WO1991002791A1

WO1991002791A1 - Enzymatic bleaching of pulp

Info

Publication number: WO1991002791A1
Application number: PCT/FI1990/000195
Authority: WO
Inventors: Ilkka Kruss; Marja Koljonen
Original assignee: Cultor Oy
Priority date: 1989-08-14
Filing date: 1990-08-13
Publication date: 1991-03-07
Also published as: PT94985A

Abstract

A method for bleaching of sulphate (kraft) pulp with decreased chemical dose and which comprises treatment of the pulp with low molecular weight xylanase having the following amino acid sequence: -Asn-Gln-Thr-Gly-Tyr-Asp-Gly-Met-Tyr-Ser-Phe-Trp-Thr-Asp-Gly-Gly-Gly-Ser-Val-Ser-Met-Thr-Leu. The enzyme is produced by Chainia sp. ATCC 53812.

Description

Enzymatic bleaching of pulp

Field of the invention The invention relates to a method for bleaching wood pulp. Specifically, it relates to a method of treating the pulp with a novel hemicellulose hydrolyzing enzyme.

Background of the invention The principal aim in the bleaching of pulp is to increase brightness. The bleaching process involves the use of oxidative compounds combined with alkaline extrac¬ tion. For a general review of pulp bleaching technology see Fengel, D. , Wegener, G. Wood: Chemistry, Ultrastruc- ture, Reactions. (1984) Berlin, DeGruyter. The use of hemicellulose hydrolyzing enzymes as an aid in the bleaching of softwood kraft pulp is previously known: Viikari, L. , Ranua, M. , Kantelinen, A., Sundqvist, J. and Linko, M. , Proceedings Biotechnology in Pulp and Paper Industry, Stockholm, (1986) . Microorganisms used as a source of such enzymes include Asperαrillus awamori, Strepto yces olivochromo enes and Bacillus subtilis.

The use of cloned he icellulose-hydrolyzing enzyme for bleaching of hardwood kraft pulp has also been sug¬ gested in Paice, M. , G. , Bernier, R. , Jr. and Jurasek, L. , Biotechn. Bioeng. 31 (1988) and Jurasek, L. , Paice, M. , G. , Biomass 15: 103-108 (1988) 103 to 108. The enzyme produced by Escherichia coli was cloned from the microor¬ ganism Bacillus subtilis.

Although the use of hemicellulose hydrolyzing enzymes has been demonstrated in laboratory scale processes, the speed and efficiency of such treatments must be improved before they can realistically be put to use in the pulp and paper industry. In practice, the use of these enzymes on an industrial scale has not been feasible. The enzyme dosages necessary for producing an adequate effect are high, the treatment times are long thus necessitating large storage capacity, yield losses of pulp are large, and viscosity decreases are often observed. Improvement of the bleaching process brought about by the addition of enzymes, as measured by increased brightness or decreased consumption of bleaching chemicals, is often insufficient to justify the cost of enzyme addition.

Summary of the invention It is therefore, the object of this invention to obtain an enzymatic bleaching process which can be used in industrial scale and which diminishes the disadvantages of the processes prior known.

This object can be obtained by means of this inven¬ tion, which is characterised by the addition to the pulp bleaching process of a small molecular weight xylanase enzyme or enzyme preparation produced by a Chainia sp. strain (ATCC 53812) which is substantially free from cellulase activity.

Description of the invention This invention is based on the discovery that a low molecular weight xylanase preparation, when used in conjunction with the traditional pulp bleaching process, can result in improved brightness, thus increasing the efficiency of the bleaching process and decreasing the necessary levels of bleaching chemicals. The small size of the enzyme enables a remarkable effect even with small dosages and with short treatment times. Because the xylanase preparation, the use of which is disclosed herein, contains no appreciable amount of cellulase, the viscosity of the pulp does not decrease and the yield losses are small.

Although not intended to be a limitation to the invention disclosed herein, it is believed that the improved bleaching process obtained by using the low molecular weight xylanase results from the fact that the small molecular weight of the enzyme permits it to pene¬ trate wood fibers so that it can perform a selective degradation of xylan. Xylanase converts xylan to xylo- oligosaccharides. It does not have any apprecia¬ ble enzymatic action against cellulose.

