5 The present invention concerns a method according to the preamble of claim 1 for

<ξ enzymatic treatment of lignocellulosic materials, in particular cellulose pulps.

According to the present invention, the material to be treated is contacted with enzymes containing hemicellulase, cellulase and/or lignin degrading activities to hydrolyze 10 hemicellulose, cellulose and/or lignin.

As far as the prior art is concerned, reference is made to the following publications:

1. Viikari L, Ranua M, Kantelinen A, Linko M and Sundquist J. Proc. 4th Int. Congr. 15 on Wood and Pulping Chemistry, Paris 1987. Vol. I, pp. 151-154.

2. Kantelinen A, Sundquist J, Linko M and Viikari L. Proc. International Symposium on Wood and Pulping Chemistry, Melbourne 1991, Vol. I, pp. 493-500.

20 3. Croon I and Enstrόm BF, Tappi J 60 (1963) 9: 151-154.

4. Sjόstrόm E, Nordic Pulp and Paper Research Journal 4 (1989) 2: 90-93.

5. Scallan AM and Grignon J, Svensk Papperstidn 82 (1979) 2: 40-47.

25

6. Scallan AM, Tappi J 66 (1983) 11: 733-75.

7. Lindstrom T and Carlsson G, Svensk Papperstidn. 85 (1982) 3: R 14-20.

30 8. Clark TA, McDonald AG, Senoir DJ, Mayers PR, Abstr. 4th Int Conf on

Biotechnology in the Pulp and Paper Industry, Raleigh 1989, pp. 39-40.

9. Tan LUL, Yu EKC, Louis-Seize GW and Saddler JN, Biotechnol. Bioeng. 30

i

(1987) 96-100.

10. Paice MG, Bernier R and Jurasek L, Biotechnol Bioeng 32 (1988) 235-239.

11. Paice MG and Jurasek L, Wood Sci Technol 4 (1984) 187-198.

12. Noe P, Chevalier J, Mora F and Comtat J, J Wood Sci Technol 6 (1986) 167-184.

13. Mora F, Comtat J, Barnoud F, Pla F, Noe P, J Wood Sci Technol 6 (1986) 147- 165.

14. Fuentes J-L and Robert M, FR Patent Application No. 8613208

15. Pommier J-C, Fuentes J-L and Goma G, Tappi J 72 (1989) 6: 187-191.

16. Rattδ M, Bacterial hemicellulases. Licentiate's Thesis, University of Helsinki, Department of Forestry and Agriculture. 1990.

17. Viikari L, Kantelinen A, Rattδ M and Sundquist J, Enzymes in Biomass Conversion. (GF Leatham, ME Himmel eds.) ACS Symp Ser 460, USA 1991, 12-22.

18. Tenkanen M, Pulps J and Poutanen K (1992) Two major xylanases of Trichoderma reesei. Enzyme Microb Technol 14 (1992) 566-574.

19. Parisi F, Advances in lignocellulosics hydrolysis and the utilization of the hydrolyzates. Adv. in Biochem. Eng. /Biotechnol. 38 (1989) 53-88.

20. Avgerinos GC, Wang DIC. Direct microbiological conversion of cellulosics to ethanol. Annual reports on fermentation processes, Vol.4, pp. 165-189.

21. Stahlbrand H. Siika-aho M, Tenkanen M, Viikari L, Purification and characterization of two jS-mannanases from Trichoderma reesei. Submitted to J. Biot.

In traditional chlorine bleaching of cellulose pulps lignin is solubilized by chlorine or chlorine dioxide and extracted with alkali. Today, pulps are also often delignified with non-chlorine chemicals, such as oxygen, hydrogen peroxide, ozone or with combinations thereof. According to methods known in the art, enzymatic treatments by hemicellulases or lignin degrading enzymes have been combined with these traditional or new bleaching methods to improve the bleachability of pulps [1, 2]. The required amounts of enzymes are small and the enzymatic treatment can easily be introduced as a part of the pulp bleaching process.

