BACKGROUND OF THE INVENTION To make paper from wood the wood material is usually chipped and wood fibres are separated from each other by a suitable process. In the industrial scale several different methods are used to separate fibres from each other. At the simplest, the chips are mechanically pulped with water in a grinder to a desired degree of defibration. Other methods include the so-called thermo-mechanical pulping (TMP), chemical treatment of chips combined with thermo-mechanical pulping (CTMP), chemical-mechanical pulping (CMP), the so-called Kraft or sulphate pulping and the sulphite process. In each of the above mentioned methods the aim is to achieve a separation of the cellulose fibres of wood from other structural material of wood to a desired degree of defibration.

Wood material is composed of polymers of various chemical structures, of which cellulose is the major structural material. Cellulose fibres are associated with hemicellulose that is associated with an aromatic polymer called lignin. In chemical pulping, lignin (and hemicellulose) is removed. In native wood, lignin is stratified around the cellulose fibres, forming a layer providing protection and mechanical support.

Treatment of wood or chips with lignin-degrading fungi before pulp making is termed biopulping. Stored unseasoned wood is rapidly attacked by microorganisms, mainly fungi. From wood, the fungi initially use the most easily utilizable nutrients such as wood extractives. Upon diminution of these nutrients in wood, growth benefits are acquired by those fungi that are capable of utilizing cellulose, hemicellulose, and lignin. White rot fungi are fungi belonging to the class Basidiomycetes capable of degrading these wood polymers. Some of these fungi degrade proportionately more lignin than wood polysaccharides. These fungi are termed as selective lignin degraders. The use of such fungi has proven useful in the pre-treatment of wood chips before mechanical pulping. By a suitable fungal treatment it is possible to lower the costs of pulping. The use of fungi

not only aims at making production more effective but also at an environmentally friendly production. In mechanical pulping it has been possible to save energy and improve paper quality by a fungal treatment.

The first article about possibilities of lignin-degrading fungi in pulp making was published in the year 1957. The first biopulping experiments were performed in the early 1960's with a spontaneously white-rotten pine that was used as raw material for chemical pulp. By the action of the fungus the strength of the paper was improved. Biopulping and research on it has been elaborated especially in Sweden during the 1970's and 1980's at Skogsindustrins Tekniska Forskningsinstitut (STFI) in the group of Prof. Karl-Erik Eriksson, and in 1987-1996 in the USA as a consortium funded for the most by 20 companies and coordinated by Dr. Richard Burgess and Prof. Kent Kirk of Forest Products Laboratory at the U. S. Department of Agriculture, Madison, Wisconsin. In the project a wide survey of white rot fungi (approx. 400) and more than 200 biopulping experiments were carried out. Among the fungi, Ceriporiopsis subvermispora became selected as the most effective one, because it seemed to be suited to the treatment of both softwood and hardwood (mainly pine and aspen). Experiments have been performed with this fungus in an industrial scale of 40-ton chips batch. Spruce (Picea abies) has been subject to much less research than aspen or pinewood. Energy savings as large as those obtained with aspen have not been achieved with spruce. Setliff et al. (1990) have reported an energy saving of 13% by a 20-day treatment with C. subvef°naispora, whereas a corresponding treatment with aspen yielded an energy saving of 20%.

The use of fungus in biopulping currently involves problems, whereby in order to reduce these problems and render the process economically more profitable a need exists to develop or find a fungus endowed with such properties that its inoculation to chips does not cause the disadvantages appearing in the use of C. subvermispora : 1. addition of the inoculum to chips requires a reduction of the chips'natural microflora before addition of the white rot fungus, for example by treatment of the chips with steam, 2. vigorous increase of fungal biomass in chips raises the temperature of the chip pile within a short period of time so high that the growth of the fungus stops, 3. the structure of chips changes in such a way that delivery of air into the chip pile, as required for fungal growth, lignin degradation and temperature adjustment, becomes difficult. It can also be considered as a prerequisite for the fungal treatment that lignin selectivity remains high

and the decrease in yield remains low. In further studies of the consortium a biopulping fungus, Phlebia subserialis, which is superior to C. subvermispora, has been found and applied for patent in the USA in the treatment of loblolly pine (Pinus taeda) and aspen (Populus tremelloides) (W09802612). Although the use of the P. subserialis fungus has appeared to reduce the abovementioned problems (Scott et al., 1998), there are no published data of its lignin selectivity and it seems that this fungus, as regards its selectivity properties, is not of the same order as C. subvermispora. There are no published data of its wood-affecting enzyme activity. In earlier studies published in 1991 (US5055159) and 1993 (Akhtar et al. ), the energy saving following treatment with P. subserialis fungus was of the same order or smaller than that found in treatment with C. subvermispora. It is obvious that from treatment with P. subserialis fungus no results have been obtained that are sufficiently good for an industrial application, possibly because of its weaker lignin selectivity.

In the invention a white rot fungus Physisporinus rivulosus, strain T241i, has been found and isolated, which for its properties with respect to growth, temperature tolerance and lignin selectivity, is superior to C subvermispora. In addition, for its temperature tolerance properties T241i is comparable with P. subserialis. Therefore, in strain T241i good properties with respect to pretreatment technology and to its mechanism of action are combined. Lignin selectivity is high and the weight loss of wood caused by the treatment is small, being comparable to the small weight loss that is important in pulp making. Already after one week of cultivation, P. rivulosus T241 i causes changes in the solubility of wood lignin in alkali, and secretes plenty of the lignin-modifying enzyme manganese peroxidase into the chips. This enzyme is secreted into the wood concomitantly with oxalic acid. The occurrence of both of these factors in wood is obviously important in lignin modification and in later processes that have an effect on the energy consumption of pulp making and on paper quality. The fungus also strongly reduces the amount of wood extractives, which is an important property regarding paper quality and runnability of paper machines. A treatment for two weeks with strain P. rivulosus T241i significantly reduces the pulping energy requirement of mechanical pulping.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Growth of C. subvermispora (CZ-3) and P. rivulosus (T241i) on malt agar medium at different temperatures. The amount of growth is expressed as millimeters per day.

