Evaluation of the Delignification and Bleaching Abilities of Selected

Chapter 27

Evaluation of the Delignification and Bleaching Abilities of Selected Laccases with HBT on Different Pulps 1,2

G. Kandioller

1,3,*

and L. Christov

Downloaded by FUDAN UNIV on March 2, 2017 | http://pubs.acs.org Publication Date: March 26, 2001 | doi: 10.1021/bk-2001-0785.ch027

1

Sappi Biotechnology Laboratory, Department of Microbiology and Biochemistry, University of the Orange Free State, 9300 Bloemfontein, South Africa Institute for Biochemical Technology and Microbiology, Technical University of Vienna, Getreidemarkt 9, Vienna, Austria Sappi Management Services, P.O. Box 3252, Springs 1560, South Africa

2

3

The laccases of Peniophora sp., Pycnoporus sanguineus, Trametes versicolor and Trametes hirsuta have been used in the presence of a mediator, 1-hydroxy-benzotriazole (HBT), to delignify and bleach various pulps. The effects induced by the laccase/HBT-treatment on pulp brightness, kappa number and viscosity were examined. It was found that delignification improved when increased enzyme and mediator dosages were used. The extent of kappa number reduction was dependent on the pulp type and enzyme source. The pulps were accessible to delignification in the following descending order: unbleached hardwood sulfite pulp>post-oxygen softwood kraft pulp>unbleached softwood kraft pulp>post-oxygen hardwood soda pulp. It was demonstrated that biobleaching with Τ. hirsuta laccase/HBT using a DED sequence could increase pulp brightness by 2.9-12.2 points, alternatively, savings of chlorine dioxide of 40-60% could be obtained. On the other hand, the pulp viscosity was influenced by the selectivity of the particular laccase/HBT system used.

© 2001 American Chemical Society Argyropoulos; Oxidative Delignification Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2001.

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428


Introduction The growing public sensitivity towards the negative environmental impacts of the chlorine-based pulp bleaching has focused research on the development of new environmentally friendly technologies. Among them biobleaching using enzymes has shown great potential in minimizing the use of chlorine-containing bleaching chemicals. In the last decade xylanases have been extensively investigated and already industrially applied (1). The use of oxidative enzymes such as laccases in pulp bleaching is still on laboratory or pilot plant scale (2). Laccases belong to the multicopper oxidases (1.10.3.2) which can reduce elemental oxygen to water in a four-electron step and simultaneously perform a one-electron oxidation of many aromatic substrates (3). Due to the redox potential, laccase alone can only oxidize phenolic lignin structures. The addition of a mediator, a small chemical compound, extends the substrate range to nonphenolic lignin structures (4-7). It is assumed that the mediator is needed because the large laccase molecule can not enter the secondary cell wall and oxidize lignin directly. The first compound described to function as a mediator was 2,2'-Azinobis(3-Ethylbenzthiazoline-6-Sufonic acid) (ABTS) (3,8). Laccase did not improve the bleachability of pulp when acting alone (9) although direct oxidation of the lignin has been reported (10). The use of ABTS and laccase demethylates and delignifies kraft pulp (11). Another compound used as a mediator was 1-hydroxybenzotriazole (HBT). HBT is more effective on pulp than ABTS (12) although HBT forms benzotriazole which is inactive as mediator (5,13). The laccase/HBT treatment of pulp was reported to increase the content of carbonyl and carboxyl groups and reduce the amount of free phenolic hydroxyl and methoxyl groups of residual lignin (14-15). Additional changes in the physical and optical properties of pulp also occurred (16). Over 50% delignification of softwood kraft pulp with Polyporus laccase/HBT was achieved (13). Using T. hirsuta laccase/HBT, 43% delignification of pine kraft pulp and 60% delignification of post-oxygen pine kraft pulp was reported (17). However, both ABTS and HBT are not available as bulk chemicals and are not completely environmentally safe. Considerable efforts have been invested in the screening for more efficient and environmentally friendly mediators (18-19). Although a number of compounds have been identified as possible mediators, one which fulfills all the requirements has not yet been identified. The extent of pulp delignification with laccase-mediator systems (LMS) was found to be dependent on both the type of mediator and laccase source. For instance, using laccase preparations from six

Argyropoulos; Oxidative Delignification Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2001.

429 different fungal strains with HBT and ABTS, pine kraft pulp was delignified between 19% and 40% (20). In this work, four microbial laccases in conjunction with HBT have been studied to evaluate their ability to delignify and bleach different pulps. In an effort to reveal the bleaching power of the LMS, the potential brightness gains and savings of chlorine dioxide using a DED bleaching sequence have been determined.


