FI112248B - New carrot fungus and its use in the preparation of wood - Google Patents

Description

112248 MY NEW WHITE SPRAYER AND ITS USE IN PRE-TREATMENT OF WOOD

FIELD OF THE INVENTION

The invention relates to a method of a papermaking process which utilizes living biological microbes. In particular, the invention provides a novel white rot fungus and its use in the treatment of wood.

BACKGROUND OF THE INVENTION

To make paper from wood, the wood material is usually chipped and the wood fibers 10 separated by a suitable process. On an industrial scale, several different methods are used to separate the fibers from one another. At its simplest, the chips are mechanically defibrated with water in the refiner to the desired degree of defibration. Other methods include the so-called. thermo-mechanical defibration (TMP), chemical chipping treatment combined with thermo-mechanical defibration (CTMP), chemo-mechanical defibration (CMP), so-called.

15 Kraft sulphate soup and sulphite process. The purpose of all of the above methods is to separate the cellulosic fibers of the wood from the other structural material of the wood to the desired level of fiberization.

Wood is composed of several types of polymers, of which cellulose is the 20 main building block. Cellulose fibers are linked to hemicellulose, which is: • linked to an aromatic polymer called lignin. Pulp manufacturing; : lignin (and hemicellulose) are being removed. In native wood, lignin is · deposited around cellulosic fibers as a protective and mechanical support layer.

. '·: 25 Treatment of wood or wood chips before making pulp with lignin-degrading fungi «· · .. is called" biopulping ". Stored fresh timber is rapidly attacked by germs, mainly fungi. Fungi initially make use of wood · ·: the most easily utilized nutrients such as wood extracts. These nutrients •; · 'Fungi capable of utilizing •': 30 cellulose, hemicellulose and lignin will benefit from a reduction in growth in wood. White fungi are fungi of the class Basidiomycetes which are capable of breaking down these wood polymers. Some of these fungi decompose relatively more lignin than wood polysaccharides. These * .: mushrooms are called selective lignin dispersants. The use of such fungi has proved to be useful in the pre-treatment of wood chips prior to mechanical 2,112,248 pulping. By suitable fungal treatment it is possible to reduce the cost of pulping. The use of mushrooms is aimed not only at improving production but also at environmentally friendly production. Mechanical pulping has been able to save energy and improve paper quality by using a sponge treatment.

5

The first article on the potential of lignin-degrading fungi in pulp production was published in 1957. The first bio-pulping experiments were conducted in the early 1960s on spontaneously decomposed pine, which was used as a chemical pulp raw material. The strength of the paper made from the pulp was improved by the fungus.

10 Biopulp and its research have been developed especially in Sweden in the 1970s and 1980s by the Skogsindustrins Tekniska Forskningsinstitut (STFI) prof. Karl-Erik in the Eriksson group and vv. 1987-1996 in the USA as a consortium funded by up to 20 companies, coordinated by Richard Burgess, Forest Products Laboratory at the University of Wisconsin, and prof. Kent Kirk. The project involved extensive mapping of white fungus (about 400) and 15 more than 200 bio-pulping experiments. Ceriporiopsis subvermispora was selected as the most potent fungus because it seemed to be suitable for treating both coniferous and deciduous trees (mainly pine and wound). This sponge has been tested on an industrial scale of 40 tons of chips. Spruce (Picea abies) is much less studied than aspen and pine. Spruce has not achieved the same amount of energy savings as wound. Setliff 20 et al. (1990) reported 13% energy savings with 20 days of C. subvermispora, · "" · treatment, whereas a similar wound treatment yielded 20% energy savings.

:, · I At present, there are problems with the use of sponge in biopulp, which need to be developed or discovered to reduce '· ... · and make the process more economically viable. '25 fungi with inoculation properties that do not cause chips inoculation · ·' C. subvermispora has the following disadvantages: 1. adding the inoculum to the chips requires reducing the natural microbial content of the chips before adding the white fungus, for example by steam treatment; * raises the internal temperature of the chips in a short time so that the growth of the fungus stops • 30, stops the structure of the 3rd chips to make it difficult to obtain the air necessary for the growth of the fungus, ... 'lignin decomposition and temperature control. In addition, lignin selectivity remains high and yield loss is a prerequisite for fungal treatment. Further research by the consortium has found Phlebia 3 112248 subserialis, a biofouling fungus superior to C. subvermispora, for which a patent has been applied for in the US in the treatment of loblolly pine (Firms taeda) and wound (Populus tremelloides) (WO9802612). Although the use of / ', subserialis fungus in consortium studies has appeared to alleviate the above problems (Scott et al., 1998), there is no published information on its lignin selectivity and it appears that the fungus 5 does not have a class of C. subvermispora. There is no published information on the enzymatic activity of its wood. In previous studies published in 1991 (US5055159) and 1993 (Akhtar et al.), The energy savings from subserialis sponge treatment were equal to or lower than those of C. subvermispora treatment. It is evident that the P. 5 foerm // 5 fungal treatment has not yielded sufficiently good results for industrial application, possibly due to its lower lignin selectivity.