Figure 2. Ability of strains P. rivulosus T241i (black bars) and C. subverniispora CZ-3 (shaded bars) to cause decay in spruce, expressed as decrease in weight of wood at three different temperatures after treatment times of 2 and 6 weeks with the fungus.

Figure 3. Amount of viable fungal hyphae in autoclaved chips during a two-week fermentation, expressed as FDA values (see Example 4) Figure 4. Changes occurring in different groups of extractives of autoclaved spruce heartwood chips after treatment with the fungus for one week. Blank bar: control chips without fungal treatment, black bar, P. rivulosus T241i, shaded bar, C. subvermispora CZ-3.

Figure 5. The upper drawing shows the changes occurring in different groups of extractives of unseasoned pine sapwood chips after treatment with the fungus for one week. From the lower drawing fatty acids and resin acids have been omitted, whereby differences between other groups of extractives become more evident.

Figure 6. Gel permeation chromatography curve of the molar mass distribution of black liquor formed in chemical pulping. The chips are spruce sapwood, treatment time with fungus, 2 weeks. S3 3, P. rivulosus T241i, S3 4, control without fungal treatment, S3 5, C. subvermispora CZ-3.

Figure 7. The energy consumed by research refiner in pulping of spruce sapwood chips at different degrees of defibration. Duration of fungal pretreatment was two weeks.

Figure 8. Effect of fungal pretreatment on the power uptake of mechanical pulp at the initial stage of refining.

Figure 9. Decay capacity of P. rivulosus T241i on aspen, birch and eucalyptus.

Figure 10. The growth habits of different P. rivulosus strains tested on agar plates at +37 °C. Plate A. T241i, Plate B. CBS-433. 48, Plate C. CBS-434. 48, Plate D. ATCC 64310 after 5 days on malt agar. Right panel (plate E): the same plates after 27 days growth, including C. subvermispora CZ-3 on the right.

Figure 11. Fragments of P-tubulin-encoding gene amplified from the genomic DNA of different P. rivulosus strains on agarose gel: lane 1: molecular weight standard; lane 2: CBS 434.48 ; lane 3: CBS 433. 48; lane 4: ATCC 64310 ; and lane 5 : T241i Figure 12. The sequence of the amplified ß-tubulin-encoding gene (740 bp) from strain T241i (SEQ ID NO : 1). The putative amino acid sequence is shown below the nucleotide sequence (SEQ ID NO : 2).

Figure 13. Eucalyptus (E. grandis and E. dunnii) and birch (Betula sp. ) wood chips shown before and after incubation with P. rivulosus T241i strain for 6 weeks. The whitening of wood chips is apparent.

DETAILED DESCRIPTION OF THE INVENTION P. rivulosus is a common wood-decaying agent causing white rot in North America and Central America, especially in western red cedar (Thujaplicata) and redwoods (Sequoia sempervirens). Other host trees include, among others, western hemlock (Tsuga heterophylla), Douglas fir (Pseudotsuga menziesii) and Oregon alder (Alnus rubra (=oregana)). Also birch (Betula sp. ) and eucalyptus (Eucalyptus sp. ) are possible host trees. The fungus is thus able to grow on both soft-and hardwood. In Europe P. rivulosus is supposed to be very rare. Strain T241i, however, was found in southern Finland. In most cases it has been found in pine (Pinus sylvestris). Usually fruiting bodies of the fungus are found on charred logs. Because many fungal genera and their designations in fungal taxonomy are unestablished, P. rivulosus has previously been associated to the genera Gelatoporia (Gloeoporus), Rigidoporus and Ceriporiopsis. The now expired generic name Poria has also been used with the fungus. P. rivulosus is related to Ceriporiopsis subvermispora, but constitutes a clearly separate species.

P. rivulosus is a poorly characterized white rot fungus. Nakasone (1981) has compared the properties of P. rivulosus (=Poria rivulosa) and C. subvermispora (--Poria subvermispora) with respect to optimum temperature, growth and microscopic characteristics. In Example 1 changes induced by P. rivulosus strain T241i and C. subvermispora strain CZ-3 in the chemical structure of wood have been investigated.

In this Example both species are shown to be capable of selective lignin degradation.

T241 i is more selective than CZ-3. In Example 2, growth of strains T24 I i and CZ-3 on agar culture plates in the laboratory has been studied. T241i possesses for its growth a clearly wider temperature range as compared to strain CZ-3. Example 3 describes the effect of temperature on wood degradation. The better temperature tolerance of strain T241i is also evident in these short-term wood decay experiments. In Example 4 a lower production of biomass in spruce chips is demonstrated for strain T241i as compared to strain CZ-3 in similar growth conditions.

The fungus T241i has also been studied in the context of mechanical pulping in experiments, in which the ability of the fungus to exert an effect on the cellulose fibers of aspen wood was studied. Using the so-called Simons staining the fungus has been shown to possess a feature that correlates with reduced energy consumption in the mechanical pulping process. Akhtar et al. (1995) have studied two strains of Ceriporiopsis rivulosa (table 2 of the article) together with C. pannocincta (Gelatoporia pannocincta) as reference material for C. subvermispora using the Simons stain. The staining result was compared with energy savings achieved in mechanical pulping in relation to untreated chips. With another strain of P. rivulosus (= C. rivulosa) no energy savings were obtained and with yet another P. rivulosus strain after two weeks'treatment in the pulping of aspenwood an energy saving of 3% was obtained as compared to uninoculated control, whereas energy savings of 7-12% were obtained with C. subvermispora strains. The result with C. pannocincta was similar to that with P. rivulosus. It is possible that the American strains of P. rivulosus used in the study differ in their properties from European strains of P. rivulosus.

