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4 , 56/2.2 mM) with two sizes and three amounts of the carriers, while .... 100. (a). (b). Figure 1. MnP production by immobilized P. chrysosporium in air at a. C/N ratio of 56/2.2 ..... Tien, M. & Kirk, T.K. 1988 Lignin peroxidase of Phanerochaete.

World Journal of Microbiology & Biotechnology (2005) 21: 323–327 DOI 10.1007/s11274-004-3571-8

 Springer 2005

Production of the ligninolytic enzymes by immobilized Phanerochaete chrysosporium in an air atmosphere Guoce Yu*, Xianghua Wen and Yi Qian State Key Joint Laboratory of Environment Simulation and Pollution Control, Department of Environmental Science and Engineering, Tsinghua University, Beijing 100084, P.R. China *Author for correspondence: Tel.: 86-10-62772838, Fax: 86-10-62771472, E-mail: [email protected] Received 17 February 2004; accepted 4 August 2004

Keywords: Immobilized culture, in air, ligninolytic enzymes, non-immersed culture, Phanerochaete chrysosporium, production

Summary The production of the ligninolytic enzymes by Phanerochaete chrysosporium immobilized on polyurethane foam cubes in air was investigated by adopting different sizes and amounts of the carriers, different medium C/N ratios and different glucose-feeding strategies. No lignin peroxidase (LiP) activity was observed under nitrogen limitation (C/N ratio, expressed as glucose/NHþ 4 , 56/2.2 mM) with two sizes and three amounts of the carriers, while comparable levels of manganese peroxidase (MnP) activities were detected only in non-immersed cultures with two sizes of the carriers. A non-immersed state also stimulated LiP formation under carbon limitation (C/N ratio 28/44 mM). High peak activities of LiP, 197 and 164 U/l, were obtained in non-immersed cultures under carbon limitation at the C/N ratios of 28/44 and 56/44 mM, respectively, the occurrence of the activities coinciding with the complete consumption of glucose. A very low level of MnP was measured at the C/N ratio of 28/44 mM compared with the similar activities at 56/2.2 and 56/44 mM. An addition of 2 g glucose/l after its complete depletion improved both the production of LiP and MnP markedly in non-immersed culture at the initial C/N ratio of 28/44 mM, whereas a replenishment of 5 g/l, still enhancing the formation of MnP, inhibited the production of LiP first before the later reactivation. It is suggested that non-immersed liquid culture under carbon limitation reinforced by a suitable glucose feeding strategy is one potential way to realize high production of the ligninolytic enzymes by P. chrysosporium in air.

Introduction White rot fungi can secrete the enzymes capable of degrading lignin as well as a variety of persistent environmental pollutants (Bumpus et al. 1985; Kirk & Farrell 1987; Barr & Aust 1994). The ligninolytic enzymes are expected to be of much importance in biopulping and biobleaching in the paper industry, water pollution control and soil bioremediation. The basidiomycete Phanerochaete chrysosporium is the most extensively characterized white rot fungus and commonly adopted in the production of the ligninolytic enzymes, its ligninolytic system consisting mainly of lignin peroxidase (LiP, EC1.11.1.14), manganese peroxidase (MnP, EC1.11.1.13) and H2O2-producing enzymes such as glyoxal oxidase. Triggered by nitrogen, carbon or sulphur limitations, the expression of these enzymes is active at high oxygen tensions (Kirk et al. 1978; Faison & Kirk 1985; Dosoretz et al. 1990a). A high partial pressure of oxygen has been proposed to help overcome difficulties of oxygen transfer into fungal mycelia, especially caused by the accumulation of extracellular

