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Whole cell immobilization of Ralstonia pickettii for lipase production
Process Biochemistry 36 (2001) 629– 633 www.elsevier.com/locate/procbio
Whole cell immobilization of Ralstonia pickettii for lipase production Capiralla Hemachander, Niranjan Bose, Rengarajulu Puvanakrishnan * Department of Biotechnology, Central Leather Research Institute, Adyar, Madras 600020, India Received 14 February 2000; received in revised form 24 February 2000; accepted 18 October 2000
Abstract Different matrices viz. agar, alginate and polyacrylamide were examined for the immobilization of whole cells of Ralstonia pickettii. The enzyme activity of whole cells immobilized in agar beads was very low. Alginate beads had the inherent disadvantage of dissolving in phosphate based media and even glutaraldehyde treatment did not have any significant effect. A Tris– HCl system was found to be the best for alginate based immobilization of whole cells from R. pickettii and 4% alginate beads gave an optimal lipase activity of 14 U/ml per min. When different concentrations of polyacrylamide were tried for immobilization of R. pickettii whole cells, 15% polyacrylamide blocks showed a retention activity of 66% (25 U/ml per min) when compared to that of the free cells (40 U/ml per min). Bis-acrylamide concentration of 0.15 g/10 ml of buffer was ideal and the optimum whole cell concentration for polyacrylamide immobilization was 2.0 g/l of saline. Optimal immobilized whole cell concentration (polyacrylamide blocks) for lipase production was 20% and polyacrylamide blocks were reused three times effectively for lipase production. Of the three matrices examined for the immobilization of whole cells from R. pickettii, polyacrylamide gave the best performance. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Whole cell; Immobilization; Lipase; Polyacrylamide; Alginate; Agar; Ralstonia pickettii
1. Introduction Traditionally, enzyme processing has been accomplished using soluble cell free preparations, but these batch processes are often not economically viable because reuse of biocatalysts involve expensive ultrafiltration processes. One of the major activities in the area of biotechnology over the past few years has been the immobilization of enzymes and another recent development is the immobilization of whole cells. The rationale for choosing whole cell immobilization is not only because it eliminates enzyme purification and extraction steps, but that it also provides higher yields of enzyme activity after immobilization, higher operational stability, greater resistance to environmental perturbations and lower effective enzyme cost [1]. Alginate and agarose, both naturally occuring * Corresponding author. Tel.: +91-44-4430273; fax: + 91-444911589. E-mail address: [email protected] (R. Puvanakrishnan).
polysaccharides extracted from seaweed, are commonly used as hydrogels for a variety of applications. Agarose has long been used to culture animal cells and more recently, it has found use as an immobilizing matrix in bioreactors for the production of pharmaceuticals [2]. There are several reports on immobilization of microbial cells by entrapment in polyvinyl alcohol cryogels [3]. Cellulose acetate fibres have also been used for immobilization of viable whole cells of microorganisms [4]. The application of immobilized biocatalysts for the production of extracellular enzymes is less well documented than their application for bioconversions or for synthesis of useful low molecular mass components [5]. Little information is available about immobilization of microbial cells for extracellular lipase production [6,7]. In an earlier study, the crude lipase from Ralstonia pickettii was shown to possess considerable stability in the presence of commercial detergents and it could be used successfully as an additive to commercial detergents in laundry and in hydrolysis of oils [8,9]. The aim
0032-9592/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 0 ) 0 0 2 5 6 - 9
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C. Hemachander et al. / Process Biochemistry 36 (2001) 629–633
of this study was to select a suitable support material for the immobilization of whole cells from R. pickettii and optimise conditions for lipase production viz. stability of the immobilized blocks, inoculum concentration and time. In addition, studies have been taken up to perform reuse experiments with the immobilized blocks and to optimise conditions of reuse.
