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Title:
METHOD FOR IMPROVING THE EFFICIENCY OF CHEMICAL PULPING PROCESSES BY PRETREATING WOOD OR PULPWOOD WITH WHITE ROT FUNGI
Document Type and Number:
WIPO Patent Application WO/1997/013025
Kind Code:
A1
Abstract:
The present invention relates to the use of certain Basidiomycette fungi, in particular, white rot fungi, such as Phlebia tremellosa, Trichaptum biforme, Schizophyllum commune, and Phanerochaete gigantea, in preconditioning wood or pulpwood to yield a more uniform and more efficient process resulting in a higher quality product. Such treatment has been found also to increase the porosity of wood substrates, including particularly non-sterilized wood substrates. Such increases in porosity are accompanied by improved liquor penetration into the cells of the more porous wood or pulpwood in a subsequent chemical treatment process. Despite the fact that select white-rot fungi may deeply penetrate and leave voids where pitch and/or resin has been removed, or the cell wall has been modified, it has been found that such voids may have substantially no effect on the lignin content in the pulp or pulpwood. Nevertheless, the resulting fungally-treated pulp, when thereafter subjected to chemical treatment, demonstrates increased brightness, increased yield and concomitant reduction in Kappa number, without significant decreases in viscosity. The invention also relates to a method for reducing the electrical energy consumption during the mechanical refining of wood or timber into pulp comprising inoculating at least one end of the wood or timber with a pitch reducing effective amount of a least one fungus selected from the group mentioned above and allowing the fungus to grow on and into the wood or timber for a time sufficient to reduce the pitch in the wood or timber, and then subjecting the thus treated wood or timber to mechanical refining.

Inventors:
BEHRENDT CHAD J (US)
BLANCHETTE ROBERT A (US)
FARRELL ROBERTA L (US)
IVERSON SARA (US)
Application Number:
PCT/EP1996/004239
Publication Date:
April 10, 1997
Filing Date:
September 27, 1996
Export Citation:
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Assignee:
CLARIANT FINANCE BVI LTD
SANDOZ AG (AT)
SANDOZ AG (AT)
BEHRENDT CHAD J (US)
BLANCHETTE ROBERT A (US)
FARRELL ROBERTA L (US)
IVERSON SARA (US)
International Classes:
D21B1/02; D21C1/00; C12S3/08; D21C5/00; D21C9/08; (IPC1-7): D21C1/00; D21B1/02
Domestic Patent References:
WO1994021854A11994-09-29
Foreign References:
AT398589B1994-12-27
US5055159A1991-10-08
US3962033A1976-06-08
US5472874A1995-12-05
US3486969A1969-12-30
EP0387187A21990-09-12
EP0470929A21992-02-12
FR2692590A11993-12-24
Other References:
L. JURASEK ET AL: "FUNGAL TREATMENT OF MECHANICAL PULPS - ITS EFFECT ON PAPER PROPERTIES", BIOTECHNOLOGY AND BIOENGINEERING, vol. 24, no. 9, September 1982 (1982-09-01), NEW YORK, pages 2063 - 2076, XP002022076
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Claims:
CLAIMS
1. A process for pulping wood or pulpwood comprising applying to the wood or pulpwood an inoculum of a Basidiomycete fungus, the inoculum being in an amount sufficient upon fungal growth therefrom to precondition the wood or pulpwood to be more efficiently cooked by a subsequent chemical treatment in the presence of heat, and maintaining the wood or pulpwood to which the inoculum has been applied under conditions which allow fungal growth from the inoculum for a time sufficient to effect the preconditioning of the wood or pulpwood by the fungal growth from the inoculum, thereafter subjecting the wood or pulpwood to a chemical treatment in the presence of heat for a time sufficient to enable chemicals of the chemical treatment to penetrate the preconditioned wood or pulpwood to yield a more efficiently cooked wood or pulpwood than nonpreconditioned wood or pulpwood.
2. A process as claimed in claim 1 , wherein the pulpwood is refined pulpwood.
3. A process as claimed in claim 2, wherein the refined pulpwood is wood chips and the inoculum is applied by spraying, the wood chips being accumulated into a mass directly after spraying.
4. A process as claimed in any one of the preceding claims, wherein the inoculum is obtained from a biologically pure fungal culture.
5. A process as claimed in any one of the preceding claims, wherein the fungus is selected from the group consisting of Phlebia tremellosa. Phanerochaete gigantea. Schizophvllum commune and Trichaptum biforme.
6. A process as claimed in claim 2, wherein a debarked or undebarked timber or log is treated to precondition wood chips made therefrom.
7. A process as claimed in any one of the preceding claims, wherein the wood or pulpwood to which the inoculum has been applied is maintained under fungal growth conditions for a period of from 4 to 20 days from inoculation.
8. A process as claimed in any one of claims 4 to 7, wherein the pulpwood is in the form of softwood wood chips or hardwood wood chips, preferably aspen wood chips.
9. A process as claimed in any one of the preceding claims, wherein the inoculum is applied in an amount of from 0.5 to lOg of mycelium per lOOg of wood or pulpwood.
10. A process as claimed in any one of claims 1 to 8, wherein the inoculum is applied in an amount of from 105 to 1010 CFU per Kg of wood or pulpwood.
11. A process as claimed in any one of the preceding claims, wherein the conditions which allow fungal growth comprise a temperature of at least 0°C.
12. A process as claimed in claim 1 1 , wherein the temperature is in the range of from 10 to 50°C.
13. A process as claimed in any one of the preceding claims, wherein the chemical treatment is selected from the group consisting of: sulfate; sulfite; bisulfite; neutral sulfite; and an alkaline solution and sulfate.
14. A process as claimed in claim 13, wherein the alkaline solution comprises sodium hydroxide.
15. A process as claimed in any one of the preceding claims, wherein the chemical treatment lasts for from 2 to 4 hours and the heat is from 160 to 180°C.
16. A process for pulping wood or pulpwood comprising applying to the wood or pulpwood an inoculum of a Basidio ycete fungus, the inoculum being in an amount sufficient upon fungal growth therefrom to produce at least one of the following effects following chemical treatment of the wood or pulpwood in the presence of heat: a) a reduction in the total amount of chlorine used; b) a reduction in the total chemical cook time; or c) a reduction in unpulped wood chip rejects.
17. A process for pulping wood or pulpwood comprising applying to the wood or pulpwood an inoculum of a Basidiomycete fungus, the inoculum being in an amount sufficient upon fungal growth therefrom to produce at least one of the following effects: a) a reduction in the total expenditure of electrical and/or mechanical energy used; or b) an increase in the porosity of the wood substrate.
18. A process as claimed in any one of the preceding claims, wherein the inoculum is admixed with one or more growth sustaining adjuvants.
19. A process as claimed in claim 18, wherein at least one of the growth sustaining adjuvant comprises an antitranspirant and/or a nutrient.
20. A method for reducing the electrical energy consumption during the mechanical refining of wood or timber into pulp comprising inoculating at least one end of the wood or timber with a pitch reducing effective amount of at least one fungus selected from the group consisting of Schizophyllum commune. Trichaptum biforme. Phanerochaete gigantea and Phlebia tremellosa. and allowing the fungus to grow on and into the wood or timber for a time sufficient to reduce the pitch in the wood or timber, and then subjecting the thus treated wood or timber to mechanical refining.
21. Use of a Basidiomycete fungus in a process as claimed in any one of the preceding claims.
22. Use as claimed in claim 21, wherein the fungus is at least one of the fungi selected from the group consisting of Schizophyllum commune. Trichaptum biforme. Phanerochaete gigantea and Phlebia tremellosa.
23. Cooked wood or pulpwood which has been prepared by a process as claimed in any one of claims 1 to 19.
Description:
METHOD FOR IMPROVING THE EFFICIENCY OF CHEMICAL PULPING PROCESSES BY PRETREATTNG WOOD OR PULPWOOD WITH WHITE ROT FUNGI

