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Immobilization of Aspergillus niger and Phanerochaete chrysosporium on polyurethane foam.
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R.H. Wijffels, R.M. Buitelaar, C. Bucke and J. Tramper (Eds) Immobilized Cells: Basics and Applications © 1996 Elsevier Science B.V. All rights reserved.
Immobilization of Aspergillus niger and Phanerochaete chrysosporium on polyurethane foam. A. Sanromanl, G. Feijoo^, and J.M. Lema^. ^Dept. of Chemical Engineering. University of Vigo. E-36200 Vigo. Galiza (Spain). ^Dept. of Chemical Engineering. University of Santiago de Compostela. E-15706 Santiago de Compostela. Galiza (Spain).
Abstract The immobilization of Aspergillus niger and Phanerochaete chrysosporium on polyurethane foam is considered. Two different methodologies, which affects the development of the fiangal hyphae, were investigated. The different morphology of the obtained bioparticles seriously modify the productivity of citric acid and extracellular peroxidases (Lignin Peroxidase, LiP, and Manganese Peroxidase, MnP) by A. niger and P. chrysosporium, respectively. The best results are obtained in both cases, when fungi developed inside the cube foam. Introduction In submerged cultures of filamentous fungi, cells form strands of interlocking hyphae resulting in a very viscous solution, which leads to poor oxygen mass transfer. Keeping filamentous fungi in pellet form or immobilizing them in solid supports reduces viscosity of the broth substantially, facilitating oxygen transfer and causing less operational problems [1]. The adequate selection of the immobilization technique, permits better productivities and stability to be obtained when operated in continuous processes. Polyurethane foam is considered a suitable carrier for immobilizing fungi due to its high porosity which allows its specific surface to be increased as well as the protection offtingifrom shear forces [2]. Aspergillus niger and Phanerochaete chrysosporium are representative fiingi of primary (citric acid) and secondary (extracellular peroxidases) metabolites production, respectively [3,4]. The objective of this work is to study the immobilization of A. niger and P. chrysosporium on polyurethane foam. Materials and methods Microorganism. A. niger CBS 733-88 and P.chrysosporium BKM-F-1767 (ATCC 24725) were maintained at 30°C and 37°C, respectively, on 2% malt agar slants.
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Support characteristics. The support for immobilization consisted of 0.5 cm^ cubes of polyurethane foam (Copo Iberica, Vigo, Spain) with a density of 20 kg m"^ and a surface area of 415±10 m^/m^. Surface area was determined by Micromeritics ASAP 2000 with degasificated foam cubes at room temperature and at pressure of 10^^ mmHg. Prior to use, the cubes were washed once in methanol, then three times in double distilled water, autoclaved and dried until use. Culture conditions. Aspergillus niger. The standard medium used was previously described [5], and contains 50 g 1" 1 of glucose and 1.14 g 1"^ of ammonia nitrogen. Erlenmeyer flasks (300 ml) containing 50 ml of medium and 1.0 g of polyurethane foam were inoculated with a final concentration of 10^ spores ml"l and sealed with rubber stoppers. They were kept in an orbital shaker (New Brunswick Scientific, NJ) at 30 °C and 50, 100, 150 and 200 rpm. Phanerochaete chrysosporwm: A Nitrogen-limited medium [6] with 10 g 1"! of glucose in a 20 mM sodium acetate buffer (pH 4.5) was used. Erlenmeyer flasks (250 ml) containing 90 ml of medium and 0.9 or 1.8 g of polyurethane foam were inoculated with 10% (vol/vol) homogeneized mycelium and sealed with rubber stoppers. The headspace was aseptically flushed with O2 gas (0.5 bar manometric pressure) for 3 min at the time of inoculation and afterwards once a day for 3 days. All cultures were performed with agitation (150 rpm) in an orbital shaker (New Brunswick Scientific, NJ) at 37 °C. Scanning electronic microscopy. Polyurethane cubes were cut tranversally and mycelia was fixed for two hours in 5% glutaraldehyde with 0.1 M phosphate buffer at 4 °C and pH 7.5. Afterwards it was dried in water:ethanol mixtures of 20, 40, 60, 80 and 100% ethanol. Samples were examined with scanning electron microscopy according to the conditions described by Basking/a/. [7]. Analytical techniques. Citric acid was determined by the Marier and Boulet method [8]. Manganese Peroxidase activity was measured as described by Kuwahara et al [9] with phenol red as substrate. Results and discussion The morphology of the bioparticles obtained by two different immobilization techniques was analyzed in two experiments according to the different agitation {A. niger) and foam.liquid ratio (P. chrysosporium) employed. The slow agitation (50 and 100 rpm) in cultures of ^. niger, and the immobilization with a low foam:liquid ratio (characteristic of submerged cultures) in P. chrysosporium, made oxygen availability very low, which implied the formation of a thick biofilm of mycelia around the foam (Figure 1).
