- Email: [email protected]
Immobilization of fungus Aspergillus sp. by a novel cryogel technique for production of extracellular hydrolytic enzymes
Process Biochemistry 35 (2000) 1177 – 1182 www.elsevier.com/locate/procbio
Immobilization of fungus Aspergillus sp. by a novel cryogel technique for production of extracellular hydrolytic enzymes Konstantin A. Lusta a, Il Kyung Chung a, Ill Whan Sul b, Hee Sung Park a, Dong Ill Shin a,* a
Faculty of Life Resources, Catholic Uni6ersity of Taegu-Hyosung, Hayang, Kyungsan, Kyungpook 712 -702, South Korea b Department of Forest Resources, Taegu Polytech. College, Taegu 706 -020, South Korea Received 9 December 1999; accepted 3 March 2000
Abstract Aerobic cells of a fungus isolate Aspergillus sp. CX-1 have been immobilized in macroporous cryoPAG and in different composite cryoPAGs — fibrous adjunct carriers. The productivity of the extracellular enzymes (exo-1.4-b-glucanase, endo-1.4-bglucanase, b-glucosidase and xylanase), and the viability, growth and ultrastructure of the immobilized fungus have been studied. The enzyme activities and stability during long-term repeated batch cultivation in the immobilized fungus were higher than in free mycelia when batch cultivated. The fungus immobilized in the composite cryoPAG, containing polypropylene non-woven fabric, possessed the highest exo-1.4-b-glucanase activity, the longest durability of enzyme production (85 days) and the most reliable mechanical strength. The fungus immobilized in porous composite cryogel possessed a variety of advantages including easy control of cryogel porosity, improved mechanical strength and durability, simplicity of construction, high enzyme productivity and high stability. © 2000 Elsevier Science Ltd. All rights reserved. Keywords: Immobilization; Fungi; Aspergillus; Cellulase; Xylanase.
1. Introduction One of the major possibilities of optimizing biotechnological processes lies in immobilization technologies, which can increase productivity and product concentrations. The use of immobilized microorganisms for the production of biologically active substances, including extracellular enzymes, has gained increasing interest because of potential in a variety of processes in industry [1,2]. The method of cell immobilization in natural and synthetic gels is currently most widely employed in biotechnology [3–5]. The main shortcomings of isotropic xerogel carriers, however, are their low durability and mass transfer problems [3,6]. These methods also impose some stress and toxic effects on the immobilized cells [7]. Examination of viable yeast cell numbers within the immobilization matrix indicated a dramatic reduction and * Corresponding author. Tel.: +82-53-8503297; fax: + 82-538503459. E-mail address: [email protected] (D.I. Shin)
ultimately the entire destruction of the submerged cell populations [7,8]. One of the feasible approaches for reducing the masstransfer resistance to the diffusion of substrate material is to immobilize cells into thin films or fixing cells on the surface of membranes [9,10]. Another possibility is to immobilize the cells in a fibrous matrix, which is attained by natural attachment or crosslinking to fibers [11]. The problem lies in anchoring the microorganisms firmly to the matrix walls [12]. Macroporous spongy carriers (like polyurethane or cellulose foams [13,14]) have been used to alleviate both problems. Although, the need to match the shape and size of the cell presents additional challenges. The investigation proposed here is aimed at developing a macroporous gel matrix as a compatible carrier for the immobilization of filamentous fungi. The specific characteristics of this gel matrix were targeted as the control of porosity and the ability to endure long-term repeated batch processing of extracellular cellulases and xylanase production.
0032-9592/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 0 ) 0 0 1 5 8 - 8
1178
K.A. Lusta et al. / Process Biochemistry 35 (2000) 1177–1182
2. Materials and methods
2.5. Culti6ation of immobilized fungus
2.1. Microorganism and culti6ation media A filamentous fungus strain exhibiting cellulolytic and xylanolytic activities was isolated from the soil around a hot spring in the Ashkhabad city (Turkmenistan) area. The fungus, identified as Aspergillus sp. (strain CX-1), was maintained on wort-agar slants and stored at 4°C. Aspergillus sp. CX-1 was cultured on the slants of a solidified Czapek – Dox mineral medium (0.3% NaNO3, 0.1% KH2PO4, 0.2% KCl and 0.05% MgSO4, pH 5.5) supplemented with 0.1% pepton, 0.2% Tween 80 and strips of filter paper on its surfaces before being used in the experiments.
The conidia immobilized in the cryoPAG were precultured in Czapek–Dox’s mineral medium supplemented with 0.1% pepton, 0.2% Tween 80 and 0.5% glucose. Cultivation was performed in Erlenmeyer flasks filled to 10% of their volume on a shaker at 41°C for 24 h. The IFB was then transferred to the same Czapek-Dox’s medium containing 2% (w/v) microcrystalline cellulose (MCC, Avicel, American Viscose Co., USA), instead of glucose, as a sole carbon source for production of cellulases and xylanase. Incubation was carried out in the same batch conditions as a repeated batch cultivation (RBC) [17], where the medium was changed periodically every 5 days.
2.2. Immobilization in cryoPAG
2.6. Electron microscopy
Acrylamide (AA), N,N%-methylene-bis-acrylamide (MBAA), N,N,N%,N%-tetramethyl-ethylenediamine (TEMED) and ammonium persulphate (APS) were purchased from Sigma (USA). For immobilisation of the fungus, the polyacrylamide gel mix (total volume 10 ml) was prepared on ice containing 2.8% AA, 0.3% MBAA, and 0.5 ml/ml TEMED along with 5 × 106 fungal conidia/ml and 0.02% Tween 80. 0.24 mg/ml APS was then added. The mixture was swirled rapidly and placed in a cryostat at different temperatures ranging from − 5 to − 80°C for 4 – 30 h. The frozen cryogel block was thawed in a warm water bath (40°C) and (monomer residues were washed from) the immobilized fungus block (IFB) with sterile tap water by squeezing the sponge.