Materials and Methods

Enzyme Preparations Prior to use, the culture supernatants of Peniophora sp., Trametes versicolor and Pycnoporus sanguineus, grown on 4% molasses for 14 days, were subjected to a 40-fold concentration using an Amicon ultrafiltration cell (10 kDa molecular mass cut-off). The laccase of Trametes hirsuta was kindly provided by Dr. Matti Siika-aho from the VTT Biotechnology and Food Research, Finland. Its production and purification has been described previously (14).

Enzyme Assays The laccase activities were determined using 2,2'-Azino-bis(3Ethylbenzthiazoline-6-Sufonic acid) (ABTS) as substrate (9). One unit of enzyme activity was defined as that amount of enzyme that can release 1 μιηοΐ of ABTS oxidized per minute. The cellulase activity was determined according to the DNS method using carboxymethyl cellulose as substrate (21). All enzyme preparations were cellulase-free.

Pulps The industrial pulps used in this work were supplied from various Sappi mills: unbleached softwood kraft pulp; post-oxygen softwood kraft pulp; unbleached hardwood sulfite pulp and post-oxygen hardwood soda pulp. The pulps were thoroughly washed with distilled water until a neutral pH of the wash waters was attained and then used in the experiments without any prior


430 pretreatment or preparation. Kappa number and brightness of these pulps are shown in Table I,


Enzyme/HBT Treatment (L) and Alkaline Extraction (E) of Pulps Washed pulps (3 g dry weight) were treated with laccase/HBT (L-treatment) at enzyme charges of 5, 10 and 15 U/g pulp (dry weight) and pH 4.5 (T versicolor, P. sanguineus and Τ hirsuta laccases) and 5.0 (Peniophora laccase). The mediator dosage was 2% and 4% (on unbleached kraft pulp), 1% and 2% (on post-oxygen kraft pulp), 0.5% and 1% (on both unbleached sulfite and postoxygen soda pulps). The pulp treatments were carried out at 55°C and 10% pulp consistency for 3 h under 3.5 bar 0 pressure. Thereafter, samples of the reaction mixture were withdrawn for determination of the residual laccase activity using the ABTS assay. Pulps were then extracted with 0.7% NaOH (on pulp) at 67°C and 10% consistency for 67 min (Ε-treatment), washed and dried. Brightness, kappa number and viscosity were determined. Alkali-extracted pulps (E-pulps) and DED-bleached pulps, omitting the L-treatment, served as controls in the delignification (LE) and biobleaching (LE-DED) experiments, respectively (Table I). All experiments were carried out in triplicate. 2

Table I. Properties of pulps used as controls Unbleached Post-oxygen kraft pulp kraft pulp Treat- Kappa Bright- Kappa Brightment number ness(%) Number Ness None 22.2 20.4 10.8 29.8 Ε 21.6 21.3 10.4 30.9 DED ND ND ND 62.2

Unbleached Post-oxygen sulfite pulp soda pulp Kappa Bright- Kappa Bright(%) number ness (%) Number ness (% 64.2 4.5 5.2 57.0 64.9 4.4 57.0 5.1 88.6 ND 91.6 ND

NOTE: ND, Not determined; E, Alkaline extraction; D, Chlorine dioxide.

Analysis of Pulp Properties Kappa number of the LE-treated pulps was determined according to TAPPI Test Method T236 cm-85. Pulps from the individual experiments were pooled together for kappa number determinations of unbleached sulfite pulp and postoxygen soda pulp. Viscosity of the LE-treated and bleached pulps was determined according to Tappi Test Method T230 om-89. Brightness of pulp handsheets was measured with an Elrepho 2000 instrument based on Tappi Test


431 Method T452 om-92. Methanol release from pulp was examined in the enzyme filtrates using a HP 5890 gas chromatograph.

Chlorine Dioxide Bleaching of LE-treated Pulp Post-oxygen kraft pulp, unbleached sulfite pulp and post-oxygen soda pulp (5 g dry weight) were treated with the laccase of T, hirsuta and P. sanguineus at a charge of 15 U/g and 2% HBT (on post-oxygen kraft pulp) or 1% HBT (on the other two pulps). The LE-treatment was followed by a D!ED bleaching performed under the following conditions: Downloaded by FUDAN UNIV on March 2, 2017 | http://pubs.acs.org Publication Date: March 26, 2001 | doi: 10.1021/bk-2001-0785.ch027

2

• • •

Dj-step: 2.63% active chlorine on pulp (or reduced as indicated); 128 min; 67°C; 10% consistency E-step: 0.7% NaOH on pulp; 82 min; 67°C; 10% consistency D -step: 1.315% active chlorine on pulp (or reduced as indicated); 195 min; 67°C; 10% consistency 2

Results and Discussion

Residual Laccase Activity The residual laccase activity was determined after completion of the Ltreatments to ensure that enough enzyme was added to successfully perform the enzymatic reactions over the entire incubation period. It was found that the amount of residual laccase activity depended on the enzyme origin, pulp type, laccase/mediator dosages used in the experiments. These activities as obtained with 1% HBT varied between 3% and 100% of the initial activity (Figure 1). Between 65% and 100% of the P. sanguineus laccase was still active after treatment. The highest residual activity was determined on post-oxygen kraft pulp, followed by unbleached sulfite pulp and post-oxygen soda pulp. The use of greater mediator and enzyme charges resulted in lower residual laccase activities (data not shown). Only trace amounts of methanol were detectable in the enzyme filtrates (data not shown).


