glicônico por Células Permeabilizadas e Imoblizadas de Zymomonas mobilis com Separação Simultânea dos Produtos por Eletrodiálise, MSc Thesis, PEQ/COPPE/UFRJ, Rio de Janeiro, Brazil, 1999. [24] M. Fürlinger, et al., Control of the association state of tetrameric glucose–fructose oxidoreductase from Zymomonas mobilis as the rationale for stabilization of the enzyme in biochemical reactors, J. Biochem. 124 (1998) 280–286.
Coupling of an electrodialysis unit to a hollow fiber bioreactor for separation of gluconic acid from sorbitol produced by Zymomonas mobilis permeabilized cells H.C. Ferraz, T.L.M. Alves, C.P. Borges∗ Chemical Engineering Program, COPPE, Federal University of Rio de Janeiro, P.O. Box 68502, 21945-970 Rio de Janeiro, Brazil Received 29 August 2000; received in revised form 11 April 2001; accepted 11 April 2001
Abstract The objective of this study was to evaluate the coupling of an electrodialysis unit to a reactor in which gluconic acid and sorbitol were produced by permeabilized and immobilized Zymomonas mobilis cells. The experimental results have shown that electrodialysis unit coupled to the bioreactor allowed an efficient removal of gluconic acid from the reaction medium, as well as an improvement in the stability of the enzyme. It was not observed any reduction in the reaction rate, even after 60 h of operation. On the other hand, when NaOH was applied to neutralize the gluconic acid produced, it was verified a reduction of 80% in the reaction rate in the same period. These results lead to the hypothesis that the enzyme stability is associated to gluconate accumulation in the reaction medium. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Zymomonas mobilis; Sorbitol; Gluconic acid; Hollow fibers; Electrodialysis
1. Introduction Sorbitol and gluconic acid production by permeabilized cells of Zymomonas mobilis has been extensively studied over the years [1–10]. Such efforts can be justified by relatively mild operation conditions of the biosynthesis when compared to the chemical route and by the enzyme specificity, which minimizes by-product formation, besides the importance of both products. Glucose–fructose oxidoreductase (GFOR) present in cells of Z. mobilis promotes simultaneous glucose oxidation to glucono-␦-lactone and fructose reduction to sorbitol. In a subsequent step, glucono-␦-lactone is hydrolyzed to gluconic acid, both spontaneously and ∗ Corresponding author. E-mail address: [email protected] (C.P. Borges).
under the action of the enzyme glucono-␦-lactonase. As shown in Fig. 1, in intact cells of Z. mobilis, sorbitol accumulates in the medium while gluconic acid is converted to ethanol, via Entner Doudoroff pathway. When cells are permeabilized, small compounds, such as metallic ions and cofactors can diffuse out of the cells, so the pathway from gluconate to ethanol is not functional anymore [3]. While this procedure is important to avoid ethanol formation, it has no effect on the activity of the enzyme, once the NADP(H) cofactor for GFOR remains bound to it. Fig. 2 shows the effect of permeabilization on cells of Z. mobilis [11]. The cytoplasmatic contents are very distinct due to intracellular compounds leakage. A better operational stability and an easier continuous operation can be achieved by immobilizing the permeabilized cells. Several methods have been applied [12,13] and most of them employed supports
0376-7388/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 6 - 7 3 8 8 ( 0 1 ) 0 0 4 4 7 - 1
44
H.C. Ferraz et al. / Journal of Membrane Science 191 (2001) 43–51
Fig. 1. Mechanism of sorbitol and gluconic acid production by permeabilized cells of Z. mobilis. When cells are fully permeabilized, the pathway from gluconate to ethanol, delimited by the broken line, becomes non functional [3].