Production of Xylanase

The production of xylanase by Chainia sp. has been disclosed e.g. in the publication Biotechnology Letters Vol. 6 (1984) No. 11 pp. 715-718, Srinivasan et al. The production of xylanase by Chainia sp. NCL 82-5-1 can be carried out as follows:

Spores of Chainia sp. NCL 82-5-1 (deposited with American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, 20852, U.S.A. under ATCC Accession No. 53812) strain preserved on PDA slants at 4 C are transferred into a sterile medium containing a xylan source (e.g. 5% wheat bran, 1% yeast extract, pH 7.0). Culturing is carried out in Erlenmeyer flasks for about 3 to 5 days at 30 °C with vigorous shaking (50-100 ml medium in 250 ml flask at 200 rp ) . On completion of the fermentation, the Chainia cells are separated from the aqueous fermentation medium by centrifugation, filtration or other procedure to obtain a clear filtrate. The yield of xylanase on wheat bran medium is about 10 U/ml, and on a medium comprised of pure xylan, at least about 25 U/ml. A unit enzyme (U) is defined as the amount which liberates one micromole of reducing sugar calculated as xylose per one minute under the assay conditions.

Alternatively, as is known in the art, the xylanase enzyme preparation used in the present invention can be isolated from other organisms carrying the gene sequences encoding the small molecular weight xylanase or active portions thereof. Such gene sequences can be those derived from the Chainia species from which the enzyme has first been isolated and identified, or they can be created synthetically and cloned into an appropriate expression system. The xylanase produced by Chainia sp. ATCC 53812 is the subject matter claimed in copending U.S. application Serial No. 224,590 which was filed on July 27, 1988 (corresponding PCT-application Serial No. PCT/US88/03334 filed on September 26, 1988) and which is incorporated by reference herein. This enzyme has specific enzymatic activity for xylan with no appreciable enzymatic activity for cellulose. It is a glycoprotein with a low molecular weight active subunit of about 25,000 daltons. The a ino acid backbone of the glycosylated protein has been tenta¬ tively sequenced as follows:

-Asn-Gln-Thr-Gly-Tyr-Asp-Gly-Met-Tyr-Ser-Phe

-Trp-Thr-Asp-Gly-Gly-Gly-Ser-Val-Ser-Met-Thr-Leu. wherein Asn, Asp, Gin, Gly, Leu, Met, Phe, Ser, Thr, Trp, Tyr, and Val, respectively are asparagine, aspartic acid, glutamine, glycine, leucine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, and valine.

The xylanase used in the present invention has important properties that make it useful in the processing of wood fibers.

Use of Xylanase In The Bleaching Process The bleaching of pulp is carried out in multi-stage processes wherein oxidative stages are combined with at least one alkaline extraction step. Typically, the bleaching of pulp is carried out in its first stage using chlorine dioxide/chlorine in combination (D/C) . The remaining oxidative stages are carried out using chlorine dioxide alone. According to the invention, the xylanase preparation can be added to the pulp before or during treatment with chlorine dioxide (D) or chlorine dioxi¬ de/chlorine (D/C) treatment stages, but most preferably it is added prior to the first D/C stage. In addition, oxygen bleaching treatment can be used in conjunction with the enzyme. Some typical sequences of enzyme addition are shown in the examples below. The dosage of enzyme used is calculated based on the amount of dry solids (d.s.) in the pulp. Levels of from about 0.1 to about 100 Units per gram of pulp dry solids have been found to be effective. Most preferably enzyme is added at a level of from about 1 to about 25 U/g of pulp dry solids. Because the xylanase contains no appre¬ ciable amount of cellulase, however, overdosing is not as significant a problem as it is with cellulase containing enzymes, where such activity can reduce viscosity and subsequent yield.

