Effects of lignin-modifying enzymes on pine kraft pulp

Bioresource Technology50 (1994) 73-77 O 1995 Elsevier Science Limited Printed in Great Britain. All rights reserved 0960-8524/94/$7.00 0960-8524(94)00043-3

ELSEVIER

EFFECTS OF LIGNIN-MODIFYING E N Z Y M E S ON PINE KRAFT PULP M.-L. Niku-Paavola, a M. Ranua, b A. SuurnS.kki & A. K a n t e l i n e n a* a l/TT/Biotechnology and Food Research, PO Box 1501, FIN-02044 I/TT, Espoo, Finland hThe Finnish Pulp and Paper Research Institute, PO Box 70, FIN-02151 Espoo, Finland

Abstract Oxygen delignified pine kraft pulps were treated with lignin-modifying enzymes: laccase (EC 1.10.3.2), Mndependent peroxidase (E C 1.11.1.13), lignin peroxidase (EC 1.11.1.14), and xylanase (EC 3.2.1.8), in order to improve bleachability. Pulps were treated either with a purified single enzyme or alternatively enzymes were successively added in different combinations. The solubilized components were analysed using HPLC, GPC and IEF. The residual pulps were delignified with alkaline hydrogen peroxide and analysed for Kappa number and ISO-brightness. Lignin-modifying enzymes did not improve bleachability when acting alone. Xylanase treatment increased the brightness by 1-2"5 ISO-units, and xylanase combined with lignin-modifying enzymes increased the brightness by a further one unit. By extending the alkaline peroxide step the final brightness increased in all samples, whereas the relative differences between reference and enzyme-treated samples remained constant.

ability of lignin by different bleaching chemicals (Viikari et al., 1986; Kantelinen et al., 1988). Higher final brightness values of pulps are obtained, or the consumption of bleaching chemicals is reduced, thus decreasing the load to the environment. The effect of hemicellulase treatment is indirect, based on limited hydrolysis of xylans facilitating the subsequent removal of lignin. The enzymatic modification of lignin itself would be a straightforward approach. The potential of lignin-modifying enzymes from white-rot fungi, lignin peroxidases (LIP), Mn-dependent peroxidases (MnP) and laccase in lignin biodegradation is still an open question (Kirk & Farrell, 1987; Sarkanen et al., 1991). Lignin-modifying enzymes abstract electrons from electron-rich aromatic compounds, converting them to unstable cation or phenoxy radicals. The reaction continues non-enzymatically according to the characteristics of the radicals created. Dimeric synthetic compounds representing the structural units of lignin undergo, for example cleavage between Ca-Cfl and r-o-4 ether bonds, oxidation of benzylic alcoholic and methylene groups, aromatic ring-opening and polymerization of phenols (Kersten et al., 1985; Schoemaker et al., 1985). The effect of the enzyme on the macromolecular lignin strongly depends on the lignin structure, and is primarily regulated by radical reactions. Depolymerization reactions catalyzed by lignin-modifying enzymes have been reported with synthetic hardwood-type DHP lignin, using veratryl alcohol as a cosubstrate, as well as with DHP-lignin containing low amounts of free phenolic hydroxyl groups (Hammel & Moen, 1991 ), or with lignin in which the hydroxyl groups have been derived (Tien & Kirk, 1983). Thus lignin-modifying enzymes seem to be a logical tool to improve the delignification of chemical pulp. Delignification of kraft pulp using lignin-modifying enzymes has been preliminarily reported (Bourbonnais & Palce, 1992; Arbeloa et al., 1992; Kantelinen et al., 1993a). In the present work this potential approach is further studied.

Key words: Lignin bleaching, lignin modifying enzymes, enzymes as bleaching aids.

NOTATION DHP GPC HPLC IEF MCC LiP MnP P

dehydrogenation polymerizate, synthetic lignin Gel permeation chromatography High pressure liquid chromatography Isoelectric focusing electrophoresis Modified continuous cooking Lignin peroxidase Manganese dependent peroxidase Alkaline hydrogen peroxide delignification

INTRODUCTION Enzymes have been used successfully to increase the bleachability of kraft pulps. Hemicellulases, especially xylanases, have been found to enhance the extract*Present address: Genencor Int., PO Box 13, FIN-42301 JSms~n_koski,Finland.

METHODS

73

Pulps In this study commercial pine kraft pulps were used (Kappa number 11.8 and 13.4 SCAN, ISO-brightness

74

M.-L. Niku-Paavola, M. Ranua, A. Suurniikki, A. Kantelinen

37.7 and 37.3%, respectively), prepared by modified continuous cooking (MCC) and delignified by oxygen.