based on k-carrageenan or alginate. The confinement of cells in the bore of hollow fiber membranes [14–16] has been used as an alternative and promising immobilization technique. Obviously, membrane pores have to be smaller than the cells and, furthermore, offer a low resistance to the substrates and products transport. As advantage, this type of system can be easily operated and regenerated. Down stream operations to concentrate and purify the products have not been taken into account in the previous studies, despite its importance to the process feasibility. In this context, techniques, such as electrodialysis (ED) and ion exchange could be useful. ED has the advantage of being easy to scale up and, in addition, it does not add any external compound to the medium. The dissociation constant K for gluconic acid is 1.99 × 10−4 corresponding to a pK of 3.7 [17]. If an aqueous solution of gluconic acid and sorbitol is pumped through an electrodialysis cell and an electrical potential is established between the anode and cathode, the gluconate ions will migrate across the anion exchange membrane toward the anode compartment. Likewise, the hydrogen ions will migrate through the cation exchange membrane to the cathode compartment. This permits the pH control without use of neutralizer [18]. Sorbitol, an uncharged molecule,
Fig. 2. Electron transmission photomicrograph of Z. mobilis cells: (A) before permeabilization; (B) after permeabilization. [11].
is not affected and will remain in the feed stream. This way, ED will promote the desired separation of gluconate from unreacted substrates. In this work, the reaction unit, consisting of a module of hollow fibers containing permeabilized cells of Z. mobilis, was coupled to the electrodialysis unit allowing the simultaneous extraction of the gluconic acid produced. A performance comparison of the systems with and without acid separation by electrodialysis is presented in terms of initial reaction rate and operational stability.
H.C. Ferraz et al. / Journal of Membrane Science 191 (2001) 43–51
2. Experimental 2.1. Microorganism and culture conditions All experiments were performed with the bacteria Z. mobilis CP4 (ATCC 31821), cultivated in a medium containing 100 g glucose/l as carbon source and 5 g yeast extract/l under controlled temperature of 30◦ C.
45
The pore size in the outer surface of the fiber is smaller than 0.1 m, while in the inner surface is smaller than 1 m. Considering cell dimensions, the range of pore size in the fiber is small enough to avoid any loss of cells during the experiment. The water permeability of these fibers was determined as 912 l/h m2 bar. 2.4. Assays and analytical methods
2.2. Preparation of permeabilized cells Z. mobilis was cultivated until the late exponential phase after about 20 h growth. After this period cetyltrimethylamonium bromide (CTAB) was added directly to the broth to give a final proportion of 0.04 g CTAB per gram of cells [12]. After gently stirring for 30 min, cells were separated by centrifugation at 5000 rpm and washed twice with distilled water, pH 5.5. 2.3. Immobilization method 2.3.1. Entrapment in alginate For the immobilization in calcium alginate, the concentrated suspension of cells was mixed with a solution of sodium alginate (8% w/v) [19]. Spherical beads were then produced by dropping the mixture into a 20 g/l calcium chloride solution through a syringe needle of 0.5 mm diameter. The mean diameter of obtained beads was ca. 2 mm. The beads were suspended in a 0.5% (v/v) glutaraldheyde solution with stirring for 30 min. After washing with water, the beads were stored at 4◦ C until further use. 2.3.2. Confinement in hollow fiber Permeabilized cells suspension was confined in the bore of microporous polycarbonate hollow fibers or in the shell side of the module. The fibers were prepared by phase inversion technique using spinning conditions reported in [20]. These fibers, about 20 cm long, were assembled in a longitudinal module of PVC as presented in Fig. 3(A). Fig. 3(B) shows a photomicrograph of a fiber cross-section, which has outer and inner diameters of 0.68 and 0.33 mm, respectively. The set up of the membrane reactor was done by filling either the bore side of the fibers or the shell side of the module with a cells suspension.
Cells concentration was determined by optical density at a wavelength of 600 nm. Protein assays were done according to the binding method of Bradford [21], slightly modified [22] using bovine serum albumin (BSA) as protein standard. Glucose, fructose, sorbitol and gluconic acid concentrations were analyzed by using a HPLC (Waters, Model 510) with refractive index detector and a Polyspher CH CA column. The column temperature was kept at 80◦ C and water was used as eluent at a flow rate of 0.5 ml/min. 2.5. Electrodialysis unit The ED stack contained three compartments made of acrylic, separated by two ion exchange membranes, 3.5 mm distant one of the other. Each membrane was ca. 6 cm of diameter, corresponding to a total effective area of 56 cm2 . Commercially available membranes designate by CR 67 HMR-412 (cationic exchange membrane) and AR 204 SZRA-412 (anionic exchange membrane), from the Ionics Inc. were used. Following the supplier, these membranes have a high water content (46%), which results in a water transport of 0.149 l/farad in 0.6N NaCl at 16 mA/cm2 . A centrifugal pump (Greycor Co model PQ-12 DC) with flow control by means of a power supply (Labo FR 3015) was used for continuously circulate the reaction medium through the feed chamber (ca. 20 ml), at flow rate of 12–24 l/h. A 20 g/l NaCl solution was used in the other two compartments of 800 ml each, for the nickel electrodes rinse. The voltage and the current across the ED stack were controlled by a dc power supply (Kepco model SM 325-2). The measured current density was always lower than 5 mA/cm2 . The temperature of the reaction medium and, consequently the temperature of the electrodialysis system, were
46
H.C. Ferraz et al. / Journal of Membrane Science 191 (2001) 43–51
Fig. 3. Hollow fiber module used to confine the permeabilized cells: (A) scheme of the hollow fiber bundle inside a module; (B) photomicrograph of the cross-section of a hollow fiber.