For scanning electron microscopy (SEM) [18] the specimens were fixed in glutaraldehyde and then in OsO4 solutions, dehydrated in ethanol and absolute amyl acetate and dried using a critical point dryer HCP-2 unit (Hitachi, Japan). Gold shadowing was performed using an ion sputter JFC-1100 (Jeol, Japan). Freeze fracture preparations [19] were performed using a Freeze-Etch/Fracture System (BAL-TEC AG, Liechtenstein). The specimens were examined with the H-600 electron microscope (Hitachi, Japan).
2.3. Viability control The viability of the immobilized conidial population was determined using the slide culture (microculture) technique described by Hartman [15] and modified by Lusta [16].
2.7. Enzyme assays The activities of exo-1,4-b-D-glucanase, endo-1,4-bglucanase, b-glucosidase and 1,4-b-D-xylan xylanohydrolase (xylanase) were examined in culture supernatants of free and immobilized Aspergillus sp. CX-1, as described previously [17].
3. Results and discussion
2.4. Porous composite cryogel carriers
3.1. Effect of freezing temperatures on the cryoPAG structure
Different fibrous adjunct materials: polypropylene nonwoven fabric (PNWF), cellulose acetate filaments (CAF), nitrocellulose filaments (NCF), polyester filaments (PEF) and glass fabric (GF) were purchased from the Institute of Nonwoven Materials (Serpukhov, Russia). For the immobilization of fungal conidia in a porous composite cryogel (PCC) 0.25 g of one of the fibrous adjuncts was added to a vial containing the polymerization mix for cryoPAG, all under sterile conditions. Further procedures were the same as those used for the cryoPAG polymerization.
Varying the freezing temperatures (between −5 and − 80°C) created the spongy cryogels with the optimal porosity appropriate for the immobilization of the filamentous fungi. Pore size decreased as the temperature of the PAG polymerization decreased (Fig. 1). The polymerization at − 20°C gave the required porosity in the cryoPAG (Fig. 2a). Freezing of the polymerization mix at temperatures above − 20°C caused the pore size to be too large, and freezing below − 20°C sharply slowed polymerization and reduced the size of the gel pore, worsening the mass exchange for the immobilized fungi.
K.A. Lusta et al. / Process Biochemistry 35 (2000) 1177–1182
1179
The use of acrylamide as a monomer for the cryopolymer carrier is suitable for immobilization at temperatures from − 10 to − 20°C, making polymerization significantly easier and safer than the method previously used [20], where hydroxyethylmethacrylate served as the monomer. The latter requires freezing to − 78oC and g-irradiation for polymerization. Cryogels formed at such low temperatures have smaller pores (occupying only 20% of the total polymer volume) than the cryoPAG, and therefore may worsen mass exchange during prolonged fungal cultures.
3.2. Effects of loading conidia and surfactant concentration
Fig. 1. The effect of freezing temperature on the cryoPAG porosity. , cryoPAG porosity,%; , cryoPAG pore size, mm.
During the immobilization of conidia in the cryoPAG, the concentration of conidia and the surfactant (Tween-80) added to the polymerization mix had significant effects on the properties of the carrier thus produced. The optimal concentration of the immobilized conidia was in the range of 106 –107/ml, while the
Fig. 2. A survey of immobilized in macroporous cryoPAG Aspergillus sp. CX-1 cultured on cellulase-production media: (a) scanning electron micrograph of the cryoPAG sponge. The macropores of about 40 mm are visible; (b) micropore structure of the ordinary PAG revealed by freeze-fracture electron microscopy, the bar represents 0.5 mm; (c) fungal conidia after immobilization in cryoPAG, the bar represents 0.5 mm; (d) immobilized fungus growth during the initial phase preculturing (12 h incubation in glucose medium), the bar represents 0.5 mm; (e) growth of mycelium after 24 h preculturing in glucose medium; (f) cryoPAG immobilized mycelium after the first 5-day cycle of repeated batch cultivation on cellulase-production media; (g) the structure of the PCC-PNWF and the mycelial growth after the first 5-day cycle of repeated batch cultivation on cellulase-production media in this carrier are demonstrated, the bar represents 50 mm.
K.A. Lusta et al. / Process Biochemistry 35 (2000) 1177–1182
1180
Table 1 Polysaccharase activities in culture filtrates measured at the end of sequential 5-day cycles of repeated batch cultivation immobilized in cryoPAG Aspergillus sp. CX-1 Number of 5-day cycle
1 2 3 4 5 6
Total enzyme activities
Enzyme activities
(U/ml)
Endo-b-glucanase
Exo-b-glucanase
b-glucosidase
Xylanase
27.3 25.7 15.8 20.8 12.0 13.4
0.52 0.46 0.54 0.70 0.44 0.63
4.3 3.7 3.1 4.6 4.0 3.2
20.1 23.2 24.6 11.8 17.7 24.6
optimal concentration of Tween-80 was 0.02%. Higher levels either prevented cryogel polymerization, or resulted in mechanical weakness and break up after just the first batch cycle of incubation.