Enzymes The enzymes used were purified xylanase pI 9 from Trichoderma reesei (Tenkanen et al., 1992), LiP pI 3.2 purified from Phlebia radiata, and laccase pI 4.0 and MnP pI 3.7 purified from Trarnetes hirsuta. Trametesculture filtrate was produced by Cultor Ltd, Finland. Purification of lignin-modifying enzymes was performed according to Niku-Paavola et al. (1988).

chemicals used were: 3% H202, 1.5% NaOH, 0.2% DTPA, 0-5% MgSO4. In some experiments the treatment was repeated. Experiments were also carried out where enzyme treatment was performed between the two delignification steps. The pulp quality after SO 3water washing was analysed by Kappa number (SCAN-C 1 : 1977) and brightness (ISO 2470).

RESULTS

Components solubilized by enzyme treatment Enzymatic treatments The enzymatic treatments were carried out in water at 3% pulp consistency with pH 5.0 and at a temperature of 50°C. The enzymes were added separately and sequentially, and were incubated for 1 h each. Enzyme doses were: xylanase 100 nkat, laccase 0.5 nkat, LiP or MnP 5 nkat/g dry pulp. Together with LiP, 0.04 mg H202/g dry pulp was introduced and when MnP was added, H202 together with 0"4 mg Mn 2÷/g dry pulp was supplied. Corresponding reference samples with a single enzyme and without enzymes were prepared for comparison.

The monophenol pattern solubilized in water during enzyme treatments was rather similar in all the samples, including the references. Only trace amounts, 10-30 ppm, of monophenols were released. Corresponding levels of acid-soluble lignin fragments with small molecular mass were detected by GPC (data not shown). Xylodextrins, amounting to 0.1-0.2% of the dry pulp were liberated by the xylanase treatment. Surprisingly, the combination of xylanase and lignlnmodifying enzymes seemed to eliminate xylose from this pattern (Fig. 1). The components extracted in alkali after enzyme treatment were macromolecular lignin and its degrada-

Extraction of iignin After enzyme treatment and washing with tap water, lignin was extracted from the pulps with 2% NaOH at 10% pulp consistency for 1 h, at a temperature of 80°C.

Analysis of solubilized components The solubilization of lignin and carbohydrates in water during the enzymatic treatment, and in alkali after the enzyme treatment, was monitored by HPLC, GPC and IEE HPLC conditions were as follows: monophenols were analysed using Nova Pak C18 at 40°C using a mixture of acetonitrile-water-tetramethylammonium hydroxide-phosphoric acid (20:80:1:1) as eluent, and a UV-detector at 240 nm (Niku-Paavola et al., 1988). Carbohydrates were analysed using a Hamilton HC-40 column with RI-detector and eluted with water at 80°C. GPC was performed using a Fractogel TSK HW 55 column at room temperature, and elution with 0"5 MNaOH. The dissolved lignin was measured at 280 nm (Hortling et al., 1990). Phenol was used as internal standard. IEF of solubilized lignin samples was performed by electrophoresis in a stabilized pH gradient of 3"5-10"0 on polyacrylamide gel (Niku-Paavola, 1991 ).

TREATMENT REF.

RI

0

I

I

I

I

I

5

10

15

20

25

XYLANASE X

0

5

X

X

I

I

I

I

10

15

20

25

X X X~

0

5

X

10

XYLANASE + LACCASE

~

15

20

25

LACCASE

Delignification Enzyme-treated and reference samples were delignified as follows: after enzyme treatment the pulp was washed with tap water. Metals were eliminated by acidifying the pulp with 1 M HCI for 1 h at 50°C. After treatment the pulp was washed to neutral pH. The pulp was delignified by alkaline hydrogen peroxide at 10% pulp consistency for 1-3 h at 80°C. The amounts of

0

J

I

J

J

I

5

10

15

20

25

RETENTION, rnin

Fig. 1. Carbohydrates solubilized in water during enzymatic treatment of pine kraft pulp. HPLC analysis in a Hamilton HC-40 cation exchanger; elution with water at 80°C, detection by RI.