controlled at 39◦ C. The absence of leaks in the system was confirmed by preliminary experiments with NaCl and sodium gluconate solutions. 2.6. Kinetic studies The experiments were operated in batch. When cells confined by hollow fibers were used, the system was operated by circulating an equimolar fructose/glucose solution through the hollow fiber module. The system
temperature was maintained constant at 39◦ C. In absence of the electrodialysis unit, the pH was also kept constant at 6.2 by automatic titration using 1 M NaOH, as shown in Fig. 4. The pH of the medium is an important parameter once if it is far from optimum for GFOR activity, the reaction stops [1]. The mass of the NaOH solution added to the system was measured during the reaction and used to monitor the gluconic acid production. In the coupled system, the setup is shown in Fig. 5.
H.C. Ferraz et al. / Journal of Membrane Science 191 (2001) 43–51
47
Fig. 4. Experimental set up for the experiments without electrodialysis: (1) digital balance; (2) NaOH tank; (3) pump; (4) pH controller; (5) magnetic stirrer; (6) substrates tank; (7) pump; (8) thermostatic bath; (9) hollow fiber module; (10) mixture vessel.
Fig. 5. Experimental set up for the experiments with electrodialysis: (1) thermostatic bath; (2) hollow fiber module; (3) pump; (4) pH controller; (5) magnetic stirrer; (6) substrates tank; (7) electrodialysis stack.
transfer. In this system, even applying same amount of protein, a comparison with the free cell system is not straight. In the gel some cells do not participate in the reaction and the mass transfer conditions are very different. Furthermore, during preparation of the beads may occur a reduction in enzyme activity. On the other hand, cells confined by hollow fibers also have a very different mass transfer condition when compared to free or immobilized cells. In this case, there is a stagnant layer of solution (including that inside of the membrane pores) which represents the main mass transfer resistance. Furthermore, the protein concentration in the layer of confined solution will be different of that used in the free cells system. A confined solution of the same concentration that is used in the free cells system would lead to different relation between protein and substrate. Table 1 shows the initial specific rate of sorbitol production and protein mass for each experiment. Ac-
3. Results In order to choose the most suitable procedure for cells immobilization, experiments using different supports were accomplished. Reactions with cells immobilized in calcium alginate or confined in hollow fibers membranes were carried out and their performances were compared to that using free cells. The alginate medium has been chosen to be the most favorable conditions of immobilization, i.e. the gel is soft and offers a minimum resistance to mass
Table 1 Performance of different cell immobilization methods without coupling ED unita Immobilization method
Initial specific reaction rate (gs /gp h)
Protein mass (mg)
Free cells Calcium alginate Bore of hollow fibers Shell side of hollow fibers module
32.8 3.8 27.9 16.5
64.5 145.7 8.6 17.4
a
A reaction volume of 100 ml was used in all experiments.
48
H.C. Ferraz et al. / Journal of Membrane Science 191 (2001) 43–51
cording to these results, the lowest initial specific reaction rate was obtained in the system utilizing cells immobilized in calcium alginate. The results presented in Table 1 also indicate qualitatively that the use of confined cells can lead to reaction rates close to that obtained with free cells. Due to evident advantages of confined or immobilized cells, and considering that immobilization by gel gives lower reaction rates, the results with hollow fiber present a very interesting perspective, despite the fact that the systems are not optimized. When cells were confined in the shell side of the module it was observed a considerable cells deposition during the reaction. This fact can account for the 40% lower specific reaction rate in this experiment compared to that using cells inside the fibers. The main reason for these results was related to less efficient pH control due to cells deposition in the shell of the module [23]. Based on these preliminary results, the use of the calcium alginate as a support for cells was ruled out and the following experiments were carried out with cells confined either in the shell side of the hollow fiber module or in the bore of the fibers.