The enzyme treatment is carried out within the pH range of from about 2 to about 10, most preferably within the range from about 4 to about 8. The enzyme is active at temperatures between about 10 to about 90 C; most preferably the reaction is carried out at temperatures of from about 25 °C to about 70 C. The time of enzyme treatment will vary with the dosage and nature of the sub¬ strate; in any event efficient bleaching will result if treatment is up to 24 hours; most preferably the treatment time is between about 0.5 and 8 hours.

The process described herein has been found to be effective on virtually all pulps on which it has been tested. Such pulps include hardwood kraft pulp, softwood kraft pulp, and sulfite pulp. The process can also be used on other xylan containing materials, e.g., grass, reeds, and bamboo, since it does not have appreciable activity against cellulose.

The invention and the advantages gained by the invention are disclosed in detail in the following exam- pies.

Examples The following bleaching conditions were used if not otherwise defined:

El=First alkaline extraction

Dl=First chlorine dioxide stage E2=Second alkaline extraction D2=Second chlorine dioxide stage

Example 1

Chlorine bleaching of pine pulp Finnish pine sulfate pulp was bleached; the original kappa number was 30. The bleaching sequence was D/C-E-D-E-D. The proportion of chlorine dioxide was 90 % of the active chlorine in the D/C stage. The control pulp was conventionally bleached but in order to make the treatment conditions as comparable as possible with the enzyme treatments, the control was treated in the same manner as the enzyme treatment by holding it at 50 C and pH 6 for two hours but without added enzymes. After that the pulp was washed and the conventional bleaching was performed by using at the D/C stage active chlorine dosage of 6 % of the d.s. of the pulp.

The enzymes that were used were the enzyme according to the invention (from Chainia sp. ATCC 53812, enzyme A), the xylanase produced by Streptomvces olivochromo enes WT-E-82157 according to Viikari et al. (1986) (enzyme B) , and the xylanase produced by Aspercrillus awamori VTT- D-79125 according to Viikari et al. (1986) (enzyme C) . The enzyme treatments of the pulp were carried out before bleaching using the following conditions: - temperature 50 C

- time 2 hours

- pH 6

- consistency 10 %

- enzyme dosage calculated as xylanase 5 U/g of the d.s. of the pulp

After the enzyme treatment, the pulp was washed and bleached by using at the D/C stage an active chlorine dosage of 6 % of the d.s. of the pulp.

At the last four stages, the chemical dosages were as follows: El, 2.7 % of NaOH of the d.s. of the pulp; DI, 3.0 % of the d.s. of the pulp; E2, 1.0 % of the d.s. of the pulp; D2, 1.5 % of the d.s. of the pulp. The results are shown in Table 1:

Table 1

Results from the bleaching experiment/pine pulp

Control Enzyme A Enzyme B Enzyme C Kappa Number After Enzyme Treatment Brightness, % ISO Viscosity, kg/dm3

yield Enzyme Treatment

+ Bleaching 94.3 92.8 92.2 92.6

These results show that the enzyme according to this invention gives better combination of brightness / viscosity / yield than the enzymes by Viikari e al.

(1986) for enhancement of pulp bleaching. With our enzyme the brightness increase was higher, the viscosity was increased rather than decreased, and the pulp yield over enzyme treatment and bleaching was higher than that observed with these known enzymes. Example 2

Chlorine Bleaching of Birch Pulp

Finnish birch sulfate pulp was bleached; the original kappa number was 18. The bleaching sequence was D/C-E-D-E-D. The proportion of chlorine dioxide was 90% and the amount of chlorine 10% calculated as active chlorine in the D/C stage. The control pulp was conven¬ tionally bleached, but in order to make the treatment conditions as comparable as possible, it was held without enzyme at 50-54 °C and at pH 6 for 4 hours. After that the pulp was washed and the control bleaching was performed by using at the D/C stage active chlorine dosages of 0.14 x kappa, 0.2 x kappa, and 0.23 x kappa % of the d.s. of the pulp. After the enzyme treatment, the pulp was washed and bleached by using an active chlorine dosage of 0.14 x kappa % of the d.s. of the pulp in D/C stage. The enzyme treatment (using three enzyme levels) of pulp was carried out before bleaching by using the following conditions: - temperature: 51-54 °C

- time: 4 hours

- pH: 6

- consistency: 10%

- enzyme dosage 1; 5; 25 U/g of calculated as the pulp d.s. xylanase:

In the four last stages, the following chemical dosages were used: El, 0.08 x kappa % of the d.s. of the pulp; Dl, 2.5% of the d.s. of the pulp; E2, 0.8 % of the pulp d.s.; D2, 1.0 % of the pulp d.s.