The present invention relates to the use of certain fungi in the treatment of cellulosic materials used in the manufacture of pulp and paper products. More specifically, the present invention relates to the use of those fungi, in particular of the class Basidiomycetes, for pre -treat ent of wood, including wood chips, in combination with a chemical cooking process, to improve the efficiency of that process and/or the quality of pulp produced.

Wood is a complex material composed of cellulose, hemi-cellulose, lignin and wood extractives or a resinous material commonly called "pitch", "resin" or "wood resin". The composition of pitch has been studied and is reported widely in the literature, e.g., Wood Extractives and Their Significance to the Pulp and Paper Industry. Chapter 10 "Wood Resins" by D.B. Mutton; W.E. Hillis, Ed, Academic Press, N.Y. (1962).

In the production of products from wood pulps, the presence of pitch is undesirable as due to its viscosity and tenacity it frequently forms deposits which are difficult to remove, causing relatively frequent and lengthy periods of down-time for cleaning, as resins tend to accumulate as deposits on strainer plates, filters, and throughout paper processing apparatus. It is well-known that pitch may also discolor pulp and paper formed therefrom if allowed to accumulate too long before cleaning. Other drawbacks resulting from pitch accumulation are known in the art, e.g. waste stream pollution.

In Nilsson, et al., US Patent 3,486,969, it is disclosed that certain fungi may be used to inoculate wood chips to reduce the resin content therein and the pulp therefrom while minimizing degradation of the other components of the wood, especially cellulose and hemicellulose. The species of fungi therein disclosed however, are apparently all mold type or surface forming fungi which, when infecting the wood, produce essentially a surface or superficial stain which may be readily planed off (see J.S. Boyce, Forest Pathology. 3rd. Ed., 1961, McGraw-Hill Book Co. at pp. 493-512, especially 496-497). Such fungi have failed to achieve practical success to our knowledge.

In published European patent application EP 03 87 187 A2 there are described the application of certain wood-penetrating fungi, generally classed as Ascomycetes or

Deuteromycetes, to pulpwoods and pulps to reduce the pitch content thereof. Similarly useful wood-penetrating fungal derivatives are also disclosed in published European patent application EP 04 70 929 A2.

In published French patent application no. 2 692 590 there are described other strain derivatives of a preferred wood-penetrating fungi, Ophiostoma piliferum. which exhibit very good pitch degrading and aggressive growth characteristics while growing white or colorless on treated substrates.

A succession of preferred and improved wood-penetrating strains of O. piliferum as above-described have demonstrated commercial capability and have achieved commercial success. In addition to substantial savings from pitch reduction, early indications of greater paper strength (translating into faster machine speeds) have been confirmed, and there are further indications of greater pulping efficiency, probably due to the ability of the fungus to substantially open up resin ducts and ray parenchyma cells. The ability of such fungi to be useful practically is in part attributed to the ability of the fungi to grow competitively on non-sterile substrates and not be excluded or dominated by other fungi or organisms which naturally infect wood sources. In retrospect, one can at least theorize why the indicated wood-penetrating fungi are able to be useful and provide the indicated advantages. For example, the indicated wood-penetrating fungi are known to be early colonizers of dead wood and hence early contributors to the process of wood decay. One might therefore imagine that a major natural purpose of such fungi is the substantial removal or reduction of resin in the wood, a process which would also open up the resin ducts and parenchyma cells to the invasion of the later colonizing rotting fungi, such as the white rots and brown rots which are, for example, commonly found in the fungal classification Basidiomycetes (Basidiomycotina). The ability of the indicated wood-penetrating fungi to dominate other fungi when substantial resin is present perhaps ensures that their pitch-degrading purpose is served and would be consistent with the theory that their primary natural purpose may be pitch degradation.

The Basidiomycetes, including particularly the white rot fungi which degrade pitch in wood without adversely affecting the quality of wood as structural wood. White rot fungi which degrade pitch and grow very well on non-sterile wood include, for example, Schizophyllum commune. Trichaptum biforme. Phanerochaete gigantea and Phlebia

tremellosa as is diclosed in US Patent Nos. 5,472,874 and 5,476,790.

Basidiomycetes remove lignin from wood in several morphologically distinct patterns. One type of decay known as "selective delignification" is apparent when greater amounts of lignin are degraded relative to the amount of cellulose. In this type of decay, lignin in the secondary wall and middle lamella may be almost entirely removed, whereas large quantities of cellulose in the S2 layer of the cell wall are left intact. White rot fungi can also cause a "simultaneous rot". This type of decay is characterized by the removal of both cellulose and lignin, leaving cells either riddled with bore holes and erosion troughs, or with extensively thinned secondary walls. Much variation exists among these decay types.

Some Basidiomycetes cause only a simultaneous rot whereas others may produce a simultaneous rot in one part of the substrate and predominantly a delignification in another. In such cases, a chemical analysis of the entire substrate can misrepresent the potential of these fungi to cause selective lignin removal. Differences in the amount of lignin, cellulose and hemicellulose degraded from wood and the sequence in which these cell wall components are attacked have been reported. Some white-rot fungi have the ability to selectively remove extensive amounts of lignin with only slight losses of cellulose and moderate to low losses of hemicellulose. Some other white rot fungi have been shown to be initially very selective for lignin and then later attack the remaining cellulose. Thus, the selectivity of some fungi for lignin can change depending on the stage of decay at which chemical analyses are done.

In the general field of research of the potential use of fungi and fungal enzymes in paper making, the Basidiomycetes, particularly white rot fungi, have been of interest for their ability to degrade lignin and produce lignin degrading enzymes. The original concept of using such fungi, referred to as "biopulping", was founded on the idea of an early treatment of pulpwood, e.g., in the form of wood chips, to begin the process of pulping or lignin removal prior to entry into the pulp mill itself.