3.1. Coupled system 3.1.1. Cells confined in the shell side of the hollow fiber module In these experiments using cells confined in the shell side of the module, the reaction medium was continuously pumped through the electrodialysis unit at a flow rate of 24 l/h, corresponding to a Reynolds number of 1230. It is important to mention that the pumping speed through HF module was varied (12–84 l/h, corresponding to a Reynolds number of 185–1300) and no alteration in the reaction rate was observed. This result indicated that the limiting step to mass transfer is diffusion through the stagnant layer (membrane pores and confined solution). The gluconic acid was removed so that its concentration was always lower than 1 g/l, the reaction evolution was accompanied by the sorbitol production. However, despite the gluconic acid removal, the pH of the reaction medium was about 5.5, a little far from that ideal for GFOR activity [1]. At first, it was used a protein mass of 36 mg for 100 ml of reaction volume. In this case, during >25 h
Fig. 6. Cells confined in the shell side of the hollow fiber module. Effect of the protein concentration on the specific reaction rate: (䊏) 36.0 mg of protein; (䊉) 73.5 mg protein. Sorbitol specific concentration (—) and sorbitol mass (----) evolution. Initial concentration: glucose = 100 g/l; fructose = 100 g/l; reaction volume = 100 ml.
the specific reaction rate remained constant at about 2.6 gsorbitol /gprotein h (gs /gp h). In another experiment, in which more cells were confined in the module, corresponding to a protein mass of 73.5 mg for 100 ml of reaction volume, the specific reaction rate decreased. Both results are shown in Fig. 6. This fact was attributed to reduction of pH near the cells, which is more critical at high concentration of the cells suspension. The result present in Table 1 (rate of 16.5 gs /gp h) for the experiment at same configuration but without electrodialysis also reinforced this conclusion. Despite the fact of different protein masses (17.4 mg) were applied in that experiment, the huge reduction in the effective reaction rates only can be explained qualitatively by unfavorable conditions for the enzymatic reaction, i.e. the reduction in pH close to the cells due to its higher concentration. These results point out the necessity of module design optimization in order to obtain better conditions for product removal and pH control. A complete process optimization is not objective of this work. Therefore, to improve the product removal, the cells were confined in the bore of the fibers, where the stagnant layer has a lower thickness and the concentration near the cells is better controlled.
H.C. Ferraz et al. / Journal of Membrane Science 191 (2001) 43–51
Fig. 7. Cells confined in the bore of the hollow fiber module. Effect of the protein concentration on the reaction rate: (䊏) one module, initial protein mass of 2.2 mg; (䊉) two modules in series, initial protein mass of 4.4 mg. Sorbitol specific concentration (—) and sorbitol mass (----) evolution. Initial concentration: glucose = 100 g/l; fructose = 100 g/l; reaction volume = 100 ml.
3.1.2. Cells confined in the bore of the hollow fibers At this time, the flow was kept at 12 l/h (Reynolds number of 185) in order to minimize the water flux through ion exchange membranes. As a consequence of the reduced volume inside fibers, a lower cells mass was used, so a protein concentration of 16 mg/l was obtained. Fig. 7 shows the result of the experiments. A specific reaction rate of 17.5 gs /gp h was observed, which is four-times higher than that obtained in the system using cells confined in the shell side of the module, indicating a better pH control. Even when two identical modules were arranged in series, duplicating the total protein mass, the specific reaction rate did not differ significantly from that using only one module. Thus, it is possible to increase reaction rate proportionally to the protein concentration by distributing the total cells mass among distinct modules in order to avoid high cells concentration in the confined solution. The most important observation is, however, related to the stability of this system. The same hollow fiber module was used twice, for >64 h, with no noticeable decrease of the enzymatic activity. The same behavior was not observed in a system without gluconic acid removal by electrodialysis. In this case, it was verified a decrease of the enzyme activity in the early stages of the reaction what should
49
Fig. 8. Experiments without electrodialysis coupling. Specific reaction rate for the first experiment (—) and after substrate solution replacement (---). Sorbitol concentration ( ). Initial concentration: glucose = 100 g/l; fructose = 100 g/l.