The results of the experiments are shown in Table 1. Table 2

Results from Bleaching Treatment/Birch Pulp

Enzyme treatment 0

D/C Stage dosage 0.14x

Kappa after

enz. treatment

Brightness %

Viscosity

₃ kg/dm

Yield

It can be seen from the results that with small enzyme dosage (1 U/g) and with moderate treatment time, 4 hours, the brightness increased from the value 81.3% to 86.1% which corresponds to about a 30 to 35% decrease in the consumption of chlorine. The yield losses in the enzyme bleaching compared to control pulp were insignificant.

Example 3 Control Example: Chlorine Bleaching of Birch Pulp Finnish birch sulfate pulp was bleached. The original kappa number was 20. A bleaching sequence D/C-E-D-E-D was used; the proportion of chlorine dioxide in the D/C stage was 10% and chlorine 90% calculated as active chlorine. Control pulp ("©-ex¬ periment") without enzyme was conventionally bleached but in order to make the treatment conditions as comparable as possible the control pulp was held at 40 °C and at pH 5 for 20 hours. After this the pulp was washed and normal bleaching was carried out by using active chlorine dosage 0.2 x kappa % of the pulp d.s. at the D/C stage.

The enzyme treatment was carried out before bleaching by using a xylanase enzyme produced by Trichoderma lonqibrachiatum fungi (Multifect K, by Cultor Ltd.) and by using the following conditions: -temperature: 40 °C

-time: 20 hours

-pH: 5 -consistency: 10%

-enzyme dosages calculated as xylanase: 56 U/g of the pulp d.s. After the enzyme treatment the pulp was washed and bleached by using at the D/C stage an active chlorine dosage 0.2 x kappa % of the pulp d.s. The chemical dosages at the .4 last stages were as follows: El, 0.08 x kappa % of the pulp d.s.; Dl, 2.5 ' of the pulp d.s. E2, 0.8 % of the pulp d.s.; D2 1.0 % of the pulp d.s.

The results of these experiments are shown in Ta¬ ble 2:

Table 3

Results From Bleaching Experiments/Birch Pulp (Control Example)

Compared with Example 2, the increase in the brightness achieved by dosing with 56 U/g of Tricho¬ derma enzyme were almost the same as by using 1 U/g dosage of enzyme according to this invention. Addi¬ tionally, by using the Trichoderma enzyme the viscosity of the pulp was decreased remarkably and the yield loss was large.

Example 4 Chlorine Bleaching of Pine Pulp

Finnish pine sulfate pulp was bleached; the origi¬ nal kappa number was 27. A bleaching sequence D/C-E-D-E-D was used. At the D/C stage, the proportion of chlorine dioxide was 10% and chlorine 90% calculated as active chlorine. The control pulp was convention¬ ally bleached but in order to make the treatment conditions as comparable as possible for the control mass, as in Example 2, the control was given a treat¬ ment corresponding to the enzyme treatment by holding it at 50-54 C and at pH 6 for 4 hours. After that the pulp was washed and the conventional bleaching was performed by using at the D/C stage active chlorine dosage 0.2 x kappa % of the d.s. of the pulp.

The enzyme treatment of the pulp was carried out before bleaching by using the following conditions:

- temperature: 51-54 C - time: 4 hours

- pH: 6

- consistency: 10%

- enzyme dosage calculated as xylanase: 25 U/g of the d.s. of the pulp.

After enzyme treatment, the pulp was washed and bleached by using at the D/C stage an active chlorine dosage of 0.2 x kappa % of the d.s. of the pulp.

