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Hydrogels obtained by radiation-induced polymerization for yeast cells immobilization
Eur. Polym.J. Vol. 29, No. 7, pp. 1013-1018, 1993 Printed in Great Britain
0014-3057/93 $6.00+0.00 Pergamon Press Ltd
H Y D R O G E L S O B T A I N E D BY R A D I A T I O N - I N D U C E D POLYMERIZATION FOR YEAST CELLS IMMOBILIZATION MARIO CARENZA,1 MASARU YOSHIDA,2 MINORU KUMAKURA2. and TAKASHI FUJIMURA2 tIstituto di Fotochimica e Radiazioni d'Alta Energia, C.N.R., Sezione di Legnaro, Via Romea 4, 35020 Legnaro (Padova), Italy 2japan Atomic Energy Research Institute, Takasaki Radiation Chemistry Research Establishment, Takasaki, Gunma-ken, Japan
(Received 13 July 1992) Abstract--Radiation-induced polymerizations of several acrylic and methacrylic esters at both room temperature and low temperature were carried out. The polymer matrices obtained under the latter conditions were examined as carriers for immobilization of yeast cells. The adhesion of the latter was found to be markedly influenced by both matrix hydrophilicity, determined by equilibrium water content measurements, and porosity, evaluated by optical microscopy and scanning electron microscopy. The ethanol productivity during batch fermentation as a function of these parameters is discussed.
INTRODUCTION
EXPERIMENTAL PROCEDURES
Hydrogels are polymer networks which absorb and retain water without dissolving [1-3]. This property makes them interesting materials as carriers for immobilization of bioactive compounds [1,4] as alternatives to others successfully used [5, 6]. Radiation-induced polymerization enables hydrogels in a wide variety of forms to be easily prepared for applications both in biomedicine and in biotechnology [7,8]. With regard to the latter topic, radiation polymerization at low temperatures of glass-forming acrylic and methacrylic esters has been successful for obtaining hydrogels suitable as carriers for immobilization of bioactive species such as enzymes, cells and antibodies as well as for controlled release of drugs [8, 9]. The method is based on radiation-induced polymerization at a temperature below 0 ° of an aqueous solution or suspension of the monomer. At this temperature, water separates in the form of microcrystals dispersed in the m o n o m e r while the latter polymerizes in a super-cooled liquid state. At the end of irradiation when the system is brought to r o o m temperature, the ice microcrystals retained by the polymer matrix give rise to small pores on the walls of which the biocomponent is immobilized mainly by adhesion. By means of this technique, immobilization of yeast cells was successfully carried out in our laboratories and a high ethanol productivity was obtained during both batch [10-13] and continuous fermentation [14, 15]. In this work, polymer matrices were prepared by g a m m a irradiation of monomers with different hydrophilicities in the presence of water and examined as carriers for immobilization of yeast cells. Moreover, the relationship between the structure and physico-chemical properties of these matrices and the effectiveness of ethanol productivity was examined.
The following monomers, with codes and structures given in Table 1, were used as received: 2-hydroxyethyl methacrylate (HEMA) and 2-hydroxyethyl acrylate (HEA) from Tokyo Kasei Co.; methoxypolyethylenegiycol methacrylate with the number of oxyethylene units, n, equal to 3 (M-3G), 9 (M-gG) or 23 (M-23G), methoxypolyethyleneglycol acrylate with n equal to 3 (AM-30G) or 9 (AM-90G), polyethyleneglycol dimethacrylate with n equal to 4 (4G) or 14 (14G) and polyethyleneglycol diacrylate with n equal to 9 (A-400) from Shin-Nakamura Chemical Co., Ltd. All polymerizations were carried out by gamma-ray irradiation with a Co-60 source (at JAERI, Takasaki) at a dose of 10 kGy after flushing N2 through the tubes. Bulk polymerizations were carried out at 25 and - 7 8 °. In the case of HEMA, HEA and M-3G, polymer films of thickness 200/~m were obtained by cast polymerization of the monomers at 25° between two polypropylene plates separated by a polycarbonate gasket. Irradiation of aqueous solutions of the monomers was conducted at - 78°. Polymer matrices in the form of discs were allowed to swell for 3 4 days in a large volume of distilled water which was changed every day. The value of equilibrium water content, We, was determined according to the equation:
*Present address: Department of Bioscience, The NishiTokyo University, Uenohara, Kitatsuru, Yamanashi 409-01, Japan.