3.3. Structural, mechanical and adhesion properties of cryoPAG The architecture of the cryoPAG was viewed using SEM. Fig. 2(a) shows that the cryoPAG has a macroporous structure with a pore size of about 40 mm. The pore space of the cryoPAG occupies 60% of the total volume. In comparison, the micropores of an isotropic PAG are not more than 0.1 mm (Fig. 2b). The cryoPAG polymer has considerable mechanical strength, elasticity, and the ability to liberate up to 70% of unbound liquid upon compression. It also can be stored for prolonged periods in a dry state, and takes up the same quantity of moisture to restore the original shape and volume. The cryoPAG pores can absorb up to 4 ml of aqueous solution per g of dry weight. The concentration of the components for PAG polymerization can be reduced ten-fold compared to a normal PAG. This is because, as the solvent freezes, the concentration of substances in the liquid phase increases, resulting in a reduction of the critical concentration required for PAG formation. The proportion of fungal conidia trapped in the carrier after immobilization in the cryoPAG was 60– 70% of the added amount.
3.4. Viability of immobilized fungal conidia The viability index of conidia embedded in the cryoPAG, as determined by the microculture technique, was 76% compared with 80% for non-immobilized conidia. The comparison of these data to those for Escherichia coli immobilization in PAG [7] indicates that in practice Aspergillus sp. conidia exhibits absolute resistance to all the stress factors of cryoPAG immobilization.
3.5. Growth of fungal mycelia in cryoPAG The successive steps for growing Aspergillus sp. CX-1 mycelium in the cryoPAG, from preculturing to the 5-day batch cycle of cultivation, are shown in Fig. 2(c–f). The cryoPAGs macroporous structure allows for abundant growth of immobilized filamentous fungus in the gel.
3.6. Variation of extracellular cellulase and xylanase acti6ities produced by cryoPAG-immobilized fungus with culture time of repeated batch process The results presented in Table 1 show that samples of culture filtrates immobilized in the cryoPAG Aspergillus sp. CX-1 had fairly high and stable polysaccharase activities. Xylanase and exo-b-glucanase activities were increased further after six cycles of batch cultivation. The total operating time was 30 days. The previous studies [17] have demonstrated that among the fungal strains used (in particular, the conventional commercial strain, Trichoderma reesei QM9414), the highest cellulolytic and xylanolytic activities were produced by the new filamentous fungus strain Aspergillus sp. CX-1. It follows that this strain, which is interesting from an economic point of view, was selected for the immobilized cell studies. Cellulases and xylanase activities in culture filtrates immobilized in cryoPAG Aspergillus sp. CX1 were higher than those of the free mycelium of the same fungal strain, described previously [17]. This could be due to the improvement of secretion and export of extracellular hydrolytic enzymes by the specific conditions of immobilization of fungal mycelia and increasing of the concentration of cellulosic substrate in cryoPAG pore matrix as compared to that of the conventional batch conditions.
3.7. Immobilization of fungus in porous composite cryogel carriers The data presented in Fig. 3 indicate exo-b-glucanase activities in culture filtrates prepared as described above
K.A. Lusta et al. / Process Biochemistry 35 (2000) 1177–1182
at the end of sequential 5-day cycles of RBC. The PCC-GF blocks proved to be the least durable. When PNWF was used, the PCC carriers retained their mechanical strength during repeated batch cultivation for an unlimited period of time. The exo-b-glucanase activities in the culture filtrates of these preparations remained at a high level for 17 cycles (85 days). The selection of the fibrous adjunct material for creating the PCC was, thus, critical for fungus immobilization because of the cryogel’s mechanical properties, fungal cell biocompatibility and resulting stability and durability of extracellular enzyme production. The culture medium remained clear and transparent during this cultivation period. Phase-contrast microscopic examination did not reveal any hyphae or conidia in the culture media. Additionally, the insoluble substrate (MCC particles) were also absorbed by the PCC and were firmly retained by the carrier throughout the RBC period. The structure of the PCC-PNWF and the mycelial growth over the course of polysaccharase production in this carrier are demonstrated in Fig. 2(g). Another positive characteristic of the PCC-immobilized fungi was that, compared to the single batch culture, the required amount of supplemental cellulose powder and other ingredients of nutrient media is ten times less. PCCs in general, and cryoPAG-PNWF in particular, were found to have better mechanical properties and stability of enzyme production than a pure cryoPAG that does not contain this fibrous adjunct filler.
1181
4. Conclusions CryoPAG was prepared by the radical-mediated copolymerization of AA (vinyl monomer) and MBAA (divinyl monomer) in the presence of initiators under freezing conditions. It had different physical and mechanical properties from the analogous isotropic xerogels formed under standard conditions at positive temperatures. Solvent (H2O) crystallization leads to the formation of porous heterophasic cryogels with an extremely macroporous structure. These properties are useful for the immobilization of filamentous fungi. The macroporous structure lends itself to the efficient mass exchange, resulting in increased fungal growth, higher enzyme producing activity and easy release of the secreted enzymes into the medium. Fungal mycelium can anchor firmly to the cryoPAG carrier because of certain peculiar conforming characteristics. The spongy cryoPAG can be used in large blocks and does not break up into granules. Porous composite cryogels were created by filling the cryoPAG with fibrous adjuncts (PHWF, GF, CAF, NCF and PEF) in order to increase the mechanical strength and durability of the cryoPAG. This also served to improve adhesion of the secondary conidia and mycelium to the carrier, and attain greater enzyme productivity and stability. Due to their high porosity and biocompatibility, macroporous cryoPAG and PCC-carriers were adopted for fungi cell immobilization and extracellular enzyme production. A valuable property of the cryoPAG and PCC carrier is its ability to absorb particles of the solid substrate (cellulose) and secondary-formed fungal conidia from the media during RBC.