The invention has found isolated white rot fungus Physisporinus rivulosus, strain T241i, which is superior in quality to growth, heat tolerance and lignin selectivity over C. subvermispora. In addition, T241i is comparable in temperature resistance to P. subserialis. Strain siis241Ϊ thus combines good qualities in terms of pretreatment technology and its mechanism of action.

The lignin selectivity is high and the processing weight loss of wood is low, which is comparable to the low yield loss important for pulp production. Already after one week of cultivation, P. rivulosus T241i 20 causes changes in the solubility of wood lignin in '': alkali and secretes a high amount of lignin-modifying enzyme, manganese peroxidase:: in chips. This enzyme is excreted in the wood at the same time as oxalic acid.

», · · The presence of both factors in wood appears to be important in the modification of lignin ... * and in subsequent processes affecting the energy consumption of pulp production and the quality of paper. Fungus also greatly reduces the amount of wood extractant, which is an important feature of paper quality and the runnability of paper machines. Two weeks treatment of the fungus with P. rivulosus T241i significantly reduces the need for mechanical pulping '·' 'pulp energy.

Γ / 30 BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Growth of C. subvermispora (CZ-3) and P. rivulosus (T241i) on malting agar medium: * .. at different temperatures. The amount of growth is expressed in millimeters per day.

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Figure 2. Decomposition ability of P. rivulosus Τ24Π (black columns) and C. subvermispora CZ-3 (shaded columns), expressed as weight loss of wood at three different temperatures after 2 and 6 weeks of fungal treatment.

5 Figure 3. Amount of live fungal mycelium in autoclaved chips during two weeks of fermentation expressed as FDA values (see Example 4).

Figure 4. Changes in different extraction groups of autoclaved spruce heartwood chips after one week of fungal treatment. Colorless column: control chips without 10 fungal treatments, black column P. rivulosus T241i, shaded column C. subvermispora CZ-3.

Figure 5. The diagram above shows the changes in different extract groups of fresh pine hardwood chips after one week of fungal treatment. Fatty acids and resin acids are omitted from the lower drawing, whereby differences with other groups of extracts are more clearly visible.

Figure 6. Gel permeation chromatography curve of molecular weight distribution of black liquor from chemical cooking. Chip spruce surface wood, mushroom treatment time two 20 weeks. S3 3, P. rivulosus T241i, S3 4, control without fungal treatment, S3 5, C. subvermispora CZ-3.

* · Figure 7. Energy consumed by the wing refiner to comfort spruce chips in different, .. · degree of defibration. Fungal pre-treatment for two weeks.

25 ... 'Figure S. Effect of mushroom pretreatment on the mechanical pulp power input during the initial phase of comfort.

DETAILED DESCRIPTION OF THE INVENTION; '. '30 The fungus Physisporinus rivulosus of the invention, strain T241i, has been found in southern Finland.

P. rivulosus is a common white-rot woodworm in North and Central America, especially the giant thuja (Thujaplicata) and redwood (Sequoia sempervirens).

Other host trees include: western hemlock (Tsuga heterophylla), douglas fir (Pseudotsuga menziesii) and oregon algae (Alnus rubra (= oregana)). Birch 5 112248 (Betula sp.) And eucalyptus (Eucalyptus sp.) Are also potential host trees. Thus, the fungus is able to grow on both coniferous and deciduous trees. P. rivulosus is probably very rare in Europe. Mostly it is found in pine {Pinus sylvestris). Usually fungal spores are found on charred tree trunks. Because many fungal genera 5 and their names are inconsistent in fungal taxonomy, P. rivulosus has also been linked previously. Gelatoporia (Gloeoporus), Rigidoporus and Ceriporiopsis. Also, the obsolete surname Poria has been used in connection with the sponge. P. rivulosus is related to C. subvermispora but is clearly a species of its own.