wo-
w,
x I00
(1)
where W, and Wa are the weights of the fully swollen matrix and of the completely dried sample, respectively. The quoted values are averages of three determinations. Portions of swollen matrices were freeze-dried, fractured at liquid N 2 temperature and analysed by scanning electron microscopy (SEM) using a Jeol Superprobe 733 apparatus. Portions of swollen matrices were sliced into thin films (10#m) with a microtome at - 1 0 ° and, after swelling again in water, examined with an Olympus light microscope. The procedure for cell immobilization has been reported [10, 11]. In short, a sample of yeast cells (Saccharomyces formosensis) precultured under aerobic conditions was incubated under agitation at 30° in the culture liquid containing portions of the polymer carrier previously swollen in the culture liquid. The ethanol produced from glucose was determined by the method of Bonichsen [16].
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Table 1. Codes and structures of the monomers 2-Hydroxyethyl methacrylate (HEMA) CH~------C(CH3)COOCH2CH2OH 2-Hydroxyethyl acrylate (HEA) CH~--'CH--COOCH2CH2OH Methoxypolyethyleneglycol methacrylate (M-nG)* CH~------'C(CH3)CO(OCH:CH2)nOCH3 Methoxypolyethyleneglycol acrylate (AM-nG)t CH~-------CH--CO(OCH2CH2).OCH 3 Polyethyleneglycol dimethacrylate (14G) CH~------C(CH~)COO(CH2CH20)t4COC(CH 3)~-CH2 Polyethyleneglycol diacrylate (A-400) CH~'CH--COO(CH2CH 20)gCOCH~"~'CH2 *The number n was 3 (M-3G), 9 (M-9G) and 23 (M-23G). tTbe number n was 3 (AM-30G) and 9 (AM-90G).
RESULTS AND DISCUSSION
Some acrylic and methacrylic esters were polymerized in bulk by irradiation at room temperature or at - 7 8 °. Some of the polymer samples were obtained by radiation-induced cast polymerization at room temperature. An irradiation dose of 10 kGy was given to the monomers so that virtually complete polymerization was achieved. We values are reported in Table 2. The swelling values of the acrylic polymers are higher than those of the methacrylic and, the longer the oxyethylene unit, the higher is We. The data referring to the polymer samples obtained at room temperature are very close to those obtained at - 7 8 °, the former being transparent and the latter showing some opacity probably due to impurities, perhaps water during the freezing step. The value of 37.3% for poly(HEMA) is similar to those recently reported [15, 17] although it markedly differs from that of 26.0% found by other authors [18]. The values for poly(HEA), in the range Table 2. Equilibrium water contents, We (%), of the polymer samples obtained by radiationinduced polymerization at room temperature (a) and at - 7 8 ° (b) of the various monomers Monomer *HEMA (a) *HEA (a) HEA (a) HEA (b) *M-3G (a) M-9G (a) M-9G (b) M-23G (b) AM-30G (a) AM-30G (b) AM-90G (a) AM-90G (b) 14G (a) 14G (b) A-400 (a) A-400 (b)
W, (%) 37.3 70.9 73.8 70.6 6.6 84.7 89.4 90.0 85.1 82.8 87.4 86.0 33.6 26.7 27.7 24.9
Dose rate = 10 kGy/hr; total dose = 10 kGy. *Polymer samples obtained by radiation-induced cast polymerization.