Fungal treatment of spruce chips with strains P. rivulosus T241i and C. subvermispora CZ-3 causes an increase in the alkali and acid solubility of wood lignin already after a treatment for one week (Examples 5 and 7). The increase in the solubility of lignin is likely to indicate that, as a consequence of the fungal treatment, changes have occurred in

the structure of lignin, promoting the liberation of lignin and its aromatic subunits to an alkaline or acidic solution, when the chips are treated either with an alkaline solution such as sodium hydroxide, or with strong acid such as sulphuric acid. These changes correlate positively with a profuse production of ligninolytic enzymes as well as with reduced pulping energy in mechanical pulping. The changes induced in lignin structure by treatment with P. rivulosus and C. subvermispora are seen as an increase in acid-soluble lignin in the pretreatment of spruce chips (Example 5), whereas in the pretreatment of unseasoned pine chips the acid solubility of lignin did not increase, despite the fact that the mount of pine extractives decreased by almost 30% (Example 6). In the treatment of pinewood, the very promising potential of strain T241i thus resides especially in being an eliminator of extractives, and could therefore enable the use of pine as raw material for mechanical pulp. The content of extractives of wood is preferably decreased in the process of the invention by more than 20%. The white rot fungus Phlebiopsis gigantea and the resin-removing fungus Ophiostoma piliferum (pale variant) studied in Example 7 do not cause an increase of lignin's alkali solubility comparable to that seen in treatments with P. rivulosus and C. subver7nispora in spruce chips. In Example 8 it is reported how the change in the alkali solubility of lignin as a result of the fungal treatment is also seen in the compound distribution of black liquor generated in chemical pulping: the amount of small molecular lignin has increased as a result of the fungal treatment. In Example 9 it is demonstrated that the amount of energy consumed by the research refiner in pulping has decreased by 10-15% as a result of fungal treatments with T241i and CZ-3, and especially the amount of energy required by the initial stage of refining has strongly decreased. In the industrial scale this already signifies a very significant energy saving.

Demonstration of the process In the process chips prepared for ordinary mechanical or chemical pulping are used. The chips are screened into evenly divided chips by a combination of a hole and bar screen in compliance with standard SCAN-CM 40: 88. The screening accept is recovered from screen plate 07. If the dry matter of chips exceeds 40%, the chips are moistened to make the chips readily colonizable by the fungus. Delivery of the fungal inoculum into the chips is most effective provided that the surface of the chips is made aseptic by a steam treatment, for example. As fungal inoculum, either solid or liquid inoculum can be used.

When using liquid inoculum, any well-known nutrient-containing cultivation medium can be used. As a most simple medium, malt extract dissolved in water can be used, for

example. The fungal inoculum is cultured separately and inoculated into the chips as a homogenized suspension. The amount of inoculum in relation to the amount of chips is minor, typically approx. 5 g on a dry weight basis per ton of chips.

The inoculated chips are incubated in a bioreactor. As bioreactor any container or structure can be used that promotes growth of the fungus. Since growth of the fungus and removal of lignin from the wood material are heat-producing and oxygen-consuming processes, regulation of temperature in the bioreactor must be taken care of. Microbial metabolism in chip piles raises especially the temperature of the inner parts of chip piles considerably. Maintenance of the temperature at different sites of the bioreactor can be adjusted by feeding air into the reactor, promoting lignin removal at the same time. In the present invention PR fungus (PR=P. rivulosus) is used, which during its growth in the chips secretes ligninolytic enzymes into the chips. Of the three enzymes affecting delignification of wood, laccase, manganese peroxidase and lignin peroxidase, PR produces manganese peroxidase in highest amounts. The intensive secretion of this enzyme in chip cultivation occurs concomitantly with increasing secretion of oxalic acid.

Upon growth in. chips, PR alters the composition of wood lignin so that the solubility of lignin in alkali increases already during a one-week cultivation. Preferably the amount of soluble lignin increases during the process by more than two-fold as compared to untreated wood. After cultivation for two weeks, the chips are pulped in a refiner to a desired degree of defibration and the energy consumed in the pulping is determined. The effect of fungal treatment on energy consumption is particularly significant in the initial stage of pulping, when the power input of the refiner into fungally treated chips is approx.

20% lower than into untreated chips. The power input in the fungally treated pulp remains for the whole time of pulping at an approx. 500 W lower level as compared to untreated pulp. Preferably the energy consumption required in the pulping of mechanical pulp is decreased by more than 15 %.

One of the preferred embodiments of the invention is to inoculate a white rot fungus of the invention to wood during its storage and transport. Pretreatment of wood according to the invention can thus take place in an environment, in which the optimal conditions of the above-described bioreactor are not necessarily achieved. In this embodiment of the invention it is also not imperative before inoculation of the fungus to chip the wood to be pretreated.

The results of our research indicate that in the fungus P. rivulosus, strain T241i, several properties are combined which are important for effective biopulping: the fungus is heat- stable, easily cultivable both in softwood chips and in liquid cultures, thus enabling easy preparation of the inoculum, the fungus is lignin selective and modifies lignin structure by producing a large amount of the lignin-modifying enzyme manganese peroxidase.

