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Performance of Luffa cylindrica as immobilization matrix in bioconversion reactions by Nicotiana tabacum BY-2
Journal of Bioscience and Bioengineering VOL. 116 No. 4, 506e508, 2013 www.elsevier.com/locate/jbiosc
NOTE
Performance of Luffa cylindrica as immobilization matrix in bioconversion reactions by Nicotiana tabacum BY-2 Hamid Bou-Saab,1 Anna Boulanger,1, * Paul Schellenbaum,2 and Serge Neunlist1 Université de Haute Alsace, Ecole Nationale Supérieure de Chimie de Mulhouse, Laboratoire de Chimie Organique et Bio-organique, 3 bis rue Alfred Werner, 68093 Mulhouse Cedex, France1 and Laboratoire Vigne, Biotechnologies et Environnement, EA3991, Université de Haute Alsace, 33 rue de Herrlisheim, 68008 Colmar Cedex, France2 Received 11 July 2012; accepted 12 April 2013 Available online 8 May 2013
The dry fruit of Luffa cylindrica was investigated as an immobilization matrix for Nicotiana tabacum cells in bioconversion reactions of exogenous substrates. Immobilized cells show high biocatalytic activity under high substrate levels. Cell growth on the dry fruit can be maintained until reaching an immobilization capacity of 1.8 g cells/gLuffa. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: Luffa cylindrica; Nicotiana tabacum; Immobilization; Bioconversion; Oxidation; Reduction]
Immobilized cell technology has been extensively studied as a mean to improve the productivity of bioconversion systems (1). In such a status, we recently found that the cell immobilization results in a 3e4 fold increase of cholesterol bioconversion by two mycobacterium species (2). Plant cell cultures and enzymes have the potential to transform substances, such as industrial by-products, adding by this way high value (3). Plant cell immobilization increases product accumulation by (i) reducing shear stress, (ii) extending viability of cells, (iii) providing high cell density within relatively small bioreactors, (iv) simplifying downstream processing (if products are secreted in the media), and (v) reusing of immobilized cells over a prolonged period (4). The technique most widely used for cell immobilization involves the entrapment of cells in some kind of gel such as calcium alginate, carrageen and pectate (5). However, cell entrapment in gels has some disadvantages; for example, the restricted diffusion of substrates within the gel (6) and the impossibility to perform cellular division with space limitation (7). Passive adsorption of cells to polymeric surfaces has also been studied (8). These polymeric surfaces such as polyurethane foams and polyester foams are often expensive, nonbiodegradable and their disposal causes a lot of environmental problems after their use (8). The dry fruit of Luffa cylindrica (DFLC) has been reported as an excellent carrier for plant cells (6) and microbial cells (9) immobilization. DFLC is a natural lignocellulosic material, biodegradable, inexpensive, non-toxic and easy to use (2). Despite his many advantages, there is so far nearly no published work on the use of DFLC in bioconversion reactions with plant cells. On the other hand, many studies reported the ability of tobacco cells culture to regio and stereoselective hydroxylation, oxidation * Corresponding author. Tel.: þ33 3 89336876; fax: þ33 3 89336815. E-mail addresses: [email protected] (H. Bou-Saab), anna.boulanger@ uha.fr (A. Boulanger), [email protected] (P. Schellenbaum), [email protected] (S. Neunlist).