Effects of lignin-modifying enzymes on pine kraft pulp tion products. GPC-analysis showed that treatment with xylanase most noticeably improved the extraction of high molecular mass lignin. A combination of xylanase with laccase did not improve this result. Lignin-modifying enzymes, alone and in combinations, produced GPC-profiles very similar to that of the reference samples (Fig. 2). IEF analysis of components solubilized in alkali showed, in all samples, the presence of macromolecular lignin similar to the marker kraft lignin (Fig. 3). The amounts of extracted lignin were highest in samples treated with xylanase.

l'OIt 1'I0

A280

REFERENCE

~XYLANASE

1'0I

XYLANASE+ CASE

1'0I~

~

CCASE I'

PHENOLSTD RELATIVEELUTIONVOLUME Fig. 2. Components solubilized in alkali after enzymatic treatment of pine kraft pulp. GPC analysis in a Fractogel TSK HW 55 molecular sieve; elution with 0"5 M NaOH, detection at 280 nm. Phenol was used as internal standard.

LACC

¢1

XYL XYL + LACC REF KRAFT

Fig. 3. Components soluble in alkali after enzymatic treatment of pine kraft pulp. IEF analysis in a stabilized pH gradient of 3.5-10-0 on polyacrylamide gel. Kraft lignin (Indulin) was used as a marker.

75

Enzyme effects on residual pulp After enzyme treatment the Kappa numbers decreased slightly, especially in the case of samples treated with MnP, xylanase and their combination (Fig. 4). After peroxide delignification the Kappa number reduction was most pronounced in samples treated with xylanase, with the combination xylanase and MnP or laccase and MnP. A lignin-modifying enzyme alone did not decrease the Kappa number (Fig. 4). Xylanase treatment improved brightness by 1-2-5 ISO-units except for the xylanase-laccase combination. The xylanase-LiP combination further increased the brightness obtained with xylanase by one unit. When the alkaline peroxide delignification was extended to 3 h, the brightness of the pulp treated with the lignin-modifying MnP-enzyme also increased by one ISO-unit (Fig. 5(a)). All the values were better after delignification for 1 h, as expected. When the peroxide treatment was repeated, the values were further improved. The highest final brightness, 77.5%, and the lowest Kappa number, 5"2, were obtained for the sample treated with the xylanase-MnP combination (Fig. 5(a) and (b)). A slight improvement was further achieved when the xylanase treatment was repeated between the two delignification steps. The first extended alkaline peroxide step was most advantageous from the point of view of the relative differences in brightness values between the reference and enzyme-treated samples. After repeated enzyme and peroxide stages the relative differences were not improved, although the final values were increased. DISCUSSION The xylanase-aided bleaching has been shown to be based mainly on the hydrolysis of re-precipitated xylan in the pulps, resulting from conventional kraft cooking. The fibre structure was rendered more permeable for the lignin extraction in the subsequent bleaching stage, and the molecular mass and amount of lignin extracted was shown to increase (Kantelinen et al., 1993a). The amount of precipitated xylan is decreased in newly developed cooking methods. The pulps used in this work were prepared by the new MCC-method -- here the xylanase treatment still improved the extractability of lignin. Lignin-modifying enzymes were assumed to facilitate delignification by increasing hydrophilicity of the residual lignin. In the present study the lignin-modifying enzymes increased the bleachability of lignin only when combined with xylanase. When xylanase and lignin-modifying enzymes acted together, the reaction products released by the xylanase, xylodextrins an6 xylose may have quenched the radicals created in the lignin structure by the lignin-modifying enzymes. The solubility of lignin may have thus increased, resulting in improved bleachability of the pulp. The bleachability was then further enhanced by the increased permeability of pulp surfaces caused by the hydrolytic

M.-L. Niku-Paavola, M. Ranua, A. Suurniikki, A. Kantelinen

76

Kappa number 15 m

10

Ori inal Brightness, %

ill

Fig. 4.

Afterenzyme / Xylanase+~ Xylanase+~ Laccase+ Afterperoxide;ref. ~ Laccase LIP MnP XYL [7777A Laccase ~ LIP XYL + MnP MnP Pulp values after enzymatic treatment and alkaline hydrogen peroxide delignification. Original K a p p a number 13.4, brightness 37-3%; peroxide treatment 1 h.

TREATMENT REF XYL XYL + MnP MnP

P-P

REF-P-P XYL+MnP-P-XYL-P XYL+MnP-P-XYL-MnP-P 65

70 Brightness, % Original brightness 37.7 %

60

~ ] After 1st peroxide treatment I

I 80

75

After 2nd peroxide treatment

]

TREATMENT

REF XYL XYL + MnP MnP

P-P

REF XYL-MnP-P-XYL-P XYL-MnP-P-XYL-MnP-P 5

I 10 Kappa number

I 15

[]

Original

•

After enzyme and 2nd peroxide treatments

Fig. 5. Pulp values after repeated, extended dehgnification and repeated enzymatic treatments. Original K a p p a number 11.8, brightness 37.7%. (a) Brightness after enzyme and two peroxide treatments. Peroxide delignification, 3 h, was performed twice after enzyme treatments or it was done between the enzyme treatments. (b) Final Kappa number after enzyme and peroxide treatments introduced in (a).