70.6-73.8%, are quite close to that of 69.9% [15] but they considerably differ from other values, viz. 45.9% [18] and 60% [17]. The main goal of this work was the establishment of a relationship between ethanol productivity (by fermentation of glucose) and the hydrophilic properties of polymeric carriers, the latter being obviously dependent on the hydrophilicity of the corresponding monomers. An important role is also played by the porosity of the polymer matrix, which in turn is brought about by the presence of water added to the reacting monomer. Thus, HEMA, HEA and AM-30G were selected as monomers capable of giving matrices over a wide We range, i.e. from 37 to 85% (see Table 2). The monomers were diluted with water in various proportions and, after flushing with N2, were rapidly cooled at - 7 8 ° . At this temperature, the mixtures were irradiated for 60 min (total dose = 10 kGy). The effect of a crosslinking agent, tetraethyleneglycol dimethyacrylate (4G), added to the polymerizing mixture in small amounts, was also examined. Table 3 shows the values of equilibrium water content, We. As for the results recently obtained for the system HEMA-water [19], We values were found to rise with increasing amount of water in the polymerizing mixture, as expected. At the same time, porosity is expected to increase. As an example, Fig. 1 shows the SEM microphotographs of the polymer matrices obtained from aqueous mixtures of HEA and AM-30G. For the more concentrating systems, Figs l(a) and (d) show that only very small pores, if any, are discernible. Correspondingly, W, values are very close to those obtained for the matrices prepared in bulk. On the other hand, as the proportion of water increases, Figs l(b), (c), (e) and (f) show that larger and larger cavities are created with the result that We values differ widely (see Table 3). A similar trend is found by light microscope examination. Figure 2 shows the microphotographs of hydrogels based on AM-90G. The closed-pore structure of the matrix prepared with a high AM90G/water ratio [Fig. 2(a)] becomes an open-pore structure in the case of a low ratio [Fig. 2(b)].
Table 3. Equilibrium water contents, We (%) of the polymer samples obtained by radiation-induced polymerization at - 7 8 ° of monomer-water mixtures (v/v) and ethanol productivity of yeast cells immobilized on the matrices Mixtures HEMA/H20 (2:1) HEMA/H20 (1 : 1) HEMA/H20 (1 : 2) HEA/H20 (2:1) HEA/H20 (1 : 1) HEA/HzO (1 : 2) AM-30G/H20 (2:1) AM-30G/H20 (1 : 1) AM-30G/H20 (1 : 2) *AM-30G/H20 ( 1: 2) AM-90G/H20 (2:1) AM-90G/H20 (1 : 2) Free cells
Wc (%) 42.8 51.3 69.4 74.7 80.7 86.8 84.0 88.2 92.5 88.2 88.7 94.1
EtOH productivity (mg/ml of gel.br) 0.49 0.49 0.60 0.78 0.83 1.48 0.81 1.10 1.78 1.06 0.60 1.75 0.31
Dose rate = 10 kGy/hr; irradiation time = 60 min. *Polymerization in the presence of 5% (v/v) of the crosslinking agent tetracthylencglycol dimethacrylate (4(3).
Hydrogels for yeast cells immobilization
Fig. 1. SEM microphotographs of polymer matrices obtained by radiation-induced polymerization at 78 ° and total dose of 10 kGy of the monomer/water mixtures (w/w): H E A / H 2 0 2:1 (a); H E A / H 2 0 l : l (b); H E A / H 2 0 l: 2 (c); AM-30G/H20 2:1 (d); AM-30G/H20 1 : 1 (e) and AM-30G/H20 1:2 (f). -
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Fig. 2. Optical microscopy microphotographs of polymer matrices obtained by radiation-induced polymerization at - 7 8 ° and total dose of 10 kGy of the monomer/water mixtures (w/w): AM-90G/H20 2:1 (a) and AM-90G/H20 1:2 (b).
Hydrogels for yeast cells immobilization
Fig. 3. SEM microphotographs of polymer matrices, with immobilized yeast cells, obtained by radiationinduced polymerization at - 7 8 ° and total dose of 10kGy of the monomer/water mixtures (w/w): HEA/H20 1:l, cross section (a); HEA/H20 1:1, surface (b); AM-30G/H20 1:1, cross section (c) and AM-30G/H20 1 : 1, surface (d).
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In order to examine the influence of the character of the carrier on the immobilization of yeast cells and hence on ethanol productivity, Fig. 3 shows SEM microphotographs of some polymer matrices on which yeast cells were allowed to multiply. The matrices were obtained by polymerization of the HEA/H20 and AM-30G/H20 mixtures and were highly swellable (see Table 3). It can be clearly seen that many colonies adhere to the polymer matrices and it is interesting to note that cell arrangement mostly takes place on the interior part (cross section) rather than on the surface. Taking into consideration the great softness of these matrices, this effect is probably due to the fact that in the former case the cells together with the nutrient liquid can easily diffuse through microchannels created inside the matrix; in the latter case, they are more susceptible to being mechanically swept away. Table 3 also gives the values of ethanol productivity obtained by batch fermentation. A very close relationship exists between EtOH productivity on the one hand and the water content and porosity of polymer carriers on the other. Actually, for matrices based on the mixtures of AM-30G and AM-90G with water in the 1:2 ratio, an EtOH productivity six times that in the case of free cells and more than three times that for the much less swellable poly(HEMA) matrices was obtained. However, it should be mentioned that the former matrices had the disadvantage of being soft and therefore weak mechanically. The addition of a small amount of a crosslinking agent greatly improved the mechanical properties of these resins. As an example, AM-30G/H20 mixture in the ratio 1:2 was polymerized in the presence of 5% 4G (see Table 3). In spite of the decrease of the water content with respect to that of the corresponding matrix obtained in the absence of crosslinking agent as well as of ethanol productivity, the greater tightness makes these matrices good candidates for continuous alcoholic fermentation.