According to present knowledge, the hemicellulases act primarily on the xylan located on the surface of the fibres [2]. This xylan has been solubilized under alkaline pulping conditions and thereafter reprecipitated on the fibre surface in a chemically and structurally modified form. This xylan is the target for hemicellulase treatments carried out prior to bleaching or between different chemical delignification steps. The chemical structure of hemicelluloses varies according to the wood species and pulping method used. Thus, for instance, the xylan in birch sulphate (kraft) pulps contains mainly xylose units with some methylglucuronic acid side groups, whereas xylan in pine sulphate pulp contains in addition to methyl glucuronic acid groups also arabinose side groups [3],

Pulp contains various ionizable groups. Of these groups only the carboxyl groups are ionized in neutral or slightly acidic conditions. The majority of the carboxyl groups in kraft pulps are methylglucuronic acid groups present on the xylan backbone [4], although the peeling reaction of the polysaccharide is stopped by the formation of metasaccharinic acid or other alkali-stable carboxyl groups. The counter-ions of the carboxylic ions influence the polyelectrolytic behavior, the extent of swelling and even the optical properties, such as the brightness stability. Furthermore, the presence of certain metal cations as counter-ions may be harmful to totally chlorine-free bleaching (TCF)-sequences (peroxide or ozone bleaching sequences) by causing degradation of the bleaching chemicals and thus decreasing the quality of the pulp. Therefore it is essential that these metals be removed prior to bleaching.

According to the prior art, enzymatic treatments have been applied directly to fibres from the cooking process which have been more or less thoroughly washed. The enzymatic treatments have also been applied to oxygen delignified pulps. In studies carried out during the work on this invention we have found that the presence and type of the counter-ions of the pulp carboxylic groups essentially affect the action of the enzymes used. In the hitherto known solutions no attention has been paid to the amount and type of the counter-ions of pulp carboxylic groups, nor have these counter-ions been modified in any way which would make the fibres more suitable for the action of enzymes and leading to optimal accessibility of the targeted substrate to the enzymes.

The present invention aims at eliminating the drawbacks of the prior art enzymatic methods by providing a new method for the treatment of lignocellulose materials, in particular cellulose pulps.

It is well known that the surface charge of lignocellulosic materials, such as pulps, primarily is affected by the carboxylic acids [4]. As stated above, the amount of carboxylic acids in the lignocellulosic materials or pulps is highly dependent on the chemical treatment of the material, i.e. the pulping method. The charge and the degree of dissociation of the carboxylic groups depend on the type of the counter-ion of the acidic group [5, 6, 7]. According to the literature it has been observed that the swelling of the fibres is essentially affected by the degree of dissociation and the type of counter-ions (metals-ions). By changing these counter-ions or by producing pulps with modified metal counter-ions, the water binding properties, i.e. the swelling of the fibres can be controlled [6]. These phenomena, however, have never been studied or exploited as a critical factor for the optimization of the enzymatic treatments. Thus, neither the metal contents of pulps nor their effect on the fibre properties (i.e. swelling or surface charge) has to date been measured with respect to the enzymatic treatments of lignocellulosic materials or pulps in order to increase the enzymatic effect on the hydrolysis of lignocellulosic materials or bleachability of pulps. In the previous studies concerning the carboxylic groups or their counter-ions, the effects of these factors have not been taken into account in combination with an enzymatic treatment or for its optimization.

According to this invention, it has unexpectedly been observed that by modifying the type of the counter-ions or by substituting carboxylic groups in the metal-free pulp with specific counter-ions, remarkable improvements of the effects of enzymatic treatments and of the bleachability of the fibres can be obtained. By changing the type of the counter-ions of the carboxylic acids or by changing their degree of dissociation, respectively, the optimal function of the enzymes in the fibre materials can be controlled. At the same time, the enzymatic action can be directed towards the most appropriate and preferred part of the fibres. This same principle, used in another way, can also be employed to increase the total hydrolysis of lignocellulosic materials.

In more detail, the method according to the present invention is mainly characterized by what is stated in the characterizing part of claim 1.