and hydrolysis of a wide variety of chemicals (4). In this report, we show the effectiveness of DFLC as immobilization matrix for Nicotiana tabacum BY-2 cells (BY-2) in the following test reactions: (i) oxidation of cyclopentanol to cyclopentanone (10) and (ii) reduction of carvone to neodihydrocarveol (11). Evaluations of bioconversion rate and of immobilized and suspended biomass are described. The cultured-cells of BY-2 were provided by Dr. M-E Chabouté, IBMP-CNRS, Strasbourg. Every two weeks, 2 mL of a previous culture were transferred in 250 mL flaks containing 100 mL of BY-2 medium as described by Quian et al. (12), and then incubated under continuous shaking (110 rpm) at 25 C in the dark. Immobilization of cells was carried out as described in our previous work (2). Briefly, DFLC (the core and peripheral parts) was cut into discs of approximately 2.5 cm diameter with different thickness (0.5e1 cm), soaked in boiling water for 30 min, thoroughly washed under tap water and left for 24 h in distilled water replaced 3e4 times. The discs were then oven dried at 70 C. DFLC discs (1.5 g) were transferred in 250 mL flasks and autoclaved at 121 C for 30 min. After sterilization, 2 mL of BY-2 cells suspension were added in 100 mL of BY-2 medium. Flasks without DFLC were kept as a control. Substrates and their bioconversion products proved stable in the MS medium and DFLC discs without biocatalysts, under the same conditions as for bioconversion. Substrates were added to the BY-2 culture on the day 10. For each experiment, triplicate samples were analyzed 5 days after feeding the substrates. Substrates and their biotransformation products were extracted and analyzed as described previously by direct comparison of GC and GCeMS with those of authentic standards (10,11). The yields of the products were determined on the basis of the peak area on the chromatogram of GC and are expressed as a relative percentage to the whole reaction mixture obtained. Immobilized biomass was evaluated by measuring the dry weight (d.w.) of DFLC discs before and after experiments. For some experiments, immobilized cells
1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.04.017
VOL. 116, 2013
NOTE
507
TABLE 1. Conversion rate of cyclopentanol and specific productivity of cyclopentanone by Nicotiana tabacum BY-2. Conversion rate (%)
Control DFLC
Specific productivity (mM d1 g1cells)
0.1 g L1 cyclopentanol
1 g L1 cyclopentanol
0.1 g L1 cyclopentanol
1 g L1 cyclopentanol
80 7.9 95 5.1
38 þ 5.5 65 6.5
0.53 0.30
2.26 2.12
were removed by vibration in deionized water with a vibrating multi-reax shaker and then filtered through a 0.2 mm mesh filter repeatedly until no cell remained on the DFLC discs. The cells on the filter were then washed three times with deionized water. After drying in an oven at 105 C until most of the water was removed, the cells were then dried again in a vacuum oven for 24 h and weighed. The oxidation of cyclopentanol to cyclopentanone was investigated with the following substrate concentrations: 0.1 and 1 g L1. After 5 days of incubation, the conversion rate of cyclopentanol (0.1 g L1) to cyclopentanone in flasks containing DFLC (95%) was slightly higher than that obtained with control (80%) (Table 1). A yield of 95% was carried out by suspended tobacco cells within 8 incubation days (10). When substrate concentration was 1 g L1, the conversion rate in control decreased dramatically to 38% while, in flasks containing DFLC, a conversion rate of 65% was observed (Table 1). However, the specific productivity of cyclopentanone in control and DFLC flasks was about 2.26 and 2.12 mM d1 g1cells respectively. Hence, the higher conversion rate of cyclopentanol by tobacco cells in DFLC flasks was probably due to the higher biomass. The effectiveness of DFLC as immobilization matrix for N. tabacum BY-2 cells was also studied in the reduction (4R)()-carvone to (1R, 2S, 4R)-neodihydrocarveol. (4R)-()-Carvone (0.1 g L1) was administered to a 10 days old preculture of suspended BY-2 cells and incubated for 5 days with shaking in the dark. The yield