World J Microbiol Biotechnol (2007) 23:599602 DOI 10.

1007/s11274-006-9264-8

TECHNICAL COMMUNICATION

Use of diluted medium in repeated-batch fermentation for production of lignin peroxidase by Phanerochaete chrysosporium
KiBeom Lee

Received: 19 May 2006 / Accepted: 11 August 2006 / Published online: 6 September 2006 Springer Science+Business Media B.V. 2006

Abstract Lignin peroxidase has been extensively studied due to the potential use of this enzyme in environmental pollution control. Important aspects of the production of the enzyme by the white rot fungus, Phanerochaete chrysosporium, include the improvement of yield results and cell maintenance. In the present work, Phanerochaete chrysosporium was immobilized in polyurethane foam and used for repeated-batch fermentations with various dilution of the initial medium (D), and lignin peroxidase production was investigated. The peak of 283 17.5 U lignin peroxidase/l production rate was obtained at a D of 1/ 5, with signicantly lower production rates seen at higher and lower dilution ratios. When six cycles of repeated-batch fermentation were conducted using a D of 1/5, the results revealed that at least four cycles of repeated-batch fermentation were possible with a high lignin peroxidase production rate under a cut-off value of 178 3.87 U/l. Furthermore, the cell-free culture broth could be successfully concentrated to 2,800 U/l by ultraltration. Thus, the present study shows that optimizing the dilution of the utilized nutritional medium can improve repeated batch production of lignin peroxidase from immobilized P. chrysosporium, in terms of both cycle number and output. Keywords Lignin peroxidase production Phanerochaete chrysosporium Repeated batch fermentation Polyurethane foam cubes Dilution of the utilized nutritional medium
KiBeom Lee (&) KeyGene Life Science Institute, 1271-11 Sa 1-dong, Sangnok-gu, Ansan-si, Gyeonggi-do 426-901, Korea e-mail: kibeomlee970@hanmail.net

Introduction Phanerochaete chrysosporium, a white rot fungus, has been extensively studied for its non-specic ability to degrade a wide range of recalcitrant and hazardous pollutants (Aust 1990; Bogan and Lamar 1996; Kennedy et al. 1990; Tsai 1991). Conditions of nitrogen or carbon limitation allow expression of the lignolytic system within P. chrysosporium triggering secretion of a secondary metabolite called lignin peroxidase (Bogan and Lamar 1996; Kirk et al. 1978; Tien and Kirk 1984). To varying degrees, pollutants are co-oxidized by the lignin peroxidase to give CO2 and largely uncharacterized polar metabolites. The xenobiotic oxidations by lignin peroxidase of the white rot fungus are not rapid or efcient, but P. chrysosporium is a natural inhabitant of soil litter. These considerations make lignin peroxidase production attractive for use in low technology bioremediation programs. Some of the previous efforts to optimize lignin peroxidase production from P. chrysosporium have involved immobilization of the fungus on porous polyurethane foam cubes (Capdevila et al. 1989; Linko et al. 1986; Linko 1988; Ruckenstein and Wang 1994; Yoshitoshi et al. 1997), which may then be used for lignin peroxidase production via repeated-batch fermentation (Capdevila et al. 1989; Linko 1988; Ruckenstein and Wang 1994). Most of these early repeated-batch studies utilized carbon and/or nitrogen limited media, with the goal of maximum production of lignin peroxidase in the nal, cell-free culture broth. However, the enzyme activity yield in cell-free culture broth previously obtained from shaken ask cultures of P. chrysosporium immobilized in polyurethane foam was very low, suggesting that repeated-batch culture

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World J Microbiol Biotechnol (2007) 23:599602

with immobilized cells might not provide the desired high-yield results. In the present work, I sought to improve repeatedbatch production of lignin peroxidase using properly diluted nutritional medium in a relatively large culture volume, followed by ultraltration-based concentration of the enzyme solution from the cell-free broth.

1 lmol of veratryl alcohol oxidized to veratraldehyde per min. Concentration of the produced enzyme by ultraltration The culture broth was concentrated on a 10,000 MW cut-off polysulfone membrane module (Millipore). The module was a plate-and-frame type with an effective area of 240 cm2, and ultraltration was performed according to the manufacturers suggested protocol.