Preferably, the fungus secretes more than 1.0 units (U) of manganese peroxidase into one gram of dry chips during 14 days. Oxalic acid is also preferably secreted over 25 µmol into one gram of dry chips during 7 days. Oxalic acid is determined by extracting it from wood by 1.5 M hydrochloric acid. Pretreatment of chips in a simple bioreactor is easy, and the saving of pulping energy obtained through the pretreatment is significant. A quantitative enlargement of the pretreatment technique to industrial scale is comparably easily achieved.

Moreover, the inventors have noticed that the incubation of wood with P. rivulosus results in the whitening of wood color. Thus, one further embodiment of the invention is the use of the white rot fungus of the invention for the whitening of wood color. The whitening phenomenon can be seen in Figure 13 showing eucalyptus and birch wood chips before and after incubation with P. rivulosus T241i strain for 6 weeks.

The publications and other materials used herein to illuminate the background of the invention, and in particular, to provide additional details with respect to its practice, are incorporated herein by reference. The invention will be described in more detail in the following Examples.

EXAMPLES Example 1 In the test the fungus is cultivated in a 100-ml erlenmeyer flask in pieces of spruce, sized 17 mm x 7 mm x 16 mm (four pcs per flask), over moist vermiculite. The fungal inoculum is prepared in malt extract broth from a mycelium grown for 10 days. The mycelium is homogenized to a suspension in a Waring-Blender power mixer for 10 s four times. A pause of 30 s is kept between homogenizations, preventing excessive heating of the mixer. The numbered, dried and weighed pieces of spruce are allowed to absorb water

for approx. 15 s by water suction and the pieces are autoclaved for 20 min at 121°C. The sterilized pieces of spruce 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 are incubated at +25°C and relative air humidity of 70% for 10 weeks. The pieces are dried for 3 days at 60°C and weighed to measure the decrease in weight of the pieces.

The dry pieces are ground by a mechanical grinder to finely graded powder, and the chemical composition of the wood powder is analyzed.

Table 1. Chemical analyses of the test for main components of the wood, and decrease in weight of the wood. The results are expressed as decrease percentages. PR=P. rivulosus, CS=C. subvernçispora. decrease fungus strain in lignin glucose xylose mannose pectin weight PR T241i 18. 7 39. 3 18. 2 19. 8 20. 2 24. 5 CS CZ-3 22.7 43.5 35.8 33.0 34.3 32. 7

Table 2. Chemical analyses of the test for main components of the wood. The results are expressed in relation to the decrease in glucose (cellulose). PR--P. rivulosus, CS=C. subvermispora decrease in fungus strain lignin/decrease lignin glucose xylose mannose pectin in weight PR T241i 2.1 2.0 1.0 0.9 1.0 1.2 CS CZ-3 1.9 1.2 1.0 0.9 1.0 0.9

The P. rivulosus strain T241i is more selective than the C. subvermispo7 » a strain CZ3, which is seen in the clearly higher decrease in lignin as compared to the decrease in cellulose in T241i-treated wood. Both fungi use also the wood's cellulose as their nutrient source, but T241i consumes two times less of it than does C. subvermispora.

C. subvermispora consumes all main components of wood in approximately similar amounts and the fungus is only slightly selective in spruce.

Example 2 In this test the growth of the fungi P. rivulosus T241i and C. subvermispora CZ-3 was studied on petri dishes at different temperatures, to obtain a picture of the temperature tolerance of the fungi. From malt extract a 1. 5% solution was prepared which was autoclaved with 1.5% agar. Onto sterile malt extract plates was made a circular inoculum having a diameter of 7 mm. The plates were incubated upside down at temperatures of 15, 20,25, 28,30, 34,37, 40 and 43 degrees Celsius and the diameter of the emerged mycelium was measured after 24 h, 48 h, 96 h and 120 h.

As can be seen in Figure 1, T241i grows within a clearly wider temperature range than strain CZ-3. The growth of strain T241i is stronger both at low (15°C) and high (37°C- 40°C) temperature. CZ-3 did not grow at all at 40°C. T241i did not grow at the highest temperature measured (43°C), but the fungus retained its viability, as could be observed by transferring the growth plate to a lower temperature: growth of the mycelium quickly reverted to the same level as the growth of a mycelium that had continuously been at that temperature. In the method of the invention the pretreatment temperature is preferably between 20 and 40°C, more preferably between 20 and 37°C, most preferably between 25 and 30°C.

Example 3 Test as in Example 1 except that cultivation of the mycelium in pieces of spruce was for two and six weeks. In the test the effect of three different temperatures, 25°C, 28°C and 37°C, was studied on the decrease in weight of wood and on the selective removal of lignin from wood as caused by the fungi. As can be observed from table 3, the ability to selectively remove lignin from wood is good during six weeks'cultivation for both of the studied fungi at 28°C, but becomes weaker at 37°C. C. subvermispora CZ-3 at this temperature removed no lignin at all. P. rivulosus T241i, however, still retained also at the high temperature its ability to selectively remove lignin from wood, i. e. the ratio was higher than 1.0. Shown in Figure 2 is the decrease in weight that has occurred in the wood during the two-week (upper figure) and the six-week (lower figure) cultivation time. It is seen from the Figure that CZ-3 is a stronger wood-decaying agent, as seen in the larger decrease in wood weight at 25°C and 28°C. T241i is clearly more thermostable than strain CZ-3, as seen in the clear-cut decrease in wood weight also at the temperature of

37°C. At this temperature the decrease in wood weight caused by strain CZ-3 remained very small after six weeks'cultivation, only 0.7%, whereas for strain T241i it was 3.2%.

Table 3. Ratio of the percentual loss of wood lignin to the percentual loss of wood weight.