In the literature, several methods and processes based on the utilization of hemicellulases and other lignocellulose degrading enzymes have been presented. All these methods aim at attaining limited or extensive hydrolysis of hemicellulose, cellulose or lignin. These methods include, e.g., partial hydrolysis of hemicellulose for reducing the consumption of chlorine chemicals or for reducing the amounts of chlorinated residues in the pulp or in the effluents [1, 8, 9]. The prior art also comprises methods for the removal of residual hemicellulose in the production of dissolving pulp [10, 11], the modification of fibre properties by a partial hydrolysis of hemicelluloses [12 to 15] and the hydrolysis of lignocellulosic materials [19, 20]. The present invention can be advantageously used for the essential improvement of all these applications, and more generally, all enzymatic applications of fibre materials.

The invention provides several important advantages. Thus, it is particularly important that, using the invented method, the extractability of lignin due to hemicellulase action can be greatly improved. By controlling the above mentioned factors, the type and amount of chemicals and enzyme dosages used for pretreatment of industrial lignocellulosic, in particular cellulose pulps, can be optimized. This allows for the use of environmentally suitable totally chlorine-free or low-chlorine bleaching methods. The invention also essentially improves the possibilities to employ said methods. When

using an enzymatic treatment combined with the method of invention, the enzyme action and thus the brightness of the pulps is improved. Another essential advantage resides in the fact that the loss of pulp yield, usually observed after the enzymatic treatment, can be remarkably decreased due to the site-directed, limited hydrolysis of pulp.

By using the method of the invention, the accessibility of lignocellulose degrading enzymes and the overall hydrolysis yield of lignocellulosic materials can be improved.

According to a preferred embodiment, the present invention provides a method for promoting the action of enzymes on essentially metal free pulps or other lignocellulosic materials. In particular, it is possible to combine said method with totally chlorine free bleaching sequences comprising an enzymatic pretreatment step. In this case, the enzymatic treatment is carried out before the metal removal or the steps of enzymatic treatment and metal removal are carried out at the same time. Within the scope of the preferred embodiment of the invention it is also possible to render the metal free pulp more susceptible to hydrolyzation by carrying out a pH adjustment by using a suitable metal hydroxide or by adding metal salts or other metal containing compounds to the pulp in order partially or totally to convert the pulp into a certain metal form.

According to another preferred embodiment, the action of the enzymatic treatment is enhanced by substituting the (monovalent) counter-ions present in the pulp by bi- or trivalent counter-ions. This may be achieved by adding salts or other compounds containg bivalent or trivalent metal ions to the pulp. The metal cations may also be added to the pulp in the enzyme preparation.

According to still a further preferred embodiment which may be combined with bleaching using oxygen chemicals, in particular (hydrogen) peroxide, the pulp counter- ions are converted to magnesium-form prior to the enzymatic hydrolysis in order to improve the enzyme action and the peroxide bleaching.

In the examples presented hereinafter, the method of invention is used for modification

of the fibre counter-ions for enhancement of the hydrolysis of lignocellulosic materials or for improving the enzymatic treatment prior to totally chlorine free bleaching sequences wherein i.e. peroxide, ozone or oxygen is used. It is noteworthy, however, that the method of invention can also be applied to other lignocellulosic raw materials, such as mechanical pulps. The method can also be utilized in connection with enzymatic treatment of lignocellulosic materials employing two or more enzymes selected from the group comprising hemicellulases, cellulases and ligninases. Although different pulp bleaching applications are particularly interesting, the invention generally may be used for enhancing the hydrolytic action of the above-mentioned enzymes.

Within the scope of the present invention, the term "enzyme preparation" denotes any product containing at least one enzyme. Thus, the enzyme preparation may be a culture liquid containing one or several enzymes, an isolated or cloned enzyme or a mixture of two or several specific enzymes acting on hemicellulose, cellulose or lignin.

