Enzymes for Pulp and Paper Processing - American Chemical Society

Chapter 18

Biological Bleaching of Kraft Pulp with Lignin-Degrading Enzymes 1

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R. Kondo , K. Harazono , K. Tsuchikawa , and K. Sakai Downloaded by STANFORD UNIV GREEN LIBR on October 7, 2012 | http://pubs.acs.org Publication Date: November 21, 1996 | doi: 10.1021/bk-1996-0655.ch018

1

Department of Forest Products, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812-81, Japan Tokushu Paper Manufacturing Company Limited, Shizuoka 411, Japan 2

In vitro bleaching of an unbleached hardwood kraft pulp was performed with manganese peroxidase from fungus Phanerochaete sordida strain YK-624. When the kraft pulp was treated with a partly purified MnP in the presence of MnSO , Tween 80 and sodium malonate with continuous H O addition at 37ºC for 24 h, pulp brightness increased by about 15 points and the kappa number decreased by about 6 points in comparison with untreated pulp. When the pulp was treated without the additon of MnSO , the pulp brightness increased by about 10 points in the presence of 2 m M oxalate, a good manganese chelator and reducing reagent, while the brightness did not significantly increase in the presence of 50 m M malonate. To establish a totally chlorine-free bleaching process, oxygenbleached kraft pulp (OKP) was treated with four-stage bleaching process consisting of sequential MnP treatment, alkaline extraction, MnP treatment and hydrogen peroxide treatment stage. Full bleached kraft pulp (brightness 91%, yield 97%) could be obtained from O K P by the combination of enzyme treatment and hydrogen peroxide bleaching. To improve in vitro bleaching of the kraft pulp with MnP, the characterization of various MnPs from some white-rot fungi was examined. MnP from Ganoderma sp. YK-505 was superior to MnPs from P. sordida YK-624 and P. chrysosporium in stabilities against high temperature and high concentration of H O . MnP from Ganoderma sp. YK-505 differed in pHactivity profile from other MnPs. These results suggest that MnP from Ganoderma sp. YK-505 has different structure from those other fungi, and may be useful for biotechnological applications and studies of the relationship between structure and function. 4

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The kraft process, at present the most common commercial chemical delignification method, produces a dark pulp because of the color of residual modified lignin residues. These residues are normally bleached or removed in multistage bleaching procedures using a combination of chlorination and alkaline-extraction stepes. The effluent from

0097-6156/96/0655-0228$15.00/0 © 1996 American Chemical Society

In Enzymes for Pulp and Paper Processing; Jeffries, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

18. KONDO ET AL.

Biological Bleaching with Lignin-Degrading Enzymes

229

such bleaching processes is of growing environmental concern because it contains numerous chlorinated organic substances, including mutagenic chlorinated phenols and dioxins (1,2). Today, general concern about the environmental impact of chlorine bleaching effluents has led to a trend toward elementary chlorine-free or totally chlorine-free bleaching methods. Considerable interest has been focussed on the white-rot basidiomycete fungi, since they are the only group of organisms known to

Downloaded by STANFORD UNIV GREEN LIBR on October 7, 2012 | http://pubs.acs.org Publication Date: November 21, 1996 | doi: 10.1021/bk-1996-0655.ch018

be capable of preferential degradation of native lignins and complete degradation of wood.

In vivo bleaching of kraft pulp with white-rot fungi Work in our laboratory has demonstrated that the white-rot fungus, IZU-154 can decrease both residual lignin color (brightness) and concentration (kappa number) in hardwood kraft pulp and softwood kraft pulp (3,4). Thus, fungal treatment yields pulps with substantially increased brightness (biobleaching). Introducing the IZU-154 treatment into the kraft pulp bleaching process made it possible to bleach unbleached kraft pulp without any chlorine-based chemicals, and the pulp bleached by this process had satisfactory optical and strength properties (5). To date, Phanerochaete sordida(6), Coriolus (Trametes) versicolor (7-9) and unidentified isolate(i0) have been shown to brighten kraft pulp remarkably.

However, the process is rather slow

compared with chemical bleaching (days instead of hours), and the attack of the cellulose by the fungus cannot be avoided completely. To overcome these drawbacks of the fungal process, the enzymolozy of fungal delignification should be determined.