A six-week fungal cultivation at three different temperatures. P. rivulosus T241i C. subvernzispora CZ-3 25 OC 2. 0 1. 3 28 °C 2. 6 2. 2 37 OC 1. 2 0* *no lignin loss, weight loss 0.7% Example 4 Growth of the fungi P. rivulosus T241i and C. subvernzispora CZ-3 in wood was studied using as growth medium stick-like spruce chips intended for plywood making. The chips were packed into plastic bags and frozen. To measure mycelial growth in chips and liquid cultures, fluorescein acetate reagent (FDA) was used, which is stable in a slightly alkaline aqueous solution and is hydrolyzed by microbially produced esterase enzymes, whereby the liberated fluorescein molecule diffuses into the aqueous solution. The amount of liberated fluorescein can be measured by a spectrophotometer. The concentration is expressed on the basis of dry weight of the wood as a function of time. The amount of liberated fluorescein correlates with viable microbial biomass.

In the method approx. 4 g fresh weight of the chips to be examined is put into a vessel and sterile 0.1 M Tris buffer, pH 8. 0 is added enough to submerge all chip pieces. In determinations the liquid is allowed to absorb into the chip pieces under aspiration vacuum for approx. 30 min, after which FDA reagent prepared in acetone at a concentration of 2 mg/ml is added to the vessel in an amount sufficient to yield a final FDA concentration of 0.02 mg/ml in the test vessel. The vessels are incubated at 28 °C until the color change is visibly detectable. The reaction is stopped by adding pure acetone into the vessel enough to make a final acetone concentration of 50%. The time of incubation is recorded. The solution is filtered through a glass fiber filter and its absorbance value is measured by a spectrophotometer at a wavelength of 490 nm. The chips in the vessel are dried at 100 °C overnight and weighed. The results are expressed

as absorbance value per gram of dry chips per minute. Due to the smallness of the numerical values, the values in the Figures have been multiplied by a factor of one hundred.

In the test freshly frozen chips were used. After thawing, the chips were sterilized by autoclaving at 121°C for 20 min. As fermentation vessels were used small 25-ml flasks, having cast on the bottom a 1% water agar layer, in the purpose of maintaining the desired humidity during fermentation in the flasks. The flasks were filled with chips (approx. 4 g fresh wt. ) and 150 1ll of mycelial suspension of the fungus was pipetted into the flasks. As can be seen in Figure 3, the growth of both strain T24 I i and strain CZ-3 starts to accelerate after three days of incubation. CZ-3 is the one producing more biomass, as shown by the larger FDA value. Despite of its stronger growth at the applied temperature of 28°C, treatment with C. subvermispora is not more advantageous, regarding mechanical pulping, than treatment with P. rivulosus. Treatment with P. rivulosus thus produces a similar beneficial effect on the defibration of chips with a smaller production of biomass.

Example 5 In this test the effect of a one-week treatment with the fungus on spruce chips was studied. The test chips were spruce heartwood chips that were screened through a 7-mm diameter screen. before use. Per vessel, 300 g of dry matter of chips were used. As incubation vessel a polypropylene vessel of 20 cm x 30 x 12 cm (height) was used that had in its lower portion at an elevation of 28 mm a perforated plate made of polycarbonate and provided with openings having a diameter of 6 mm. The distance between openings was approx. 30 mm. The screened and autoclaved (121 °C, 20 min) chips were placed on top of the plate. The vessel was supplied with approx. 0.03 liters/min of compressed filter-sterilized air from beneath the perforated plate for the whole time of incubation. The chips were inoculated with 150 ml of similar mycelial suspension as in Example 1. The malt broth draining through the chips was left on the vessel bottom for the time of incubation. Incubation temperature was 28°C. The humidity of the chips during cultivation was 42-44% (of dry matter). After the cultivation the chips were ground as in Example 1 and its chemical composition was analyzed.

Shown in Table 4 are the amounts of the so-called Klason lignin and soluble lignin as well as the contents of wood extractives after the incubation. The contents of extractives are expressed also in relation to chips treated without the fungus. As can be seen from Table 4, already in one week both fungi strongly increase the amount of acid-soluble lignin of the wood, the amount of which in untreated wood is negligible. P. rivulosus T241i forms less biomass as compared to C subvermispora strain CZ-3, as shown by the weaker lignin degradation at the initial stage of cultivation. T2411 is instead a more effective decayer of spruce heartwood extractives: more than 30% of the extractives have disappeared in a one-week treatment. The composition of extractives after the treatment is shown in Figure 4. It can be seen from the Figure that treatment with T241i has, better than the treatment with CZ-3, caused removal of all distinct groups of extractives, fatty acids and lignans in particular.

Table 4. Amount of autoclaved spruce heartwood lignin and extractives in a fungal treatment for one week. PR=P. rivulosus, CS=C. subvermispora. lignin extractives treatment Klason (mg/g) soluble (%) mg/g loss in % no fungus 273. 2 0. 005 3. 8 0 PR (T241i) 274. 8 0. 065 2. 6 31. 6 CS (CZ-3) 267. 4 0. 080 3. 0 21. 1 Example 6 In this test it was studied how P. rivulosus T241i and C. subvermispora CZ-3 grow on fresh pine chips without pretreatment (Pinus sylvestris). Pine sapwood was used in the test. The fungi were inoculated and cultivated as in Example 5. Both fungi grew also on pine chips, as could be observed using the PDA reagent in measuring growth according to Example 4. After an incubation of one week the growth of strain CZ-3 was approx. 30% greater than that of strain T241i, but after two weeks'growth the amount of viable biomass of strain CZ-3 had decreased, which could indicate a weaker ability of the fungus to grow on fresh pine chips as compared to strain T241i, whose biomass had increased in two weeks. After one week's incubation the cultures were analyzed for the contents of

extractives and lignin. As can be noted from Table 5, the content of extractives of pine is significantly higher that that of spruce (see Example 5). Both fungi are also able to effectively degrade extractives of pine: in one week almost 30% of pine sapwood extractives have disappeared. Figure 5 shows the loss of extractives for each group of extractives. It can be noted from the figure that especially the amount of resin acids has strongly fallen (approx. 50%), and the amount of fatty acids has similarly fallen. Already after treatment for one week T241i has decreased the content of Klason lignin of pine, whereas treatment with CZ-3 has not substantially affected the lignin content of pine.

