Gottfried Kandiollerand Lew Christov1,2

Companies and addresses

1Sappi Biotechnology Laboratory, Dept. of Microbiology, Biochemistry & Food Science, University of the Free State, PO Box 339, Bloemfontein 9300, South Africa
2Sappi Management Services, PO Box 3252, Springs 1560, South Africa

emails and


Trametes versicolor, laccase, mediators, HBT, NHA, VA, sulfite dissolving pulp, soda-aq pulp, heteropolyacid, HPA-5, POM


The delignification and bleaching potential of laccase-mediator systems (LMS) and transition metal polyoxometalates to enhance oxygen bleaching efficiency has been examined on hardwood soda-aq pulp and sulfite dissolving pulp, 1-Hydroxybenzotriazole (HBT), N-hydroxy-acetanilide (NHA), violuric acid (VA) and potassium-octacyanomolybdate(IV) (PCM) were used as mediators in combination with the laccase of Trametes versicolor at various dosages. Following alkaline extraction, kappa number reductions between 7.1% and 50% and brightness increases of up to 6.4 points, depending on pulp type, enzyme and mediator charge, were attained without effecting negatively pulp viscosity. In contrast to LMS treatments, conducted at 55°C, the thermostable heteropolyacid Na8(PMo7V5O40), HPA-5, was used at elevated temperatures (up to 140°C). The polyoxometalate(POM) reinforced oxygen bleaching of soda-aq pulp reduced kappa number by 52% and improved brightness by 10 points whereas kappa number reductions of up to 45.5% and brightness gains of up to 4.4 points were obtained on sulfite dissolving pulp. Compared to LMS treatment, the use of HPA-5 at the oxygen stage resulted in significant viscosity losses on both pulps.


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. The use of oxidative enzymes such as laccases in pulp bleaching is still on laboratory or pilot plant scale1.

Laccases belong to the multicopper oxidases ( which can reduce elemental oxygen to water in a four-electron step and simultaneously perform a one-electron oxidation of many aromatic substrates2. 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 non-phenolic lignin structures. It is assumed that the mediator is needed because the large laccase molecule can not enter the secondary cell wall and oxidize lignin directly. Most of the mediators found contain N-hydroxy-groups, such as 1-Hydroxybenzotriazole (HBT), Violuric acid (VA) and N-Hydroxy-Acetanilide (NHA). Recently, transition metal complexes, such as K4Mo(CN)8 or K4W(CN)8), were suggested  as a new class of laccase mediator for pulp bleaching3.

Another approach to enhance the efficiency of oxygen bleaching is the use of inorganic metal-oxygen cluster anions (polyoxometalates, POM). This class of oxidation catalysts is known to be effective in different organic syntheses4. Two approaches for the use of POM in pulp bleaching have been published. According to the first one, pulp may be bleached with POM under anaerobic conditions and the regeneration of the POM occurs in a separate stage at temperatures higher than those applied in the bleaching stage5-6. However, the high POM concentrations required (0.5M) and the need for a separate appear as drawbacks of this approach. Alternatively, the use of POMs as catalysts in the oxygen delignification stage has been described7. The aerobic lignin oxidation in the presence of POM allows the catalyst to regenerate in the same process stage.


3.1 Pulps
Post-oxygen hardwood soda-aq pulp (kappa number 10.7, 31.2% brightness and 882 cm3/g viscosity), unbleached hardwood sulfite dissolving pulp (kappa number 5.5, 52.5% brightness and 20.25 mPa. s viscosity) and unbleached hardwood soda-aq pulp (kappa number 4.5, 64.2% brightness) were used in the bleaching experiments with LMS and POMs.

3.2 Enzyme
The laccase from Trametes versicolor (optimum pH 4.5 and 55C temperature optimum) was kindly provided by the Consortium für elektrochemische Industrie GmbH (Wacker-Chemie, Munich, Germany).

3.3 Mediators
Potassium Octacyano-Molybdate(IV) (PCM) was synthesized according to literature8. HBT was supplied by Fluka Chemie, Buchs, Switzerland. NHA and VA were kindly provided by Wacker-Chemie, Munich, Germany.

3.4 Laccase mediator treatment (L) and alkaline extraction (E) of pulps
Various charges of mediator (0.5-1.0% on both soda-aq pulp and sulfite pulp) and laccase (5 and 15 U/g) were used at pH 4.5, 55ºC, 3.5 bar oxygen pressure and 10% consistency for 3 h. E-Step: Alkaline extraction has been conducted with 0.7% NaOH at 67ºC and 10% consistency for 67 min.

3.5 HPA-5 synthesis and bleaching of pulp
The synthesis of HPA-5 was conducted as published7. The conditions applied during HPA-5 bleaching of the pulps are indicated in the corresponding chapters of results and discussion.

3.6 Pulp tests
Kappa number of pulps was determined according to Tappi Test Method T236. Brightness and viscosity of pulp handsheets were determined according to Tappi Test Method T452 and T230, respectively. All bleaching experiments and pulp tests were conducted in duplicate and the results presented were the averages of these values.


The goal of this study was to compare the effects of oxidative enzymes and heteropolyacids (POM) as two approaches for catalyzed oxygen delignification of pulp. A subsequent alkaline extraction following LMS pretreatment was conducted due to the fact that the bulk of the lignin removal occurs during the alkaline extraction stage9. As can be seen in Table 4.1, the unbleached hardwood sulfite dissolving pulp was significantly more susceptible to delignification than the post-oxygen hardwood soda-aq pulp. The application of VA resulted in superior results compared to the other mediators, with kappa number reduction of 50% and brightness increase of 6.3 points. In terms of both kappa number reduction and brightness gain, HBT was the second most efficient mediator (45.2% kappa number reduction and 3.5 points brightness gain), followed by NHA and PCM. The application of T. versicolor laccase in combination with NHA, HBT or VA on post-oxygen soda-aq pulp reduced kappa number by 14.6%. Regarding brightness boost, HBT was more efficient (6.4 points brightness gain) than VA (5.4 points), PCM (3.7 points) and NHA (2.1 points).