10 P. rivulosus is a poorly studied white rot fungus. Nakasone (1981) compared the properties of P. rivulosus {= Poria rivulosa) and C. subvermispora (= Poria subvermispora) with respect to temperature optimum, growth and microscopic features. Example 1 investigates changes in the chemical structure of wood caused by P. rivulosus strain T241i and C. subvermisporan CZ-3. The example demonstrates that both species are capable of selective lignin degradation. Τ241Ϊ is more selective than CZ-3. Example 2 examines the growth of strains T241i and CZ-3 in growth plates in a laboratory. The T241i has a much wider temperature range compared to the CZ-3 strain. Example 3 illustrates the effect of temperature on the decomposition of wood. The superior heat resistance of the T241i strain is also evident in these short wood decomposition tests.

Example 4 demonstrates lower biomass yield of strain T241i in spruce chips: under the same growth conditions as in strain CZ-3.

«* •: The T241i fungus has also been studied in mechanical pulping processes in ... * tests on the ability of the fungus to affect aspen cellulose fibers. The so-called Simonsin t t • ·. ”· 25 dyeing sponge has a proven property that correlates with reduced energy consumption in mechanical pulping. Akhtar et al. (1995) have studied two Ceriporiopsis rivulosa strains (Table 2 of the article) together with C.

; pannocinctan {Gelatoporiapannocincta) as reference material for C. subvermisporalle% * * '.' using Simons staining. The staining result was compared with mechanical • · *; .'30 energy savings in pulp production relative to untreated 'chips. The second P. rivulosus strain (= C. rivulosa) did not achieve any of the following: '· energy savings and the second P. rivulosus strain after two weeks treatment' · / · ': only 3% energy savings were achieved in aspen consolation when compared to non-inoculated controls The strains of C. subvermispora achieved 7-6 112248 12% energy savings. The result for C. pannocincta was similar to that for P. rivulosus. It is possible that the American strains of P. rivulosus used in the study differ in their properties from the European strains of P. rivulosus.

Fungal treatment of spruce chips with strains of P. rivulosus T241i and C. subvermispora CZ-3 causes an increase in alkaline and acid solubility of wood lignin even after one week of treatment (Examples 5 and 7). An increase in the solubility of lignin is likely to indicate that the fungal treatment has resulted in changes in the structure of the lignin that promote the release of lignin and its aromatic subunits into a basic or acidic solution when treated with either an alkaline solution such as sodium hydroxide or a strong acid. These changes are positively correlated with high production of ligninolytic enzymes and reduced pulping energy in mechanical pulping. Changes in the structure of lignin caused by the treatment of P. rivulosus and C. subvermispora are reflected in an increase in 15 acid-soluble lignin in spruce chips pre-treatment (Example 5), whereas in fresh pine chips pre-treatment did not increase in lignin acid solubility (30%). Thus, in the treatment of pine wood, the very promising potential of T24li strain is in particular for the removal of wood extractants, and thus could allow the use of pine as a raw material for mechanical pulp. Preferably, the wood extractant content in the process of the invention is reduced by more than 20%. Phlebiopsis gigante and white resin fungus studied in Example 7! : Removing fungus Ophiostomapiliferum (light variant) does not induce an increase in alkaline solubility of lignin in spruce chips similar to P. rivulosus and \ · C. subvermispora. Example 8 describes how the change in the alkali solubility of lignin. * i 25 fungus treatment also shows chemical pulp ... "black liquor compound distribution: low molecular weight lignin has increased as a result of fungal treatment. Example 9 demonstrates how 'the amount consumed by the wing refiner': the amount of comforter is due to T24li and CZ-3 fungal treatments · · * 10-15%, and in particular the amount of energy required for the initial defibration phase has been reduced: '.30 strongly, which means a very significant ... * energy savings on a factory scale.

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Presentation of the process

The process involves the use of chips, which are made for ordinary mechanical or chemical pulping. The chips are screened into even chips by a combination of hole and bar screens according to SCAN-CM 40:88. The screening accept 5 is recovered from the screen plate 07. If the dry matter content of the chips exceeds 40%, chop. moistened to allow the fungus to colonize the chips well. Bringing the sponge transfer onto the chips is most effective if the surface of the chips is aseptic, for example by steam treatment. The fungal inoculum may be either solid or liquid. When using a liquid passage, any of 10 known nutrient media can be used. In its simplest form, for example, malt extract, dissolved in water, can be used. The fungal graft is grown separately and inoculated into chips as a homogenized suspension. The amount of transition is small compared to the amount of chips, typically about 0.5% of the dry matter content of the chips.