Materials and methods Organism, media, and initial culture conditions Phanerochaete chrysosporium strain BKM-F-1767 (ATCC 24725) was used throughout the investigation. Spores were grown on malt-agar plates, suspended in sterile water by vortexing and ltered through glass wool. For experiments, spore cultures were grown in 500-ml asks containing 300 ml of liquid medium (Tien and Kirk 1988) at an initial spore concentration of 5 106 spores/ml. Samples were cultured in a rotary shaker at 120 rev/min at 39C. Preparation of fungi for immobilization A polyurethane foam sheet was cut into cubes (2 2 1 cm3), and 20 g of polyurethane foam cubes were placed in each 500 ml ask, followed by sterilization in an autoclave. P. chrysosporium spores were inoculated into the asks in 300 ml medium, and the cultures were incubated in a shaking incubator for cell immobilization. Repeated-batch cultures with various medium dilutions The spores were immobilized as described above, and then the polyurethane foam cubes were exposed to the circulation of medium and oxygen. When the maximum activity level was reached by testing supernatant enzyme activity at various time points, the culture supernatant was decanted and asks were lled with medium diluted with sterile water to ratios of 1/2, 1/3, 1/5, 1/7 and 1/9 (designated D1, D2, D3, D4 and D5, respectively). This medium was N and C decient. For repeated-batch culture experiments, the culture broth was harvested after 24 h, and 300 ml fresh medium of the same dilution was added to the asks for continued culture. Enzyme activity assay Lignin peroxidase activity was measured essentially as described by Tien and Kirk (1988), with 1 U dened as

Results and discussion To conrm this system, I rst compared the growth rates and lignin peroxidase activity levels in pellet and foam-immobilized shaken cultures of P. chrysosporium. The P. chrysosporium cells were completely attached to the polyurethane foam cubes, as shown by the presence of cells on surface or within polyurethane foam cubes. Comparison of glucose consumption rates indicated that the immobilized cells had a higher growth rate than the pelletized suspended cells (data not shown). As shown in Fig. 1, comparison of lignin peroxidase activities revealed that the maximum lignin peroxidase production was 10-fold higher in P. chrysosporium immobilized on polyurethane foam cubes versus pellets (283 U/l vs. 28 U/l). In addition, maximum production was achieved more quickly in immobilized versus pellet cultures (9 days vs. 11 days). These results indicate that P. chrysosporium cells for use in lignin peroxidase production were successfully immobilized.

Fig. 1 Lignin peroxidase production by P. chrysosporium in single batch culture inoculated with pelleted or foam-immobilized cells. Data represent the means of triplicate and error bars indicate the standard deviation

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World J Microbiol Biotechnol (2007) 23:599602 Table 1 Repeated batch lignin peroxidase production by foam cube-immobilized P. chrysosporium grown in medium diluted to the given ratios

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Repeated batch no.

Lignin peroxidase activity (U/l) Dilution of the initial medium 1/2 (D1) 1/3 (D2) 265 15.1 71 0.78 62 40 0 1/5 (D3) 283 219 200 183 178 77 18 17.5 10.5 4.63 4.4 3.87 0.82 1/7 (D4) 258 17 63 52 40 0 1/9 (D5) 267 5.2 67 2 0

Each batch was incubated for 24 h. Data are presented as the means of triplicate and denotes standard deviation among triplicate experiments

1 2 3 4 5 6 7

273 17.2 52 0

In an effort to increase the efciency and output of lignin peroxidase production in this system for industrial use, the impact of diluted nutrients on cultures of P. chrysosporium was next examined and the use of these dilutions during repeated-batch shaken cultures with the immobilized cells was tested. Five different dilutions (1/2, 1/3, 1/5, 1/7 and 1/9, designated D1, D2, D3, D4 and D5, respectively) were examined for their ability to increase and/or maintain lignin peroxidase production over six 24 h batch cultures. As shown in Table 1, D1 produced the same level of enzyme activity as the initial batch culture, and the enzyme activity decreased signicantly between the rst and second batches. Use of D2, D4 and D5 decreased the observed enzyme activity across all batches. In contrast, cultures grown with D3 yielded good enzyme activity (above 178 U/l) for up to 5 batches, with decreased enzyme activity seen thereafter. These results indicate that the 1/5 dilution was most appropriate for repeated-batch culture. It seems reasonable to suggest that the weaker dilutions (1/7 and 1/9) did not provide sufcient nutrients for cell maintenance and enzyme production, whereas the stronger dilutions (1/2 and 1/3) probably inhibited the secondary metabolism required for production of lignin peroxidase.