2.5. Preparation of alginate beads
2. Materials and methods
Fifty ml of the cell suspension (0.4 g/l) was mixed with 50 ml of 4% alginate solution prepared in saline. The slurry was extruded through a syringe into 250 ml of 0.2 M calcium chloride solution with constant stirring and the stirring was continued for 4 h. The beads were allowed to cure in 0.2 M calcium chloride solution for 24 h at 4°C. The beads were washed in sterile saline, transferred to NB and stored at 4°C. The beads were comparable in size to the agar beads.
2.1. Culture of cells
2.6. Preparation of polyacrylamide blocks
Soil bacteria, isolated from a rice field and identified earlier as R. pickettii was chosen for this study. A loopful of R. pickettii cells from the LB slant were inoculated into 50 ml of nutrient broth medium and incubated overnight at 37°C at 125 rpm. From this culture, 1 ml was added to each of the flasks containing production medium (50 ml) and incubated for 96 h at 30°C at 125 rpm. Production medium [10] contained the following: olive oil: 5 g/l, ammonium sulphate: 2 g/l, magnesium sulphate: 2.5 g/l, manganese sulphate: 5 mg/l, calcium chloride: 480 mg/l, ferrous sulphate: 15 mg/l, disodium hydrogen phosphate: 10 g/l, potassium dihydrogen phosphate: 20 g/l, pH: 7.0, distilled water: 1.0 l. Calcium chloride, ferrous sulphate and sodium and potassium phosphates were autoclaved separately to avoid precipitation. Production conditions were the same for immobilized cells.
To a sterile 15% solution of acrylamide monomer (14.25 g of acrylamide and 0.75 g of bis-acrylamide) in 0.05 M phosphate buffer (pH 6.5), 10 mg of ammonium persulphate was added. Ten microliters of tetramethyl ethylene diamine (TEMED) was then added followed by the addition of 10 ml of cell suspension (0.4 g/l) and the solution was mixed and poured onto a sterile plate. The contents were allowed to polymerise and cut into blocks (10 mm× 10 mm).
2.2. Har6esting of cells The culture was centrifuged at 12 000 rpm for 15 min at 4°C. The supernatant was discarded and the cell pellet was stored in saline (0.85% sodium chloride) solution at 4°C.
2.3. Estimation of lipase acti6ity Lipase activity was estimated by following the method of Ota and Yamada [11] and one unit of lipase activity is defined as 1 mmol of free fatty acid liberated per ml of enzyme per minute at 37°C.
2.4. Preparation of agar beads A total of 2.5 ml of the cell suspension (0.4 g/l) was mixed with 47.50 ml of 2.5% agar solution and the slurry was extruded through a syringe into a 250 ml of cold mixture of toluene:chloroform (3:1). The beads were washed with sterile saline and stored in NB at 4°C. The bead sizes were 0.5 cm3.
3. Results
3.1. Lipase acti6ity in 2.5% agar beads 2.5% agar was by far the most effective out of the different concentrations tried (data not shown). Enzyme activity in 2.5% agar beads was 4 U/ml per min at 72 h and 8.5 U/ml per min at 120 h. Activity increased between 96 and 120 h, but decrease at 144 h to 4 U/ml per min.
3.2. Immobilization of R. pickettii whole cells in alginate The alginate beads have an inherent problem of dissolving in phosphate solution, and hence, in order to harden the beads, they were treated with glutaraldehyde. The 4% alginate beads treated with 0.5% glutaraldehyde dissolved in the production medium after 48 h (data not shown). The 4% alginate beads dissolved in production medium containing 2 and 1% KH2PO4 and Na2HPO4, respectively, and 1 and 0.5% KH2PO4 and Na2HPO4, respectively, but they were observed to be stable in production medium containing 0.5 and 0.25% KH2PO4 and Na2HPO4, respectively, and 0.25 and 0.125% KH2PO4 and Na2HPO4, respectively. The activity of the enzyme was higher when production medium containing 0.5 and 0.25% KH2PO4 and Na2HPO4, respectively, was used (Fig. 1). The alginate beads dissolved in the presence of maleate, succinate and citrate salts much before the completion of 48 h.
