METHOD FOR SAVING ENERGY IN PAPER PRODUCTION

Abstract

The present invention is in the field of paper production, more in particular it relates to the process of wood pulping. It provides useful biological methods and compounds for reducing the energy requirements of the production of wood pulp. It describes a method for reducing the energy requirement of a thermo-mechanical pulping (TMP) process wherein cellulose fibers are recovered from a biomass comprising lignocellulosic material, wherein the lignocellulosic material is treated with a CotA laccase before recovering the cellulose from the lignocellulosic material.

Claims

1.-20. (canceled).

21. A vector comprising: a nucleic acid molecule encoding a protein having laccase activity and having a primary amino acid sequence having at least 99% sequence identity with the sequence of COT1 (SEQ ID NO:1) or COT2 (SEQ ID NO:2); and a polynucleotide heterologous to the nucleic acid molecule encoding a protein having laccase activity.

22. The vector of claim 21, wherein the protein having laccase activity is COT1 (SEQ ID NO:1) or COT2 (SEQ ID NO:2).

23. Wood chips comprising the vector of claim 21.

Description

LEGEND TO THE FIGURES

[0087] FIG. 1 shows an ESEM microscopy image of a section at the edge of a wood chip treated with CotA laccase.

[0088] FIG. 2 shows an ESEM microscopy image of a section at the center of a wood chip treated with CotA laccase.

[0089] FIG. 3 shows an ESEM microscopy image of a section at the edge of a wood chip treated with a fungal laccase from Trametes versicolor. FIG. 4 shows an ESEM microscopy image of a section at the center of a wood chip treated with a fungal laccase from Trametes versicolor.

[0090] FIG. 5 shows an ESEM microscopy image of a section at the edge of a wood chip treated with an Escherichia coli laccase CuEO.

[0091] FIG. 6 shows an ESEM microscopy image of a section at the center of a wood chip treated with an Escherichia coli laccase CuEO.

[0092] FIG. 7 Diagram of pulp manufacturing. Solid arrows indicate the positions in the process where CotA laccase treatment is beneficial for energy saving. The term screened chips means that the chips are selected for the appropriate and desired size. Washing means that they are cleaned with water to remove unwanted dust and dirt. Steaming means that the chips are subjected to steam. The figure shows three different pretreatment steps. First (position marked with (3)) the pretreatment consists of contacting the biomass containing lignocellulose with a CotA laccase (grey solid arrow). Second, the pretreatment may consist of contacting the biomass containing lignocellulose with a CotA laccase before or after low energy mechanical treatment (position marked with (2)). Thirdly, the pretreatment may consist of contacting the biomass containing lignocellulose with a CotA laccase before or after impregnation, for instance with a chemical reagent or a biological reagent such as an enzyme. Via an optional steaming step, the biomass containing the lignocellulose is then fed into the refining stage where the biomass is then heated to a temperature above 100 degrees Celsius and refined, i.e. subjected to mechanical defibration. After the refining step the resulting pulp is screened for residual lignocellulosic material where wood fibers were not sufficiently separated. That material (reject) is then fed into the reject refining process. CotA may advantageously be applied to the reject pulp in order to save energy in the reject refining process.

[0093] FIG. 8: Energy curves (Pulp freeness plotted against specific energy consumption) from refining experiment (example 2).

[0094] The graph shows a shift of the energy line to the left (or down) in the samples treated with COTA laccase, meaning that energy saving is achieved. In other words, the same freeness (as for example taken here at 300 ml, indicated in the figure by a horizontal line) can be obtained with less energy. Curves correspond to the following samples 1Trametes versicolor (0.05 u/ml), 2Reference, 3Trametes versicolor (0.1 u/ml), 4COT1 (0.05 u/ml), 5COT2 (0.05 u/ml), 6COT2 (0.1 u/ml), 7COT1 (0.1 u/ml)

EXAMPLES

Example 1: Microscopy

[0095] Wood chips of 50 g dry matter content (DMC) with a largest size between 35 mm and 40 mm mm were washed to remove residual dirt. and thereafter treated with steam for 10 minutes. Chips were than equilibrated to 50 degrees C., placed in a cylindrical device with a press. Mechanical pressure was applied from the top 63,5 kPa/cm.sup.2 , until the layer of chips was half of the original height. Any liquid coming out of the chips was drained though small holes at the bottom of the cylinder. After that, the pressure was slowly released and the enzyme solution fed from the bottom of the cylinder. The enzyme solution contained 1 unit/ml of one of the laccase enzymes (as indicated) in 20 mM Succinic acid pH 5.0. A catalytic unit is defined as the amount of enzyme needed to convert 1 micromole of substrate (ABTS) in 1 min.

[0096] The following laccases were used herein: (1) Spore coat protein from Bacillus subtilis CotA (COT2 (SEQ ID NO:2, recombinantly expressed in E.coli), (2) commercially available fungal laccase from from white-rot-fungi Trametes versicolor (available from Sigma-Aldrich), and (3) laccase from E. coli termed CUEO, recombinantly expressed in E. coli.