The inoculated chips are fermented in a bioreactor. As a bioreactor, any vessel or structure that promotes fungal growth can be used. Because fungal growth, the removal of lignin from wood is a heat-generating and oxygen-consuming process, temperature control in the bioreactor must be ensured. Microbial metabolism in chips heaps, in particular, raises the temperature of the chips heaps considerably. Temperature retention at the various locations of the 20 bioreactors can be controlled by introducing air into the reactor, which at the same time facilitates lignin removal. This invention employs a PR fungus «· * * (PR = P. rivulosus) which, when chipped, secretes ligninolytic enzymes into wood. Of the three enzymes that affect wood delignification, laccase, ... * manganese peroxidase and lignin peroxidase, PR produces by far the largest amount of «· •" · 25 manganese peroxidase. the lignin composition of the wood such that the alkalinity solubility of the lignin is already increased during the first week of cultivation. Preferably, the amount of soluble lignin increases * · a '' during the process compared to untreated wood. After two weeks of cultivation The effect of the sponge treatment on the energy consumption is particularly '* · noticeable at the initial stage of defibration, whereby the efficiency of the refiner on the sponge-treated * <.' * · chips is approx. Power consumption in the sponge-treated pulp remains at about 500 W lower at 8112248 throughout the defibration process compared to the untreated pulp. Preferably, the energy consumption required for torture of mechanical pulp is reduced by more than 15%.

One of the preferred embodiments of the invention is to transfer the white rot sponge according to the invention to wood when it is stored or transported. According to the invention. the pre-treatment of the wood may thus take place in an environment where the optimal conditions for the bioreactor described above may not be achieved. Also, in this embodiment of the invention, the wood to be treated is not necessary to chop before the sponge is inoculated.

10 Our research results show that P. rivulosus strain T241i combines several properties important for efficient biopulp: the fungus is thermostable, easy to grow in both coniferous chips and solution cultures, thus allowing easy inoculation, fungal is lignin-selective, and modifiable lignin-modifying enzyme 15 manganese peroxidase. Preferably, the fungus secretes more than 1.0 units (U) of manganese peroxidase per gram of dry chips and oxalic acid to a concentration greater than 10 mM when determined by extracting the oxalic acid from the wood with 1.5 M hydrochloric acid. Chip pretreatment in a simple bioreactor is easy, and the fiberization energy savings achieved by pretreatment are significant. Quantifying the pre-treatment technology to an industrial scale is relatively easy to accomplish.

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25 EXAMPLES

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Example 1 • · '; t * 'In the test, the fungus is grown in a 10 ml Erlenmeyer flask on moist vermiculite t ·'; '17mm x 7mm x 16mm (four pieces per bottle).

1 * *. * 30 The sponge graft is prepared from the mycelium grown in malt malt broth for 10 days.

The mycelium is homogenized to a suspension in a Waring-Blender blender for 10 sec. There is a 30 sec pause between homogenisations to prevent the mixer from> t: · overheating. The numbered, dried and weighed fir pieces 9112248 are soaked in water for about 15 s and autoclaved for 20 min at 121 ° C. The sterilized spruce pieces are infiltrated as above with the mycelial suspension for 30 s and any mycelium on the pieces is scraped off. The pieces are placed in flasks and the flasks incubated at + 25 ° C for 70 weeks at 70% RH. The pieces 5 are dried for 3 days at 60 ° C and weighed to measure the weight loss of the pieces. Dry cuts. is ground to a fine chop by a mechanical refiner, and the chemical composition of the wood chips is analyzed.