C. Hemachander et al. / Process Biochemistry 36 (2001) 629–633
631
Fig. 3. Effect of polyacrylamide (total monomer) concentration on lipase production.
Fig. 1. Effect of different salt systems in production medium on 4% alginate beads.
The 4% alginate beads were stable and exhibited a higher activity in Tris – HCl system than that observed in the presence of phosphates. The 5% inoculum was ideal for lipase production at 96 h (Fig. 2). The immobilized calcium alginate beads were reused thrice and a lipase activity of 14, 12 and 11.5 U/min per ml were observed for first, second and third uses respectively. An activity loss of 10% was observed after each consecutive use (data not shown).
Fig. 2. Effect of inoculum concentration on lipase production using calcium alginate beads.
3.3. Polyacrylamide as matrix for immobilization of R. pickettii whole cells A decreased activity was observed when a polyacrylamide concentration of 10% was used and optimal lipase production was achieved when 15 or 20% gels were used. Further increase in polyacrylamide concentration to 30% led to a high release of heat during polymerisation and hence, higher concentrations were not tested. 15% w/v of total monomers was chosen as the optimum concentration. The amount of lipase produced by 15% w/v polyacrylamide gels was nearly 66% of the free cell activity (40 U/ml) and the lipase activity increased till 96 h, beyond which there was a fall in activity (Fig. 3). Increase in concentration of crosslinking agent (bis-acrylamide) resulted in a highly crosslinked polymeric network, which in turn reduced leakage of cells and porosity. There was an increase in lipase production when the bis-acrylamide concentration was increased to 0.15 g/10 ml of buffer at 72 h, but no change was noticed upon further increase (Fig. 4). Lipase production increased with increase in cell concentration till 2.0 g/l of saline, and a decrease in activity was observed beyond 2.0g/l. The optimum cell concentration was found to be 2.0 g/l of saline and an optimal activity of 24.75 U was achieved at the end of 96 h (Fig. 5). There was a significant increase in activity at 96 h when the concentration of immobilized cells (polyacrylamide blocks) was increased up to 20% and above that level, a decrease in activity was observed (Fig. 6). Lipase activity was retained during three successive uses with just 10% loss in activity for each use. The first use resulted in an activity of 24.25 U/ml per min and second, third and fourth uses resulted in 22.75, 19.75 and 13.25 U/ml per min, respectively (data not shown).
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C. Hemachander et al. / Process Biochemistry 36 (2001) 629–633
Fig. 6. Effect of immobilized whole cell concentration on lipase production using polyacrylamide blocks.
Fig. 4. Effect of crosslinker concentration on lipase production.
4. Discussion Agar was used for the entrapment of cells in the form of spherical beads, blocks and membranes [12]. When R. pickettii cells were immobilized in 2.5% agar beads, there was no substantial production of lipase probably due to cell death caused by thermal shock, while suspending cells in molten agar which is at a higher temperature. Thus, entrapment in agar does not seem to be a viable alternative for immobilization of R. pickettii cells for lipase production. The optimum pH for lipase production by R. pickettii is 6.5 and to maintain this pH in the medium, a mixture of dibasic and monobasic phosphate solution
Fig. 5. Effect of inoculum concentration on lipase production using polyacrylamide blocks.