Lignin-degrading enzymes contributed to biobleaching Paice et al. and we have reported that manganese peroxidase (MnP) and laccase activities but not lignin peroxidase (LiP) activity are detected during biobleaching of unbleached hardwood kraft pulp by white-rot fungi with bleaching ability(#,i7). Furthermore, we developed a cultivation system in which a membrane filter was used to prévoit direct contact between hyphae and kraft pulp while allowing extracellular enzymes to attack the kraft pulp. By using this system we found that the level of secreted MnP activity in the filterable components was substantial during in vitro bleaching with P. sordida YK-624 (12). Some reports have also shown that MnP plays an important role in the bleaching of the pulp by white rot fungi (13-15). MnP first discovered in P. chrysosporium (16,17) is known to be secreted by many lignin-


230

ENZYMES FOR PULP AND PAPER PROCESSING

degrading fungi, including T. versicolor (75), P. sordida (18), and others(19,20). MnP is a heme-containing enzyme and oxidizes M n

2 +

to M n

3 +

which chelated with an

organic acid, in turn, oxidizes phenolic substrates, including lignin model compounds (21,22), dehydropolymerizate (23) and high-molecular-weight chlorolignin (24).


In vitro bleaching of kraft pulp with MnP There have been several reports of using in vitro enzyme treatments to bleach hardwood kraft pulp. Arbeloa et al. showed that treatment of unbleached kraft pulp with LiPs facilitated subsequent chemical bleaching (25). Bourbonnais et aL demonstrated that unbleached kraft pulp was delignified with isolated laccase from T. versicolor in the presence of 2,2-azmobis-(3-emylbenztliiazoline-6-sulphonate) and that methanol was released (26). It has also been reported that some delignification of hardwood kraft pulp by MnP was observed, but the extensive brightening observed with the fungus was not achieved with MnP (15). Direct utilization of LiP and laccase of Phlebia radiata in kraft pulp bleaching was not successful (27). We performed in vitro bleaching of the kraft pulp with MnP from the fungus P. sordida YK-624, which was isolated from decayed wood obtained from a forest and exhibited remarkable bleaching ability with the pulp (11,12). When the kraft pulp was treated with a partly purified MnP in the presence of MhSC>4, Tween 80 and sodium malonate with continuous

H2Q2

addition at 3 7 ^ for 24 h, pulp brightness

increased by about 15 points and the kappa number decreased by about 6 points in comparison with untreated pulp. When the pulp was treated with MnP without Tween 80, the brightness increased by only about 4 points, to 35.5%. Therefore, the surfactant is also an important factor on bleaching of pulp with MnP. The effect of various surfactant on the brightening of the pulp with MnP is shown in Fig. 1 (28). It was observed that the use of Tween 80 or the combination with an unsaturated fatty acid and Tween 20 improved the brightness of the pulp after bleaching with MnP compared with the use of Tween 20 only. Recently, Bao et al. indicated that nonphenolic lignin was oxidized by a lipid peroxidation system that consisted of MnP, Mn

2 +

and unsaturated fatty acid esters present in Tween 80 (29). The detergent action

of Tween 80 and/or the unsaturated fatty acids it contains may be responsible for the enhancement of MnP's bleaching effect



50

Time (h)

Figure 1. Effect of surfactant on bleaching of U K P with MnP. U K P was suspended at a consistency of 1% in 50 m M malonate buffer (pH 4.5) containing 100 U of MnP, 0.1 m M MnS04 and 0.05% surfactant, and 10 m M aqueous H2O2 was added at a rate of 3 ml/h at 3 0 Ό for 24 h. Symbols: • , Tween 20; Δ , Tween 20 + linolenic acidjO , Tween 80;B , linolenic acid; A , CHAPS; · , without surfatant. (Reproduced with permission from Ref. 28. Copyright 1996. Japan Tappi.)