[0097] Approximately 30 ml of this solution was absorbed by the chips. Any residual solution was drained. These chips were then placed in a sealed container to prevent evaporation and incubated for 2 hours. Samples with COT2 and CUEO laccases were incubated at their optimal temperature, i.e. 70 degrees Celsius, sample with Trametes versicolor laccase was incubated at 50 degrees Celsius since Trametes versicolor laccase would be quickly inactivated at 70 degrees Celsius.

[0098] From each sample, four chips were taken out and cut approximately in the middle. The cut surface was then sectioned manually by a razor blade and left to dry at room temperature.

[0099] The chip cross-sections were imaged using a Philips XL30 ESEM-FEG (Environmental Scanning Electron Microscope-Field Emission Gun). Working conditions were as follows: low vacuum mode, 0.7 mbar pressure in the sample chamber, BSE detector (backscattered electrons) and 15 kV acceleration voltage. The magnifications used were 200, 250, 500 and 1000.

Example 2: Energy saving in TMP

[0100] A series of laboratory scale refining experiments was performed in order to evaluate the effect of laccase pretreatment on refining energy.

[0101] Screened wood chips with an average maximum size of approximatelly 40 mm were impregnated with a solution containing either one of two CotA laccases (COTA laccases (COT1 and COT2, SEQ ID NO: 1 and SEQ ID NO: 2 resp.) or a commercially obtained fungal laccase from from white-rot-fungi Trametes versicolor (available from Sigma-Aldrich.

[0102] The impregnation of wood chips was done in portions of 50 g DMC as described in example 1, with the exception that two concentrations of impregnation solution were used; 0.1 u/ml or 0,5 u/ml of laccase in 20 mM Succinic acid pH 5. Reference sample was impregnated with the same solution without laccase.

[0103] Three portions of 50 gram (DMC) of impregnated chips were produced with each dosage of each laccase. After impregnation, the portions treated with the same enzyme were combined to a single sample of 150 gram DMC.

[0104] Impregnated chips were then placed in a sealed container to prevent evaporation and incubated for 1 hour. Samples with COTA laccases were incubated at their optimal temperature, i.e. 70 degrees Celsius, sample with Trametes versicolor laccase was incubated at 50 degrees Celsius since Trametes versicolor laccase would be quickly inactivated at 70 degrees Celsius.

[0105] The treated wood chips described above were portioned into 125 g DMC batches and refined in a low-intensity wing refiner comprising a wing defibrator chamber. The wing defibrator chamber consisted of two rotating blades which rotate in opposite axial directions. A 20 blade cylindrical structure rotated within a distance of 1 mm from 4 wing-like rotating blades.

[0106] The refiner was heated and three empty runs were used as blank. The steaming temperature was 124 C.0.6 C. The wood chips were steamed for 5 minutes, during which the 4 wing-like blades were rotated 90 every 1.25 minutes to heat the chips evenly. After 2 minutes of steaming the condensate was let out during 10 seconds. After 4 minutes and 50 seconds of steaming the valve was closed, puls-meter zeroed and the run started after 5 minutes of steaming. The experiments were done for 2, 4, 6, and 8 minutes of refining. When the time had passed, the experiment was stopped directly when the puls-meter changed value. The pressure in the chamber was 1.9 -2.6 bars and the temperature rising from about 124 C. to 136 C., depending on how long the experiment was continued. All enzymatic trials were run in singles, as well as 2, 6, and 8 minutes for the reference. 4 minutes of refining for the reference was run 4 times.

[0107] After refining, the pulps were centrifuged and measured for dry matter content DMC (according to SCAN-C 3:78) and freeness was determined with a Canadian Standard Freeness tester. The results are shown in table 1. The total amount of consumed energy was plotted against freeness (FIG. 8) and trend lines were calculated for the reference, COT1, COT2 and Trametes versicolor (R=0.99; 0.97; 0.99; and 0.99 respectively). The energy consumption was compared at constant levels of freeness (300 ml).

[0108] Fungal laccase had no significant effect of energy consumption in this refining experiment. In contrast, COT1 and COT2 samples showed very similar significant reduction in energy consumption about 7-8% for 0.05 u/ml dosage and 13-15% for 0.1 u/ml dosage. This energy saving values are highly industrially relevant and considering the low dosing of enzyme, this may be considered of high commercial importance.

TABLE-US-00001 TABLE 1 refining energy at 300 ml freeness from reference and laccase treated samples. SEC at 300 ml Curve No Sample freeness Energy saving (%) 2 Reference 1.73 0 1 Tv* (0.05 u/ml) 1.75 0.61 3 Tv (0.1 u/ml) 1.74 0.10 4 COT1 (0.05 u/ml) 1.62 6.8 7 COT1 (0.1 u/ml) 1.47 14.9 5 COT2 (0.05 u/ml) 1.59 8.3 6 COT2 (0.1 u/ml) 1.50 13.2 *Tv = Trametes versicolor laccase.