Acknowledgements This work was financially supported by the 1999 Russian Scientist Exchange Program’ through the Korean Institute of Science and Technology Evaluation and Planning (KISTEP).
References
Fig. 3. Exo-b-glucanase activities of culture filtrates at the end of sequential 5-day cycles of repeated batch cultivation of Aspergillus sp. CX1 immobilized in various porous composite cryoPAG (PCC) carriers: , Cryo-PAG — polypropylene nonwoven fabric (PNWF); ", Cryo-PAG — cellulose acetate filaments (CAF); , Cryo-PAG — glass fabric (GF); , Cryo-PAG — nitrocellulose filaments (NCF); , Cryo-PAG — polyester filaments (PEF).
[1] Katzbauer B, Narodoslawsky M, Moser A. Classification system for immobilization techniques. Bioprocess Eng 1995;12:173–9. [2] Virkajarvi I, Linko M. Immobilization: a revolution in traditional brewing. Naturwissenschaften 1999;86:112 – 22. [3] Gough S, McHale AP. Continuous ethanol production from molasses at 45°C using alginate-immobilized Kluy6eromyces marxianus IMB3 in a continuous-flow bioreactor. Bioprocess Eng 1998;19:33 – 6. [4] Quirasco M, Remaud-Simeon M, Monsan P, Lopez- Munguia A. Experimental behavior of a whole cell immobilized dextransucrase biocatalyst in batch and packed bed reactors. Bioprocess Eng 1999;20:289 – 95.
1182
K.A. Lusta et al. / Process Biochemistry 35 (2000) 1177–1182
[5] Slokoska L, Angelova M, Pashova S, Petricheva E, Konstantinov Ch. Production of acid proteinase by Humicola lutea 120-5 immobilized in mixed photo-cross-linked polyvinyl alcohol and calcium-alginate beads. Process Biochem 1999; 34:73 – 6. [6] Roisin C, Bienaime C, Nava Saucedo JE, Barbotin JN. Influence of microenvironment on immobilised Gibberella fujikuroi. Prog Biotechnol 1996;11:189–95. [7] Lusta KA, Starostina NG, Fikhte BA. Immobilization of microorganisms: cytophysiological aspects. In: de Bont JAM, Visser J, Mattiasson B, Tramper J, editors. Proceedings of an International Symposium: Physiology of Immobilized Cells. Amsterdam: Elsevier, 1990:557–62. [8] Barbotin JN, Nava Saucedo JE. Bioencapsulation of living cells by entrapment in polysaccharide gels. In: Dumitriu S, editor. Polysaccharides: structural diversity and functional versatility. New York: Marcel Dekker, 1998:749–74. [9] Tonkova A, Ivanova V, Dobreva E, Stefanova M, Spasova D. Thermostable alpha-amylase production by immobilized Bacillus licheniformis cells in agar gel and on acrylonitrile/acrylamide membranes. Appl Microbiol Biotechnol 1994;41:517–22. [10] Ascencio F, Johansson G, Wadstrom T. Cell-surface charge and cell-surface hydrophobicity of collagen-binding Aeromonas and Vibrio strains. Arch Microbiol 1995;164:223–30. [11] Cho HY, Yousef AE, Yang ST. Continuous production of pediocin by immobilized Pediococcus acidilactici PO2 in a packed-bed bioreactor. Appl Microbiol Biotechnol 1996;45:589 – 94. [12] Busscher HJ, Poortinga AT, Bos R. Lateral and perpendicular interaction forces involved in mobile and immobile adhesion of
.
[13]
[14]
[15]
[16]
[17]
[18]
[19] [20]
microorganisms on model solid surfaces. Curr Microbiol 1998;37:319 – 23. Lapadatescu C, Feron G, Vergoignan C, Djian A, Durand A, Bonnarme P. Influence of cell immobilization on the production of benzaldehyde and benzyl alcohol by the white-rot fungi Bjerkandera adusta, Ischnoderma benzoinum and Dichomitus squalens. Appl Microbiol Biotechnol 1997;47:708 – 14. Chiou TW, Wang YC, Liu HS. Utilizing the macroporous packed bed for insect cell/baculovirus expression — part 2: the production of human interleukin-5 in polyurethane foam and cellulose foam packed bed bioreactors. Bioprocess Eng 1998;18:91 – 100. Hartman PA. Miniaturized microbiological methods. In: Umbreit WW, editor. Advances in Applied Microbiology (Suppl. I). New York: Academic Press, 1968:65 – 84. Lusta KA. A microculture method for the determination of viability of immobilized microbial cells. In: Koshcheenko KA, editor. Immobilized Microbial Cells. Pushchino: NCBI, Russian Academy of Sciences, 1979:164 – 73. Lusta KA, Chung IK, Sul IW, Park HS, Shin DI. Characterization of extracellular cellulose-hydrolyzing enzyme complex from a thermotolerant strain of Aspergillus sp. J Microbiol Biotechnol 1999;9:873 – 6. Goldstein JI, Newbury DE, Echlin P, Joy DC, Fiori C, Lifshin E. Scanning Electron Microscopy and X-ray Microanalysis. New York: Plenum Press, 1992. Hayat MA. Principles and Techniques of Electron Microscopy: Biological Applications. New York: CRC Press, 1989. Kumakura M, Tamada M, Kaetsu I. Properties of Trichoderma reesei cells immobilized by the irradiation technique. Enzym Microbiol Technol 1984;6:411 – 4.