was used. However, this posed the problem of alginate dissolving in phosphate solution and calcium precipitation as calcium phosphate. To avoid such precipitation and to strengthen the beads, crosslinking with glutaraldehyde was tested. Even with 1% glutaraldehyde, which can be toxic to the cells, the beads tended to dissolve. This suggested that the phosphate concentration needed to be reduced and by reducing the phosphate concentration in the production medium to 0.5 and 0.25% KH2PO4 and Na2HPO4, respectively, 4% Ca-alginate beads were found to be stable and they showed higher activity. Replacing the phosphates in production medium with maleate, citrate and succinate did not have much effect on the chemical stability of the Ca-alginate beads while the Tris –HCl system was much better than the other systems tested. Some of the observations of this study showing calcium alginate beads dissolving in phosphate buffer are in accordance with an earlier report on immobilized Candida rugosa cells for lipase production by Ferrer and Sola [13].The merits and demerits of immobilization of whole cells using Ca-alginate system have been critically reviewed [14]. Polyacrylamide, the most commonly used matrix for the entrapment of enzymes, has the property of being non-ionic [15]. A number of procedures for the entrapment of enzymes in polyacrylamide gels have been reported [16,17]. Systems similar to those described for enzymes could be used for immobilization of whole cells, both viable and non viable [15]. However, loss in viability is encountered as a result of the toxicity of the monomers used to form polyacrylamide gel and the heat evolved during polymerization. In an attempt to improve the production capability of immobilized R. pickettii cells, polyacrylamide gels have been tested. It was observed that with 15% polyacrylamide, R. pickettii cells produced nearly 66% of the free cell activity which
C. Hemachander et al. / Process Biochemistry 36 (2001) 629–633
was an improvement over alginate immobilized cells. The decrease in activity observed with 10% polyacrylamide gels could possibly be due to a leakage of cells as a result of the large pore size. At acrylamide concentrations higher than 20%, considerable heat was generated during polymerization and this could adversely affect cell viability and consequently lipase production [17]. Increase in bis-acrylamide concentration to 0.15 g/10 ml resulted in an increase in production but concentrations above this level had no effect probably because a concentration of 0.15 g/10 ml could have resulted in an ideal pore size to entrap R. pickettii cells with minimum leakage. From the reuse experiments with both alginate and acrylamide immobilized cells, it was clear that immobilized whole cells were reasonably stable because there was only a loss of 10% activity between each use. This loss in activity after each use could be countered by replenishing the required amount of cells into the medium. This study shows that a polyacrylamide matrix was better than alginate and agar for whole cell immobilization. Lipase production was around 24.75 U/ml per min. R. pickettii whole cells immobilized in a polyacrylamide matrix could be reused effectively three times for lipase production with a loss of 10% activity for each consecutive use.
Acknowledgements The authors thank Dr T. Ramasami, Director, Central Leather Research Institute, Madras for his kind permission to publish this work. The financial assistance extended by the Department of Biotechnology, Government of India to Mr C. Hemachander is gratefully acknowledged.
References [1] Kierstan MPJ, Coughlan MP. Immobilization of cells and enzymes by gel entrapment. In: Woodward J, editor. Immobilized Cells and Enzymes: A Practical Approach. Oxford, UK: IRL Press, 1985.
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[2] Li HR, Altreuter HD, Gentile TF. Transport characterization of hydrogel matrices for cell encapsulation. Biotechnol Bioeng 1996;50:365– 73. [3] Rainina EI, Pusheva AM, Ryabokon MA, Bolotina PN, Lozinsky IV, Varfolomeyev DS. Microbial cells immobilized in poly (vinyl alcohol) cryogels: biocatalytic reduction of CO2 by the thermophilic homoacetogenic bacterium Acetogenium ki6ui. Biotechnol Appl Biochem 1994;19:321– 9. [4] Kozlyak EI, Solomon ZG, Yakimov MM, Fadyushina TV, Germanskii GI, Utkin IB, Rogozhin IS, Biber BL, Bezborodov AM. Entrapment of Pseudomonas fluorescens 16N2 cells into cellulose triacetate fibers during the forming process. Trans Prikladnaya Biokhim Mikbiol 1991;27:210– 5. [5] Linko YY, Li GX, Zhong LC, Linko S, Linko P. Enzyme production by immobilized cells. Methods Enzymol 1985;137:686– 96. [6] Lechner M, Markl