Acknowledgement--The authors thank Mr H. Itoh for help in taking SEM microphotographs. REFERENCES
1. V. Kudela, Hydrogels. In Encyclopedia of Polymer Science and Technology (edited by H. F. Mark, N. M. Bikales, C. G. Overberger and N. Menges), Vol. 7, 2nd edn, pp. 783-806. Wiley, New York (1987). 2. N. A. Peppas and A. G. Mikos. Preparation methods and structure of hydrogels. In Hydrogels in Medicine and Pharmacy, Vol. L Fundamentals, Chap. 1, pp. 1-25 (edited by N. A. Peppas). CRC Press, Boca Raton, FL. (1986). 3. J.-P. Montheard, M. Chatzopoulos and D. Chappard. J. Macromolec. Sci., Rev. Macromolec. Chem. Phys., C 32, 1 (1992). 4. W. R. Gombotz and A. S. Hoffman. Immobilization of biomoleculesand cells on and within synthetic polymer hydrogels. In Hydrogels in Medicine and Pharmacy Vol. I. Fundamentals, Chap. 5, pp. 95-126 (edited by N. A. Peppas). CRC Press, Boca Raton, FL. (1986). 5. J. Klein and H. Ziehr. J. Biotechn. 16, 1 (1990). 6. G. J. Salter and D. B. Kell. Curr. Opin. Biotechn. 2, 385 (1991). 7. A. S. Hoffman. Adv. Polym. Sci. 57, 141 (1984). 8. M. Carenza. Radiat. Phys. Chem. 39, 485 (1992). 9. I. Kaetsu. Radiat. Phys. Chem. 18, 343 (1981). 10. T. Fujimura and I. Kaetsu. Appl. Biochem. Biotechn. 8, 145 (1983). I1. T. Fujimura and I. Kaetsu. Biotechn. Bioengng 29, 171 (1987). 12. T. Fujimura, M. Carenza and I. Kaetsu. Radiat. Phys. Chem. 31, 653 (1988). 13. M. Carenza, T. Fujimura and I. Kaetsu. Chim. Oggi 8, 41 (1990). 14. M. Carenza, E. de Alteriis, S. Lora, P. Parascandola and V. Scardi. Ann. Microbiol. 39, 257 (1989). 15. M. Carenza, E. de Alteriis, S. Lora, P. Parascandola and V. Scardi. Ann. iV. Y. Acad. Sci. (Enzyme Eng. 10) 613, 547 (1990). 16. R. Bonichsen.Methods of Enzymatic Analysis (edited by H. V. Bergmeyer), p. 285. Academic Press, New York (1971). 17. P. H. Corkhill and B. J. Tighe. Polymer 31, 1526(1990). 18. M. Yoshida, M. Kumakura and I. Kaetsu. J. Macromolec. Sci. Chem. A. 14, 555 (1980). 19. M. Carenza, S. Lora, G. Palma, E. Boccu', R. Largajolli and F. M. Verones¢. Radiat. Phys. Chem. 31, 657 (1988).
0014-3057/93 $6.00+0.00 Pergamon Press Ltd
H Y D R O G E L S O B T A I N E D BY R A D I A T I O N - I N D U C E D POLYMERIZATION FOR YEAST CELLS IMMOBILIZATION MARIO CARENZA,1 MASARU YOSHIDA,2 MINORU KUMAKURA2. and TAKASHI FUJIMURA2 tIstituto di Fotochimica e Radiazioni d'Alta Energia, C.N.R., Sezione di Legnaro, Via Romea 4, 35020 Legnaro (Padova), Italy 2japan Atomic Energy Research Institute, Takasaki Radiation Chemistry Research Establishment, Takasaki, Gunma-ken, Japan
(Received 13 July 1992) Abstract--Radiation-induced polymerizations of several acrylic and methacrylic esters at both room temperature and low temperature were carried out. The polymer matrices obtained under the latter conditions were examined as carriers for immobilization of yeast cells. The adhesion of the latter was found to be markedly influenced by both matrix hydrophilicity, determined by equilibrium water content measurements, and porosity, evaluated by optical microscopy and scanning electron microscopy. The ethanol productivity during batch fermentation as a function of these parameters is discussed.