At the last four stages, the chemical dosages were as follows: El, 0.09 x kappa % of the d.s. of the pulp; Dl, 3.0 % of the d.s. of the pulp; E2, 1.0 % of the d.s. of the pulp; D2, 1.5% of the d.s. of the pulp. The results are shown in Table 4:

Table 4

Results From the Bleaching Experiment/Pine Pulp

Control Enzyme Treated

Kappa Number After Enzyme Treatment 25.0 25.1

Brightness % 80.0 87.0 Viscosity kg/dm 3: 820 850

TCI mg/kg 330 225

Yield Enzyme Treatment

+ Bleaching 93.2 93.1

Despite the fact that the time of enzyme treatment was moderate (4 hours) , the brightness increased a re¬ markable 7% units. Additionally, no yield loss was observed.

Example 5

Oxygen Bleaching of Birch Pulp Finnish birch sulfate pulp was bleached; the original kappa number was 20.5. A bleaching sequence 0 -D-E-D-E-D was used. The chlorine dioxide used contained about 8% chlorine.

The control pulp was conventionally bleached but in order to make the treatment conditions as comparable as possible, the control was treated in a manner corresponding to the enzyme treatment (but without enzyme) by holding the control pulp at 55 °C and at pH 6 for 4 hours. The enzyme treatment was performed for the pulp before bleaching by using the following conditions:

After enzyme treatment (incubation of the control pulp) the pulp was washed and conventional oxygen bleaching was performed. At the oxygen stage, the following conditions were used:

- temperature: 90 °C - time: 30 min.

- 0₂: 3 bar

- consistency: 10%

- NaOH: 2%

At the first chlorine dioxide stage, an active chlorine dosage 0.2 x kappa % of the d.s. of the pulp was used.

At the last four stages chemical dosages were as follows: El, 0.1 x kappa % of the d.s. of the pulp; Dl, 2.5 % of the d.s. of the pulp; E2, 0.8 % of the d.s. of the pulp; D2, 1.0 % of the d.s. of the pulp. The results are shown in Table 5:

Table 5

Results From Bleaching Experiment/Oxygen Bleaching/Birch Pulp

With a time of enzyme treatment of only 4 hours, the brightness increased 1.8% units even though the original brightness was over 90%. In addition no yield loss was observed.

Example 6

Oxygen Bleaching of Birch Pulp Finnish birch sulfate pulp was bleached; the original kappa number was 20.5. A bleaching sequence 0 -enzyme-D/C-ED-E-D was used. The proportion of chloride dioxide at the D/C stage was 90% and of chlorine 10% calculated as active chlorine.

The oxygen stage was performed for pulp by using following conditions:

- temperature: 90 °C - time: 30 min.

^" °2 3 bar

- consistency: 10%

- NaOH: 2%

After the oxygen stage the pulp was divided into two portions. The control pulp was conventionally bleached, but in order to make the conditions of the treatment as comparable as possible, the treatment corresponding to the enzyme treatment was performed by holding the control pulp at 55 °C and at pH 6 for 4 hours. Enzyme treatment was performed for the pulp after the oxygen stage but before the D/C stage by using following conditions:

- temperature:

- time: - pH:

- consistency:

- enzyme dosage calculated as xylanase:

After enzyme treatment (incubation of the control pulp) the pulp was washed and the final bleaching was performed with the sequence mentioned above. Active chlorine dosage 0.2 x kappa % of the d.s. of the pulp was used at the D/C stage.

At the four last stages, the chemical dosages were as follows: El, 0.1 x kappa % of the d.s. of the pulp; Dl, 2.5 % of the d.s. of the pulp; E2, 0.8% of the d.s. of the pulp; D2, 1.0 % of the d.s. of the pulp. The results are shown in Table 6:

Table 6

Results From Bleaching Experiments/ Oxygen Bleaching II/Birch Pulp

Example 7

Finnish birch kraft pulp (original kappa 22,9) was bleached after enzyme treatment and for reference without enzyme treatment (control) with the bleaching sequence D/C-E1-D1-E2-D2. The amount of chlorine dioxide in D/C-stage was 90 % of act. cl.