H, Gotz F. Lipase production of Staphylococcus carnosus in a dialysis fermentor. Appl Microbiol Biotechnol 1988;28:345– 9. [7] Jhori BN, Alurrald JD, Klein J. Lipase production by free and immobilized protoplasts of Sporotrichum (Chrysopodium) thermophile Apinis. Appl Microbiol Biotechnol 1990;33:367–71. [8] Hemachander C, Puvanakrishnan R. Lipase from Ralstonia pickettii as an additive in laundry detergent formulations. Process Biochemistry, 1999. Process Biochem 2000;35:809–14. [9] Hemachander, C., Naidu, R.B., Puvanakrishnan, R., 1999. A process for the production of a novel lipase. Indian Patent no. 1555 DEL 99. [10] Christenson L, Dionne K, Lysaught M. Biomedical application of immobilized cells. In: Goosen MFA, editor. Fundamentals of Animal Cell Encapsulation and Immobilization. Boca Raton, FL: CRC Press, 1993:7– 41. [11] Ota Y, Yamada K. Lipase from Candida paralipolytica Part I. Anionic surfactants as the essential activator in the systems emulsified by polyvinyl alcohol. Agric Biol Chem 1966;30:351–8. [12] Ishihara K, Suzuki T, Yamane T, Shimuzu S. Effective production of Pseudomonas fluorescens lipase by semi-batch culture with turbidity dependent automatic feeding of both olive oil and iron ion. Appl Microbiol Biotechnol 1989;31:45– 8. [13] Ferrer P, Sola C. Lipase production by immobized Candida rugosa cells. Appl Microbiol Biotechnol 1992;37:737– 41. [14] Mattiasson B. Immobilized Cells and Organelles, vol. I/II. Boca Raton, FL: CRC Press, 1982. [15] Trevan MD. Immobilized Enzymes: An Introduction and Application in Biotechnology. New York: Wiley, 1980. [16] Pizarro C, Fernandez-Torroba MA, Benito C, Gonzalez-Saiz JM. Optimization by experimental design of polyacrylamide gel composition as support for enzyme immobilization by entrapment. Biotechnol Bioeng 1997;53:497– 510. [17] Freeman A, Aharonowitz Y. Immobilization of microbial cells in crosslinked, prepolymerized, linear polyacrylamide gels: antibiotic production by immobilzed Streptomyces daruligerus cells. Biotechnol Bioeng 1981;23:2747– 50.
Whole cell immobilization of Ralstonia pickettii for lipase production Capiralla Hemachander, Niranjan Bose, Rengarajulu Puvanakrishnan * Department of Biotechnology, Central Leather Research Institute, Adyar, Madras 600020, India Received 14 February 2000; received in revised form 24 February 2000; accepted 18 October 2000
Abstract Different matrices viz. agar, alginate and polyacrylamide were examined for the immobilization of whole cells of Ralstonia pickettii. The enzyme activity of whole cells immobilized in agar beads was very low. Alginate beads had the inherent disadvantage of dissolving in phosphate based media and even glutaraldehyde treatment did not have any significant effect. A Tris– HCl system was found to be the best for alginate based immobilization of whole cells from R. pickettii and 4% alginate beads gave an optimal lipase activity of 14 U/ml per min. When different concentrations of polyacrylamide were tried for immobilization of R. pickettii whole cells, 15% polyacrylamide blocks showed a retention activity of 66% (25 U/ml per min) when compared to that of the free cells (40 U/ml per min). Bis-acrylamide concentration of 0.15 g/10 ml of buffer was ideal and the optimum whole cell concentration for polyacrylamide immobilization was 2.0 g/l of saline. Optimal immobilized whole cell concentration (polyacrylamide blocks) for lipase production was 20% and polyacrylamide blocks were reused three times effectively for lipase production. Of the three matrices examined for the immobilization of whole cells from R. pickettii, polyacrylamide gave the best performance. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Whole cell; Immobilization; Lipase; Polyacrylamide; Alginate; Agar; Ralstonia pickettii
1. Introduction Traditionally, enzyme processing has been accomplished using soluble cell free preparations, but these batch processes are often not economically viable because reuse of biocatalysts involve expensive ultrafiltration processes. One of the major activities in the area of biotechnology over the past few years has been the immobilization of enzymes and another recent development is the immobilization of whole cells. The rationale for choosing whole cell immobilization is not only because it eliminates enzyme purification and extraction steps, but that it also provides higher yields of enzyme activity after immobilization, higher operational stability, greater resistance to environmental perturbations and lower effective enzyme cost [1]. Alginate and agarose, both naturally occuring * Corresponding author. Tel.: +91-44-4430273; fax: + 91-444911589. E-mail address: [email protected] (R. Puvanakrishnan).