A white rot fungus judged particularly suitable for such purpose is Ceriporiopsis subvermispora. as described in U.S. Letters Patent No. 5,055,159. While the cause or

mechanism of action of such fungus in obtaining its desirable effects are indicated in the patent to be related to selective lignin degradation, we have noted that some reported benefits are also suggestive of those obtained by our above-indicated pitch degrading fungi. Consistent with our general understanding concerning Basidiomycetes, the fungus Ceriporiopsis subvermispora does not grow well on non-sterile substrates and the subject patent discloses the sterilization of the substrates prior to inoculation with the fungus.

Several reports have been made of attempts to create biopulping systems using white-rot fungi on a variety of wood fibers. Previous research has concentrated on a single, or relatively few, species of fungi. The most commonly utilized fungus in such prior systems is the white-rot fungus, Phanerochaete chrysosporium. The prior art is generally cognizant of the fact that attempts have been made to use biological organisms, such as white-rot fungi, as part of a process of treating wood, in combination with a step of either mechanical or thermal mechanical pulping of cellulose fiber.

There are many white-rot fungi that are not selective for lignin degradation and large amounts of polysaccharides also are removed. Coriolus versicolor is an example of a white-rot fungus that causes a simultaneous degradation of all cell wall components. It is a species that has been repeatedly used in assays as a representative of all white-rot fungi. However, there is a great deal of variation among the white rots. In addition to those that are either selective or nonselective for lignin degradation, it is possible to find fungi thai cause both types of white-rot attack within one substrate. This results in a mottled pattern of delignified wood and simultaneous white-rotted wood. Other types of white-rot attack, e.g., on cellulose or hemicellulose, also have been reported.

The degradation of lignin by white-rot fungi, especially those that selectively degrade lignin from wood, is a characteristic that makes them ideally suited for industrial applications where lignin or various phenolic compounds must be altered or removed such as in the making of paper. The potential use of white-rot fungi for biopulping, biobleaching and treatment of pulp mill waste effluents has also been suggested, but practically effective results remain elusive.

In fact, while a certain amount is known about the interaction of lignin and cellulose in wood fibers, because of the extreme complexity of the relationships, and the variation in the enzymes produced by varieties of the white-rot fungi, it is not readily possible to

predict from the action of a given fungus on wood whether or not the pulp or paper made from wood partially digested with such fungi will have desirable qualities or not. The selection of white-rot fungi for biopulping applications on the basis of selective lignin degradation may seem a rational one, but it has proven to be a poor predictor of the quality of the resultant paper. The exact relationship between the degradation of lignin, and the resulting desirable qualities of pulp or paper produced at the end of the pulping process, are not at all clear. Accordingly, given present standards of technology and the present understanding of the complex interaction of lignin and cellulose, it is only possible to determine empirically the quality of paper produced through a given biological pulping process and the amount of any energy or chemical savings achieved through such a process.

An objective of the present invention is to expand the field of fungi useful for treating pulps and pulpwoods, and particularly in non-sterile wood substrates.

Another object of the present invention is to provide a fungal pretreatment to a chemical pulping process whereby the wood chips may be more easily and uniformly pulped and are productive of a higher quality paper product.

In accord with the present invention, it has been found that certain Basidiomycete fungi, in particular, white rot fungi, such as Phlebia tremellosa. Trichaptum biforme. Schizophyllum commune, and Phanerochaete gigantea. are desirably effective in preconditioning the thus treated wood or pulpwood to yield a more uniform and more efficient process resulting in a higher quality product. Such treatment has been found also to increase the porosity of wood substrates, including particularly non-sterilized wood substrates. Such increases in porosity are accompanied by improved liquor penetration into the cells of the more porous wood or pulpwood in a subsequent chemical treatment process. Despite the fact that select white-rot fungi may deeply penetrate and leave voids where pitch and/or resin has been removed, or the cell wall has been modified, it has been found that such voids may have substantially no effect on the lignin content in the pulp or pulpwood. Nevertheless, the resulting fungally-treated pulp, when thereafter subjected to chemical treatment, demonstrates increased brightness, increased yield and concomitant

reduction in Kappa number, without significant decreases in viscosity.

The potential utility of white rot fungi in the pulping process has generated much interest. In fact, some limited success has been observed on sterilized (e.g., autoclaved) wood in the laboratory [see, e.g., AT-B-397 589; U.S. Pat. No. 5,055,159; Messner et al., "Biopulping: An overview of developments in an environmentally safe paper-making technology", FEMS MICRO. REV, 13: 351-364 (1994)] but efficacy on non-sterile wood has not heretofore been demonstrated, nor is it predictable from such laboratory results. Virtually all lignin-degrading fungi, given the chance to colonize a pure nutrient source, whether agar, sterilized wood chips or other sterile substrate, would be expected to grow on wood, and to some degree continue to grow successfully when later challenged by competing fungi.

In order for a fungus to compete successfully with other microorganisms on unsterilized wood as found in a paper mill setting, however, it must possess certain characteristics of growth virulence necessary to inhibit the growth of competing microorganisms already present on the substrate. Otherwise, the fungi will be of little or no practical utility for rendering the wood more easily pulped.

Accordingly, the invention provides a method of softening the wood or pulpwood such that it is preconditioned to produce a better pulp following a subsequent chemical cook than pulp which has not been biologically preconditioned. Such fungal pretreatment is believed to modify the cell wall and/or increase the porosity of wood, particularly pulpwoods and pulps, rendering the wood or pulpwood more easily penetrated by cook chemicals, and thus more easily and uniformly pulped, said method comprising applying to pulpwood or pulp an inoculum of a Basidiomycetes fungus, in particular, a white rot fungus, and thereafter maintaining the inoculated pulpwood or pulp under conditions allowing growth of the fungus for a time sufficient to precondition the cellulosic substrate (e.g., wood or pulpwood) to produce a better pulp, e.g., by modifying the cell wall and/or degrading the pit membranes and/or increasing the porosity of the pulpwood or pulp for penetration by cook chemicals , so as to soften the wood to make it easier to pulp chemically. Such "preconditioning effects" are clearly visible by SEM inspection. The present invention is also directed toward the biological pretreatment of

unsterilized wood or wood chips for pulp making for paper manufacture. It has been found here that through the use of particular species of white rot fungi, such as Phlebia tremellosa. Phanerochaete gigantea. Schizophvllum commune, or Trichaptum biforme. and the maintenance of relatively forgiving conditions during the treatment of wood or wood chips by said fungus, it is possible to utilize a biological treatment or pretreatment as a part of a chemical pulping process. It has further been found that the process results in a paper which has a strength which is increased over paper made by purely chemical pulping but which, at the same time, results in a dramatic savings in the energy expended during the mechanical and/or chemical pulping process. Moreover, chemical pulping processes are more efficient as evidenced by chemical savings, shortened cook times, and/or improved yields.