Immobilization of fungus Aspergillus sp. by a novel cryogel technique for production of extracellular hydrolytic enzymes Konstantin A. Lusta a, Il Kyung Chung a, Ill Whan Sul b, Hee Sung Park a, Dong Ill Shin a,* a
Faculty of Life Resources, Catholic Uni6ersity of Taegu-Hyosung, Hayang, Kyungsan, Kyungpook 712 -702, South Korea b Department of Forest Resources, Taegu Polytech. College, Taegu 706 -020, South Korea Received 9 December 1999; accepted 3 March 2000
Abstract Aerobic cells of a fungus isolate Aspergillus sp. CX-1 have been immobilized in macroporous cryoPAG and in different composite cryoPAGs — fibrous adjunct carriers. The productivity of the extracellular enzymes (exo-1.4-b-glucanase, endo-1.4-bglucanase, b-glucosidase and xylanase), and the viability, growth and ultrastructure of the immobilized fungus have been studied. The enzyme activities and stability during long-term repeated batch cultivation in the immobilized fungus were higher than in free mycelia when batch cultivated. The fungus immobilized in the composite cryoPAG, containing polypropylene non-woven fabric, possessed the highest exo-1.4-b-glucanase activity, the longest durability of enzyme production (85 days) and the most reliable mechanical strength. The fungus immobilized in porous composite cryogel possessed a variety of advantages including easy control of cryogel porosity, improved mechanical strength and durability, simplicity of construction, high enzyme productivity and high stability. © 2000 Elsevier Science Ltd. All rights reserved. Keywords: Immobilization; Fungi; Aspergillus; Cellulase; Xylanase.
1. Introduction One of the major possibilities of optimizing biotechnological processes lies in immobilization technologies, which can increase productivity and product concentrations. The use of immobilized microorganisms for the production of biologically active substances, including extracellular enzymes, has gained increasing interest because of potential in a variety of processes in industry [1,2]. The method of cell immobilization in natural and synthetic gels is currently most widely employed in biotechnology [3–5]. The main shortcomings of isotropic xerogel carriers, however, are their low durability and mass transfer problems [3,6]. These methods also impose some stress and toxic effects on the immobilized cells [7]. Examination of viable yeast cell numbers within the immobilization matrix indicated a dramatic reduction and * Corresponding author. Tel.: +82-53-8503297; fax: + 82-538503459. E-mail address: [email protected] (D.I. Shin)
ultimately the entire destruction of the submerged cell populations [7,8]. One of the feasible approaches for reducing the masstransfer resistance to the diffusion of substrate material is to immobilize cells into thin films or fixing cells on the surface of membranes [9,10]. Another possibility is to immobilize the cells in a fibrous matrix, which is attained by natural attachment or crosslinking to fibers [11]. The problem lies in anchoring the microorganisms firmly to the matrix walls [12]. Macroporous spongy carriers (like polyurethane or cellulose foams [13,14]) have been used to alleviate both problems. Although, the need to match the shape and size of the cell presents additional challenges. The investigation proposed here is aimed at developing a macroporous gel matrix as a compatible carrier for the immobilization of filamentous fungi. The specific characteristics of this gel matrix were targeted as the control of porosity and the ability to endure long-term repeated batch processing of extracellular cellulases and xylanase production.
0032-9592/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 0 ) 0 0 1 5 8 - 8
1178
K.A. Lusta et al. / Process Biochemistry 35 (2000) 1177–1182
2. Materials and methods
2.5. Culti6ation of immobilized fungus
2.1. Microorganism and culti6ation media A filamentous fungus strain exhibiting cellulolytic and xylanolytic activities was isolated from the soil around a hot spring in the Ashkhabad city (Turkmenistan) area. The fungus, identified as Aspergillus sp. (strain CX-1), was maintained on wort-agar slants and stored at 4°C. Aspergillus sp. CX-1 was cultured on the slants of a solidified Czapek – Dox mineral medium (0.3% NaNO3, 0.1% KH2PO4, 0.2% KCl and 0.05% MgSO4, pH 5.5) supplemented with 0.1% pepton, 0.2% Tween 80 and strips of filter paper on its surfaces before being used in the experiments.
The conidia immobilized in the cryoPAG were precultured in Czapek–Dox’s mineral medium supplemented with 0.1% pepton, 0.2% Tween 80 and 0.5% glucose. Cultivation was performed in Erlenmeyer flasks filled to 10% of their volume on a shaker at 41°C for 24 h. The IFB was then transferred to the same Czapek-Dox’s medium containing 2% (w/v) microcrystalline cellulose (MCC, Avicel, American Viscose Co., USA), instead of glucose, as a sole carbon source for production of cellulases and xylanase. Incubation was carried out in the same batch conditions as a repeated batch cultivation (RBC) [17], where the medium was changed periodically every 5 days.
2.2. Immobilization in cryoPAG
2.6. Electron microscopy
Acrylamide (AA), N,N%-methylene-bis-acrylamide (MBAA), N,N,N%,N%-tetramethyl-ethylenediamine (TEMED) and ammonium persulphate (APS) were purchased from Sigma (USA). For immobilisation of the fungus, the polyacrylamide gel mix (total volume 10 ml) was prepared on ice containing 2.8% AA, 0.3% MBAA, and 0.5 ml/ml TEMED along with 5 × 106 fungal conidia/ml and 0.02% Tween 80. 0.24 mg/ml APS was then added. The mixture was swirled rapidly and placed in a cryostat at different temperatures ranging from − 5 to − 80°C for 4 – 30 h. The frozen cryogel block was thawed in a warm water bath (40°C) and (monomer residues were washed from) the immobilized fungus block (IFB) with sterile tap water by squeezing the sponge.