INTRODUCTION
EXPERIMENTAL PROCEDURES
Hydrogels are polymer networks which absorb and retain water without dissolving [1-3]. This property makes them interesting materials as carriers for immobilization of bioactive compounds [1,4] as alternatives to others successfully used [5, 6]. Radiation-induced polymerization enables hydrogels in a wide variety of forms to be easily prepared for applications both in biomedicine and in biotechnology [7,8]. With regard to the latter topic, radiation polymerization at low temperatures of glass-forming acrylic and methacrylic esters has been successful for obtaining hydrogels suitable as carriers for immobilization of bioactive species such as enzymes, cells and antibodies as well as for controlled release of drugs [8, 9]. The method is based on radiation-induced polymerization at a temperature below 0 ° of an aqueous solution or suspension of the monomer. At this temperature, water separates in the form of microcrystals dispersed in the m o n o m e r while the latter polymerizes in a super-cooled liquid state. At the end of irradiation when the system is brought to r o o m temperature, the ice microcrystals retained by the polymer matrix give rise to small pores on the walls of which the biocomponent is immobilized mainly by adhesion. By means of this technique, immobilization of yeast cells was successfully carried out in our laboratories and a high ethanol productivity was obtained during both batch [10-13] and continuous fermentation [14, 15]. In this work, polymer matrices were prepared by g a m m a irradiation of monomers with different hydrophilicities in the presence of water and examined as carriers for immobilization of yeast cells. Moreover, the relationship between the structure and physico-chemical properties of these matrices and the effectiveness of ethanol productivity was examined.
The following monomers, with codes and structures given in Table 1, were used as received: 2-hydroxyethyl methacrylate (HEMA) and 2-hydroxyethyl acrylate (HEA) from Tokyo Kasei Co.; methoxypolyethylenegiycol methacrylate with the number of oxyethylene units, n, equal to 3 (M-3G), 9 (M-gG) or 23 (M-23G), methoxypolyethyleneglycol acrylate with n equal to 3 (AM-30G) or 9 (AM-90G), polyethyleneglycol dimethacrylate with n equal to 4 (4G) or 14 (14G) and polyethyleneglycol diacrylate with n equal to 9 (A-400) from Shin-Nakamura Chemical Co., Ltd. All polymerizations were carried out by gamma-ray irradiation with a Co-60 source (at JAERI, Takasaki) at a dose of 10 kGy after flushing N2 through the tubes. Bulk polymerizations were carried out at 25 and - 7 8 °. In the case of HEMA, HEA and M-3G, polymer films of thickness 200/~m were obtained by cast polymerization of the monomers at 25° between two polypropylene plates separated by a polycarbonate gasket. Irradiation of aqueous solutions of the monomers was conducted at - 78°. Polymer matrices in the form of discs were allowed to swell for 3 4 days in a large volume of distilled water which was changed every day. The value of equilibrium water content, We, was determined according to the equation:
*Present address: Department of Bioscience, The NishiTokyo University, Uenohara, Kitatsuru, Yamanashi 409-01, Japan.
wo-
w,
x I00
(1)
where W, and Wa are the weights of the fully swollen matrix and of the completely dried sample, respectively. The quoted values are averages of three determinations. Portions of swollen matrices were freeze-dried, fractured at liquid N 2 temperature and analysed by scanning electron microscopy (SEM) using a Jeol Superprobe 733 apparatus. Portions of swollen matrices were sliced into thin films (10#m) with a microtome at - 1 0 ° and, after swelling again in water, examined with an Olympus light microscope. The procedure for cell immobilization has been reported [10, 11]. In short, a sample of yeast cells (Saccharomyces formosensis) precultured under aerobic conditions was incubated under agitation at 30° in the culture liquid containing portions of the polymer carrier previously swollen in the culture liquid. The ethanol produced from glucose was determined by the method of Bonichsen [16].