of neodihydrocarveol in control experiments was less than 2% and the biomass was estimated at 3.54 0.44 g L1 (d.w.). Adding DFLC in flasks, under the same conditions, increased the biomass to 7.50 0.35 g L1 (d.w.) and by then allowed yields of 30% of neodihydrocarveol (Table 2). However, it appears that the better yields in flasks with DFLC were not only due to the increasing of biomass but also to a better activity of immobilized cells. In fact, the specific productivity of neodihydrocarveol was about 53.12 mM d1 g1cells in DFLC flasks compared to 7.53 mM d1 g1cells in control experiments (Table 2). The immobilization capacity of DFLC and viability of immobilized cells were investigated through 4 incubation cycles. The MS medium was renewed every two weeks and triplicate flasks were analyzed. As shown in Fig. 1A, the amount of immobilized cells continues to increase within the 4 incubation cycles to reach an immobilization capacity of 1.8 0.1 gcells/gDFLC (d. w.). Further incubation of DFLC didn’t increase its immobilization capacity. Fig. 1B illustrates a disc of DFLC before and after 15 days of incubation with tobacco cells. The whole fibers of DFLC were colonized by BY-2 cells. The efficiency of DFLC as a cell carrier can be attributed to its high porosity (79e93%) and low density allowing use of a small amount of DFLC to fulfill the medium volume and then provide a large surface for cell adhesion. Generally, cells with hydrophobic properties prefer material with hydrophobic surfaces for adhesion, and TABLE 2. Conversion rate of carvone (0.1 g L1) and specific productivity of neodihydrocarveol by Nicotiana tabacum BY-2.
Control DFLC
Conversion rate (%)
Specific productivity (mM d1 g1cells)
2 0.3 30 1.9
7.53 53.12
FIG. 1. (A) Cell immobilization capacity of DFLC during 6 incubation cycles. Flask volume: 250 mL. MS medium volume: 100 mL. Inoculum: 2%. Temperature: 25 C. Shaking: 110 rpm in the dark. After each cycle (15 days) MS medium was renewed. (B) Photograph of segments of DFLC before (left) and after (right) 15 days incubation with tobacco cell culture.
those with hydrophilic characteristics prefer hydrophilic surfaces. Hydrophilic polysaccharides are the main components of BY-2 cell wall (13). On the other hand, fibers of DFLC are composed by hydrophilic holocellulose (84%) (14). As a consequence, the suspended tobacco cells adhere passively to surface fibers of DFLC. Moreover, the surface roughness of DFLC allowed the adhesion of cell aggregates. In conclusion, the use of DFLC as immobilization matrix for plant cells in bioconversion reactions offer many advantages. Adding DFLC to bioconversion medium allows a high volumetric and specific productivity by maintaining biocatalytic activity of cells under high substrate levels. Furthermore, DFLC is natural, biodegradable, inexpensive, non-toxic and doesn’t need a chemical pretreatment. References 1. Kourkoutas, Y., Bekatorou, A., Banat, I. M., Marchant, R., and Koutinas, A. A.: Immobilization technologies and support materials suitable in alcohol beverages production: a review, Food Microbiol., 21, 377e397 (2004). 2. Bou Saab, H., Fouchard, S., Boulanger, A., Llopiz, P., and Neunlist, S.: Performance of Luffa cylindrica as an immobilization matrix for the biotransformation of cholesterol by Mycobacterium species, Biocatal. Biotransfor., 28, 387e394 (2010). 3. Giri, A., Dhingra, V., Giri, C. C., Singh, A., Ward, O. P., and Lakshmi Narasu, M.: Biotransformations using plant cells, organ cultures and enzyme systems: current trends and future prospects, Biotechnol. Adv., 19, 175e199 (2001). 4. Ishihara, K., Hamada, H., Hirata, T., and Nakajima, N.: Biotransformation using plant cultured cells, J. Mol. Catal. B-Enzym., 23, 145e170 (2003). 5. Ramachandra Rao, S. R. and Ravishankar, G. A.: Plant cell cultures: chemical factories of secondary metabolites, Biotechnol. Adv., 20, 101e153 (2002). 6. Liu, Y. K., Seki, M., Tanaka, H., and Furusaki, S.: Characteristics of loofa (Luffa cylindrica) sponge as a carrier for plant cell immobilization, J. Ferment. Bioeng., 85, 416e421 (1998).