For scanning electron microscopy (SEM) [18] the specimens were fixed in glutaraldehyde and then in OsO4 solutions, dehydrated in ethanol and absolute amyl acetate and dried using a critical point dryer HCP-2 unit (Hitachi, Japan). Gold shadowing was performed using an ion sputter JFC-1100 (Jeol, Japan). Freeze fracture preparations [19] were performed using a Freeze-Etch/Fracture System (BAL-TEC AG, Liechtenstein). The specimens were examined with the H-600 electron microscope (Hitachi, Japan).
2.3. Viability control The viability of the immobilized conidial population was determined using the slide culture (microculture) technique described by Hartman [15] and modified by Lusta [16].
2.7. Enzyme assays The activities of exo-1,4-b-D-glucanase, endo-1,4-bglucanase, b-glucosidase and 1,4-b-D-xylan xylanohydrolase (xylanase) were examined in culture supernatants of free and immobilized Aspergillus sp. CX-1, as described previously [17].
3. Results and discussion
2.4. Porous composite cryogel carriers
3.1. Effect of freezing temperatures on the cryoPAG structure
Different fibrous adjunct materials: polypropylene nonwoven fabric (PNWF), cellulose acetate filaments (CAF), nitrocellulose filaments (NCF), polyester filaments (PEF) and glass fabric (GF) were purchased from the Institute of Nonwoven Materials (Serpukhov, Russia). For the immobilization of fungal conidia in a porous composite cryogel (PCC) 0.25 g of one of the fibrous adjuncts was added to a vial containing the polymerization mix for cryoPAG, all under sterile conditions. Further procedures were the same as those used for the cryoPAG polymerization.
Varying the freezing temperatures (between −5 and − 80°C) created the spongy cryogels with the optimal porosity appropriate for the immobilization of the filamentous fungi. Pore size decreased as the temperature of the PAG polymerization decreased (Fig. 1). The polymerization at − 20°C gave the required porosity in the cryoPAG (Fig. 2a). Freezing of the polymerization mix at temperatures above − 20°C caused the pore size to be too large, and freezing below − 20°C sharply slowed polymerization and reduced the size of the gel pore, worsening the mass exchange for the immobilized fungi.
K.A. Lusta et al. / Process Biochemistry 35 (2000) 1177–1182
1179
The use of acrylamide as a monomer for the cryopolymer carrier is suitable for immobilization at temperatures from − 10 to − 20°C, making polymerization significantly easier and safer than the method previously used [20], where hydroxyethylmethacrylate served as the monomer. The latter requires freezing to − 78oC and g-irradiation for polymerization. Cryogels formed at such low temperatures have smaller pores (occupying only 20% of the total polymer volume) than the cryoPAG, and therefore may worsen mass exchange during prolonged fungal cultures.
3.2. Effects of loading conidia and surfactant concentration
Fig. 1. The effect of freezing temperature on the cryoPAG porosity. , cryoPAG porosity,%; , cryoPAG pore size, mm.
During the immobilization of conidia in the cryoPAG, the concentration of conidia and the surfactant (Tween-80) added to the polymerization mix had significant effects on the properties of the carrier thus produced. The optimal concentration of the immobilized conidia was in the range of 106 –107/ml, while the
Fig. 2. A survey of immobilized in macroporous cryoPAG Aspergillus sp. CX-1 cultured on cellulase-production media: (a) scanning electron micrograph of the cryoPAG sponge. The macropores of about 40 mm are visible; (b) micropore structure of the ordinary PAG revealed by freeze-fracture electron microscopy, the bar represents 0.5 mm; (c) fungal conidia after immobilization in cryoPAG, the bar represents 0.5 mm; (d) immobilized fungus growth during the initial phase preculturing (12 h incubation in glucose medium), the bar represents 0.5 mm; (e) growth of mycelium after 24 h preculturing in glucose medium; (f) cryoPAG immobilized mycelium after the first 5-day cycle of repeated batch cultivation on cellulase-production media; (g) the structure of the PCC-PNWF and the mycelial growth after the first 5-day cycle of repeated batch cultivation on cellulase-production media in this carrier are demonstrated, the bar represents 50 mm.
K.A. Lusta et al. / Process Biochemistry 35 (2000) 1177–1182
1180
Table 1 Polysaccharase activities in culture filtrates measured at the end of sequential 5-day cycles of repeated batch cultivation immobilized in cryoPAG Aspergillus sp. CX-1 Number of 5-day cycle
1 2 3 4 5 6
Total enzyme activities
Enzyme activities
(U/ml)
Endo-b-glucanase
Exo-b-glucanase
b-glucosidase
Xylanase
27.3 25.7 15.8 20.8 12.0 13.4
0.52 0.46 0.54 0.70 0.44 0.63
4.3 3.7 3.1 4.6 4.0 3.2
20.1 23.2 24.6 11.8 17.7 24.6
optimal concentration of Tween-80 was 0.02%. Higher levels either prevented cryogel polymerization, or resulted in mechanical weakness and break up after just the first batch cycle of incubation.