The fungi used in the process of the invention may, therefore, be selective for lignin or not. Rather, the important biologically induced effect, referred to herein as preconditioning, renders the wood more easily pulped due to one or more of a cell wall modification and/or porosity increase and/or a pit membrane degradation, which allows the chemical pulping to proceed more effectively leading to the unexpected chemical pulping result. Such fungi may be generally applied to accelerate wood "seasoning" resulting in a shorter, more efficient cook time and pulping process, attributable to a reduction in the pitch content and/or modification of the cell wall and/or degradation of the pit membrane of pulpwoods and pulps used in the manufacture of improved pulp and paper products.

The use of such white rot fungi in a biopulping process not only results in fewer wood chips being rejected by mills as being inadequately or unevenly cooked, but also results in quicker reductions in Kappa number due to more efficient solubilization of lignin during chemical treatment. Surprisingly, such a process is effective even when the white rot fungal pretreatment SSL ___ produces no significant decrease in lignin content. Such a discovery expands the scope of fungi useful in such a process to those non-lignin degrading fungi, e.g., white rot fungi, which may, nevertheless, precondition the cellulosic substrate to produce an improved product or process from a subsequent chemical cook. Such biopulping processes also reduce the need for environmentally harmful chlorine- containing compositions.

Such chemical "biopulping" processes, in particular kraft (sulfate) or sulfite processes, wherein wood or pulpwood are chemically treated in the presence of heat with, e.g. sulfate, acid sulfite, bisulfite and/or neutralsulfite treatment at temperatures ranging from 100°c to 200°C, appear to yield many additional benefits, including reduced chlorine or chlorine-containing chemical usage, shorter cooking time, a reduction in H-factor, a quicker reduction in Kappa number (delignification) and a greater chemical penetration into the wood resulting from increased porosity. In a particularly preferred embodiment of the invention the preconditioned wood or pulpwood is subjected to a chemical treatment comprising a sulfate and an alkaline solution at a temperature in the range of from 160 to 180°C. Accordingly, subsequent chemical penetration into the wood or pulpwood is facilitated, as is diffusion of ions and solubilized lignin product out of the wood or pulpwood, resulting in a more efficient pulping process.

In addition, application to a wood substrate of the white rot fungi of the invention, appears generally to "soften" the wood and yield significant savings of electrical and/or mechanical energy normally expended by the paper industry in the pulping process. A reliable model for evaluating such energy savings is the Simons' stain. The Simons' staining procedure has been presented and discussed by Blanchette et al. (Using Simons' Stain to Evaluate Fiber Characteristics of Biomechanical Pulps, TAPPI Journal 75: 121 -124, 1992) and Yu et al. 1995 (Mechanism of Action of Simons' Stain TAPPI Journal 78: 175-180). The intensity of the color change to ends of refined fibers can reliably be used to predict energy savings. The orange-yellow coloration is an indicator that significant electrical energy savings would occur during the mechanical refining of the wood into pulp (Akhtar, M., R.A. Blanchette, and T.A. Burnes, 1995; Using Simons' Stain to Predict Energy Savings During Biomechanical Pulping, Wood and Fiber Science 27: 258-264).

By the terms "resin" or "pitch" (which are used interchangeably) is meant that complex mixture of hydrophobic substances in wood, also known as extractives, which is soluble in neutral organic solvents, such as methylene chloride, diethyl ether, benzyl alcohol and the like. These extractives include the terpenes, the diterpene ("resin") acids, fatty acids and esters, such as steryl esters, glycerides and waxes as well as alcohols, hydrocarbons and other compounds associated therewith. For purposes of this invention,

the standard Tappi extraction analysis using dichloromethane will suffice for measuring the reduction in undesirable wood resins which is the object of the invention. However, other recognized solvent systems such as ethanol toluene are essentially equally representative.

Resin or pitch is a significant constituent of both softwood, such as southern pine, conifers and cedars, and hardwoods, such as Betula and Populus, and including aspen, maple, birch and oak, and it may comprise as much as 4% weight percent or even more of the feed sent to mechanical or chemical pulping processes, generally 1.5 to 4.0% for most woods used for pulping. Softwoods generally contain more resin than hardwoods, with pines having among the highest resin content among softwoods. In hardwoods, resin is located primarily in the ray parenchyma cells which form much of the fiber fraction when wood is pulped. In softwoods, resin is contained in both the ray parenchyma cells and also in resin ducts.

The term "pulpwood" as used herein means any harvested (cut down) form of a tree material used in making paper, cardboard or other cellulosic products such as viscose, but prior to pulping, and includes such forms as timber, logs, wood chips, sawdust and the like. The term "refined pulpwood" means a pulpwood resulting from the application of mechanical and/or shearing forces to whole pulpwood forms such as logs to obtain a multiplicity of high surface area, small pieces, such as wood chips and sawdust, which are introducible into a pulping process. The invention may also be applied to lignin-containing cellulosic materials classifiable as pulps which have yet to undergo sufficient treatment to significantly reduce its lignin content (and liberate contained pitch), in particular pulp which still retains 60% or more of its original lignin content, such as first stage mechanical pulp.

The invention may therefore be utilized in one aspect thereof to at least partially reduce the pitch and/or resin component of unsterilized, refined pulpwood and incompletely refined pulps by applying to the pulpwood or pulp an inoculum of a white rot fungus, in particular, a white rot gungus selected from the group consisting of Phlebia tremellosa. Phanerochaete gigantea. Schizophyllum commune, and Trichaptum biforme. accumulating the inoculated pulpwood or pulp in a mass and maintaining the accumulated

mass under conditions which allow or promote fungal growth in the mass for a time sufficient to effect a reduction in the pitch and/or resin component of the pulpwood or pulp by the fungus. Moreover, application of an inoculum of such fungi in connection with the chemical and/or kraft pulping process of the invention renders the wood or pulpwood preconditioned for a subsequent chemical treatment. Whereas the precise nature of the preconditioning or seasoning is not known, it is believed to include rendering the cellulosic substrate more amenable to subsequent chemical penetration during cooking wherein the inoculum is applied in an amount effective to precondition, soften and/or season the wood or pulpwood by, inter alia, increasing the porosity and/or degrading the pit membrane and/or cell wall of the wood or pulpwood. The thus treated wood or pulpwood is more easily and uniformly cooked than non-preconditioned wood or pulpwood.

The fungus may be applied to unsterilized, unrefined pulpwoods such as cut timber in debarked or undebarked form by inoculating the timber, desirably at least partially scored in the case of undebarked timber, and maintaining the timber for a time sufficient to allow growth of the fungus on and into the wood substrate to yield a preconditioned wood substrate. Such preconditioning is believed to include cell wall modification and/or a reduction in the pitch and/or resin component thereof and/or to effect a degradation of the pit membrane and/or increase the porosity of the cellulosic substrate. Moreover, though the fungus j)£i ≤e need not necessarily remove the lignin component of the cellulosic substrate, it may modify it such that the lignin chromophore, or other lignin degradation products, are released and/or modified. Such effect would also account for the greater brightening effect.