508
BOU-SAAB ET AL.
7. Perullini, M., Rivero, M. M., Jobbágy, M., Mentaberry, A., and Bilmes, S. A.: Plant cell proliferation inside an inorganic host, J. Biotechnol., 127, 542e548 (2007). 8. Hideno, A., Ogbonna, J. C., Aoyagi, H., and Tanaka, H.: Acetylation of loofa (Luffa cylindrica) sponge as immobilization carrier for bioprocesses involving cellulose, J. Biosci. Bioeng., 103, 311e317 (2007). 9. Scott, J. A. and O’Reilly, A. M.: Co-Immobilization of selected yeast and bacteria for controlled flavour development in an alcoholic cider beverage, Process Biochem., 31, 111e117 (1996). 10. Suga, T., Hamada, H., and Hirata, T.: Oxidation-reduction relationship between cycloalkanones and the corresponding cycloalkanols in a cell suspension culture of Nicotiana tabacum, Plant Cell Rep., 2, 66e68 (1983).
J. BIOSCI. BIOENG., 11. Hirata, T., Hamada, H., Aoki, T., and Suga, T.: Stereoselectivity of the reduction of carvone and dihydrocarvone by suspension cells of Nicotiana tabacum, Phytochemistry, 21, 2209e2212 (1982). 12. Qian, D., Zhou, D., Ju, R., Cramer, C. L., and Yang, Z.: Protein farnesyltransferase in plants: molecular characterization and involvement in the cell cycle control, Plant Cell, 8, 2381e2394 (1996). 13. Blaschek, W., Haass, D., Koehler, H., and Franz, G.: Cell wall regeneration by Nicotiana tabacum protoplast: chemical and biochemical aspects, Plant Sci. Lett., 22, 47e57 (1981). 14. Guimarães, J. L., Frollini, E., da Silva, C. G., Wypych, F., and Satyanarayana, K. G.: Characterization of banana, sugarcane bagasse and sponge gourd fibers of Brazil, Ind. Crops Prod., 30, 407e415 (2009).
NOTE
Performance of Luffa cylindrica as immobilization matrix in bioconversion reactions by Nicotiana tabacum BY-2 Hamid Bou-Saab,1 Anna Boulanger,1, * Paul Schellenbaum,2 and Serge Neunlist1 Université de Haute Alsace, Ecole Nationale Supérieure de Chimie de Mulhouse, Laboratoire de Chimie Organique et Bio-organique, 3 bis rue Alfred Werner, 68093 Mulhouse Cedex, France1 and Laboratoire Vigne, Biotechnologies et Environnement, EA3991, Université de Haute Alsace, 33 rue de Herrlisheim, 68008 Colmar Cedex, France2 Received 11 July 2012; accepted 12 April 2013 Available online 8 May 2013
The dry fruit of Luffa cylindrica was investigated as an immobilization matrix for Nicotiana tabacum cells in bioconversion reactions of exogenous substrates. Immobilized cells show high biocatalytic activity under high substrate levels. Cell growth on the dry fruit can be maintained until reaching an immobilization capacity of 1.8 g cells/gLuffa. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: Luffa cylindrica; Nicotiana tabacum; Immobilization; Bioconversion; Oxidation; Reduction]
Immobilized cell technology has been extensively studied as a mean to improve the productivity of bioconversion systems (1). In such a status, we recently found that the cell immobilization results in a 3e4 fold increase of cholesterol bioconversion by two mycobacterium species (2). Plant cell cultures and enzymes have the potential to transform substances, such as industrial by-products, adding by this way high value (3). Plant cell immobilization increases product accumulation by (i) reducing shear stress, (ii) extending viability of cells, (iii) providing high cell density within relatively small bioreactors, (iv) simplifying downstream processing (if products are secreted in the media), and (v) reusing of immobilized cells over a prolonged period (4). The technique most widely used for cell immobilization involves the entrapment of cells in some kind of gel such as calcium alginate, carrageen and pectate (5). However, cell entrapment in gels has some