134
Figure 1. A. niger immobilized in cultures at low (left) and high (right) agitated conditions. As a result, the lower external surface increased diffusional problems and disabled substrate exchange within the medium which could explain the low citric acid production and and the low peroxidases activities achieved (Figure 2).
2
4 6 8 Time (days)
10
12
50
100 150 200 Time (hours)
250
Figure 2. Citric acid production by A. niger and Peroxidases activities (LiP and MnP) by P. chrysosporium immobilized at low (open symbols) and high (closed symbols) oxygen availability conditions. In contrast, when a high foam:liquid ratio (non-submerged culture) or higher agitation speed (150 and 200 rpm) were considered, oxygen transfer is considerably increased, which allowed the fungal hyphae became entrapped inside the foam (Figures 1 and 3); consequently both citric acid and peroxidases production were improved (Figure 2).
135
Figure 3. Scanning electronic microscope photograph of entrapped mycelium of P. chrysosporium within foam cube Besides, this thin biofilm entrapped into foam cubes allowed a proteolytic system of P. chrysosporium to be secreted, which finally released available nitrogen into the extracellular medium, and induced C-starvation conditions (data not shown). These conditions triggered again the synthesis of ligninolytic enzymatic complex, at a similar or even higher level of activity to that of the beginning of secondary metabolism (Figure 2). Acknowlegements This work was funded by the Spanish Commission of Science and Technology (CICYT), (Project BI095-377). References 1 2 3 4 5 6 7 8 9
Prosser JI, Tough AJ. Crit Rev Biotechnol 1991; 10: 253-274. Asther M, Bellon-Fontaine M, Capdevila C, Corrieu G. Biotechnol Bioeng 1990; 35: 477482. Kubicek CP, Rohr M.. CRC Crit Rev Biotechnol 1986; 3: 331-373. Reddy CA. J Biotechnol 1993;. Sanroman A, Pintado J, Lema JM. Biotechnol Tech 1994; 8: 389-394. Tien M, Kirk TK. Methods Enzymol 1988; 161: 238-248. Baskin DG, Henderson M, Kenneth CG, Fujimoto WT. J Histochem Cytochem 1982; 30: 710-712. Marier JR, Boulet M J. Dairy Sci. 1958; 41: 1683-1692. Kuwahara M, Glenn JK, Morgan MA, Gold HH. FEBS Lett 1984; 169: 247-250.
R.H. Wijffels, R.M. Buitelaar, C. Bucke and J. Tramper (Eds) Immobilized Cells: Basics and Applications © 1996 Elsevier Science B.V. All rights reserved.
Immobilization of Aspergillus niger and Phanerochaete chrysosporium on polyurethane foam. A. Sanromanl, G. Feijoo^, and J.M. Lema^. ^Dept. of Chemical Engineering. University of Vigo. E-36200 Vigo. Galiza (Spain). ^Dept. of Chemical Engineering. University of Santiago de Compostela. E-15706 Santiago de Compostela. Galiza (Spain).
Abstract The immobilization of Aspergillus niger and Phanerochaete chrysosporium on polyurethane foam is considered. Two different methodologies, which affects the development of the fiangal hyphae, were investigated. The different morphology of the obtained bioparticles seriously modify the productivity of citric acid and extracellular peroxidases (Lignin Peroxidase, LiP, and Manganese Peroxidase, MnP) by A. niger and P. chrysosporium, respectively. The best results are obtained in both cases, when fungi developed inside the cube foam. Introduction In submerged cultures of filamentous fungi, cells form strands of interlocking hyphae resulting in a very viscous solution, which leads to poor oxygen mass transfer. Keeping filamentous fungi in pellet form or immobilizing them in solid supports reduces viscosity of the broth substantially, facilitating oxygen transfer and causing less operational problems [1]. The adequate selection of the immobilization technique, permits better productivities and stability to be obtained when operated in continuous processes. Polyurethane foam is considered a suitable carrier for immobilizing fungi due to its high porosity which allows its specific surface to be increased as well as the protection offtingifrom shear forces [2]. Aspergillus niger and Phanerochaete chrysosporium are representative fiingi of primary (citric acid) and secondary (extracellular peroxidases) metabolites production, respectively [3,4]. The objective of this work is to study the immobilization of A. niger and P. chrysosporium on polyurethane foam. Materials and methods Microorganism. A. niger CBS 733-88 and P.chrysosporium BKM-F-1767 (ATCC 24725) were maintained at 30°C and 37°C, respectively, on 2% malt agar slants.