Effects of lignin-modifying enzymes on pine kraft pulp xylanase action. It has been proposed that bleaching always ends without complete penetration of bleaching chemicals, leaving a fraction of the original lignin intact (Gellerstedt & Lindfors, 1991; Gellerstedt & Backman, 1992). The improvement of penetration is thus one of the key questions in bleaching. Agents such as xylanase which can increase the permeability of pulp will therefore improve the bleachability. ACKNOWLEDGEMENTS Financial support was provided by the Technology Development Centre of Finland, and by the Foundation for Biotechnical and Industrial Fermentation Research. REFERENCES

Arbeloa, M., de Leseleuc, J., Goma, G. & Pommier, J.-C. (1992). An evaluation of the potential of lignin peroxidases to improve pulps. Tappi J., 215-21. Bourbonnais, R. & Paice, M. G. (1992). Demethylation and delignification of kraft pulp by Trametes versicolor laccase in the presence of 2,2-azinobis-(3-ethylbenzthiazoline-6sulphonate). AppL MicrobioL BiotechnoL, 36, 823-7. Gellerstedt, G. & Backman, L. (1992). Reactions of alkaline hydrogen peroxide with kraft pulp lignin. Paper presented at the 2nd European Workshop on Lignocellulosics and Pulp, Grenoble, France, 2-4 September 1992. Geilerstedt, G. & Lindfors, E.-L. (1991). On the structure and reactivity of residual lignin in kraft pulp fibers. In Proc. Int. Pulp Bleaching Conf., SPCI. Vol. I, 1991, pp. 73-88. Hammel, K. E. & Moen, M. A. (1991 ). Depolymerization of a synthetic lignin in vitro by lignin peroxidase. Enzyme MicrobioL TechnoL, 13, 15-18. Hortling, B., Ranua, M. & Sundquist, J. (1990). lnvestiga-

77

tions of the residual lignin in chemical pulps. Part I. Enzymatic hydrolysis of the pulps and fractionation of the products. Nordic Pulp Paper Res. J., 5(1), 33-7. Kantelinen, A., Ra'tt6, M., Sundquist, J., Ranua, M., Viikari, L. & Linko, M. (1988). Hemicellulases and potential role in bleaching, Tappi J., i-9. Kantelinen, A., Hortling, B., Sundquist, J., Linko, M. & Viikari, L. (1993a). Proposed mechanism of the enzymatic bleaching of kraft pulp with xylanases. Holzforschung, 47, 318-24. Kantelinen, A., Hortling, B., Ranua, M. & Viikari, L. (1993b). Effects of fungal and enzymatic treatments on isolated lignins and on pulp bleachability. Holzforschung, 47, 29-35. Kersten, P. J., Tien, M., Kalyanaraman, B. & Kirk, T. K. (1985). The ligninase of Phanerochaete chrysosporium generates cation radicals from methoxybenzenes. J. BioL Chem., 260, 2609-12. Kirk, T. K. & Phrrell, R. L. (1987). Enzymatic 'combustion': the microbial degradation of lignin. Ann. Rev. MicrobioL, 41,465-505. Niku-Paavola, M.-L. (1991). Isoelectric focusing electrophoresis of lignin. AnaL Biochem., 197, 101-3. Niku-Paavola, M.-L., Karhunen, E., Salola, P. & Raunio, V. (1988). Ligninolytic enzymes of the white-rot fungus Phlebia radiata. Biochem. J., 254, 877-84. Sarkenen, S., Razal, R. A., Piccariello, T., Yamamoto, E. & Lewis, N. G. (1991). Lignin peroxidase: towards a clarification of its role in vivo. J. BioL Chem., 266, 3636-43. Schoemaker, H. E., Harvey, P. J., Bowen, R. M. & Palmer, J. M. (1985). On the mechanism of enzymatic lignin breakdown. FEBSLett., 183, 7-12. Tenkanen, M., Puls, J. & Poutanen, K. (1992). Two major xylanases of Trichoderma reesei. Enzyme MicrobioL TechnoL, 14,566-74. Tien, M. & Kirk, T. K. (1983). Lignin degrading enzyme from the hymenomycete Phanerochaete chrysosporium Burds. Science, 221,661-3. Viikari, L., Ranua, M., Kantelinen, A., Sundquist, J. & Linko, M. (1986). Bleaching with enzymes. In Proc. 3rd Int. Conf. Biotechnology in the Pulp and Paper Industry, pp. 67-9.