disadvantages; for example, the restricted diffusion of substrates within the gel (6) and the impossibility to perform cellular division with space limitation (7). Passive adsorption of cells to polymeric surfaces has also been studied (8). These polymeric surfaces such as polyurethane foams and polyester foams are often expensive, nonbiodegradable and their disposal causes a lot of environmental problems after their use (8). The dry fruit of Luffa cylindrica (DFLC) has been reported as an excellent carrier for plant cells (6) and microbial cells (9) immobilization. DFLC is a natural lignocellulosic material, biodegradable, inexpensive, non-toxic and easy to use (2). Despite his many advantages, there is so far nearly no published work on the use of DFLC in bioconversion reactions with plant cells. On the other hand, many studies reported the ability of tobacco cells culture to regio and stereoselective hydroxylation, oxidation * Corresponding author. Tel.: þ33 3 89336876; fax: þ33 3 89336815. E-mail addresses: [email protected] (H. Bou-Saab), anna.boulanger@ uha.fr (A. Boulanger), [email protected] (P. Schellenbaum), [email protected] (S. Neunlist).
and hydrolysis of a wide variety of chemicals (4). In this report, we show the effectiveness of DFLC as immobilization matrix for Nicotiana tabacum BY-2 cells (BY-2) in the following test reactions: (i) oxidation of cyclopentanol to cyclopentanone (10) and (ii) reduction of carvone to neodihydrocarveol (11). Evaluations of bioconversion rate and of immobilized and suspended biomass are described. The cultured-cells of BY-2 were provided by Dr. M-E Chabouté, IBMP-CNRS, Strasbourg. Every two weeks, 2 mL of a previous culture were transferred in 250 mL flaks containing 100 mL of BY-2 medium as described by Quian et al. (12), and then incubated under continuous shaking (110 rpm) at 25 C in the dark. Immobilization of cells was carried out as described in our previous work (2). Briefly, DFLC (the core and peripheral parts) was cut into discs of approximately 2.5 cm diameter with different thickness (0.5e1 cm), soaked in boiling water for 30 min, thoroughly washed under tap water and left for 24 h in distilled water replaced 3e4 times. The discs were then oven dried at 70 C. DFLC discs (1.5 g) were transferred in 250 mL flasks and autoclaved at 121 C for 30 min. After sterilization, 2 mL of BY-2 cells suspension were added in 100 mL of BY-2 medium. Flasks without DFLC were kept as a control. Substrates and their bioconversion products proved stable in the MS medium and DFLC discs without biocatalysts, under the same conditions as for bioconversion. Substrates were added to the BY-2 culture on the day 10. For each experiment, triplicate samples were analyzed 5 days after feeding the substrates. Substrates and their biotransformation products were extracted and analyzed as described previously by direct comparison of GC and GCeMS with those of authentic standards (10,11). The yields of the products were determined on the basis of the peak area on the chromatogram of GC and are expressed as a relative percentage to the whole reaction mixture obtained. Immobilized biomass was evaluated by measuring the dry weight (d.w.) of DFLC discs before and after experiments. For some experiments, immobilized cells
1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.04.017
VOL. 116, 2013
NOTE
507
TABLE 1. Conversion rate of cyclopentanol and specific productivity of cyclopentanone by Nicotiana tabacum BY-2. Conversion rate (%)
Control DFLC
Specific productivity (mM d1 g1cells)
0.1 g L1 cyclopentanol
1 g L1 cyclopentanol
0.1 g L1 cyclopentanol
1 g L1 cyclopentanol
80 7.9 95 5.1
38 þ 5.5 65 6.5