Table 1. Chemical analyzes of the main components of the test and the weight loss of the wood 10. Results are expressed as percentage loss. PR = P. rivulosus, CS = C. subvermispora. mycelium strain lignin glucose xylose mannose pectin loss ~ PR T241i 18J 393 182 19.8 2Ö2 243 ~ CS CZ3 22/7 4Ϊ5 3Ϊ8 3Ϊ0 343 32/7

Table 2. Chemical analyzes of the main wood components of the test. The results are expressed in relation to the loss of glucose (cellulose). PR = P. rivulosus, CS = C. subvermispora. lignin loss / sponge strain lignin glucose xylose mannose pectin weight loss • · · ·! .. 1 “PR Τ24ΪΪ 2Λ IÖ Γ0 Ö9 LÖ L2“ CS CZ3 L9 L2 LÖ Ö9 LÖ Ö9 «·

* · · I_ I I _ I I

1 a

I I

: 15 P. rivulosus strain T241i is more selective than C. subvermispora strain CZ3, which. · · ·, Exhibits a significantly higher lignin loss compared to cellulose loss in T241 i-treated wood. Both mushrooms also use wood pulp. 1.: As a source of food, but T241 i consumes twice as much as C.

• '' 1: subvermispor. C. subvermispora consumes approximately all major wood components. /. 20 is equal and the fungus is only slightly selective in spruce.

• · «« · • · · ·

Example 2 »· · ^ t •. , The assay examined the growth of P. rivulosus T241i and C. subvermispora CZ-3 in petri dishes at various temperatures to gain insight into the fungal heat tolerance.

A 1.5% solution of malt extract was autoclaved with 1.5% agar.

10 112248

The sterile malt agar plates were placed in a 7 mm diameter round sponge graft. Plates were incubated upside down at 15, 20, 25.28, 30, 34, 37.40 and 43 degrees Celsius and the resulting mycelial diameter was measured after 24 h, 48 h, 96 h and 120 h.

As can be seen in Figure 1, the T24li clearly grows beyond strain CZ-3. temperature range. Strain T241i exhibits stronger growth at both low (15 ° C) and high (37 ° C to 40 ° C) temperatures. CZ-3 did not grow at all at 40 ° C. At the highest measured temperature (43 ° C), Τ24Π did not grow, but the fungus retained its viability, which could be observed by moving the culture dish to a lower temperature: 10 fungal growths quickly returned to the same level at that time. with growth of mycelium at temperature. Preferably, in the process of the invention, the pretreatment temperature is between 20 and 40 ° C, more preferably between 20 and 37 ° C, most preferably between 25 and 30 ° C.

15 Example 3

Test as in Example 1 but growing mycelium in six pieces for two and six weeks. The test investigated the effect of three different temperatures, 25 ° C, 28 ° C and 37 ° C, on the weight loss of wood caused by fungi and the selective removal of lignin from the wood. As can be seen in Table 3, the ability to selectively remove lignin from the 20 · wood material is good during six weeks of cultivation in both studied fungi at I I I · · · · 28 ° C, but degrades at 37 ° C. C. subvermispora CZ-3 did not remove lignin at this temperature. However, P. rivulosus T241i also remained high; ' '. the ability to selectively remove lignin from the wood at a temperature of greater than 1.0.

! .. 'Figure 2 shows the weight loss in the tree during the two (top) and six weeks 25 (bottom) growth period. The picture shows that CZ-3 is a more powerful wood splitter,. . which is reflected in the greater weight loss of wood at 25 ° C and 28 ° C. The T241 i has a clear strain. · ·, CZ-3 is more heat stable, which is also evident in the weight loss of wood at 37 ° C. At this temperature, the wood weight loss caused by strain CZ-3 remained well I · · small after six weeks of growth, only 0.7%, whereas it was 3.2% with strain T241i.

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Table 3. Relation of wood lignin loss to wood weight loss. Six weeks of mushroom growing at three different temperatures.

P. rivulosus T241 i C. subvermispora CZ-3 25 ° C 20 L3 28 ° C 26 22. 37 ° C L 2 Ö 5 Example 4

The growth of P. rivulosus T241i and C. subvermispora CZ-3 in wood was investigated using stubble spruce chips for plywood production. The chips were packed in plastic bags and frozen. To measure fungal mycelial growth in chips and solution cultures, fluorescein diacetate reagent (FDA) was used, which is stable in mild 10 basic aqueous solutions and is hydrolyzed by microbial esterase enzymes to diffuse the released fluorescein molecule. The amount of fluorescein released can be measured with a spectrophotometer. The concentration is expressed as dry weight of wood as a function of time. The amount of fluorescein released correlated with the living microbial biomass.