Time (h)

50



232


8.0

5.0H .0.0

1 0.1

,

,

0.2

0.3

H 0.4

P A M (%)

Figure 2. Effect of P A M addition on breaking length. Symbols: • , OM E M P ; O , O - M E M ; O, O-CED; V , O P ; Δ , OKP. [MnP treatment (M): 20 g of oxygen bleached kraft pulp (OKP) (pulp consistency, 2%), 2000 U of MnP, 0.1 m M MnS04, 200 U of glucose oxidase and 2.5 m M glucose at 3 0 Ό and 150 rpm for 24 h. Alkaline extraction (E): 2.5% aqueous NaOH at a pulp consistency of 10% for 1 h at room temperature. Peroxide treatment (P): 4% aqueous H2O2 at a pulp consistency of 15%. C E D treatment: 2.1%Cl2, 1.5%NaOH, 0.5%ClO2.] (Reproduced with permission from Ref. 30. Copyright 1996. Japan Tappi.)

5.5

P A M (%)

Figure 3. Effect of P A M addition on burst index. Symbols are same as Figure 2.


18. KONDO ET AL.


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TCF bleaching process with introduction of enzyme treatment of MnP To establish a totally chlorine-free bleaching process, oxygen-bleached kraft pulp (OKP) was treated with four-stage bleaching process consisting of sequential MnP treatment, alkaline extraction, MnP treatment and hydrogen peroxide treatment stage. Full bleached kraft pulp (brightness 91%, yield 97%) could be obtained from OKP by


the combination of enzyme treatment and hydrogen peroxide bleaching. The results suggest that it should be possible to establish a chlorine-free bleaching process. The physical properties of this in vitro bleached pulp showed higher values in breaking length and burst index than those of a pulp bleached with chlorine-based chemicals. Moreover, the addition of polyacrylamide to the in vitro bleached pulp resulted the great improvement on strength properties as shown in Fig. 2-4 (30).

In vitro bleaching of kraft pulp with MnP without addition of MnS04 There are some differences between in vivo bleaching with fungi and in vitro bleaching with MnP with respect to treatment conditions. Major differences between the two bleaching systems were the use of additional MnSOt and high concentrations 3+

of malonate as a Mn -chelator during in vitro bleaching with MnP. Since the solidstate fermentation system used in our work were added only water and mycelia to the pulp (3-5,11), the manganese in the pulp must have been used by the MnP catalytic system. We reported that unbleached hardwood kraft pulp was not brightened by P. sordida YK-624 when metal ions had previously been removed from the pulp by acid treatment but that the ability of the fungus to bleach this pulp was restored when Mh salts were added back to the pulp (6). Chemical species of Mh present in the pulp is unclear. Insoluble MnQ2 accumulates in wood decayed by some white rot fungi (31). Mn

3 +

oxidized by MnP is transformed to M n

4 +

(MnCte) and M n

2 +

due to the

hydrolysis and disproportionation (32). Organic acids such as lactate, malonate and oxalate, which chelate M h generated by MnP, stabilizing M n

3 +

3 +

in aqueous solution, play important roles in the

MnP system. The white rot fungi, including P. chrysosporium and T. versicolor, secrete organic acids which function as M n

3 +

chelators (33-35). It was reported that

millimolar concentrations of oxalate are produced by various fungi such as P. chrysosporium and T. versicolor (34,36-38). The physiological concentrations of


234



350

P A M (%)

Figure 4. Effect of P A M addition on folding endurance. Symbols are same as Figure 2.

55

30H

,

0

5

H -h 10 0

H2O2 added (mM)

. 5

r H 10 0

H2O2 added (mM)

.

1

5

HH 10 0

H2O2 added (mM)

r

1

1

5

10

H2O2 added (mM)

Figure 5. Effect of the addition of MnS04 on the brightness of U K P treated with MnP. U K P was suspended at a consistency of 1% in 50 m M malonate buffer (pH 4.5) containing 100 U of MnP, 0.1 m M MnS04 and 0.05% surfactant, and various concentrations of aqueous H2O2 were added at a rate of 3 ml/h at 3 0 Ό for 24 h. Symbols: · , without MnS04; O , 0.1 m M MnS04 added. (Reproduced with permission from Ref. 40. Copyright 1996. American Society for Microbiology.)