"Enzymatic bleaching" means a bleaching process which comprises at least one treatment step, during which the material to be treated is subjected to the action of a hydrolytic enzyme. Typically the enzymatic treatment step is carried out before contacting the material with conventional bleaching chemicals.

The terms "hydrogen form" and "acid form" are in the following used interchangeably to denote the carboxlic acid form of the carboxylic groups.

In the following, the invention will be examined in with the aid of a detail description and non-limiting examples.

According to the invention, the counter-ions of pulp carboxylic groups are converted before the enzymatic treatment to hydrogen form or to mono- bi- or trivalent metal cation form or to a known mixture thereof. Pulps in different metal forms can also be obtained by adjusting the cooking processes or by modifying the pulp counter-ions with known methods [5,6]. The monovalent metal cations may be selected from the group comprising Na ⁺ , Li ⁺ and K ⁺ , the bivalent cations may be selected from the group

ft comprising Ca ²⁺ , Mg ⁺ , Ba ²⁺ and Fe (H), and the trivalent cations may be selected from the group comprising Al ³⁺ and Fe(HT.). Pulps which are in the hydrogen-form are essentially lacking metal counter-ions (metal-free). Such metal-free hydrogen-form pulp can be obtained by acidifying the pulp to a sufficiently low pH, which causes the counter-ions to dissociate, and by subsequently washing the pulp, or by treating the pulp with known complexing agents (e.g. EDTA, DTP A). As mentioned above, the degree of dissociation and the type of the counter-ion affect the swelling of the fibres. According to the invention, the swelling of the fibres can also be modified by increasing the ionic strength by adding the salt corresponding to the counter-ion of the pulp.

The pulps can be analyzed for metal counter-ions by conventional analytical methods used for determining metals. The amounts of carboxylic groups in pulp can be determined by titration (i.e. conductometric or potentiometric), and the swelling can be measured by the WRV method (SCAN-M 102 X, proposal). The zeta potential can be determined on basis of the microelectrophoresis principle. The enzymatic action in fibres or other lignicellulosic materials can be monitored by for instance, determining the amounts sugars released during the treatment or by extraction of lignin fragments or by determining the result of the bleaching carried out after the enzymatic treatment. In this invention, the pulp brightness values were measured by ISO 2470, the kappa number by SCAN Cl: 1977 and the viscosity by SCAN-C15: 1988.

In the following examples, the applicable hemicellulases, cellulases and lignin modifying enzymes are usually exemplified by using purified xylanases, mannanases and cellulases from the well-known fungus Trichoderma reesei and laccase from

Phlebia radiata. These enzymes represent typical enzymes, but the method of invention is not limited to them.

Example 1

Effect of counter-ions on swelling, surface charge and hydrolysis

Birch kraft pulp from a pulp mill (kappa 15.5) was modified by the method of Scallan and Grignon [5]. The pulps were converted into acid (hydrogen) form at 2 % consistency in 0.1 M HC1 overnight at room temperature. The pulp was subsequently washed with distilled water until no chlorides were present in the washings. The acid pulp was converted to different metal forms in 0.1 M metal chloride solutions (NaCl, LiCl, KC1, CaCl ₂ , BaCl ₂ ) and the pH of the suspensions was adjusted to 9.5 with the corresponding metal hydroxides. The pulps were incubated at room temperature overnigth with occasional shaking. The WRV-values and zeta potentials were measured from the modified pulps before the enzyme addition. The modified pulps were used as subtrates which were hydrolyzed with Bacillus circulans VTT-E-87305 xylanase [16]. The enzyme dose was 500 nkat/g and the hydrolysis time 1 h at 50 °C. The consistency of the hydrolyzation was 2 %.

The results are presented in Table 1.

Table 1. The effect of different counter-ions on hydrolysis, swelling and surface charge of an industrial pulp

It)

From the results above it can be noted that the higher the valency of the metal cation, the lower the absolute value of the zeta potential and the more sugars are released during the hydrolysis.