432

M Peniophora Φ P. sanguineus • T. versicolor • Τ hirsuta


post-oxygen kraft pulp

unbleached sulfite pulp

post-oxygen soda pulp

Pulp type Figure 1: Impact ofpulp type on residual laccase activity following Ltreatment with 15U/g and 1% HBT

LE-treatment of Pulps

Unbleached Kraft Pulp The delignifying effect of the LE-treatment on unbleached kraft pulp, expressed as kappa number reduction over the Ε-treated unbleached kraft pulp, revealed notable differences related to the treatment conditions used (Figure 2). A further delignification of pulp was achieved by increasing the enzyme and mediator dosages. For instance, the kappa number reductions attained using the highest enzyme (15 U/g) and mediator dosage (4%) were 18.3% (Peniophora laccase), 15.7% (P. sanguineus laccase), 21.3% (T versicolor laccase) and 19% (T. hirsuta laccase). The LE-treatment induced no brightness gains on unbleached kraft pulp (Figure 3). The brightness change was negative in all experiments except when 5 U/g of Peniophora laccase and 2 % HBT were used. The brightness loss was more pronounced when increased enzyme and mediator dosages were used. A brightness decrease of up to 2.7 points was obtained with 15 U/g of P. sanguineus laccase and 4% HBT. Apparently the darkening effect observed is due to chromophore changes in pulp which occur during the laccase/HBT treatment.


433

ΕΠΟ Peniophora fàP. sanguineus


Τ3

MT. versicolor •

T. hirsuta

2

I / / / / / / *^ ^ ^^ (

s

s

Charge of laccase/mediator (U/g / %)

Figure 2: Impact of LE treatment on kappa number of unbleached kraft pulp

M Peniophora P. sanguineus ϋ 71 versicolor • T. hirsuta

*r

&

a*

y y y y yy

&

Charge of laccase/ mediator (U/g / %)

Figure 3: Impact of LE treatment on brightness of unbleached kraft pulp


434


Post-oxygen Kraft Pulp The L-treatment of post-oxygen kraft pulp was carried out with 1% and 2% HBT so to keep a ratio between kappa number and mediator dose similar to that used in the experiments with unbleached kraft pulp. Compared to unbleached kraft pulp, however, the effect of the LE-treatment on post-oxygen kraft pulp was more profound, as shown in Figure 4. Treatment of post-oxygen kraft pulp with the Peniophora, Τ hirsuta and P. sanguineus laccases resulted in kappa number reductions similar to those observed on unbleached kraft pulp. However, the Τ versicolor laccase was able to decrease kappa number by 19.2% (5 U/g and 1% HBT) as compared to 14.4% (5 U/g and 2% HBT) obtained on unbleached kraft pulp. Apparently, the rate of delignification could be improved by increasing the enzyme and mediator dosage as judged by the results obtained on all pulps with 15 U/g and 2% HBT. Treatment with the two Trametes laccases brought about the greatest reductions in kappa number: 29% with Τ versicolor laccase (10 and 15 U/g with 2% HBT and 15 U/g with 1% HBT) and 26% with Τ hirsuta laccase (15 U/g and 2% HBT ).

H Peniophora MP. sanguineus M 71 versicolor • T. hirsuta

Charge of laccase/mediator (U/g / %)

Figure 4: Impact of LE treatment on kappa number ofpost-oxygen kraft pulp In most instances, in contrast to unbleached kraft pulp, post-oxygen kraft pulp gained brightness following LE-treatment (Figure 5). The enzymes induced different brightness effects on pulp. While 7! hirsuta and T. versicolor laccases increased brightness by up to 2.3 points (with 15 U/g and 2% HBT), however, the P. sanguineus enzyme in fact darkened the pulp by 1.1 points (15 U/g and 1% HBT) and 0.6 points (15 U/g and 2 % HBT). On the other hand, the laccase of Peniophora could not cause any substantial change in brightness under the conditions used (Figure 5). Because of the poor performance of this enzyme, results obtained on the other pulps are not shown.


435

M Peniophora

S 'δ

MP. sanguineus • Γ versicolor

Ο

• Γ hirsuta

I

# # ^

\V

^

^

Evaluation of the Delignification and Bleaching Abilities of Selected

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