Neither of the treatments has increased the acid solubility of pine lignin.

Table 5. Amount of lignin and extractives of fresh pine sapwood in a one-week treatment with the fungus. PR=P. rivulosus, CS=C. subvermispora. lignin extractives treatment klason (mg/g) soluble (%) mg/g loss in % no fungus 269. 4 0 64. 6 0 PR (T241i) 260. 5 0 47. 2 26. 9 CS (CZ-3) 267. 6 0 46. 3 28. 3 Example 7 In the test the ability of the fungi to affect the solubility of spruce lignin in alkali was tested.

On one hand, the method makes it possible to evaluate the effect of the fungus on lignin structure and on the refinability of wood during mechanical pulping, on the other hand it is possible by the method to predict, for example, the consumption of chemicals in chemical pulping. In the method fungi were cultivated on pieces of wood sawed to fixed size of : length, 25 mm, width, 15 mm, thickness, 8 mm. The pieces were made of spruce sapwood.

Pieces, 20 g (dry matter) were placed into a culture flask of glass and the flasks were autoclaved as in Example 1. The fungal treatment was performed as two parallel culture flasks. The flasks were aerated with filter-sterilized air three times per week by inserting via the rubber stopper an inlet duct, to which an air tube was mounted for the time of aeration.

Cultivation time was 14 days. The pieces were impregnated with 100 ml of 1 M sodium hydroxide for two hours at 80°C. The alkali soluble compounds of the impregnation solutions were measured by a spectrophotometer at a wavelength of 280 nm and quantitated

by using commercial alkali lignin Indulin AT as standard. As seen in Table 6, of the studied fungi only P. rivulosus T241i and C. subvermispora CZ-3 increase lignin's alkali solubility. Also the consumption of sodium hydroxide increased, which may result from organic acids produced by strains T241i and CZ-3 into the chips.

Table 6. Effect of fungal treatments on the absorption of alkali into the wood and on the liberation of alkali-soluble compounds. PR=P. rivulosus, CS=C. subvermispora. NaOH uptake Alkali-soluble compounds (A 280 nm) Sample (mg/g wood) (mg/g wood) Control (no fungus) 72. 02. 0 4~0 CS (CZ-3) 81. 5~1. 5 12~0. 3 PR (T241i) 81. 5i1. 5 90. 9 P. gigantea I 71. 5i1. 5 40 Cartapip*2 73. 5~1. 5 30. 1 *lPhlebiopsis gigantea, *ZCartapip Ophiostoma piliferum, pale variant.

Example 8 Pretreatment as in Example 5, but spruce sapwood as wood raw material and fungal treatment time of 2 weeks. To ascertain the increase in lignin solubility induced by the fungus, the chips treated with fungus were cooked and the relative molar mass distribution of the compounds was determined from the resulting black liquor by gel permeation chromatography. Conditions of cooking in the air bath boiler utilized were as follows: sulphidity 30%, cooking temperature 170°C, elevation time 80°C-170°C 40 min.

The effective alkali portion was 22%, H factor 1250 and liquor-to-wood ratio 4.

As can be seen from Figure 6, a fungal treatment of two weeks with strains P. rivulosus T241i (S3-3) and C. subvermispora CZ-3 (S3-5) decreases the amount of the large molecular fraction (molecular weight over 10 000) and correspondingly increases the amount of the small molecular fraction (molecular weight less than 1 000) as compared to untreated wood (S3-4).

Example 9 Pretreatment and chip material as in Example 8. In the test the effect of fungal treatment on the amount of energy required for the pulping of chips was determined. The chips (125 g, dry matter) were ground by a research defibrator. Shown in Figure 7 is the energy saving in mechanical pulping, which in the fungal treatment is more than 20% as compared to untreated chips, and compared to uninoculated control, 10-15%. The power demand of mechanical pulping is shown in Figure 8. Power intake at the start of pulping is significantly lower in chips treated with P. rivulosus T241i and C. subvermispora CZ-3 and remains for the whole time of pulping at an approx. 500W lower level as compared to the control.

Example 10 The experiment was done as in Example 1 with the following exceptions: (i) Wood substrate used was either aspen (Populus tremula) or silver birch (Betula pendula) wood blocks or Eucalyptus grandis or E. dunnii wood chips; (ii) Incubation temperature was +28°C ; (iii) Incubation time was 6 weeks. In the experiment, 11 blocks of aspen, 9 blocks of birch and 10 chips of eucalyptus were used.

The results are shown in Figure 9, P. rivulosus T241i is efficient degrader of all hardwood species tested, namely aspen, birch and eucalyptus. The fungus caused 9-13% weight loss in six weeks.

Example 11 Experiment was done as in Example 1, except that the incubation temperature for aspen blocks was +28°C, and for the spruce blocks +25°C, and the incubation time was six weeks for aspen blocks, and 10 weeks for spruce blocks. In the experiment aspen (Populus tremula) and spruce (Picea abies) wood blocks were used in order to determine the decay capacity of the species Physisporinus rivulosus. Strain Ceriporiopsis rivulosa ATCC64310 was obtained from American Type Culture Collection, Ceriporiopsis rivulosa CBS 433.48 and C. rivulosa CBS 434.48 from Centraalbureau for Schimmelcultures, Baarn, the Netherlands. These Physisporinus rivulosus strains were used in addition to P. rivulosus T241i.