133
Support characteristics. The support for immobilization consisted of 0.5 cm^ cubes of polyurethane foam (Copo Iberica, Vigo, Spain) with a density of 20 kg m"^ and a surface area of 415±10 m^/m^. Surface area was determined by Micromeritics ASAP 2000 with degasificated foam cubes at room temperature and at pressure of 10^^ mmHg. Prior to use, the cubes were washed once in methanol, then three times in double distilled water, autoclaved and dried until use. Culture conditions. Aspergillus niger. The standard medium used was previously described [5], and contains 50 g 1" 1 of glucose and 1.14 g 1"^ of ammonia nitrogen. Erlenmeyer flasks (300 ml) containing 50 ml of medium and 1.0 g of polyurethane foam were inoculated with a final concentration of 10^ spores ml"l and sealed with rubber stoppers. They were kept in an orbital shaker (New Brunswick Scientific, NJ) at 30 °C and 50, 100, 150 and 200 rpm. Phanerochaete chrysosporwm: A Nitrogen-limited medium [6] with 10 g 1"! of glucose in a 20 mM sodium acetate buffer (pH 4.5) was used. Erlenmeyer flasks (250 ml) containing 90 ml of medium and 0.9 or 1.8 g of polyurethane foam were inoculated with 10% (vol/vol) homogeneized mycelium and sealed with rubber stoppers. The headspace was aseptically flushed with O2 gas (0.5 bar manometric pressure) for 3 min at the time of inoculation and afterwards once a day for 3 days. All cultures were performed with agitation (150 rpm) in an orbital shaker (New Brunswick Scientific, NJ) at 37 °C. Scanning electronic microscopy. Polyurethane cubes were cut tranversally and mycelia was fixed for two hours in 5% glutaraldehyde with 0.1 M phosphate buffer at 4 °C and pH 7.5. Afterwards it was dried in water:ethanol mixtures of 20, 40, 60, 80 and 100% ethanol. Samples were examined with scanning electron microscopy according to the conditions described by Basking/a/. [7]. Analytical techniques. Citric acid was determined by the Marier and Boulet method [8]. Manganese Peroxidase activity was measured as described by Kuwahara et al [9] with phenol red as substrate. Results and discussion The morphology of the bioparticles obtained by two different immobilization techniques was analyzed in two experiments according to the different agitation {A. niger) and foam.liquid ratio (P. chrysosporium) employed. The slow agitation (50 and 100 rpm) in cultures of ^. niger, and the immobilization with a low foam:liquid ratio (characteristic of submerged cultures) in P. chrysosporium, made oxygen availability very low, which implied the formation of a thick biofilm of mycelia around the foam (Figure 1).
134
Figure 1. A. niger immobilized in cultures at low (left) and high (right) agitated conditions. As a result, the lower external surface increased diffusional problems and disabled substrate exchange within the medium which could explain the low citric acid production and and the low peroxidases activities achieved (Figure 2).
2
4 6 8 Time (days)
10
12
50
100 150 200 Time (hours)
250
Figure 2. Citric acid production by A. niger and Peroxidases activities (LiP and MnP) by P. chrysosporium immobilized at low (open symbols) and high (closed symbols) oxygen availability conditions. In contrast, when a high foam:liquid ratio (non-submerged culture) or higher agitation speed (150 and 200 rpm) were considered, oxygen transfer is considerably increased, which allowed the fungal hyphae became entrapped inside the foam (Figures 1 and 3); consequently both citric acid and peroxidases production were improved (Figure 2).
135
Figure 3. Scanning electronic microscope photograph of entrapped mycelium of P. chrysosporium within foam cube Besides, this thin biofilm entrapped into foam cubes allowed a proteolytic system of P. chrysosporium to be secreted, which finally released available nitrogen into the extracellular medium, and induced C-starvation conditions (data not shown). These conditions triggered again the synthesis of ligninolytic enzymatic complex, at a similar or even higher level of activity to that of the beginning of secondary metabolism (Figure 2). Acknowlegements This work was funded by the Spanish Commission of Science and Technology (CICYT), (Project BI095-377). References 1 2 3 4 5 6 7 8 9
Prosser JI, Tough AJ. Crit Rev Biotechnol 1991; 10: 253-274. Asther M, Bellon-Fontaine M, Capdevila C, Corrieu G. Biotechnol Bioeng 1990; 35: 477482. Kubicek CP, Rohr M.. CRC Crit Rev Biotechnol 1986; 3: 331-373. Reddy CA. J Biotechnol 1993;. Sanroman A, Pintado J, Lema JM. Biotechnol Tech 1994; 8: 389-394. Tien M, Kirk TK. Methods Enzymol 1988; 161: 238-248. Baskin DG, Henderson M, Kenneth CG, Fujimoto WT. J Histochem Cytochem 1982; 30: 710-712. Marier JR, Boulet M J. Dairy Sci. 1958; 41: 1683-1692. Kuwahara M, Glenn JK, Morgan MA, Gold HH. FEBS Lett 1984; 169: 247-250.