polysaccharides, to induce the synthesis of the ligninolytic enzymes (Dosoretz et al. 1990a; Michel et al. 1992). However, a high oxygen level may also lead to a rapid decay of the ligninolytic enzymes due to the increased protease activity (Dosoretz et al. 1990a,b). Most studies on the production of the ligninolytic enzymes in liquid cultures of P. chrysosporium have been conducted in pure oxygen or in an oxygen-enriched environment, though various kinds of culture systems have been employed. Rothschild et al. (1995) first demonstrated the formation of LiP and glyoxal oxidase by P. chrysosporium in a non-immersed culture system in an air atmosphere, and later it was proposed that the high oxygen level required for LiP formation by P. chrysosporium can be substituted by the absence of manganese in shallow stationary culture (Rothschild et al. 1999). Zacchi et al. (2000) obtained high LiP activities (200–400 U/l) in submerged agitated liquid culture in air by using cellulose instead of glucose as the carbon source. The effective synthesis of the ligninolytic enzymes under an air condition implies lower costs and greater feasibility, which are essential for enzyme production on a large

324 scale. More efforts on enhancing formation of the ligninolytic enzymes in air are required to facilitate their realistic production. Immobilized culture can improve the availability of oxygen to fungal mycelia by providing a great surface area and enabling efficient mass transfer, and thus may be taken as a possible culture mode for enzyme fermentation in air. By using an immobilized culture system of P. chrysosporium, this work investigated the effects of sizes and amounts of the carriers, medium C/N ratios and fed-batch operation on the formation of the ligninolytic enzymes, and proposed a possible way to realize high enzyme production in air.

Materials and methods Strain and medium P. chrysosporium strain BKM-F-1767 was maintained at 30 C on PDA (200 g potato extract/l, 20 g glucose/l and 20 g agar/l) plates. The culture medium was based on that described by Tien & Kirk (1988), containing 0.02 M acetate buffer (pH 4.4) instead of dimethyl succinate buffer. The glucose concentration and the nitrogen (supplied as diammonium tartrate) concentration were modified as indicated. 1.5 mM veratryl alcohol was introduced from the beginning of cultures and no surfactant was added.

G. Yu et al. Chemicals Veratryl alcohol and nitrilotriacetate (used in medium) were from Fluka (Buchs, Switzerland). The glucose assay kit was from Shanghai Institute of Biological Products (Shanghai, China). All other chemicals used were of analytical grade.

Results Effect of sizes and amounts of the carriers No LiP activity was observed under nitrogen limitation (C/N ratio, expressed as glucose/NHþ 4 , 56/2.2 mM) throughout the cultures, no matter what sizes (0.5 and 1.5 cm) and amounts (0.4, 0.8 and 1.6 g) of the carriers were used (data not shown). Not detected with a less amount (0.4 g and 0.8 g) of the carriers, MnP activity could be measured with 1.6 g carriers corresponding to a non-immersed state, both occurring on day 2, peaking at comparable levels (67 U/l for the 0.5 cm carriers and 93 U/l for the 1.5 cm) on day 4 with two sizes of the carriers (Figure 1). Under carbon limitation (C/N ratio 28/44 mM) in another experiment, LiP activity was also (a) 70

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Assays LiP activity was measured as described by Tien & Kirk (1988), with 1 U defined as 1 lmol of veratryl alcohol oxidized to veratraldehyde per min. A molar extinction coefficient of 9300 M)1 cm)1 was used for veratraldehyde. MnP activity was measured as described by Paszczynski et al. (1988) with Mn2+ as the substrate, with an extinction coefficient of 6500 M)1 cm-1 used for the Mn3+-tartaric acid complex. One unit of activity was defined as 1 lmol of Mn2+ oxidized per min. Glucose was measured by using a glucose assay kit.

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Cubes of polyurethane foam (average pore diameter 0.031 cm, density 0.039 g/cm3), 0.5 and 1.5 cm per side, respectively, were adopted as the carriers after boiling for 30 min, rinsing three times and drying. Their amounts were organized so that the carriers corresponded to an immersed (0.4 g), a non-immersed (1.6 g) and the critical state (the liquid level was largely equal to the height of the piled cubes) (0.8 g), respectively, in a 250 ml Erlenmeyer flask containing 100 ml medium. The spore concentration after inoculation was 1 · 105 spores/ml. Cultures were incubated in air at 37 C in a rotary shaker with agitation at 160 rev/min with a 2.5-cm-diameter throw. Experiments were carried out in triplicate and results are expressed as the mean values.