0.53 0.30
2.26 2.12
were removed by vibration in deionized water with a vibrating multi-reax shaker and then filtered through a 0.2 mm mesh filter repeatedly until no cell remained on the DFLC discs. The cells on the filter were then washed three times with deionized water. After drying in an oven at 105 C until most of the water was removed, the cells were then dried again in a vacuum oven for 24 h and weighed. The oxidation of cyclopentanol to cyclopentanone was investigated with the following substrate concentrations: 0.1 and 1 g L1. After 5 days of incubation, the conversion rate of cyclopentanol (0.1 g L1) to cyclopentanone in flasks containing DFLC (95%) was slightly higher than that obtained with control (80%) (Table 1). A yield of 95% was carried out by suspended tobacco cells within 8 incubation days (10). When substrate concentration was 1 g L1, the conversion rate in control decreased dramatically to 38% while, in flasks containing DFLC, a conversion rate of 65% was observed (Table 1). However, the specific productivity of cyclopentanone in control and DFLC flasks was about 2.26 and 2.12 mM d1 g1cells respectively. Hence, the higher conversion rate of cyclopentanol by tobacco cells in DFLC flasks was probably due to the higher biomass. The effectiveness of DFLC as immobilization matrix for N. tabacum BY-2 cells was also studied in the reduction (4R)()-carvone to (1R, 2S, 4R)-neodihydrocarveol. (4R)-()-Carvone (0.1 g L1) was administered to a 10 days old preculture of suspended BY-2 cells and incubated for 5 days with shaking in the dark. The yield of neodihydrocarveol in control experiments was less than 2% and the biomass was estimated at 3.54 0.44 g L1 (d.w.). Adding DFLC in flasks, under the same conditions, increased the biomass to 7.50 0.35 g L1 (d.w.) and by then allowed yields of 30% of neodihydrocarveol (Table 2). However, it appears that the better yields in flasks with DFLC were not only due to the increasing of biomass but also to a better activity of immobilized cells. In fact, the specific productivity of neodihydrocarveol was about 53.12 mM d1 g1cells in DFLC flasks compared to 7.53 mM d1 g1cells in control experiments (Table 2). The immobilization capacity of DFLC and viability of immobilized cells were investigated through 4 incubation cycles. The MS medium was renewed every two weeks and triplicate flasks were analyzed. As shown in Fig. 1A, the amount of immobilized cells continues to increase within the 4 incubation cycles to reach an immobilization capacity of 1.8 0.1 gcells/gDFLC (d. w.). Further incubation of DFLC didn’t increase its immobilization capacity. Fig. 1B illustrates a disc of DFLC before and after 15 days of incubation with tobacco cells. The whole fibers of DFLC were colonized by BY-2 cells. The efficiency of DFLC as a cell carrier can be attributed to its high porosity (79e93%) and low density allowing use of a small amount of DFLC to fulfill the medium volume and then provide a large surface for cell adhesion. Generally, cells with hydrophobic properties prefer material with hydrophobic surfaces for adhesion, and TABLE 2. Conversion rate of carvone (0.1 g L1) and specific productivity of neodihydrocarveol by Nicotiana tabacum BY-2.
Control DFLC
Conversion rate (%)
Specific productivity (mM d1 g1cells)
2 0.3 30 1.9
7.53 53.12
FIG. 1. (A) Cell immobilization capacity of DFLC during 6 incubation cycles. Flask volume: 250 mL. MS medium volume: 100 mL. Inoculum: 2%. Temperature: 25 C. Shaking: 110 rpm in the dark. After each cycle (15 days) MS medium was renewed. (B) Photograph of segments of DFLC before (left) and after (right) 15 days incubation with tobacco cell culture.