In contrast to LMS, alkaline extraction of HPA-5 delignified pulps did not enhance pulp delignification and bleaching (Data not shown). It has been described previously that water is not the most efficient solvent for phosphomolybdovanadate oxidation of aromatic substances7. Consequently, HPA-5 treatment of the pulps was conducted in an ethanol/ water medium (50% v/v). One of the advantages of using POM in pulp bleaching is their good thermostability, which in contrast is one of the setbacks of using LMS for lignin oxidation.

Table 4.1. Comparison of the delignification and bleaching abilities of various mediators and T. versicolor laccasea following LE treatment of pulps


Hardwood soda-aq pulp

Hardwood sulfite dissolving pulp

Kappa no reduction (%)

Brightness change (points)

Kappa no reduction (%)

Brightness change (points)





















a Ratio of kappa number:mediator dose of 5:1 was applied for treatment of different pulps. A laccase charge of 15 U/g was used in conjunction with VA, HBT and PCM and 5 U/g with NHA.

As can be seen in Figure 4.1, increased reaction temperature had a beneficiary effect on both kappa number reduction and brightness gain of the POM treated hardwood soda-aq pulp. Delignification at 127C produced pulp with kappa number of 2.5 (76.6% reduction) compared to 6.2 at 90C (42.1% reduction). A further increase of the reaction temperature to 140C reduced kappa number even further to 1.5 (86.0% reduction) within 2 h. Brightness of the pulps showed a similar trend with improved brightness at higher temperatures (62.9% brightness at 140 compared to 40.8% at 90C). However, viscosity of the HPA-5 bleached soda-aq pulp dropped from 589cm3/g (90C) to 257 cm3/g (140C). The severe viscosity loss is a major disadvantage of HPA-5 compared to LMS action where viscosity remains unchanged.

Figure 4.1

However, the improved delignification efficiency of HPA-5 at high temperatures enables the use of short incubation times to limit viscosity losses and increase throughput. As can be seen in Figure 4.2, the majority of both kappa number reduction and viscosity loss during HPA-5 action at 127C, occurred within the first 60 min of treatment. Kappa number was reduced from 10.7 (untreated pulp) to 5.2 (40min), 3.8 (60 min) and 2.3 (120 min), respectively. Viscosity dropped in a similar trend from 882 cm3/g (untreated) to 532 cm3/g (40 min) and 439 cm3/g (60 min) and reached 338 cm3/g after 2h. Brightness however increased from 31.2% (untreated pulp) to 42.4% (40 min), 48.9% (60 min) and reached 56.7% after 2 h.

The optimum HPA-5 concentration under the reaction conditions applied (45 min; 7.4 bar O2; 20% consistency and 127C) on soda-aq pulp was 1.5 mM. Compared to LE treatment of soda-aq pulp, HPA-5 action achieved significantly higher kappa number reduction (51.4% compared to 14.6% with HBT) together with higher brightness gains (12.3 points vs 6.4 with HBT) at reduced treatment time (45 min compared to 3h).

Figure 4.2

HPA-5 treatment of unbleached hardwood sulfite dissolving pulp was conducted at medium consistency (10%) in ethanol/water (50% v/v) at pH 1.9; 90C and 4.5 bar O2 for 75 min. POM bleaching with 0.75mM HPA-5 produced pulp with kappa number 3.0 (45.5%), 56.9% brightness (4.4 points gain) and 15.75 mPa.s viscosity (22.2% reduction) (Figure 4.3). Compared to LE treatment of sulfite dissolving pulp, HPA-5 application produced pulp with lower brightness than VA (6.3 points gain) but with significantly higher brightness values than HBT, NHA and PCM. LE treatment using VA as mediator produced pulp with lower kappa number as compared to HPA-5 application (50% reduction compared to 45.5%). However, a longer treatment time was necessary using LMS (3h compared to 90 min with HPA-5).

Figure 4.3


The efficiency of LMS systems in pulp delignification depends on the pulp and mediator choice. Thermal inactivation of the enzyme limits the application temperature of LMS. The main advantage of LMS is their high selectivity towards lignin and consequently low impact on pulp viscosity.

In contrast, POMs are highly thermostable and applicable at high temperatures (up to 140C). The higher application temperature enables the use of reduced treatment times resulting in enhanced throughput in the delignification stage. However, the significant viscosity loss represents a setback of the system.


The authors wish to acknowledge Sappi Management Services and Sappi Saiccor for funding this project, Sappi Management Services for granting permission to publish this work, and Wacker-Chemie (Germany) for making the T. versicolor laccase, NHA, and VA available.


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6. Springer EL, Atalla RH, Weinstock IA and Ahmed A, Delignfication of high kappa number kraft and soda-anthraquinone pulps with a polyoxometalate in 2000 Tappi Pulping/ Process & Product Quality Conference, Boston, November 5-8, 2000

7. Evtuguin DV, Neto CP, Roch J and de Jesus JDP, Oxidative delignification in the presence of molybdovanadophosphate heteropolyanions: mechanism and kinetic studies. Appl. Cat. A: General 167: 123-139 (1998).

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9. Sealey JE, Runge TM and Ragauskas AJ, Laccase N-hydroxybenzotriazole full sequence bleaching with hydrogen peroxide and chlorine dioxide. Tappi J. 83(9) (2000).


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