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Figure 1. MnP production by immobilized P. chrysosporium in air at a C/N ratio of 56/2.2 mM with different amounts of (a) the 0.5 cm cube and (b) the 1.5 cm cube carriers. Cultures were incubated at 37 C in a rotary shaker with agitation at 160 rev/min. Experiments were performed in triplicate and values are means ± standard deviations.

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Ligninolytic enzyme production in air

The effect of medium C/N ratio on the production of the ligninolytic enzymes by immobilized P. chrysosporium in air in a non-immersed culture is presented in Figure 2. In contrast with no production of LiP at the C/N ratio of 56/2.2 mM, high LiP activities were detected under carbon limitation conditions at the C/N ratios of 28/44 and 56/44 mM (Figure 2a). The activities occurred on day 2 and day 4, respectively, coinciding with the complete consumption of glucose (Figure 2c), and achieved the maxima, 197 U/l on day 3 at 28/44 mM and 164 U/l on day 5 at 56/44 mM. Though MnP was produced at the three C/N ratios, the peak activity was very low at 28/44 compared with the similar levels at 56/2.2 mM and 56/44 mM (Figure 2b). The formation of MnP also coincided with the depletion of glucose under carbon limitation conditions (Figure 2c).

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only found in non-immersed culture states and not in immersed states when adopting the 0.5 cm carriers, and no MnP was produced during the cultures with any carrier amount used at this time (data not shown).

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In non-immersed cultures with 1.6 g 1.5 cm carriers at an initial C/N ratio of 28/44 mM, two strategies of glucose feeding, i.e. restoring the glucose concentration to 2 and 5 g/l, respectively after the complete consumption of glucose, were adopted to check their effect on the formation of the ligninolytic enzymes. Figure 3 depicts the effect of glucose feeding as well as the utilization of glucose. 2 g glucose/l was fed on day 3 and day 5 and depleted within one day, and 5 g/l on day 3 and day 6 and consumed within two days (Figure 3c). Compared with no glucose feeding, an addition of 2 g/l reactivated the synthesis of LiP and MnP immediately, as seen clearly from the elevated enzyme activities on day 4 and day 6 (Figure 3a and b). A replenishment of 5 g/l first decreased the activity of LiP, and then restored it to the approximately original level on day 5 and day 6 with the depletion of added glucose, and in the meantime it improved the MnP production on day 4 and day 7 apparently, as somewhat similar to the effect of the 2 g/l feeding (Figure 3a and b).

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Time (day) Figure 2. Production of the ligninolytic enzymes by P. chrysosporium in air in non-immersed culture with 1.6 g 1.5 cm carriers at different C/N ratios. (a) LiP, (b) MnP, (c) glucose. Cultures were incubated at 37 C in a rotary shaker with agitation at 160 rev/min. Experiments were performed in triplicate and values are means ± standard deviations.

Discussion The production of the ligninolytic enzymes by P. chrysosporium has rarely been reported in liquid cultures in an air environment (Rothschild et al. 1995; 1999; Zacchi et al. 2000). The present work showed some detailed characteristics of the production of the ligninolytic enzymes by immobilized P. chrysosporium in air concerning the effects of sizes and amounts of the carriers, medium C/N ratios and glucose feeding. The results obtained imply a great possibility for the

accomplishment of high ligninolytic enzyme production by P. chrysosporium in liquid culture in an air atmosphere. It was shown in this work that the carrier size did not cause distinct effects on the formation of the ligninolytic enzymes by P. chrysosporium immobilized on a porous support, while the carrier amount played a critical role. Non-immersed culture (Dosoretz et al. 1993), in which a higher quantity of the carriers were used for the