By the term "inoculum" and the like as used herein is meant any fungal material which is sufficiently viable to result in growth of the fungus when applied to the substrate. Typical fungal inoculums include fungal cultures or preparations obtained from a fungal culture, desirably from a biologically pure culture. The basic structural unit of most fungi in the fungal filament or "hypha". In aggregate, these filaments comprise a fungal body called the "mycelium". Fungi typically reproduce asexually by means of spores called conidia which are given off by the mycelia, have resting structures called chlamydiospores

or may reproduce sexually by means of basidiospores. All such forms and fungal elements, e.g., mycelia and spores, may be suitably used as inoculum in the invention. An inoculum form may be provided by culturing the fungus in any of several conventional ways. Solid or liquid culturing media may be used as desired or required, preferably liquid media. Culturing of the fungus under conditions favoring spore formation is usually preferred when possible, and the generally preferred inoculum will contain a large number of spores resulting from the fungal culture. When spores are not produced, mycelial fragments may serve as the inoculum.

The inoculum may be in solid or liquid form. Whole liquid cultures or portions thereof may be used, e.g., mixtures of mycelia and spores. When a high content of spores is available in the culture, the product may be lyophilized (freeze-dried) or spray-dried to obtain a dry inoculum in which spores constitute the viable component to generate the fungus after inoculation. Inocula in the form of concentrates to be diluted with water for application are generally stored at temperatures which will preserve desired viability. Liquid forms are usually stored frozen, typically at temperatures of from -5°C to -80°C, more usually -10°C to -75°C. Dry forms are similarly stored although lyophilized forms containing spores as the operable inoculum are often more stable and may be stored at higher temperatures than counterpart liquid forms. Inoculum compositions may comprise other ingredients such as preservatives and stabilizing agents or inert carriers introduced in certain types of drying processes.

The fungal inoculum may be admixed with or applied concurrently with one or more growth sustaining adjuvants for various purposes. For example, an anti-transpirant (to inhibit desiccation) may be applied with the inoculum to ensure the suitable early growth conditions for the inoculum in cases of low humidity or high temperatures. Also, materials which act as stickers and/or nutrients may be used to ensure or sustain germination and provide a conducive environment for growth. Carboxymethyl-cellulose is preferred for these purposes, although a variety of materials may also be used.

The inoculum may be applied to the wood substrate in a variety of manners. Typically, the inoculum is applied in a systematic or methodical manner. For example, the inoculum is distributed at intervals in the mass of refined pulpwood, or on the outer

surface of a cut timber, preferably at regular intervals. More preferably, the inoculum is distributed in a homogeneous or uniform manner, i.e., substantially throughout the mass of refined pulpwood. A common example would include spraying the wood. However, it is not necessary that each individual wood chip, sawdust particle and the like be inoculated. As little as 10% or even less, but preferably about at least 20%, more preferably at least about 50%, of the individual pieces can be inoculated since the uninoculated pieces are accumulated in contact with the inoculated pieces. For example, inocula may be incorporated into a suitable medium, such as vegetable and/or mineral oil, which can be used in lubricating chain saws that cut the wood. Upon growth, the infection will spread very easily.

A thorough or uniform inoculation of a mass of wood chips is generally reflected by the fact that the fungus grows substantially throughout the mass. However, it may happen that some part of the mass, particularly the outer layer of a pile of refined wood or pulpwood, will show little growth compared to the rest of the mass, or no growth at all, although it has been inoculated.

In one preferred embodiment, the inoculum is sprayed onto wood chips or sawdust as they are discharged from the refining operation but before being accumulated into piles. For example, a wood chipping apparatus is generally provided with conveyor means which receive the newly prepared chips and convey them to the accumulating pile. A spray applicator containing the inoculum preparation may be conveniently adapted to the conveyor, preferably at the junction with the chipper when the chips are airborne, e.g., free falling or tumbling, or at the very end of the conveyor so that chips are sprayed just before falling from the conveyor. Alternatively, the inoculum may be applied to the wood chip pile in the course of its accumulation by more or less continuous spraying over the accumulating pile.

When treating pulps or refined pulpwood, the dosage applied may vary depending upon several factors such as the wood being treated, condition or age of the wood, growth conditions, desired treatment time and the like. In general, satisfactory results can be obtained upon application of an inoculum containing from 0.5 to 10 g of mycelia (wet weight of dewatered mycelia) per 100 g of pulp or pulpwood, preferably from 1 to 5 g of mycelia per 100 g of substrate to be treated. Such mycelia prior to dewatering may be

prepared as described in Example 1 or Example A, below, preferably Example A, and may contain spores. Dosage of an inoculation based predominantly or solely on spores may be routinely determined and can be indicated to range from 10 s to 10 10 CFU (colony forming units) per kg of substrate, more usually from 10 6 to 10 9 CFU per kg. Similarly, expressed dosages of mycelia may be determined and applied. For example, mycelia may be homogenized, e.g., 5-10 minutes, and the number of colonies formed from the fragments when grown on a nutrient medium may be approximated in a conventional manner to determine CFUs for a given volume.

The inoculum dosage will generally be applied in a water-diluted sprayable composition, for example, a composition to be applied in a volume of from 20 to 60 ml per kg of substrate. The fungus is preferably applied to freshly cut or refined pulpwood or freshly cut substrates frozen or stored at reduced temperatures until treatment, or the substrate may be sterilized. When applied to non-sterile pulpwood which has been allowed to age before treatment, e.g., wood chips which were produced about 5 days or more before treatment, it may be desirable to increase the inoculum dosage to the higher end of the dosage range in order to avoid, suppress or overcome the background growth effects of fungi which naturally infected the wood prior to inoculation.

The fungus may be applied to the log ends in any of a variety of forms and ways. The fungus may be applied in any inoculum form giving rise to growth of the fungus, for example, in the form of mycelia or spores. Such inoculum may also be in liquid or dry form. For example, aqueous suspensions of mycelia and/or spores may be used, or the mycelia and/or spores may be dried or lyophilized to produce dry forms. Liquid aqueous forms of dilute or medium concentrations are generally preferred. Hence, the inoculum of the fungus may be applied as a powder in dry form or sprayed or smeared by hand when in liquid form. The log ends will be completely covered with the inoculum such as by spraying the log ends to run off or smearing a medium concentrated liquid, e.g. of mycelia, over the entire log end.

In another embodiment, chips which have been previously inoculated and incubated according to the process of the invention may be dispersed into fresh chips to effect or enhance inoculation. Such an inoculum is likely to be not biologically pure. However, it reflects the previous inoculation as at least 20%, preferably at least 40%, more preferably

at least 50% of the inoculum is the desired fungus.