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Table 1. Codes and structures of the monomers 2-Hydroxyethyl methacrylate (HEMA) CH~------C(CH3)COOCH2CH2OH 2-Hydroxyethyl acrylate (HEA) CH~--'CH--COOCH2CH2OH Methoxypolyethyleneglycol methacrylate (M-nG)* CH~------'C(CH3)CO(OCH:CH2)nOCH3 Methoxypolyethyleneglycol acrylate (AM-nG)t CH~-------CH--CO(OCH2CH2).OCH 3 Polyethyleneglycol dimethacrylate (14G) CH~------C(CH~)COO(CH2CH20)t4COC(CH 3)~-CH2 Polyethyleneglycol diacrylate (A-400) CH~'CH--COO(CH2CH 20)gCOCH~"~'CH2 *The number n was 3 (M-3G), 9 (M-9G) and 23 (M-23G). tTbe number n was 3 (AM-30G) and 9 (AM-90G).
RESULTS AND DISCUSSION
Some acrylic and methacrylic esters were polymerized in bulk by irradiation at room temperature or at - 7 8 °. Some of the polymer samples were obtained by radiation-induced cast polymerization at room temperature. An irradiation dose of 10 kGy was given to the monomers so that virtually complete polymerization was achieved. We values are reported in Table 2. The swelling values of the acrylic polymers are higher than those of the methacrylic and, the longer the oxyethylene unit, the higher is We. The data referring to the polymer samples obtained at room temperature are very close to those obtained at - 7 8 °, the former being transparent and the latter showing some opacity probably due to impurities, perhaps water during the freezing step. The value of 37.3% for poly(HEMA) is similar to those recently reported [15, 17] although it markedly differs from that of 26.0% found by other authors [18]. The values for poly(HEA), in the range Table 2. Equilibrium water contents, We (%), of the polymer samples obtained by radiationinduced polymerization at room temperature (a) and at - 7 8 ° (b) of the various monomers Monomer *HEMA (a) *HEA (a) HEA (a) HEA (b) *M-3G (a) M-9G (a) M-9G (b) M-23G (b) AM-30G (a) AM-30G (b) AM-90G (a) AM-90G (b) 14G (a) 14G (b) A-400 (a) A-400 (b)
W, (%) 37.3 70.9 73.8 70.6 6.6 84.7 89.4 90.0 85.1 82.8 87.4 86.0 33.6 26.7 27.7 24.9
Dose rate = 10 kGy/hr; total dose = 10 kGy. *Polymer samples obtained by radiation-induced cast polymerization.
70.6-73.8%, are quite close to that of 69.9% [15] but they considerably differ from other values, viz. 45.9% [18] and 60% [17]. The main goal of this work was the establishment of a relationship between ethanol productivity (by fermentation of glucose) and the hydrophilic properties of polymeric carriers, the latter being obviously dependent on the hydrophilicity of the corresponding monomers. An important role is also played by the porosity of the polymer matrix, which in turn is brought about by the presence of water added to the reacting monomer. Thus, HEMA, HEA and AM-30G were selected as monomers capable of giving matrices over a wide We range, i.e. from 37 to 85% (see Table 2). The monomers were diluted with water in various proportions and, after flushing with N2, were rapidly cooled at - 7 8 ° . At this temperature, the mixtures were irradiated for 60 min (total dose = 10 kGy). The effect of a crosslinking agent, tetraethyleneglycol dimethyacrylate (4G), added to the polymerizing mixture in small amounts, was also examined. Table 3 shows the values of equilibrium water content, We. As for the results recently obtained for the system HEMA-water [19], We values were found to rise with increasing amount of water in the polymerizing mixture, as expected. At the same time, porosity is expected to increase. As an example, Fig. 1 shows the SEM microphotographs of the polymer matrices obtained from aqueous mixtures of HEA and AM-30G. For the more concentrating systems, Figs l(a) and (d) show that only very small pores, if any, are discernible. Correspondingly, W, values are very close to those obtained for the matrices prepared in bulk. On the other hand, as the proportion of water increases, Figs l(b), (c), (e) and (f) show that larger and larger cavities are created with the result that We values differ widely (see Table 3). A similar trend is found by light microscope examination. Figure 2 shows the microphotographs of hydrogels based on AM-90G. The closed-pore structure of the matrix prepared with a high AM90G/water ratio [Fig. 2(a)] becomes an open-pore structure in the case of a low ratio [Fig. 2(b)].