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Figure 3. Production of the ligninolytic enzymes by P. chrysosporium in air in non-immersed culture with 1.6 g 1.5 cm carriers at an initial C/N ratio of 28/44 mM with different glucose feeding strategies. (a) LiP, (b) MnP, (c) glucose. Cultures were incubated at 37 C in a rotary shaker with agitation at 160 rev/min. Experiments were performed in triplicate and values are means ± standard deviations.

exposure of the immobilized mycelia to air, was demonstrated to be advantageous for enzyme production. Resembling the wild state of cell growth to an extent, a non-immersed culture system may strongly enhance the oxygen availability to the fungi by generating much more air contact area and thus can satisfy the high oxygen demand for the expression of the ligninolytic enzymes. In the meantime, good mass transfer of liquid

nutrients may be guaranteed with agitation close to that in submerged culture. Somewhat similar to semi-solid state culture (Rodriguez et al. 1997; 1998), non-immersed culture is usually characterized by holding a far larger proportion of free liquid medium which can also enable an easy harvest of the fermented product. Medium C/N ratio exerted different influence on the production of LiP and MnP in non-immersed culture of P. chrysosporium in air. LiP was synthesized under carbon limitation and not under nitrogen limitation, indicating that a low C/N ratio was advantageous for LiP production. On the other hand, the significant production under nitrogen limitation or high carbon conditions (56 mM/44 mM) and the repressed formation at a low carbon concentration (28 mM/44 mM) suggested that the MnP synthesis preferred a high carbon level. These results were consistent with some observations by Rothschild et al. (1995). The discrepancy between the two enzymes may result from the different regulatory mechanisms in the expression of LiP and MnP, which were closely related to the physiological effects caused by carbon and nitrogen starvation, such as nitrogen or carbon catabolic repression. In addition, the possible formation of extracellular polysaccharides under nitrogen limitation may result in lower oxygen availability and affect enzyme synthesis to some degree (Rothschild et al. 1995). It is deduced that C/N ratio control can be taken as a measure to regulate the relative production of LiP and MnP in non-immersed culture in air. Enzyme instability seriously damaged the high production of the ligninolytic enzymes, for which protease was thought to be responsible (Dosoretz et al. 1990b,c). The production of protease was closely related to glucose metabolism. The addition of a suitable amount of glucose upon its complete depletion may prevent cell autolysis, inhibit protease activity (Dosoretz et al. 1990b; Chen et al. 1992), and thus stabilize and enhance the production of the ligninolytic enzymes. An excess supplement, however, may greatly destroy the carbon starvation condition required for enzyme expression and reduce its production during a short time as seen for LiP with an addition of 5 g glucose/l. The improvement of MnP activity at this time (5 g/l addition) reflected its different response to the same carbon level from LiP as indicated above. An optimal glucose feeding strategy, probably one continuous feeding mode, simultaneously maintaining carbon limitation and inhibiting protease activity, should exist for the maximum and stable production of LiP and MnP. In conclusion, this paper has presented some characteristics of the production of the ligninolytic enzymes by immobilized P. chrysosporium in air. Non-immersed liquid culture under carbon limitation reinforced by a suitable glucose feeding strategy may be one promising way to accomplish high production of the ligninolytic enzymes by P. chrysosporium in air. Further optimization of the culture parameters as well as scale up in a suitable bioreactor is required for its practice.