After inoculation, the accumulated mass is maintained under conditions which will allow or promote the growth of the fungus substantially throughout the mass. Given the fact that the invention will in most cases be likely to be practiced in open air and the mass therefore subjected to a wide variety of weather conditions, the maintenance of any given set of ideal conditions throughout the entire treatment period is usually too difficult to achieve and is often unnecessary in practice. It is generally sufficient that the mass be substantially maintained at a temperature at which the fungus grows while avoiding higher temperatures at which the fungus dies. While our fungi may exhibit some reasonable growth at or below 0°C, it will generally be more suitable to have a temperature of at least 5°C, preferably a temperature of from 10°C to 50°C, more preferably of from 15°C to 45°C, most preferably of from 20°C to 40°C.

In mild or warm weather conditions, it is not necessary to influence the environmental temperature and the inoculated mass may be left to stand in open air without special maintenance. In cold weather conditions, it may be desirable to provide the inoculated mass with means for maintaining the more suitable temperatures. This may be a heat-retaining covering placed over or on the inoculated mass such as a large plastic sheet. Alternatively, the ground base on which is placed the inoculated mass may be provided with heating and/or cooling pipes or a plurality of openings for releasing warm air or steam or coursing cool air or other fluid. In a similar manner, a concrete "igloo" or similar structure which can be internally heated and emit radiant heat may be used to support the accumulated mass of pulpwood. When providing heating means, it would also be desirable to control the moisture conditions to avoid an excessive dryness. In view of this, means for venting the heat or steam would be adequate. However, due to the heat generated in an accumulated mass from fungal growth and other microbial or natural effects, operation under many cold weather conditions may proceed satisfactorily with little or no assistance, other than may be venting the interior of the mass to release heat build up, if needed.

The period of time during which the inoculated refined pulpwood mass is treated may vary considerably depending upon a number of factors including the desired extent of resin and/or pitch removal, the desired preconditioning, the temperature and moisture

conditions, the extent of inoculation and the like. However, satisfactory results may generally be obtained after 2 days, preferably from 3 to 40 days, more preferably from 4 to 30 days. Under preferred conditions, very effective results, e.g., a pitch reduction of about 20% or more, may be obtained 4 to 20 days after the inoculation, more usually 5 to 15 days.

Treatment of unrefined pulpwood, such as cut timbers, will usually be somewhat longer than that of refined pulpwood and may extend up to 2 months. However, treatment of pulps and pulpwoods with the indicated fungus generally should be conducted for periods which effect desired pitch reduction and/or preconditioning while avoiding excessive periods which might result in any substantial attack on the cellulose component of the substrate(s). Dosages for unrefined pulpwood may be similar to those for refined pulpwood and applied over from 10% to 100% of available surfaces, more usually over 15% to 50% of the available surfaces.

The fungi used in carrying out the invention are previously known species and may be obtained in a known manner, e.g., from public culture collections or by isolation from wood or other sources on which they grow in nature. While some variation among strains can be expected depending on factors such as the source from which they may be isolated, our fungi demonstrated remarkable growth on both unsterilized Southern Yellow Pine, Red Pine, or loblolly pine, and also on hardwoods, such as maple, oak, aspen and birch, and can be expected to grow well on other wood types commonly used in making cellulosic products. Nevertheless, we believe that the observed preconditioning effect is common to each species, rather than strain-specific. Naturally occurring isolates of our fungus can be modified by various known means of strain selection, mating and mutation without losing their identifying species characteristics.

Our preferred natural isolates have been deposited with the Northern Regional Research Center (NRRL), as detailed below, but it will be apparent that the same can be modified and that preferred fungal strains will include not only such isolates but also all other isolates and modifications which substantially possess at least the pitch degrading and/or growth properties on sterilized Southern Yellow Pine that are possessed by any of the deposited strains. The fungi used in the invention will grow white or essentially

colorless on pulpwood and pulp. Since they may be used to largely or completely dominate other darker growing fungus which naturally infect unsterilized substrates, the fungi used in the process of the invention may be used to produce a superior product requiring less bleaching to obtain the final paper product.

DEPOSITS

We have, under the Budapest Treaty, deposited with the Northern Regional Research Center (NRRL) at Peoria, Illinois, U.S.A., a biologically pure specimen of ten isolates, which deposits were assigned the Accession Numbers given below along with their date of deposit. All specimens have been tested and found viable upon receipt by the depository, and all restrictions on the availability of the specimens to the public will be irrevocably removed upon the granting of a patent from this application.

The strains in the above Phlebia tremellosa deposits are identified below as isolate BRI-94 and isolate BRI-1 18.

The above deposits were obtained as natural isolates from fallen timber in the State of Minnesota, U.S.A., but other isolates can be obtained from a variety of other global locations. The fungus, Phlebia tremellosa. was isolated from hardwood. The classification of our fungus as Phlebia tremellosa is in accord with Ainsworth & Bisby's dictionary of

the Fungi, 7th Edition, 1983 D.L. Hawksworth, B.C. Sutton, & G.C. Ainsworth, Commonwealth Mycological Institute Kew, Surrey UK.

The S. commune and T. biforme were also isolated from a hardwood and the E. gigantea was isolated from a red pine. It is noted that Trichaptum biforme has in the past also been referred to as Polyporus pergamenus and Hirschioporus pargamenus. see Gilbertson et al., North American Polypores, Vol. 2, Fungiflore, Oslo, Norway 1987, pages 770-772 and Otjen et al., "Selective Delignification of Birch Wood (Betula papyrifera. by Hirschioporus pargamenus in the Field and Laboratory", Holzforschung 40 (1986), 183-189. Also, Phanerochaete gigantea has also been known in the past as Peniophora gigantea. see Burdsall, H.H., Jr., "A Contribution to the Taxonomy of the Genus Phanerochaete", Mycological Memoir, No. 10, J. Cramer Publishers, Braunschweig, Germany (1985).

The following examples are merely illustrative of the invention and its practice and are not intended to limit the same in any respect.

General Experimental Procedures: Cultures and Inoculation:

Various evaluations are made on pulpwood substrates to determine pitch reduction and growth. For evaluation of softwood characteristics, sterile and non-sterile Southern Yellow Pine wood chips were used. For evaluation of hardwood characteristics, non-sterile wood chips comprising aspen, oak, birch and maple were used. Wood chips are stored at 5°C prior to evaluation. Each evaluation was performed on substrates of the same wood species and upon wood chip samples which were obtained from the same wood chip source. For each test, individual sample lots of wood chips were first weighed, after which the wood chip samples to be sterilized were heated in an autoclave at 121°C for about 20 minutes and allowed to cool to room temperature prior to the initiation of a test. The wood chip samples which were to be in non-sterile form were untreated and used in their natural condition. Individual sample lots were prepared by placing measured amounts of wood chips into individual transparent plastic bags; the bags were of sufficient

size such that they were closeable (although not hermetically sealable). The use of a transparent bag allowed for the visual inspection of the growth of chips, and to further allow for admission of ambient light to the sample of wood chips being evaluated. A YNPD liquid culture medium was prepared using the following constituents (amounts are grams per liter of liquid culture medium produced):

which are added in sequential order to one liter of distilled water, and subsequently autoclaved at 121°C for about 20 minutes, and allowed to cool to room temperature. Afterwards, 1 mg of thiamine is added to the other constituents, after which the YNPD media was ready for use.