Table 7. Weight loss (%) of aspen and spruce wood blocks by different Physisporinus rivulosus strains. strain spruce aspen T241i 16. 3 3. 0 8. 8 4 2. 9 CBS 434. 48 10. 3 4. 0 4. 6'2. 0 ATCC64310 3. 0 i 3. 0 3. 3 + 2. 7 CBS 433. 48 0. 2 + 0. 3 1. 7'0. 5

Table 7 shows the weight loss values after 10 weeks (spruce blocks) and six weeks (aspen blocks) incubation, Strain T241i is clearly the most efficient strain to degrade spruce and aspen, although all the investigated strains were able to colonise both woods and to degrade the wood components at least to some extent.

Example 12 The temperature sensitivity of the species Physisporinus rivulosus was tested on agar plates supplemented with 2% (w/v) malt extract. The same strains as in Example 11 were tested. Moreover, Ceriporiopsis subvermispora CZ-3 was used as a comparison strain.

Growth habit was evaluated visually after 5 and 27 days. The results are shown in Fig. 10 and in Table 8. All of the P. rivulosus-strains were able to grow at +37°C, whereas C. subvermispora CZ-3 was not able to grow at this elevated temperature. The most tolerant strain of the P. rivulosus-species is T241i, and it retained its normal hyphal morphology at 37°C temperature, whereas other investigated strains show some alteration in the hyphal morphology; especially the growth of CBS-434. 48 is altered with concomitant production of pigments.

Table 8. Growth of the mycelia of different P. rivulosus-strains at malt agar plates in two different temperatures. fungal strain 1 day 3 days 11 days 28°C 37°C 28°C 37°C 28°C 37°C ATCC64310 + + 50 2 24 ND >90 CBS 433. 48 24 + 2 14 ND >90 CBS 434. 48-236 8 ND 43 T241i 43 22 ND >90 C. subvermispora CZ 3 + 3345 + ND + _

Example 13 In this experiment the growth rate of the fungi within the spruce (Picea abies) wood was evaluated using spruce sapwood blocks, sized 12 x 15 x 27mm. A chemical reagent, 2,2'- azinobis- (3-ethylbenzthiazoline-6-sulphonate) (ABTS) was supplemented to the bottom agar, where the blocks were placed, in order to facilitate the hyphal outgrowth from the block to the medium. Most wood-decay fungi are able to oxidize ABTS. Oxidized ABTS will appear as a greenish product in the medium. The bottom medium was prepared adding the following compounds to the suitable container and adding water to 1000ml : glucose lOg KH2P04 2g MgS04-7 H20 0. 5g CaCl2 2 H20 0.13g (NH4) 2-tartrate 0. 5g Dimethylsuccinate 2.2g yeast extract 0. 2g agar 25g 250 mg ABTS was dissolved into methanol : water (1 : 1)-mixture (lOml) and the solution was added to the rest of the medium after medium sterilization.

Prior to the use the fresh blocks were sterilized by autoclaving them for 20 min at 121 degrees Celcius. The sides of the blocks were covered by DuraSeal Laboratory Sealing Film, and the space between the film and wood was sealed with a mixture of glue and nail varnish in order to hinder the hyphal growth on the surfaces of the block. The seal was left to harden, and the inoculum was placed on the top of the block. Cultures were kept at +28°C and investigated daily by eye to see the color change in the bottom agar medium, which was an indication of the outgrowth of the mycelium.

Results from the experiment are shown in Table 9. As can be seen, no significant differences in the growth rates of the P. rivulosus strains were noted. Strain ATCC64310 appears to have the fastest penetration ability: 27mm of the wood was penetrated within 4 days; other investigated strains grew through the block within 5-7 days. ABTS was oxidized by all strains, although there were differences between the oxidation rate.

P. rivulosus strain ATCC64310 oxidized ABTS only slightly.

Table 9. Growth rate through spruce blocks with ABTS oxidation capacity

rate of penetration (days) intensity of ABTS fungal strain through the block in oxidation below the fibre direction block* ATCC64310 4 + CBS 433. 48 4-5 +++ CBS 434. 48 7 +++ T241i 5-6 +++ C. subvermispora CZ-3 5-6 +++ +, weak color change and ABTS oxidation, ++, clear greenish color and ABTS oxidation, +++, strong greenish color and effient ABTS oxidation Example 14 For further identifying characteristics, a test to bleach a chemical polymeric dye poly R- 478 was performed. The basal medium described in Example 13 was used, except the ABTS was replaced by adding 0. 5g (w/v) Poly R-478 to 1000ml of the liquid medium, and 1% w/v D-glucose was added for the carbon source. Medium was solidified by adding 25g of agar. The fungi were inoculated with a 4 mm diameter full-grown agar plug to the centre of the test plates. Plates were incubated in the dark at 25°C and the decolorisation was followed by eye every second day. The results are reported in Table 10. All P. rivulosus strains were able to decolorise the dye after 8 days, although there was differences in the decolorisation rates.