Example 2

Effect of ionic strength on swelling and hydrolysis

Birch kraft pulp was converted to sodium-form according to the procedure of Example 1. Sodium chloride was added to the pulp prior to enzymatic hydrolysis. The hydrolysis was carried out as described in Example 1. The hydrolysis time was 20 h and the hydrolysis temperature 50 °C. The results are presented in Table 2. The WRV was measured after one our incubation before the enzyme addition.

Table 2. The effect of addition of metal chloride on the swelling and hydrolysis of birch kraft pulp.

From the results it appears that by increasing the ionic strength by adding the corresponding metal salt to the pulp (e.g. metal chloride), the swelling can be decreased while the hydrolysis levels are increased.

li

Example 3

Hydrolysis of metal-free pulp

Birch kraft pulp was converted to metal-free form as described in Example 1. The pul thus obtained was used as substrate for the hydrolytic actions of xylanase and mannanase enzymes isolated from Trichoderma reesei [18, 21]. The enzyme dose was 500 nkat g and the consistency during hydrolysis was 5 %. The degree of hydrolysatio was determined on basis of the amounts of reducing sugars obtained after 1 h. Conventional pine kraft pulp was used as reference. The pH of the hydrolysis was 4.5 to 5.

The results are presented in Table 3.

Table 3. The hydrolysis of metal-free pulp with

Trichoderma reesei xylanase and mannanase enzymes

It may be noticed from the data of Table 3 that chemical pulp in metal-free form is generally not hydrolyzable with hemicellulases.

Example 4

Combination of metal removal (EDTA treatment) with enzymatic treatment

For the purpose of totally chlorine free bleaching sequences metals contained in the pulp can be removed by lowering the pH or by complexing the metals with, for instance, EDTA or DTPA or similar complexing agents. In this example pulp was treated with EDTA at 5 % consistency and at pH 5, the amount of EDTA being 0.2 % (of pulp d.w.). The treatment was carried out for 1 h. The peroxide delignification was carried out at 80 °C for 1 h. The chemical dosages were 3 % of H,O ₂ , 1.5 % of NaOH, 0.5 % of MgSO ₄ , and 0.2 % of EDTA. Washing was carried out after the enzymatic treatment as well as after the EDTA treatment. 200 nkat/g T. reesei xylanase was used for the enzymatic treatment. The hydrolysis time was 2 h and the consistency was 5 %.

The results are presented in Table 4.

Table 4. Combination of metal removal and enzymatic treatment.

It will appear from Table 4 that the steps of removing the metal and treating the pulp with an enzyme can be carried out simultaneously. Should this not be the case, then the enzymatic treatment must preceed the metal removal. The improved enzyme action is evidenced by increased levels of reducing sugars, by lower kappa numbers of peroxide delignified pulps as well as by higher brightness values.

Example 5

Improving the hydrolysis of metal-free pulps by adding Al containing salts

This example shows how metal-free pulps can be rendered more hydrolyzable by adding Al containing salts and how this affects the hydrolysis and bleachability of said pulps.

Birch kraft pulp was converted to metal-free form as described in Example 1. The pulp in hydrogen-form was then converted to aluminium form by suspending it in a 0.25 M

A1C1 ₃ solution at 1 % consistency without pH-adjustment. The pulp was incubated at room temperature for 15 hours with occasional shaking. Thereafter the pulp was washed according to the procedure of Example 1. The aluminium-form and unmodified (reference) pulp were used as substrates for xylanase treatment. The xylanase dosage was 500 nkat/g, the consistency during hydrolysation being 2 %. The degree of hydrolysation was measured by determining the amounts of reducing sugars released after 1 h.

The results are presented in table 5.

Table 5. Hydrolysis of hydrogen- and aluminium-form pulps with two Trichoderma reesei xylanases.

According to the results obtained, it is possible to improve the xylanase hydrolysis of metal-form pulp by adding aluminium salts. This is shown for both of the xylanases. Depending on the type of xylanase the improvement can be as dramatical as 300 %.