15 • «• · · ♦

In the method, about 4 g of fresh chips to be examined are placed in a vessel and '' * / sterile 0.1M Tris buffer, pH 8.0, is added until all chips are on the liquid surface ';;.' below. In chips assays, the liquid is soaked in chips • · i ". In aspiration vacuum for about 30 minutes, then FDA reagent, which is prepared in acetone at a concentration of 2 mg / ml, is added to the vessel to a concentration of The final concentration in the test vessel is 0.02 mg / ml. The dishes are incubated at 28 ° C until discoloration is observed. ·.; The reaction is terminated by the addition of pure acetone to a final concentration of 50%. · · · Incubation time Take the solution through a glass fiber filter and measure the absorbance at 490 nm with a spectrophotometer. The vessel chips are dried at 100 ° C overnight and weighed. The results are expressed as the absorbance per gram of dry chips per minute. Due to the small size, the values in the pictures are multiplied by 100.

»· I2 112248

The test used fresh frozen chips. After thawing, the chips were sterilized by autoclaving at 121 ° C for 20 min. The fermentation vessel was used as small 25 ml bottles with a 1% aqueous agar layer poured on the bottom to maintain the desired moisture during fermentation in the bottles. Flasks were filled with chips (about 4 g tp) and 5 sponge suspension of mycelium were pipetted into 150 μΐ flasks. As can be seen in Figure 3, both. strain Τ24Π that the growth of strain CZ-3 begins to increase after three days of incubation. CZ-3 is more biomass producing, which is reflected in the higher FDA value. Despite stronger growth at the temperature of 28 ° C studied, C. subvermispora treatment is not more favorable than P. rivulosus for mechanical pulping. P. The Nvu / osus treatment thus provides the same advantageous effect on the chipping of the wood chips with lower biomass yield.

Example 5

The effect of a week-long treatment of fungus on spruce chips was investigated. Test chips 15 were spruce heartwood chips, which were screened through a 7 mm diameter sieve before use. 300 g of chips were used. per container. The incubation vessel used was a 20 cm x 30 x 12 cm (high) polypropylene dish with a bottom plate of 6 mm diameter openings made of polycarbonate at 28 mm height. The distance between the openings was about 30 mm. Screened autoclaved (121 ° C, / 20 20 min) chips were placed on top of the plate. Compressed air was introduced from below the perforated plate throughout the incubation period.

. 0.03 liters / min. The chips were inoculated with 150 mL of the same suspension of mycelium as !!. 'In Example 1. The malt broth that had passed through the chips was left on the bottom of the vessel for incubation. for the duration of. The incubation temperature was 28 ° C. The moisture content of the chips during cultivation was 42-44% (k.a.). After culturing, the chips were ground as in Example 1 and analyzed for chemical composition.

. · · \ Table 4 shows the so-called. Quantities of Klason lignin and soluble lignin and the extract content of wood after fermentation. The extract content is also shown in relation to '..! for chips treated without fungus. As can be seen from the table, already in one week, both fungi strongly increase the amount of lignin soluble in wood acid: * * the amount in untreated wood is non-existent. P. rivulosus T241i is less biomass-forming than '*': C. subvermispora strain CZ-3, which is reflected by less lignin degradation in the early stages of cultivation. In contrast, T241i i3 112248 is a more effective decomposer of spruce heartwood extracts: over 30% of the extracts have been lost in a week's treatment. The extract composition after treatment is shown in Figure 4. From the figure it can be seen that the T241 i treatment has removed all the different groups of extractants, but especially fatty acids and lignans, better than the CZ-3 treatment.

5

Table 4. Amount of autoclaved spruce heartwood lignin and extracts in weekly fungal treatment. PR = P. rivulosus, CS = C. subvermispora.

lignin extracts treatment ____

Klason (mg / g) soluble (%) mg / g loss% no fungus 273.2 0.005 3.8 PR (T24 li) 274.8 δ65 2 ^ 6 3R6 CS (CZ-3) 267.4 δ8 8 ΪΤ 10

Example 6

The experiment investigated how P. rivulosus T241i and C. subvermispora CZ-3 grow on fresh, untreated pine chips (Pinus sylvestris). The surface of the pine was used in the experiment. The fungi were inoculated and grown as in Example 5. Both fungi also grew on pine chips, which could be observed using the FDA reagent in Example 4 to measure growth. After one week of fermentation, the growth of strain CZ-3 ''. Was about 30% greater than that of strain T241i, but after two weeks of growth, the amount of live biomass of strain CZ-3 was reduced, indicating poor fungal growth of the fungus on fresh pine chips. T241i, with 20 biomass increases in two weeks After weekly fermentation, the wood, ·: extractant and lignin content was measured in the cultures, As can be seen in Table 5, the content of pine · · · extract is significantly higher than that of spruce (see example,. Both mushrooms are also capable of effectively breaking down pine extractives: '..! Per week almost 30% of the pine tree extract has disappeared. Figure 5 shows' · * 25 loss of extractives for each group of extractives. the amount has dropped drastically (about 50%) and the amount of fatty acids '·' · is clearly Already a week after treatment, T241i has reduced the concentration of clonegrignin in pine, whereas treatment with CZ-3 has had little effect on the concentration of lignin in pine 14 112248. Neither treatment has increased the acid solubility of pine lignin.