Table 10. Plate test for poly R-478 decolorisation. ,,.... decolonsatton of poly R* decolorisation of ol R* fungal strain P Y day4 day6 day 8 ATCC64310 + ++ ++ CBS-433. 48 + +++ CBS-434. 48--+ T241i + + +++ C. subvermispora ++ +++ CZ-3 **- : no decolorisation, +: slight decolorisation, ++: medium decolorisation through mi part of the petri plate/strong decolorisation restricted to small part of the plate, +++: strong decolorisation through the complete plate

Example 15 Identifying of the species Physisporinus rivulosus : Physisporinus rivulosus is a fungus which belongs to the class Basidioriaycetes of filamentous fungi. As a dikaryotic state the mycelium has clamp connections. In the past the fungus has also been named as Polyporus rivulosus (Berkeley & Curtis), Poria rivulosa (Berk. & Curt.) Cooke, Poria albipellucida Baxter, Rigidoporus rivulosus (Berk.

& Curt. ) David and Ceriporiopsis rivulosa Gilbertson & Ryvarden.

The molecular data from the species was obtained by isolating genomic DNA from the mycelium of the strain T241i and three other Physisporinus rivulosus-strains from culture collections (CBS 433. 48, CBS 434.48 and ATCC64310). Genomic DNA was extracted from young, 4-5 days old mycelia by standard methods. A pair of oligonucleotide primers was utilised to amplify a region from the p-tubulin-encoding gene. The primers were: A. 5'-TAYTGYATTGAYAAYGARGC-3' (SEQ ID NO : 3) B. 5'-RAAYTCCATYTCRTCCAT-3' (SEQ ID NO : 4) where"Y"codes for either C or T and"R"codes for either A or G.

These primers amplified a fragment from the P. rivulosus genomic DNA of about 740 bp. In addition, another product of about 1250 bp was obtained by the strains T241i, CBS 433.48 and CBS 434. 48 (Fig. 11). In general, PCR included 50 pmol of each primer, 50-100 ng of genomic DNA, 2 mM dNTPs, 1.5 mM MgCl2, and 2.5 units of DNA-polymerase (Promega) per 100 gl reaction. Cycling conditions typically included an initial denaturation at 94 °C for 95 s and 34 cycles of 94 °C for 35 s, 52 °C for 1 min, and 72 °C for 2 min, with a final elongation of 10 min at 72 °C. Slow ramping (1 °C for every 5 s) was used from annealing to extension temperatures. Resulting products were cut from the agarose gel and cloned into the TOPO cloning vector (Invitrogen). For sequencing, cloned products were cycle- sequenced with an automated ABI sequencer in both directions using the M13 universal primers that flank the cloning site.

The sequence of the amplified product (740 bp) from the strain T241 i is shown in Fig. 12 (SEQ ID NO: 1). The putative amino acid sequence is shown below the nucleotide sequence (SEQ ID NO : 2). The fragment has two introns, based on the sequence comparison with other known P-tubulin gene sequences from fungi. Introns are indicated in the Figure 12 by small letters. Table 11 shows the sequence similarities between the fragments of four P. rivulosus isolates and P-tubulin encoding genes of four other fungal species calculated using the program"Matcher"included in the European Molecular Biology Open Software Suite (EMBOSS) package for sequence analysis. Schizophyllum commune and Coprinus cinereus belong to the class Basidiomycetes, and Gibberellafujikuroi and Aspergillus nidulans to the class Ascomycetes.

Three P. rivulosus strains, CBS 433.48, CBS 434.48 and T241i share a very high (3-tubulin gene sequence homology between 95.5 and 98.5%, whereas the strain ATCC64310 shares only 77-78% homology to the other P. rivulosus-strains. This is a significant difference, as the comparison with the p-tubulin genes from other basidiomycete species share about 80% homology, and the ascomycete p-tubulins about 70% homology. p-tubulins are conserved proteins in eucaryotes, and not much variation would be expected within a species. The detected variation, about 5%, within P. rivulosus p-tubulin gene at the nucleotide level, can be regarded as the normal variation between species isolates. Therefore it is assumed that the strain ATCC64310, possessing the homology (and identity) below 80%, does not represent Physisporinus rivulosus. Thus, preferably Physisporinus rivulosus strain of the invention, i. e. a strain having essentially same biopulping properties as strain T241i, is a fungal strain having p-tubulin gene sequence homology of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (i. e. 80%- 100%) with P. rivulosus strain T241i. More preferably, Physisporinus rivulosus of the invention is a fungal strain having (3-tubulin gene sequence homology of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (i. e. 90%-100%) with P. rivulosus strain T241i. Most preferably, Physisporinus rivulosus of the invention is a fungal strain having P- tubulin gene sequence homology of 95%, 96%, 97%, 98%, 99%, or 100% (i. e. 95%-100%) with P. rivulosus strain T241i. Table 11. Sequence homologies of the putative p-tubulin gene fragment fromP. rivulosus strains and four other fungi at the nucleotide level. CBS CBS ATCC S. C. G. A. 433. 48 434. 48 l 64310 coml cine2 fuiiM nidu4 CBS 433. 48 95. 8 98. 5 77. 4 80. 0 81. 1 69. 1 68. 3 CBS 434. 48 95. 5 78. 4 80 81. 1 63. 8 72. 2 T241i 77. 6 79. 7 80. 6 69. 9 69. 6 ATCC64310 79. 7 80. 6 69. 9 69. 6 S. commlme ND ND ND C. cinereus ND ND G. u'ikuroi-ND

P-tubulin gene (tub-2) from Schizophyllum commune, gene bank accession number X63372, 2, B-1 tubulin gene from Coprinus cinereus, AB000116, 3ß-tubulin gene (tub2) from Gibberellafujikuroi, U27303, 4ß-tubulin gene (benS) fromAspergillus nidulans, M17519.

DEPOSITED MICROORGANISM The following microorganism was deposited according to the Budapest treaty in depository authority DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1 b, D-38124 Braunschweig, Germany): Microorganism Accession number Date of deposition Physisporinus rivulosus T241i DSM 14618 14 November, 2001

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