In order to obtain a higher enzyme activity, the mycelial mats by centrifugation were separated and ultraltration at 5C was carried out. The ultraltration enabled to concentrate the enzyme to 2,800 U/l at a ux of 120 l/h m2 atm without a signicant ux decline, indicating minimal membrane fouling. During ultraltration, the concentrated product is likely to have lost a part of its total enzyme activity due to the shear force of the recirculation pump. Concentrated product stored at 20C was found to retain nearly 100% of its initial activity over 5 weeks. As shown in Table 2, this work is somewhat different from those previously reported in similar matrices (Capdevila et al. 1989; Ruckenstein and Wang 1994). The enzyme activity obtained in this study was lower than that found in the previous reports. The lower enzyme activity observed in the present study might be due to a lower concentration of the nitrogen and/or a lower efciency of oxygen transfer in the larger culture volume. However, more enzyme could be concentrated from each larger batch culture volume in the present study, so the novelty of this study is clear. In terms of using results to improve lignin peroxidase production in the laboratory or for industrial applications, it is likely that the optimal dilution may

Table 2 Comparison of P. chrysosporium lignin peroxidase production by repeated batch fermentation in three studies Method of immobilization Polyurethane foam Major carbon and nitrogen sources Glycerol 2.5 g/l Diammonium tartrate 0.45 g/l Yeast extract 0.25 g/l Potassium 2.2-dimethyl succinate 1.46 g/l Glucose 1 g/l NH4Cl 0.12 g/l Glycerol 2 g/l Ammonium tartrate 0.04 g/l Thiamine trace Culture Volume (ml) 30 Harvest Time (h) 24 Maximum activity (U/l) 1,800 Repetitive batch no. 8 Reference Capdevila et al. 1989

Porous polystyrene Polyurethane foam

75 300

48 24

600 283

16 7

Ruckenstein and Wang 1994 This work (D3)

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World J Microbiol Biotechnol (2007) 23:599602 Kennedy DW, Aust SD, Bumpus JA (1990) Comparative biodegradation of alkyl halide insecticides by the white rot fungus Phanerochaete chrysosporium (BKM-F1767). Appl Environ Microbiol 56:23472353 Kirk TK, Schultz F, Connors WJ, Lorenz LF, Zeikus JG (1978) Inuence of culture parameters in lignin metabolism by Phanerochaete chrysosporium. Arch Microbiol 177:277 285 Linko YY, Leisola M, Lindholm N, Troller J, Linko P, Fiechter A (1986) Continuous production of lignin peroxidase by Phanerochaete chrysosporium. J Biotechnol 4:283 291 Linko S (1988) Production and characterization of extracellular lignin peroxidase from immobilized Phanerochaete chrysosporium in a 10 l bioreactor. Enzyme Microb Technol 10:410417 Ruckenstein E, Wang XB (1994) Production of lignin peroxidase by Phanerochaete chrysosporium immobilized on porous poly (styrene-divinyl benzene) carrier and its application to the degrade of 2-chlorophenol. Biotechnol Bioeng 44:79 86 Tien M, Kirk TK (1984) Lignin-degrading enzyme from Phanerochaete chrysosporium: purication, characterization and catalytic properties of a unique H2O2-requirig oxygenase. Proc Natl Acad Sci USA 81:22802284 Tien M, Kirk TK (1988) Lignin peroxidase of Phanerochaete chrysosporium. Meth Enzymol 161:238249 Tsai TS (1991) Biotreatment of red watera hazardous waste stream from explosive manufacture-with fungal system. Hazard Waste Hazard Mater 8:231244 Yoshitoshi N, Sawada T, Sungusia GM, Kobayash F (1997) Lignin peroxidase production by Phanerochaete chrysosporium immobilized on polyurethane foam. J Chem Eng Jpn 30:16

differ based on the composition of the initial medium and the harvest time, so additional optimization studies may be required for specic applications. In addition, further studies will be required to examine whether immobilized cells can be rejuvenated by introduction of a high-nutrient medium at certain points during repeated-batch production.

Conclusions It was shown herein for the rst time that properly diluted medium can be used to maximize lignin peroxidase production in repeated-batch culture of P. chrysosporium and that the cell-free broth from such cultures can be concentrated by ultraltration to produce a high enzyme activity solution.

References
Aust SD (1990) Degradation of environmental pollutants by Phanerochaete chrysosporium. Microb Ecol 20:197209 Bogan BW, Lamar RT (1996) Polycyclic aromatic hydrocarbondegrading capabilities of Phanerochaete laevis HHB-1625 and its extracellular ligninolytic enzymes. Appl Environ Microbiol 62:15971603 Capdevila C, Corrieu G, Asther M (1989) A feed harvest culturing method to improve lignin peroxidase production by Phanerochaete chrysosporium INA-12 immobilized on polyurethane foam. J Ferment Bioeng 68:6063

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