Ligninolytic enzyme production in air Acknowledgments This work was supported by a grant from the National High Technology Research and Development Program of China (863 Program) (No. 2002AA649100). References Barr, D.P. & Aust, S.D. 1994 Mechanisms white rot fungi use to degrade pollutants. Environmental Science and Technology 28, 78A–87A. Bumpus, J.A., Tien, M., Wright, D. & Aust, S.D. 1985 Oxidation of persistent environmental pollutants by a white rot fungus. Science 228, 1434–1436. Chen, A.H.C., Dosoretz, C.G. & Grethlein, H.E. 1992 Strategy for the production and stabilization of lignin peroxidase from Phanerochaete chrysosporium in air-lift and stirred tank bioreactors. In Frontiers in Bioprocessing II, eds. Todd, P., Sikdar, S.K. & Bier, M. pp. 181–187. Washington, DC: American Chemical Society. ISBN 0-8412-2181-2. Dosoretz, C.G., Chen, A.H.C. & Grethlein, H.E. 1990a Effect of oxygenation conditions on submerged cultures of Phanerochaete chrysosporium. Applied Microbiology and Biotechnology 34, 131–137. Dosoretz, C.G., Chen, H.C. & Grethlein, H.E. 1990b Effect of environmental conditions on extracellular protease activity in ligninolytic cultures of Phanerochaete chrysosporium. Applied and Environmental Microbiology 56, 395–400. Dosoretz, C.G., Dass, S.B., Reddy, C.A. & Grethlein, H.E. 1990c Protease-mediated degradation of lignin peroxidase in liquid cultures of Phanerochaete chrysosporium. Applied and Environmental Microbiology 56, 3429–3434. Dosoretz, C.G., Rothschild, N. & Hadar, Y. 1993 Overproduction of lignin peroxidase by Phanerochaete chrysosporium (BKM-F-1767) under nonlimiting nutrient conditions. Applied and Environmental Microbiology 59, 1919–1926.

327 Faison, B.D. & Kirk, T.K. 1985 Factors involved in the regulation of a ligninase activity in Phanerochaete chrysosporium. Applied and Environmental Microbiology 49, 299–304. Kirk, T.K. & Farrell, R.L. 1987 Enzymatic ‘‘combustion’’: the microbial degradation of lignin. Annual Review of Microbiology 41, 465–505. Kirk, T.K., Schultz, E., Connors, W.J., Lorenz, L.F. & Zeikus, J.G. 1978 Influence of culture parameters on lignin metabolism by Phanerochaete chrysosporium. Archives of Microbiology 117, 277– 285. Michel, F.C., Grulke, E.A. & Reddy, C.A. 1992 Determination of the respiration kinetics for mycelial pellets of Phanerochaete chrysosporium. Applied and Environmental Microbiology 58, 1740–1745. Paszczynski, A., Crawford, R.L. & Huynh, V.-B. 1988 Manganese peroxidase of Phanerochaete chrysosporium: purification. Methods in Enzymology 161, 264–270. Rodriguez, C.S., Santoro, R., Cameselle, C. & Sanroman, A. 1997 Laccase production in semi-solid cultures of Phanerochaete chrysosporium. Biotechnology Letters 19, 995–998. Rodriguez, S., Santoro, R., Cameselle, C. & Sanroman, A. 1998 Effect of the different parts of the corn cob employed as a carrier on ligninolytic activity in solid state cultures by P. chrysosporium. Bioprocess Engineering 18, 251–255. Rothschild, N., Hadar, Y. & Dosoretz, C. 1995 Ligninolytic system formation by Phanerochaete chrysosporium in air. Applied and Environmental Microbiology 61, 1833–1838. Rothschild, N., Levkowitz, A., Hadar, Y. & Dosoretz, C.G. 1999 Manganese deficiency can replace high oxygen levels needed for lignin peroxidase formation by Phanerochaete chrysosporium. Applied and Environmental Microbiology 65, 483–488. Tien, M. & Kirk, T.K. 1988 Lignin peroxidase of Phanerochaete chrysosporium. Methods in Enzymology 161, 238–249. Zacchi, L., Burla, G., Zuolong, D. & Harvey, P.J. 2000 Metabolism of cellulose by Phanerochaete chrysosporium in continuously agitated culture is associated with enhanced production of lignin peroxidase. Journal of Biotechnology 78, 185–192.

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