18. KONDO ET AL.


oxalate stimulate optimal MnP activity (33-35,37). Oxalate is also known to have reductive activity (39). Therefore, we tried to bleach kraft pulp with MnP without 3+

addition of MnS04 by using oxalate with a good Mn -chelating and reductive ability. The effect of the addition of MnS04 on bleaching of kraft pulp with MnP in the presence of various organic acids was examined by changing the concentration of aqueous H2O2 added (Fig. 5) (40). In 50 m M malonate buffer, an increase in


brightness was observed with the addition of MnSOt but not without additional MnSQt. When the pulp was treated with the continuous addition of 3 and 5 m M aqueous H2O2 in the presence of 2 m M oxalate, the brightness increased by about 13 points without the addition of MnSQ*. This indicates that MnP bleaching of kraft pulp can be performed without the addition of MnS04 if we use oxalate, which can also reduce insoluble Mn02. Oxygen-bleached hardwood kraft pulp was bleached with MnP with addition of MnSGi for the establishment of a totally chlorine-free bleaching process as previously mentioned. The addition of hydrogen peroxide bleaching was needed after treatment with MnP. The metals in pulp should be controlled by using chelating agents, e. g., EDTA, prior to hydrogen peroxide bleaching. Since manganese contait in the pulp decreases by bleaching with MnP without the addition of MnSOt, the use of oxalate during bleaching with MnP will reduce the amount of EDTA or allow omission of the EDTA treatment step during the subsequent hydrogen peroxide bleaching stage.

Characterization of MnPs from various white-rot fungi for improvement of enzyme bleaching

the

Many ligninolytic fungi produce MnP in various cultures such as synthetic liquid and solid-state media (19,20). For the ubiquity of MnP among various fungi, a MnP isozyme which has a different function from well-known MnP from P. chrysosporium (41-43) may be present Enzymological and kinetic studies of the isozyme could be useful for understanding catalytic characteristics and improving stability of peroxidases. In previous study, higher selectively ligninolytic fungi was screened and isolated from decayed wood samples, and biological bleaching of hardwood kraft pulp with those fungi was carried out (11). The result showed the relationship between pulp brightness increases and MnP activities was observed during the bleaching with almost fungi which remarkably brightened hardwood kraft pulp. On the other hand, since Ganoderma sp. YK-505 deviated from the relationship,


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236


Temperature (°C) Figure 6. Effect of temperature on the activity of MnP from Ganoderma sp. YK-505 ( · ) , P. sordida YK-624 ( Δ ) and P. chrysosporium O.Reaction mixtures contained 1 m M 2, 6-dimethoxyphenol, 1 m M MnSCto, 0.2 m M H2O2 and 50 m M malonate (pH4.5). Control activity(100%) was obtained at37°C.

100-

80>* "> *-· Ο 05

I V

ce

604020-

0-

0

2

4

6

H2O2

(mM)

8

10

Figure 7. Effect of H2O2 concentration on the activity of MnP from Ganoderma sp. YK-505 ( · ) , P. sordida YK-624 ( Δ ) and P. chrysosporium (D).Reaction mixtures contained 1 m M 2, 6dimethoxyphenol, 1 m M MnS04 and 50 m M malonate (pH 4.5). Control activity (100%) was obtained with 0.2 m M H2OZ


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KONDO ET AL.


237

we expected MnP from the fungus might have a different property from those of other white rot fungi. Therefore, properties of partially purified MnPs from liquid cultures of three white rot fungi, Ganoderma sp. YK-505, P. sordida YK-624 and P. chrysosporium were compared. Thermostability of MnPs from three fungi was determined. Enzyme reaction was done at various temperatures for 3 minutes after incubation at each temperature


for 10 minutes (Fig. 6). The temperature profile of MnP from P. sordida YK-624 was similar to that of MnP from P. chrysosporium. High activities were exhibited in the temperature range 40-50^. The activities of MnPs from both fungi were not significantiy detected at temperatures more than 50*€. In the case of MnP from Y K 505, higher the temperature was, higher the activity was observed. Optimum activity was exhibited at 55*0. About 50% activity was also observed at 65

Enzymes for Pulp and Paper Processing - American Chemical Society

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