Table 3. Equilibrium water contents, We (%) of the polymer samples obtained by radiation-induced polymerization at - 7 8 ° of monomer-water mixtures (v/v) and ethanol productivity of yeast cells immobilized on the matrices Mixtures HEMA/H20 (2:1) HEMA/H20 (1 : 1) HEMA/H20 (1 : 2) HEA/H20 (2:1) HEA/H20 (1 : 1) HEA/HzO (1 : 2) AM-30G/H20 (2:1) AM-30G/H20 (1 : 1) AM-30G/H20 (1 : 2) *AM-30G/H20 ( 1: 2) AM-90G/H20 (2:1) AM-90G/H20 (1 : 2) Free cells
Wc (%) 42.8 51.3 69.4 74.7 80.7 86.8 84.0 88.2 92.5 88.2 88.7 94.1
EtOH productivity (mg/ml of gel.br) 0.49 0.49 0.60 0.78 0.83 1.48 0.81 1.10 1.78 1.06 0.60 1.75 0.31
Dose rate = 10 kGy/hr; irradiation time = 60 min. *Polymerization in the presence of 5% (v/v) of the crosslinking agent tetracthylencglycol dimethacrylate (4(3).
Hydrogels for yeast cells immobilization
Fig. 1. SEM microphotographs of polymer matrices obtained by radiation-induced polymerization at 78 ° and total dose of 10 kGy of the monomer/water mixtures (w/w): H E A / H 2 0 2:1 (a); H E A / H 2 0 l : l (b); H E A / H 2 0 l: 2 (c); AM-30G/H20 2:1 (d); AM-30G/H20 1 : 1 (e) and AM-30G/H20 1:2 (f). -
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Fig. 2. Optical microscopy microphotographs of polymer matrices obtained by radiation-induced polymerization at - 7 8 ° and total dose of 10 kGy of the monomer/water mixtures (w/w): AM-90G/H20 2:1 (a) and AM-90G/H20 1:2 (b).
Hydrogels for yeast cells immobilization
Fig. 3. SEM microphotographs of polymer matrices, with immobilized yeast cells, obtained by radiationinduced polymerization at - 7 8 ° and total dose of 10kGy of the monomer/water mixtures (w/w): HEA/H20 1:l, cross section (a); HEA/H20 1:1, surface (b); AM-30G/H20 1:1, cross section (c) and AM-30G/H20 1 : 1, surface (d).
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In order to examine the influence of the character of the carrier on the immobilization of yeast cells and hence on ethanol productivity, Fig. 3 shows SEM microphotographs of some polymer matrices on which yeast cells were allowed to multiply. The matrices were obtained by polymerization of the HEA/H20 and AM-30G/H20 mixtures and were highly swellable (see Table 3). It can be clearly seen that many colonies adhere to the polymer matrices and it is interesting to note that cell arrangement mostly takes place on the interior part (cross section) rather than on the surface. Taking into consideration the great softness of these matrices, this effect is probably due to the fact that in the former case the cells together with the nutrient liquid can easily diffuse through microchannels created inside the matrix; in the latter case, they are more susceptible to being mechanically swept away. Table 3 also gives the values of ethanol productivity obtained by batch fermentation. A very close relationship exists between EtOH productivity on the one hand and the water content and porosity of polymer carriers on the other. Actually, for matrices based on the mixtures of AM-30G and AM-90G with water in the 1:2 ratio, an EtOH productivity six times that in the case of free cells and more than three times that for the much less swellable poly(HEMA) matrices was obtained. However, it should be mentioned that the former matrices had the disadvantage of being soft and therefore weak mechanically. The addition of a small amount of a crosslinking agent greatly improved the mechanical properties of these resins. As an example, AM-30G/H20 mixture in the ratio 1:2 was polymerized in the presence of 5% 4G (see Table 3). In spite of the decrease of the water content with respect to that of the corresponding matrix obtained in the absence of crosslinking agent as well as of ethanol productivity, the greater tightness makes these matrices good candidates for continuous alcoholic fermentation.
Acknowledgement--The authors thank Mr H. Itoh for help in taking SEM microphotographs. REFERENCES
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