3.3. Structural, mechanical and adhesion properties of cryoPAG The architecture of the cryoPAG was viewed using SEM. Fig. 2(a) shows that the cryoPAG has a macroporous structure with a pore size of about 40 mm. The pore space of the cryoPAG occupies 60% of the total volume. In comparison, the micropores of an isotropic PAG are not more than 0.1 mm (Fig. 2b). The cryoPAG polymer has considerable mechanical strength, elasticity, and the ability to liberate up to 70% of unbound liquid upon compression. It also can be stored for prolonged periods in a dry state, and takes up the same quantity of moisture to restore the original shape and volume. The cryoPAG pores can absorb up to 4 ml of aqueous solution per g of dry weight. The concentration of the components for PAG polymerization can be reduced ten-fold compared to a normal PAG. This is because, as the solvent freezes, the concentration of substances in the liquid phase increases, resulting in a reduction of the critical concentration required for PAG formation. The proportion of fungal conidia trapped in the carrier after immobilization in the cryoPAG was 60– 70% of the added amount.
3.4. Viability of immobilized fungal conidia The viability index of conidia embedded in the cryoPAG, as determined by the microculture technique, was 76% compared with 80% for non-immobilized conidia. The comparison of these data to those for Escherichia coli immobilization in PAG [7] indicates that in practice Aspergillus sp. conidia exhibits absolute resistance to all the stress factors of cryoPAG immobilization.
3.5. Growth of fungal mycelia in cryoPAG The successive steps for growing Aspergillus sp. CX-1 mycelium in the cryoPAG, from preculturing to the 5-day batch cycle of cultivation, are shown in Fig. 2(c–f). The cryoPAGs macroporous structure allows for abundant growth of immobilized filamentous fungus in the gel.
3.6. Variation of extracellular cellulase and xylanase acti6ities produced by cryoPAG-immobilized fungus with culture time of repeated batch process The results presented in Table 1 show that samples of culture filtrates immobilized in the cryoPAG Aspergillus sp. CX-1 had fairly high and stable polysaccharase activities. Xylanase and exo-b-glucanase activities were increased further after six cycles of batch cultivation. The total operating time was 30 days. The previous studies [17] have demonstrated that among the fungal strains used (in particular, the conventional commercial strain, Trichoderma reesei QM9414), the highest cellulolytic and xylanolytic activities were produced by the new filamentous fungus strain Aspergillus sp. CX-1. It follows that this strain, which is interesting from an economic point of view, was selected for the immobilized cell studies. Cellulases and xylanase activities in culture filtrates immobilized in cryoPAG Aspergillus sp. CX1 were higher than those of the free mycelium of the same fungal strain, described previously [17]. This could be due to the improvement of secretion and export of extracellular hydrolytic enzymes by the specific conditions of immobilization of fungal mycelia and increasing of the concentration of cellulosic substrate in cryoPAG pore matrix as compared to that of the conventional batch conditions.
3.7. Immobilization of fungus in porous composite cryogel carriers The data presented in Fig. 3 indicate exo-b-glucanase activities in culture filtrates prepared as described above
K.A. Lusta et al. / Process Biochemistry 35 (2000) 1177–1182
at the end of sequential 5-day cycles of RBC. The PCC-GF blocks proved to be the least durable. When PNWF was used, the PCC carriers retained their mechanical strength during repeated batch cultivation for an unlimited period of time. The exo-b-glucanase activities in the culture filtrates of these preparations remained at a high level for 17 cycles (85 days). The selection of the fibrous adjunct material for creating the PCC was, thus, critical for fungus immobilization because of the cryogel’s mechanical properties, fungal cell biocompatibility and resulting stability and durability of extracellular enzyme production. The culture medium remained clear and transparent during this cultivation period. Phase-contrast microscopic examination did not reveal any hyphae or conidia in the culture media. Additionally, the insoluble substrate (MCC particles) were also absorbed by the PCC and were firmly retained by the carrier throughout the RBC period. The structure of the PCC-PNWF and the mycelial growth over the course of polysaccharase production in this carrier are demonstrated in Fig. 2(g). Another positive characteristic of the PCC-immobilized fungi was that, compared to the single batch culture, the required amount of supplemental cellulose powder and other ingredients of nutrient media is ten times less. PCCs in general, and cryoPAG-PNWF in particular, were found to have better mechanical properties and stability of enzyme production than a pure cryoPAG that does not contain this fibrous adjunct filler.
1181
4. Conclusions CryoPAG was prepared by the radical-mediated copolymerization of AA (vinyl monomer) and MBAA (divinyl monomer) in the presence of initiators under freezing conditions. It had different physical and mechanical properties from the analogous isotropic xerogels formed under standard conditions at positive temperatures. Solvent (H2O) crystallization leads to the formation of porous heterophasic cryogels with an extremely macroporous structure. These properties are useful for the immobilization of filamentous fungi. The macroporous structure lends itself to the efficient mass exchange, resulting in increased fungal growth, higher enzyme producing activity and easy release of the secreted enzymes into the medium. Fungal mycelium can anchor firmly to the cryoPAG carrier because of certain peculiar conforming characteristics. The spongy cryoPAG can be used in large blocks and does not break up into granules. Porous composite cryogels were created by filling the cryoPAG with fibrous adjuncts (PHWF, GF, CAF, NCF and PEF) in order to increase the mechanical strength and durability of the cryoPAG. This also served to improve adhesion of the secondary conidia and mycelium to the carrier, and attain greater enzyme productivity and stability. Due to their high porosity and biocompatibility, macroporous cryoPAG and PCC-carriers were adopted for fungi cell immobilization and extracellular enzyme production. A valuable property of the cryoPAG and PCC carrier is its ability to absorb particles of the solid substrate (cellulose) and secondary-formed fungal conidia from the media during RBC.