Table 5. Amount of fresh pine lignin lignin and extractants in week 5 fungal treatment. PR = P. rivulosus, CS = C. subvermispora.

lignin extracts treatment _____ clone (mg / g) soluble (%) mg / g loss% no fungus 269.4 0 64.6 PR (T241i) 260.5 O 47 ^ 2 26 ^ 9 CS (CZ-3) 267 , 6 Ö 46 ^ 3 28 ^ 3

Example 7

The test examined the ability of fungi to influence the alkali solubility of spruce lignin. Method 10, on the one hand, enables the evaluation of the effect of a fungus on the lignin structure and the fiber massability of the mechanical pulp, on the other hand, it can be used to predict, for example, chemical consumption in chemical pulping. In the method, the fungi were grown on quantitatively cut pieces of wood of size: length: 25 mm, width: 15 mm, thickness: 8 mm. The pieces were made of spruce surface wood. The pieces were placed in a 20 g · · 15 (k.a.) glass culture flask and the flasks autoclaved as in Example 1. The sponge treatment “·”> was done in duplicate culture flasks. The flasks were aerated three times a week by inserting a through-tube through a rubber stopper to which the air tubing was attached during aeration. * Growth time was 14 days. IM sodium hydroxide was soaked in 100 ml x 2 for two hours at 80 ° C. Compounds were measured by 20 spectrophotometry at 280 nm and quantified using commercially available alkaline lignin Indulin AT. As shown in Table 6, only P. rivulosus T241i and C. subvermispora CZ-3 of the fungi examined increase the alkaline solubility of lignin. consumption of sodium hydroxide increased, which may be a consequence of organic acids produced by chips of strains T241i and CZ-3.

'*:' 25 t · 15 1 12248

Table 6. Effect of fungal treatments on absorption of alkali into wood and release of alkali-soluble compounds. PR = P. rivulosus, CS = C. subvermispora.

NaOH uptake Alkaline soluble compounds (λ 280 nm) Sample (mg / g wood) (mg / g wood)

Control (no fungus) 72 ± 2 4 ± 0 CS (CZ-3) 81.5 ± 1.5 12 ± 0.3 PR (T241i) 81.5 ± 1.5 9 ± 0.9 P. gigantea * 1 71.5 ± 1.5 4 ± 0

Cartapip * 2 73.5 ± 1.5 3 ± 0.1

White variant of Phlebiopsis gigantea, Cartapip Ophiostomapiliferum.

5

Example 8

Pretreatment as in Example 5, but as a wood raw material for spruce surface and in fungal treatment period for two weeks. To confirm the increase in solubility of the fungal lignin, the sponge-treated chips were cooked and the relative molecular weight distribution of the compounds was determined from: ..: 10 black liquor »·: ·. by gel permeation chromatography. Cooking conditions in the used air bath

'Were as follows: 30% sulphide, boiling point 170 ° C, rise time 80 ° C to 170 ° C

• «» t .1 '*. 40 min The effective alkali dose was 22%, H-factor 1250, and liquid ratio 4.

• · »* ·: '': 15 As can be seen in Figure 6, two weeks treatment of fungi with strains« * · P. rivulosus T241i (S3 -3) and C. subvermispora CZ-3 (S3-5) reduces: '·. ; high molecular weight fraction (molecular weight greater than 10,000) and correspondingly increases the amount of low molecular weight fraction (molecular weight less than 1000) to the crude: '. ·. compared to wood (S3-4).

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Tl · • · * · ·. ·. : Example 9 i »· • ·

Pretreatments for wood chips as in Example 8. The effect of the sponge treatment on the amount of energy needed to defibrate the chips was investigated. The chips (125 g, c.a.) 1662248 were ground in a wing refiner. Figure 7 shows the energy saving in mechanical torture, which is over 20% in fungal treatment compared to untreated chips and 10-15% in uninoculated control. The power requirement for mechanical defibration is shown in Figure 8. The power uptake at the onset of torture is 5 significantly lower for P. rivulosus T241i and C. subvermispora CZ-3. in chips and remains at about 500W lower than the control throughout the defibration.