The enzyme preparations used in the experiment were Multifect K (Cultor Ltd.) produced by the fungus Trichoderma lonqibrachiatum and the xylanase produced by Chainia sp ATCC 53812 as described above. The reaction conditions used in the enzyme treat¬ ment before conventional bleaching were as follows:

- temperature: 50 C

- time 4 hours

- pH 6,5 (Chainia), 5,5 (Multifect K)

- consistency 10%

- enzyme dosage 5 xylanase U/g pulp d.s.

The control pulp was treated before bleaching using the same conditions as in the enzyme treatment but no enzyme was added to the mixture. After the enzyme treatment the pulps were bleached using following chemical dosages: D/C-stage dosage for control 3,2 %, for Multifect K 3,2 % and for Chainia 2,9 % of active chlorine per pulp d.s. In El-stage the chemical dosage was for control 1,8 %, for Multifect K 1,8 % and for Chainia 1,7 % of NaOH per pulp d.s. In other stages the chemical dosages were the same for all pulps. The chemical dosages were in the final stages following: Dl 2,5 % active chlorine, E2 0,8 % NaOH and D2 1,0 % active chlorine of pulp d.s.

The results of experiments are shown in table 7.

Table 7

Results from Bleaching treatment/Birch pulp

Enzyme Control Chainia Tricho¬ derma

Enzyme dosage 0 5 5 (xylanase U/g pulp) Kappa after enz. 23,0 20,8 22,6 treatment

Brightness (%) VViissccoossiity (dm 3 ty /kg) Yield (%)

Chainia enzyme has a remarkable effect on bright¬ ness compared to Trichoderma despite of the fact that the dosage in D/C-stage has been smaller. Both enzyme treatments improved clearly final brightness compared to the control.

In spite of the fact that enzyme treatment was for only four hours, the brightness increased 2.5% units, even though the original brightness was almost 90%. In addition the yield losses were insignificant.

From Examples 5 and 6, the conclusion can be drawn that by using the enzyme in combination with oxygen, it may be possible to omit the final .bleaching stages E2 and D2. This could lead to considerable savings when new bleaching plants are built.

Claims

1. In a process for the bleaching of pulp the im¬ provement comprising adding to the pulp a low molecular weight xylanase having a specific activity for xylan with no appreciable activity for cellulose.

2. The method according to claim 1 wherein said xylanase has the following 23 a ino acid sequence:

-Asn-Gln-Thr-Gly-Tyr-Asp-Gly-Met-Tyr-Ser-Phe -Trp-Thr-Asp-Gly-Gly-Gly-Ser-Val-Ser-Met

-Thr-Leu. wherein Asn, Asp, Gin, Gly, Leu, Met, Phe, Ser, Thr, Trp, Tyr, and Val, respectively are asparagine, aspar¬ tic acid, glutamine, glycine, leucine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, and valine.

3. The method according to claim 1, wherein said low molecular weight xylanase is produced by Chainia sp. ATCC strain 53812 or strains derived from it by means of mutation, selection or gene transfer.

4. The method according to claim 1, 2 or 3, wherein said xylanase is added prior to any chemical bleaching stages.

5. The method according to claim 1, 2 or 3, wherein said xylanase is added following an oxygen bleaching stage.

6. The method according to claims 1, 2 or 3 wherein the pulp is selected from the group consisting of hardwood kraft pulp, softwood kraft pulp and sulfite pulp.

7. The method of claims 1, 2 or 3, wherein said xylanase preparation is added in the amount of about 0.1 to about 100 U/g calculated on the dry solids of the pulp.

8. The method according to claim 7, wherein said xylanase preparation is added in the amount of about 1 to 25 U/g calculated on the dry solids of the pulp.

9. The method according to claims 1, 2 or 3, wherein the xylanase treatment is carried out within the pH range from about 2 to about 10, at a temperature from about 10 to about 90 °C and in less than 24 hours.

10. The method according to claim 9, wherein the xylanase treatment is carried out within the pH range from about 4 to about 8, at a temperature of between about 25 to 70 C and in about 0.5 to 8 hours.