polysaccharides extracted from seaweed, are commonly used as hydrogels for a variety of applications. Agarose has long been used to culture animal cells and more recently, it has found use as an immobilizing matrix in bioreactors for the production of pharmaceuticals [2]. There are several reports on immobilization of microbial cells by entrapment in polyvinyl alcohol cryogels [3]. Cellulose acetate fibres have also been used for immobilization of viable whole cells of microorganisms [4]. The application of immobilized biocatalysts for the production of extracellular enzymes is less well documented than their application for bioconversions or for synthesis of useful low molecular mass components [5]. Little information is available about immobilization of microbial cells for extracellular lipase production [6,7]. In an earlier study, the crude lipase from Ralstonia pickettii was shown to possess considerable stability in the presence of commercial detergents and it could be used successfully as an additive to commercial detergents in laundry and in hydrolysis of oils [8,9]. The aim
0032-9592/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 0 ) 0 0 2 5 6 - 9
630
C. Hemachander et al. / Process Biochemistry 36 (2001) 629–633
of this study was to select a suitable support material for the immobilization of whole cells from R. pickettii and optimise conditions for lipase production viz. stability of the immobilized blocks, inoculum concentration and time. In addition, studies have been taken up to perform reuse experiments with the immobilized blocks and to optimise conditions of reuse.
2.5. Preparation of alginate beads
2. Materials and methods
Fifty ml of the cell suspension (0.4 g/l) was mixed with 50 ml of 4% alginate solution prepared in saline. The slurry was extruded through a syringe into 250 ml of 0.2 M calcium chloride solution with constant stirring and the stirring was continued for 4 h. The beads were allowed to cure in 0.2 M calcium chloride solution for 24 h at 4°C. The beads were washed in sterile saline, transferred to NB and stored at 4°C. The beads were comparable in size to the agar beads.
2.1. Culture of cells
2.6. Preparation of polyacrylamide blocks
Soil bacteria, isolated from a rice field and identified earlier as R. pickettii was chosen for this study. A loopful of R. pickettii cells from the LB slant were inoculated into 50 ml of nutrient broth medium and incubated overnight at 37°C at 125 rpm. From this culture, 1 ml was added to each of the flasks containing production medium (50 ml) and incubated for 96 h at 30°C at 125 rpm. Production medium [10] contained the following: olive oil: 5 g/l, ammonium sulphate: 2 g/l, magnesium sulphate: 2.5 g/l, manganese sulphate: 5 mg/l, calcium chloride: 480 mg/l, ferrous sulphate: 15 mg/l, disodium hydrogen phosphate: 10 g/l, potassium dihydrogen phosphate: 20 g/l, pH: 7.0, distilled water: 1.0 l. Calcium chloride, ferrous sulphate and sodium and potassium phosphates were autoclaved separately to avoid precipitation. Production conditions were the same for immobilized cells.
To a sterile 15% solution of acrylamide monomer (14.25 g of acrylamide and 0.75 g of bis-acrylamide) in 0.05 M phosphate buffer (pH 6.5), 10 mg of ammonium persulphate was added. Ten microliters of tetramethyl ethylene diamine (TEMED) was then added followed by the addition of 10 ml of cell suspension (0.4 g/l) and the solution was mixed and poured onto a sterile plate. The contents were allowed to polymerise and cut into blocks (10 mm× 10 mm).
2.2. Har6esting of cells The culture was centrifuged at 12 000 rpm for 15 min at 4°C. The supernatant was discarded and the cell pellet was stored in saline (0.85% sodium chloride) solution at 4°C.
2.3. Estimation of lipase acti6ity Lipase activity was estimated by following the method of Ota and Yamada [11] and one unit of lipase activity is defined as 1 mmol of free fatty acid liberated per ml of enzyme per minute at 37°C.