not be attributed only to the reduction of substrate concentration in the reaction medium. To confirm the hypothesis of enzyme inactivation, after about 100 h of reaction the medium was replaced by a fresh sugar solution. The specific reaction rates presented in Figs. 8 and 9 were calculated from the sorbitol
Fig. 9. Specific reaction rate for the system with (---) and without (—) electrodialysis.
50
H.C. Ferraz et al. / Journal of Membrane Science 191 (2001) 43–51
concentration in the reaction medium. As shown in Fig. 8, the specific reaction rate for the new substrate solution was almost the same as that observed in the final stages of the previous reaction, which ratifies the enzyme inactivation hypothesis. Fig. 9 compares the specific reaction rate for this experiment and that obtained with electrodialysis coupling. It is clear the enzyme stability improvement when gluconic acid was removed by electrodialysis. It must be clarified that the absolute value of specific reaction rates is not very reliable when cells are confined in the bore of hollow fibers. This is due to the difficulty in determining the volume of cells suspension actually contained inside the fibers, for once it was not assured that fibers were totally filled with the suspension. Anyway, the removal of gluconate by electrodialysis seems to be responsible for the greater stability of the GFOR. The presence of gluconic acid causes an inhibition of the lactone hydrolysis and its accumulation in the reaction medium. According to Fürlinger et al. [10,24] the interaction with the lactone produced is responsible for the enzyme GFOR inactivation. In this way, the removal of gluconate ion by electrodialysis has a favorable effect once it prevents the lactone accumulation.
4. Conclusions During the reaction, the pH of the medium decreases quickly due to gluconic acid production. In order to prevent the enzyme inhibition, it is usually employed an alkali solution, such as NaOH or KOH. With electrodialysis, the acid produced was continuously removed and the pH was maintained constant, without alkali addition. It was found that a lower enzyme activity for cells confined in the shell side of hollow fiber module, which was attributed to local reduction of pH due to cell deposition. The experiments with different cells loading made evident the importance of pH control. The highest specific reaction rates were obtained by using low protein concentrations, which points out that the enzyme activity is affected by the pH near the cells, as it was indicated previously. The electrodialysis unit coupled to the reactor allowed an efficient removal of gluconic acid. Its concentration in the reaction medium was kept as low as 1 g/l. Furthermore, an improvement in the stability of
the enzyme was observed. No reduction in the reaction rate was found, even after 60 h of reaction. On the other hand, when NaOH was applied to neutralize the gluconic acid produced, a reduction of 80% in the reaction rate in the same period was observed. These results emphasize the hypothesis that the enzyme stability is associated to lactone concentration in the reaction medium. Although, the experimental condition were not optimized, the performance of different investigated systems leads to the conclusion that coupling of a separation unit has very good perspective to be applied in a technical scale.