Acknowledgements This work was financially supported by the 1999 Russian Scientist Exchange Program’ through the Korean Institute of Science and Technology Evaluation and Planning (KISTEP).
References
Fig. 3. Exo-b-glucanase activities of culture filtrates at the end of sequential 5-day cycles of repeated batch cultivation of Aspergillus sp. CX1 immobilized in various porous composite cryoPAG (PCC) carriers: , Cryo-PAG — polypropylene nonwoven fabric (PNWF); ", Cryo-PAG — cellulose acetate filaments (CAF); , Cryo-PAG — glass fabric (GF); , Cryo-PAG — nitrocellulose filaments (NCF); , Cryo-PAG — polyester filaments (PEF).
[1] Katzbauer B, Narodoslawsky M, Moser A. Classification system for immobilization techniques. Bioprocess Eng 1995;12:173–9. [2] Virkajarvi I, Linko M. Immobilization: a revolution in traditional brewing. Naturwissenschaften 1999;86:112 – 22. [3] Gough S, McHale AP. Continuous ethanol production from molasses at 45°C using alginate-immobilized Kluy6eromyces marxianus IMB3 in a continuous-flow bioreactor. Bioprocess Eng 1998;19:33 – 6. [4] Quirasco M, Remaud-Simeon M, Monsan P, Lopez- Munguia A. Experimental behavior of a whole cell immobilized dextransucrase biocatalyst in batch and packed bed reactors. Bioprocess Eng 1999;20:289 – 95.
1182
K.A. Lusta et al. / Process Biochemistry 35 (2000) 1177–1182
[5] Slokoska L, Angelova M, Pashova S, Petricheva E, Konstantinov Ch. Production of acid proteinase by Humicola lutea 120-5 immobilized in mixed photo-cross-linked polyvinyl alcohol and calcium-alginate beads. Process Biochem 1999; 34:73 – 6. [6] Roisin C, Bienaime C, Nava Saucedo JE, Barbotin JN. Influence of microenvironment on immobilised Gibberella fujikuroi. Prog Biotechnol 1996;11:189–95. [7] Lusta KA, Starostina NG, Fikhte BA. Immobilization of microorganisms: cytophysiological aspects. In: de Bont JAM, Visser J, Mattiasson B, Tramper J, editors. Proceedings of an International Symposium: Physiology of Immobilized Cells. Amsterdam: Elsevier, 1990:557–62. [8] Barbotin JN, Nava Saucedo JE. Bioencapsulation of living cells by entrapment in polysaccharide gels. In: Dumitriu S, editor. Polysaccharides: structural diversity and functional versatility. New York: Marcel Dekker, 1998:749–74. [9] Tonkova A, Ivanova V, Dobreva E, Stefanova M, Spasova D. Thermostable alpha-amylase production by immobilized Bacillus licheniformis cells in agar gel and on acrylonitrile/acrylamide membranes. Appl Microbiol Biotechnol 1994;41:517–22. [10] Ascencio F, Johansson G, Wadstrom T. Cell-surface charge and cell-surface hydrophobicity of collagen-binding Aeromonas and Vibrio strains. Arch Microbiol 1995;164:223–30. [11] Cho HY, Yousef AE, Yang ST. Continuous production of pediocin by immobilized Pediococcus acidilactici PO2 in a packed-bed bioreactor. Appl Microbiol Biotechnol 1996;45:589 – 94. [12] Busscher HJ, Poortinga AT, Bos R. Lateral and perpendicular interaction forces involved in mobile and immobile adhesion of
.
[13]
[14]
[15]
[16]
[17]
[18]
[19] [20]
microorganisms on model solid surfaces. Curr Microbiol 1998;37:319 – 23. Lapadatescu C, Feron G, Vergoignan C, Djian A, Durand A, Bonnarme P. Influence of cell immobilization on the production of benzaldehyde and benzyl alcohol by the white-rot fungi Bjerkandera adusta, Ischnoderma benzoinum and Dichomitus squalens. Appl Microbiol Biotechnol 1997;47:708 – 14. Chiou TW, Wang YC, Liu HS. Utilizing the macroporous packed bed for insect cell/baculovirus expression — part 2: the production of human interleukin-5 in polyurethane foam and cellulose foam packed bed bioreactors. Bioprocess Eng 1998;18:91 – 100. Hartman PA. Miniaturized microbiological methods. In: Umbreit WW, editor. Advances in Applied Microbiology (Suppl. I). New York: Academic Press, 1968:65 – 84. Lusta KA. A microculture method for the determination of viability of immobilized microbial cells. In: Koshcheenko KA, editor. Immobilized Microbial Cells. Pushchino: NCBI, Russian Academy of Sciences, 1979:164 – 73. Lusta KA, Chung IK, Sul IW, Park HS, Shin DI. Characterization of extracellular cellulose-hydrolyzing enzyme complex from a thermotolerant strain of Aspergillus sp. J Microbiol Biotechnol 1999;9:873 – 6. Goldstein JI, Newbury DE, Echlin P, Joy DC, Fiori C, Lifshin E. Scanning Electron Microscopy and X-ray Microanalysis. New York: Plenum Press, 1992. Hayat MA. Principles and Techniques of Electron Microscopy: Biological Applications. New York: CRC Press, 1989. Kumakura M, Tamada M, Kaetsu I. Properties of Trichoderma reesei cells immobilized by the irradiation technique. Enzym Microbiol Technol 1984;6:411 – 4.