2.4. Preparation of agar beads A total of 2.5 ml of the cell suspension (0.4 g/l) was mixed with 47.50 ml of 2.5% agar solution and the slurry was extruded through a syringe into a 250 ml of cold mixture of toluene:chloroform (3:1). The beads were washed with sterile saline and stored in NB at 4°C. The bead sizes were 0.5 cm3.
3. Results
3.1. Lipase acti6ity in 2.5% agar beads 2.5% agar was by far the most effective out of the different concentrations tried (data not shown). Enzyme activity in 2.5% agar beads was 4 U/ml per min at 72 h and 8.5 U/ml per min at 120 h. Activity increased between 96 and 120 h, but decrease at 144 h to 4 U/ml per min.
3.2. Immobilization of R. pickettii whole cells in alginate The alginate beads have an inherent problem of dissolving in phosphate solution, and hence, in order to harden the beads, they were treated with glutaraldehyde. The 4% alginate beads treated with 0.5% glutaraldehyde dissolved in the production medium after 48 h (data not shown). The 4% alginate beads dissolved in production medium containing 2 and 1% KH2PO4 and Na2HPO4, respectively, and 1 and 0.5% KH2PO4 and Na2HPO4, respectively, but they were observed to be stable in production medium containing 0.5 and 0.25% KH2PO4 and Na2HPO4, respectively, and 0.25 and 0.125% KH2PO4 and Na2HPO4, respectively. The activity of the enzyme was higher when production medium containing 0.5 and 0.25% KH2PO4 and Na2HPO4, respectively, was used (Fig. 1). The alginate beads dissolved in the presence of maleate, succinate and citrate salts much before the completion of 48 h.
C. Hemachander et al. / Process Biochemistry 36 (2001) 629–633
631
Fig. 3. Effect of polyacrylamide (total monomer) concentration on lipase production.
Fig. 1. Effect of different salt systems in production medium on 4% alginate beads.
The 4% alginate beads were stable and exhibited a higher activity in Tris – HCl system than that observed in the presence of phosphates. The 5% inoculum was ideal for lipase production at 96 h (Fig. 2). The immobilized calcium alginate beads were reused thrice and a lipase activity of 14, 12 and 11.5 U/min per ml were observed for first, second and third uses respectively. An activity loss of 10% was observed after each consecutive use (data not shown).
Fig. 2. Effect of inoculum concentration on lipase production using calcium alginate beads.
3.3. Polyacrylamide as matrix for immobilization of R. pickettii whole cells A decreased activity was observed when a polyacrylamide concentration of 10% was used and optimal lipase production was achieved when 15 or 20% gels were used. Further increase in polyacrylamide concentration to 30% led to a high release of heat during polymerisation and hence, higher concentrations were not tested. 15% w/v of total monomers was chosen as the optimum concentration. The amount of lipase produced by 15% w/v polyacrylamide gels was nearly 66% of the free cell activity (40 U/ml) and the lipase activity increased till 96 h, beyond which there was a fall in activity (Fig. 3). Increase in concentration of crosslinking agent (bis-acrylamide) resulted in a highly crosslinked polymeric network, which in turn reduced leakage of cells and porosity. There was an increase in lipase production when the bis-acrylamide concentration was increased to 0.15 g/10 ml of buffer at 72 h, but no change was noticed upon further increase (Fig. 4). Lipase production increased with increase in cell concentration till 2.0 g/l of saline, and a decrease in activity was observed beyond 2.0g/l. The optimum cell concentration was found to be 2.0 g/l of saline and an optimal activity of 24.75 U was achieved at the end of 96 h (Fig. 5). There was a significant increase in activity at 96 h when the concentration of immobilized cells (polyacrylamide blocks) was increased up to 20% and above that level, a decrease in activity was observed (Fig. 6). Lipase activity was retained during three successive uses with just 10% loss in activity for each use. The first use resulted in an activity of 24.25 U/ml per min and second, third and fourth uses resulted in 22.75, 19.75 and 13.25 U/ml per min, respectively (data not shown).