Acknowledgements The authors would like to thank CNPq (Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnológico) for financial support. References [1] M. Zachariou, R.K. Scopes, Glucose–fructose oxidoreductase, a new enzyme isolated from Zymomonas mobilis that is responsible for sorbitol production, J. Bacteriol. 167 (1986) 863–869. [2] J. Hardman, R.K. Scopes, The kinetics of glucose–fructose oxidoreductase from Zymomonas mobilis, Bur. J. Biochem. 173 (1988) 203–209. [3] U.H. Chun, P.L. Rogers, The simultaneous production of sorbitol from fructose and gluconic acid from glucose using an oxidoreductase of Zymomonas mobilis, Appl. Microbiol. Biotechnol. 29 (1988) 19–24. [4] H.S. Ro, H.S. Kim, Continuous production of gluconic acid and sorbitol from sucrose using invertase and an oxidoreductase of Zymomonas mobilis, Enzyme Microbiol. Biotechnol. 13 (1991) 920–924. [5] B. Rehr, C. Wilhelm, H. Sahm, Production of sorbitol and gluconic acid by permeabilized cells of Zymomonas mobilis, Appl. Microbiol. Biotecnhol. 35 (1991) 144–148. [6] K.H. Jang, C.J. Park, U.H. Chun, Improvement of oxidoreductase stability of cethyltrimethylammoniumbromide permeabilized cells of Zymomonas mobilis through glutaraldheyde crosslinking, Biotechnol. Lett. 14 (1992) 311–316. [7] H. Loos, R. Krämer, H. Sahm, et al., Sorbitol promotes growth of Zymomonas mobilis in environments with high concentrations of sugar: evidence for a physiological function of glucose–fructose oxidoreductase in osmoprotection, J. Bacteriol. 176 (1994) 7688–7693. [8] D. Gollhofer, B Nidetzky, M. Furlinger, et al., Efficient protection of glucose–fructose oxidoreductase from
H.C. Ferraz et al. / Journal of Membrane Science 191 (2001) 43–51
[9]
[10]
[11]
[12] [13]
[14]
[15]
[16]
Zymomonas mobilis against irreversible inactivation during its catalytic action, Enzyme Microb. Technol. 17 (1995) 235–240. C. Parker, N. Peekhaus, X. Zhang, et al., Kinetics of sugar transport and phosphorylation influence glucose and fructose cometabolism by Zymomonas mobilis, Appl. Environ. Microbiol. 63 (1997) 3519–3525. M. Fürlinger, D. Haltrich, D. Kulbe, et al., A multistep process is responsible for product-induced inactivation of glucose–fructose oxidoreductase from Zymomonas mobilis, Eur. J. Biochem. 251 (1998) 955–963. K.Q. Wilberg, T.L.M. Alves, R. Nobrega, Enzymatic catalysis by permeabilized cells, Brazilian J. Chem. Eng. 14 (1997) 347–352. J. Woodward, Methods of immobilization of microbial cells, J. Microbiol. Methods 8 (1988) 91–102. H.W. Doelle, L. Kirk, R. Crittenden, et al., Zymomonas mobilis, science and industrial application, Critical. Rev. Biotechnol. 13 (1993) 57–98. A.W. Bunch, The uses and future potential of microbial hollow-fibre bioreactors, J. Microbiol. Methods 8 (1988) 103– 119. D.M.F. Prazeres, J.M.S. Cabral, Enzymatic membranes bioreactors and their applications, Enzyme Microb. Technol. 16 (1994) 738–750. S.L. Paterson, A.G. Fane, C.J.D. Fell, et al., Sorbitol and gluconate production in a hollow fibre membrane reactor by immobilized Zymomonas mobilis, Biocatalysis 1 (1988) 217–229.
51
[17] S. Novalic, T. Kongbangkerd, K.D. Kulbe, Separation of gluconate with conventional and bipolar electrodialysis, Desalination 114 (1997) 45–50. [18] H. Strathmann, Electrodialysis, in: W.S.W. Ho, K.K. Sirkar, (Eds), Membrane Handbook, Van Nostrand Reinhold, New York, NY, pp. 219–262. [19] J.E. Bailey, D.F. Ollis, Biochemical Engineering Fundamentals, McGraw-Hill, New York, NY, 1986. [20] M. Di Luccio, C.P. Borges, R. Nobrega, A.C. Habert, Spinning of polycarbonate hollow fibers, in: Proceedings of the 1996 International Congress on Membranes and Membrane Processes, ICOM’96, Yokohama, Japan, August 1996, pp. 972–973. [21] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [22] C. Cordeiro, A.P. Freire, Protein determination in permeabilized yeast cells using the coomassie brilliant blue dye binding assay, Anal. Biochem. 223 (1994) 321–323. [23] H.C. Ferraz, Produção de Sorbitol e Ácido Glicônico por Células Permeabilizadas e Imoblizadas de Zymomonas mobilis com Separação Simultânea dos Produtos por Eletrodiálise, MSc Thesis, PEQ/COPPE/UFRJ, Rio de Janeiro, Brazil, 1999. [24] M. Fürlinger, et al., Control of the association state of tetrameric glucose–fructose oxidoreductase from Zymomonas mobilis as the rationale for stabilization of the enzyme in biochemical reactors, J. Biochem. 124 (1998) 280–286.