Using the YNPD culture media prepared as indicated above, each of the fungi was prepared under the following general conditions:

(a) samples of the particular fungus were used to inoculate sterile petri dishes which contained the YNPD culture media as prepared above, and the dishes were covered;

(b) the inoculated YNPD culture media was maintained at room temperature (approximately 20°C) until it was visually discernible that the inoculated fungus had grown well upon the YNPD culture media in the form of mycelial mats (about 5 days);

(c) after good growth had been observed, the mycelial mats were then removed in

hand (covered with a rubber glove) from the petri dish, the mat squeezed in hand until essentially no further water was emitted and the squeezed mat weighed to determine the "wet weight". The squeezed or dewatered mat was introduced into a clean laboratory beaker where it was then homogenized with the addition of between 5 - 10 ml of distilled water to form a pipetteable slurry which could then be removed from the beaker and used to inoculate a substrate; and

(d) the contents of the beaker were then introduced into a graduated cylinder to determine the volume of the pipetteable slurry, and once determined, the contents were returned to the laboratory beaker, from whence they were withdrawn for inoculation of samples.

The inoculation of a sample of wood chips was done by injecting the contents of the pipette containing 2-5 grams wet weight of the mycelial mat for each 100 grams of wood chips, after which the open end of the bag was folded over, and the contents of the bag shaken and tumbled so to maximize the number of chips that came into contact with the inoculant. The folded over end of the bag was stapled at two places. All inoculated wood chip samples were then placed on a laboratory benchtop at room temperature for the periods indicated in each specific test.

Each test was performed on two to five samples; reports of the growth of fungi reported herein are the average of these plural results.

Pitch Content Evaluations:

Evaluation of the pitch content of substrates was determined according to standard TAPPI Procedure T204 OM-88 which provides results expressible as milligrams (mg) of pitch content per gram (g) of substrate extracted with "DCM" which is methylene chloride (dichloromethane). In accordance with the TAPPI Procedure, as used on a substrate such as wood chips, the treated chips are dried overnight at 60°C and then ground into sawdust using a Thomas-Wiley Mill with 10-mesh screen (10 gauge wire screen). Three grams of the dried sawdust are combined with 30 ml of DCM and the resulting mixture is agitated overnight (about 15 hours) at room

temperature (approximately 20°C). The liquid medium is pipetted from the mixture, filtered through an organic filter having a pore size of 0.45μm, and then the liquid is allowed to evaporate at room temperature overnight in a tared (preweighed) dish. The dish residue is then heated in an air-circulation oven at 60°C for 30 minutes to further remove any residual DCM, after which the dish is allowed to cool to room temperature and reweighed; the weight of the remaining residue, viz., the remaining pitch, is determined and expressed in units of mg and correlated to the amount of the original sample being evaluated so to provide an expression of mg of pitch per g of substrate wood chip, or in the alternative as the percent DCM extractables present in the substrate wood chip sample, which result is equated to and taken as the percent of pitch in the substrate (% extractives).

Pitch evaluations may be conducted on both sterile and non-sterile substrates. Evaluations on sterilized substrates will usually eliminate any possible influence of other organisms which naturally infect the substrate. An evaluation on a sterilized substrate can be generally considered the more objective measure of the fungus to reduce pitch on a particular substrate. However, whether conducted on a sterilized or non-sterilized substrate, pitch reduction is generally evaluated relative to an untreated control which is sterilized (for sterilized or substrate tests) held in the frozen state during the test period (non-sterilized substrate evaluation). In general, it is desired to achieve a pitch reduction relative to such a control of at least 20% in no more than 21 days after inoculation, preferably in no more than 14 days. Particularly good results are indicated when pitch is reduced 25% in no more than 21 days, and especially when such reduction is achieved in no more than 14 days.

Growth Evaluations:

Evaluations of the growth of the fungus is made as uniformly as possible and in a manner as nearly identical as possible for all of the individual samples being evaluated for each of the several tests where the growth is to be determined. Evaluation is done using simple visual observation with a protocol applied on a consistent basis and carried out at each evaluation interval (where an intermediate evaluation is performed during a test) and at the end of each test. The protocol is based on color categories of possible fungal growth which can be observed or ascertained on each individual wood chip or substrate with the unaided eye at normal reading distance. When the substrate is sterilized, only one color category, that of the invention candidate, will be recognized and the protocol involves simple visual inspection of all wood chips to determine the number or percentage of chips which show visible growth of candidate fungus. When the growth evaluation is carried out on non-sterile substrates, different color categories will be usually recognized to distinguish between the invention or inoculated fungus and those which naturally infected the substrate. The inoculated candidate, typically the lightest color, will be identified and the number or percentage of wood chips visibly exhibiting such growth will be counted. Results reported below are given in terms of the percentage of the wood chips observed to exhibit growth of our desired fungus in each test case. Treated, non-sterile wood chips may show growth in other areas of the chips of other organisms, such as a black coloring fungus, and such background growth coloring may be separately recorded in a similar fashion. Such background growth should not be taken as negating otherwise positive growth results with the inoculated fungus, but the more desired fungal candidates are clearly those which best suppress or dominate over such background growth.

BIOPULPING GROWTH EVALUATIONS

The pitch content of substrates is determined in accord with the standard TAPPI Procedure T204 OM-88 and may be expressed as mg of pitch content per gram of substrates which had been extracted with DCM (a.k.a., methylene chloride).

The Kappa number of the substrates is determined in accord with the standard TAPPI procedure T 236 cm-85 to indicate the lignin content of the wood chips after cooking (partial cook by the kraft process). The kraft (chemical) process involves the heating of the wood chips with the cooking chemicals characterized by active alkali (AA) (NaOH + Na-S), and Sulphidity,

Na.S or Na.S

NaOH + Na-S active alkali (AA) .

By convention (Vroom, K.E., "The H Factor: The Means of Expressing Cooking Times and Temperatures as a Single Variable, "Pulp and Paper Magazine of Canada, £_ . (3):228-231 (1957)), a relative reaction rate of 1 has been assigned for 100°C to express cooking time and temperature as a single variable known as the H-factor. When the relative reaction rate is plotted against the cooking time in hours, the area under the curve is characterized as the H-factor.