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C. Hemachander et al. / Process Biochemistry 36 (2001) 629–633
Fig. 6. Effect of immobilized whole cell concentration on lipase production using polyacrylamide blocks.
Fig. 4. Effect of crosslinker concentration on lipase production.
4. Discussion Agar was used for the entrapment of cells in the form of spherical beads, blocks and membranes [12]. When R. pickettii cells were immobilized in 2.5% agar beads, there was no substantial production of lipase probably due to cell death caused by thermal shock, while suspending cells in molten agar which is at a higher temperature. Thus, entrapment in agar does not seem to be a viable alternative for immobilization of R. pickettii cells for lipase production. The optimum pH for lipase production by R. pickettii is 6.5 and to maintain this pH in the medium, a mixture of dibasic and monobasic phosphate solution
Fig. 5. Effect of inoculum concentration on lipase production using polyacrylamide blocks.
was used. However, this posed the problem of alginate dissolving in phosphate solution and calcium precipitation as calcium phosphate. To avoid such precipitation and to strengthen the beads, crosslinking with glutaraldehyde was tested. Even with 1% glutaraldehyde, which can be toxic to the cells, the beads tended to dissolve. This suggested that the phosphate concentration needed to be reduced and by reducing the phosphate concentration in the production medium to 0.5 and 0.25% KH2PO4 and Na2HPO4, respectively, 4% Ca-alginate beads were found to be stable and they showed higher activity. Replacing the phosphates in production medium with maleate, citrate and succinate did not have much effect on the chemical stability of the Ca-alginate beads while the Tris –HCl system was much better than the other systems tested. Some of the observations of this study showing calcium alginate beads dissolving in phosphate buffer are in accordance with an earlier report on immobilized Candida rugosa cells for lipase production by Ferrer and Sola [13].The merits and demerits of immobilization of whole cells using Ca-alginate system have been critically reviewed [14]. Polyacrylamide, the most commonly used matrix for the entrapment of enzymes, has the property of being non-ionic [15]. A number of procedures for the entrapment of enzymes in polyacrylamide gels have been reported [16,17]. Systems similar to those described for enzymes could be used for immobilization of whole cells, both viable and non viable [15]. However, loss in viability is encountered as a result of the toxicity of the monomers used to form polyacrylamide gel and the heat evolved during polymerization. In an attempt to improve the production capability of immobilized R. pickettii cells, polyacrylamide gels have been tested. It was observed that with 15% polyacrylamide, R. pickettii cells produced nearly 66% of the free cell activity which
C. Hemachander et al. / Process Biochemistry 36 (2001) 629–633
was an improvement over alginate immobilized cells. The decrease in activity observed with 10% polyacrylamide gels could possibly be due to a leakage of cells as a result of the large pore size. At acrylamide concentrations higher than 20%, considerable heat was generated during polymerization and this could adversely affect cell viability and consequently lipase production [17]. Increase in bis-acrylamide concentration to 0.15 g/10 ml resulted in an increase in production but concentrations above this level had no effect probably because a concentration of 0.15 g/10 ml could have resulted in an ideal pore size to entrap R. pickettii cells with minimum leakage. From the reuse experiments with both alginate and acrylamide immobilized cells, it was clear that immobilized whole cells were reasonably stable because there was only a loss of 10% activity between each use. This loss in activity after each use could be countered by replenishing the required amount of cells into the medium. This study shows that a polyacrylamide matrix was better than alginate and agar for whole cell immobilization. Lipase production was around 24.75 U/ml per min. R. pickettii whole cells immobilized in a polyacrylamide matrix could be reused effectively three times for lipase production with a loss of 10% activity for each consecutive use.
Acknowledgements The authors thank Dr T. Ramasami, Director, Central Leather Research Institute, Madras for his kind permission to publish this work. The financial assistance extended by the Department of Biotechnology, Government of India to Mr C. Hemachander is gratefully acknowledged.
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