SAVED MICRO-ORGANISM

10

The following microorganism was deposited with the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany) pursuant to the Budapest Treaty: 15 Microorganism Deposit number Deposit date

Physisporinus rivulosus T241i DSM 14618 November 14, 2001 (t I * «·» · * · »*> *»

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REFERENCES

Akhtar, M., Attridge, M.C., Myers, G.C & Blanchette, R.A. (1993). Biomechanical Pulping of loblolly pine chips with selected white-rot fungi. Holzforschung 47, pp. 36-40.

5. Akhtar, M., Blanchette, R.A. & Bumes, T.A. (1995). Using Simons Stain to Predict Energy Savings During Biomechanical Pulping. Wood and Fiber Science 27, pp. 258-264.

Nakasone, K.K. (1981). Cultural studies on Poria cinerascens, P. rivulosa, and P.

10 subvermispora (Aphyllophorales, Basidiomycotina). Mycotaxon 13, pp. 105-111.

Scott, G.M., Akhtar, M, Lentz, M.J. & Swaney, R.E. (1998). Engineering, scale-up, and economic aspects of fungal pretreatment of wood chips. In: R.A. Young and M. Akhtar (Eds.): Environmentally Friendly Technologies for the Pulp and Paper Industry, 15 pp. 341-383. ISBN 0-471-15770-8, John Wiley & Sons.

Setliff, E.C., Marton, R., Granzow, S.G. & Eriksson, K.L. (1990). Biomechanical Pulping with white-rot fungi. TAPPI Journal 73, issue 8, pp. 141-147.

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Claims (18)

A method of pretreatment of wood using a carrot fungus, in which the process of carrot fungus is inoculated into wood, characterized in that the carrot fungus is The method of claim 1, wherein the wood is chopped into wood chips prior to the inoculation of the carrot fungus in the wood. 10 The method of claim 2, wherein the chipped wood is moved to a bioreactor prior to the inoculation of the carrot fungus in the wood. A method according to any of the preceding claims, wherein the wood is pre-treated for the production of mechanical pulp. 15 5. A method according to claim 4, characterized in that the energy consumption required in defibrating the mechanical pulp is reduced by more than 15%. 5 Physisporinus rivulosus. A method according to any of the preceding claims, wherein the wood chips are made from softwood, which is preferably spruce (Picea abies) or tali (Pinus sylvestris), or hardwood, such as; | Preferably, aspen (Populus tremelloides), birch (Betula sp.) or eucalyptus (Eucalyptus: Y: sp.). 7. A method according to any of the preceding claims, characterized in that the liquid root fungus selectively removes lignin from the wood. * 8. A process according to claim 7, characterized in that the amount of soluble lignin rises above double compared to untreated wood. ♦ 30 Method according to any of the preceding claims, characterized in that the Physporinus rivulosus white root fungus is grown in a continuously aerated bioreactor. * 21 1 1224b 10. A method according to any of the preceding claims, characterized in that the Physporinus rivulosus carrot fungus reduces the wood's extract content already in 7 days. 5 11. A method according to claim 10, characterized in that the extractive material content of the wood is reduced by more than 20%. Process according to any of the preceding claims, characterized in that the Physporinus rivulosus white root fungus secretes manganese peroxidase and oxalic acid in the chip 10 during the pre-treatment. Process according to any of the preceding claims, characterized in that the pretreatment temperature is between 20 ° C and 40 ° C, preferably between 20 ° C and 37 ° C, between 25 ° C and 30 ° C. 15 14. A method according to claim 1, characterized in that the carrot fungus is a sebum that has been found and isolated in Europe, i.e. Physisporinus rivulosus. 15. A method according to claim 14, characterized in that the carrot fungus is the strain 20 Τ241Ϊ of Physisporinus rivulosus. : · 16. Pure cultivation of Physporinus rivulosus T241i carrot fungus strain (DSM 14618). . . · 17. Use of Physisporinus rivulosus carrot fungus for wood pretreatment. • At 25 Use according to Claim 17, characterized in that the carrot fungus is the strain T241i of Physisporinus rivulosus (DSM 14618).