those with hydrophilic characteristics prefer hydrophilic surfaces. Hydrophilic polysaccharides are the main components of BY-2 cell wall (13). On the other hand, fibers of DFLC are composed by hydrophilic holocellulose (84%) (14). As a consequence, the suspended tobacco cells adhere passively to surface fibers of DFLC. Moreover, the surface roughness of DFLC allowed the adhesion of cell aggregates. In conclusion, the use of DFLC as immobilization matrix for plant cells in bioconversion reactions offer many advantages. Adding DFLC to bioconversion medium allows a high volumetric and specific productivity by maintaining biocatalytic activity of cells under high substrate levels. Furthermore, DFLC is natural, biodegradable, inexpensive, non-toxic and doesn’t need a chemical pretreatment. References 1. Kourkoutas, Y., Bekatorou, A., Banat, I. M., Marchant, R., and Koutinas, A. A.: Immobilization technologies and support materials suitable in alcohol beverages production: a review, Food Microbiol., 21, 377e397 (2004). 2. Bou Saab, H., Fouchard, S., Boulanger, A., Llopiz, P., and Neunlist, S.: Performance of Luffa cylindrica as an immobilization matrix for the biotransformation of cholesterol by Mycobacterium species, Biocatal. Biotransfor., 28, 387e394 (2010). 3. Giri, A., Dhingra, V., Giri, C. C., Singh, A., Ward, O. P., and Lakshmi Narasu, M.: Biotransformations using plant cells, organ cultures and enzyme systems: current trends and future prospects, Biotechnol. Adv., 19, 175e199 (2001). 4. Ishihara, K., Hamada, H., Hirata, T., and Nakajima, N.: Biotransformation using plant cultured cells, J. Mol. Catal. B-Enzym., 23, 145e170 (2003). 5. Ramachandra Rao, S. R. and Ravishankar, G. A.: Plant cell cultures: chemical factories of secondary metabolites, Biotechnol. Adv., 20, 101e153 (2002). 6. Liu, Y. K., Seki, M., Tanaka, H., and Furusaki, S.: Characteristics of loofa (Luffa cylindrica) sponge as a carrier for plant cell immobilization, J. Ferment. Bioeng., 85, 416e421 (1998).
508
BOU-SAAB ET AL.
7. Perullini, M., Rivero, M. M., Jobbágy, M., Mentaberry, A., and Bilmes, S. A.: Plant cell proliferation inside an inorganic host, J. Biotechnol., 127, 542e548 (2007). 8. Hideno, A., Ogbonna, J. C., Aoyagi, H., and Tanaka, H.: Acetylation of loofa (Luffa cylindrica) sponge as immobilization carrier for bioprocesses involving cellulose, J. Biosci. Bioeng., 103, 311e317 (2007). 9. Scott, J. A. and O’Reilly, A. M.: Co-Immobilization of selected yeast and bacteria for controlled flavour development in an alcoholic cider beverage, Process Biochem., 31, 111e117 (1996). 10. Suga, T., Hamada, H., and Hirata, T.: Oxidation-reduction relationship between cycloalkanones and the corresponding cycloalkanols in a cell suspension culture of Nicotiana tabacum, Plant Cell Rep., 2, 66e68 (1983).
J. BIOSCI. BIOENG., 11. Hirata, T., Hamada, H., Aoki, T., and Suga, T.: Stereoselectivity of the reduction of carvone and dihydrocarvone by suspension cells of Nicotiana tabacum, Phytochemistry, 21, 2209e2212 (1982). 12. Qian, D., Zhou, D., Ju, R., Cramer, C. L., and Yang, Z.: Protein farnesyltransferase in plants: molecular characterization and involvement in the cell cycle control, Plant Cell, 8, 2381e2394 (1996). 13. Blaschek, W., Haass, D., Koehler, H., and Franz, G.: Cell wall regeneration by Nicotiana tabacum protoplast: chemical and biochemical aspects, Plant Sci. Lett., 22, 47e57 (1981). 14. Guimarães, J. L., Frollini, E., da Silva, C. G., Wypych, F., and Satyanarayana, K. G.: Characterization of banana, sugarcane bagasse and sponge gourd fibers of Brazil, Ind. Crops Prod., 30, 407e415 (2009).