Example 6

Improving the hydrolysis of metal-free pulps by adding magnesium-containing compounds

In this example, the counter-ions of metal-free birch kraft pulp were modified by adjusting the pH to 5 with Mg(OH> ₂ and MgCl, or mixtures of Mg(OH) ₂ and MgSO ₄ . The molalities of the solutions used were 0.25 M. Metal-free pulp whose pH had been adjusted with NaOH was used as a reference. The enzyme dosage (T. reesei xylanase) was 200 nkat/g and the hydrolysis time was 2 h. The results obtained are presented in Table 6.

Table 6. Effect of magnesium salts on the hydrolyzability of metal-free birch kraft pulp.

As indicated by the data of table above, the hydrolyzability of metal-free pulp can be increased by adding Mg-salts to the pulp.

Example 7 Improving the hydrolysis of metal-free pulps by adjusting the pulp pH with suitable hydroxides

The effect of pH adjustment of metal-free birch kraft pulp was assassed by using different metal hydroxides. The metal-free hydrogen-form pulp was prepared as described in Example 1. The pulp was hydrolyzed with T. reesei xylanase for 2 h at 5 % consistency using an enzyme dosage of 200 nkat/g.

Table 7. Effect of pH adjustment with different hydroxides on the hydrolysis.

Thus, it appears that by proper choice of hydroxide for adjustment of the pulp pH, the enzymatic hydrolysis of the metal-free pulp can be enhanced.

Example 8

Improving the hydrolysis of metal-free pulps by adding various cations to pulp

The metal cations of the pulp were removed by acidifying the pulp as described in Example 1. The pH of the metal-free pulp was adjusted with a solution of A1C1, and with mixtures of LiOH and LiCl ₂ , Ba(OH), and BaCl ₂ , Ca(OH) ₂ and CaCl,, and KOH and KCl, respectively, to pH 7.5, Then, the pulp was washed with distilled water and treated with T. reesei xylanase (pi 9). The enzyme dosage was 500 nkat/g and the hydrolysis time 2 h at 45 °C in 5 % consistency.

Table 8. Effect of various cations on pulp bleachability

A significant decrease of the kappa numbers after one-stage peroxide delignification and xylanase treatment was obtained when the pulp counter-ions were substituted by bi- or trivalent metal cations. Similar effects can be obtained by treating the pulp with the salts of any bi- or trivalent metals (e.g. MgSO ₄ or MgCl).

Example 9

Improving hydrolysis of metal-free pulps by adding CaCl ₂ to sulphite pulp

Sulphite pulp (made by the acid Mg process) was used as a substrate for Trichoderma reesei xylanase, the enzyme dosage being 500 nkat/g and the hydrolysis time 2 h at 45 °C. The pH of the pulp was adjusted with NaOH to pH 5 and CaCl ₂ was added to the mixture (0.5 % of pulp d.w.).

Table 9. Effect of CaCl ₂ addition on the action of xylanases on sulphite pulps

The action of xylanases on sulphite pulp can be increase by adding bi- or trivalent cations to the pulp. Above, the use of CaCl ₂ is described but it is clear that Mg, Al and Ba containing compounds can be used, as well.

Example 10

Effect of counter-ions on the total hydrolysis of cellulose

In this example the effect of the counter-ion of the lignocellulosic substrate on the enzymatic hydrolysis of cellulose is presented. Birch kraft pulp was converted to aluminium form by using the method described in Example 1. Then the pulp was subjected to enzymatic hydrolysis carried out at 2 % consistency for 24 h. The cellulase employed comprised a mixture of Trichoderma reesei cellulases (endoglucanase 1 and CBH I) .The enzyme dosages were 100,000 nkat/g of the endoglucanase and 50 mg/g of the cellobiohydrolase. Conventional birch kraft pulp which had been subjected to pH adjustment with NaOH to pH 5 was used as reference.

The results are given in table 10.

Table 10. Effect of the pulp counter-ion on the hydrolysis of xylan.

As will appear from Table 10, the hydrolysis yield was increased by 50 % , when the pulp was in Al-form. Aluminium cations can also be added to conventional pulp in the form of various aluminium salts.