As used on a substrate such as wood chips, the treated chips are dried overnight at 60°C and then ground into sawdust using a Thomas-Wiley Mill with a 10-mesh screen (10 gauge wire screen). Sawdust is extracted with DCM or other solvents as described in TAPPI Procedure T 204 OM-88. The weight of the residue is determined in mg as the pitch content and expressed either as mg of pitch content per g of substrate or as a percentage of pitch in the original substrate (% extractives). Pitch reduction is generally indicated when the inoculated fungus show a statistically significant reduction in pitch content compared with the control. Preferably, the pitch is reduced at least 10%, and more preferably at least 15%, compared to the control.

EXAMPLE 1

Removal of Pitch in Non-Sterile Softwood (Pine.:

The two different isolates of the fungus Phlebia tremellosa designated BRI-94 and BRI-1 18 were evaluated for their efficacy in the removal of pitch in non-sterile Southern Yellow Pine and other characteristics. Control samples were also evaluated to provide a comparative indication. Control samples included a non-inoculated control sample which was maintained frozen (-20°C) throughout the period of the test, and a water inoculated ambient control sample which was maintained at room temperature. The ambient temperature control was used as an indicator of the effect on pitch reduction of background organisms present on the non-sterile wood chip samples, and pitch removal of the fungal isolates was measured as a percent reduction below that of the ambient control. All evaluations were performed on 500 g samples of non-sterile Southern Yellow Pine wood chip samples after 14 days of growth after inoculation, with each test run in triplicate and the results averaged. The wood chips were of unknown age, but had at time of inoculation 20% blue stain and 2% Yellow stain background growth, again suggesting an aged wood source and substrates which are difficult challenges for pitch removal. For comparison, the tests also involved the fungal species Ophiostoma piliferum in the form of the product available under the registered trademark CARTAPIP ® 97 (Clariant Corp.) which normally performs very well on non-sterile Southern Yellow Pine.

Each of the samples were evaluated for the amount of DCM extractables in accordance with the protocol described TAPPI Procedure T204 OS-76. Analysis of the Klason lignin was performed upon selected wood chip samples to provide an indicator of the degradation of lignin in the sample chips; quantitative determination of five principal monosaccharides (glucan, mannan, arabinan, xylan and galactan) was performed on an absolute basis so to define the carbohydrate composition of the wood. The Klason lignin analysis was performed generally in accordance with the testing protocol of TAPPI T222 om-85. In summary, Klason lignin analysis according to the TAPPI T222 om-88 protocol is as follows; the samples of extractive-free wood is disintegrated in a blender or mill; the

carbohydrates are hydrolyzed and solubilized by sulfuric acid; the acid insoluble lignin is filtered off, dried and weighed.

Further, for selected wood chip samples an analysis of the carbohydrates was performed so as to evaluate the extent of cellulose and hemicellulose degradation. The carbohydrate analysis was performed generally in accordance with the testing protocol of TAPPI T249 cm 85, "Carbohydrate composition of extractive-free wood and good pulp by gas-liquid chromatography". In summary, samples are hydrolyzed with sulfuric acid using a two-step technique; a portion of the hydrolyzate is then neutralized and the sugars present in the sample reduced with sodium borohydrate to the alditols, which are then acetylated with acetic anhydride and pyridine, and the alditol acetates then dissolved in methylene chloride and then used for injection into the gas chromatograph. In this Example, the inoculum involved 15 g of mycelial mat (wet weight) representing a CFU count of 2.3 X 10 6 /g of homogenized mycelial mat in the case of BRI-94 and a CFU count of 3.5 X 10 6 /g of homogenized mat in the case of BRI-1 18.

Results of the samples being evaluated, %DCM extractives, pitch reduction and %Klason lignin are reported on Table 1, and the carbohydrate analysis of selected samples are reported on Table 2, both below.

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a) Dose of 5 X 108 CFU per 500 g of chips represents colony forming Units based solely on spore count for O. piliferum (product only contains spores).

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As may be seen from the Klason lignin test results, the fungi, Phlebia tremellosa. per se were found not to appreciatively effect the lignin content of the pine wood chip samples. A pronounced effect was observed, however, on kraft pulp generated from P. tremellosa-treatment on aspen wood chips, followed by a chemical pulping treatment (Example 4).

As may be seen from the results of Table 2, there was not an appreciable loss in the amount of carbohydrates in samples of pine wood chips which were treated with our fungus as compared to the ambient control sample. Hence, no reduction of cellulose and or hemicellulose was indicated as a result of the pitch reducing treatments.

In growth experiments conducted in connection with this Example 1, it was difficult to detect growth of the fungi even after 12 days with CARTAPIP ® 97 showing virtually no easily detectable growth, BRI-94 showing only 20% and BRI-1 18 only 10%. Various possible explanations for this phenomenon include aged condition of the chips, the tendency of these fungi to grow colorless and/or penetration and internal action by the fungi.

EXAMPLE 2

Removal of Pitch in Hardwoods (Aspen):

The fungal strains deposited in connection with this invention were evaluated for their efficacy in the removal of pitch in aspen and other characteristics. Control samples were also evaluated to provide a comparative indication. Control samples included a non-inoculated control sample which was maintained frozen (-20°C) throughout the period of the test, and a non-inoculated control sample which was maintained at room temperature. The ambient temperature control was used as an indicator of the effect on pitch reduction of background organisms present on the non-sterile wood chip samples. All evaluations were performed on 400 g samples of non-sterile aspen wood chip samples after 14 days of growth after inoculation, with each test run in triplicate and the results

averaged (the wood chips had been aged about 5 days prior to inoculation). For comparison, the tests also involved treatment with CARTAPIP ® 97 (Clariant Corp.). Each of the samples were evaluated for the amount of DCM extractables in accordance with the protocol described TAPPI Procedure T204 OS-76. Analysis of the Klason lignin was performed upon selected aspen wood chip samples to provide an indicator of the degradation of lignin in the sample chips, as before. Quantitative determination of five principal monosaccharides (glucan, mannan, arabinan, xylan and galactan) was performed, as before, on an absolute basis so to define the carbohydrate composition of the wood.

Results of the samples being evaluated, %DCM extractives and %Klason lignin are reported on Table 3, and the carbohydrate analysis of selected samples are reported on Table 4, both below.

a) CFU is colony forming Units based solely on spore count for O. piliferum (product only contains spores).

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As may be seen from the Klason lignin test results, the white rot fungal species of the invention were found not to appreciatively affect the lignin content of the wood chip samples. Hoewver, the fungal species of the invention caused a significant reduction in the pitch content of the samples, it being noted that CARTAPIP ® 97 is regarded as a potent degrader of pitch.

As may be seen from the results of Table 4, there was not an appreciable loss in the amount of carbohydrates in samples of aspen wood chips which were treated with our fungi as compared to the ambient control sample. Hence, no reduction of cellulose and/or hemicellulose was indicated as a result of the pitch reducing treatments.

EXAMPLE 3

Red pine trees, Pinus resinosa. approximately 25 to 40 years old, were felled at the Cloquet Forestry Center, Cloquet, MN. The trees were cut into logs approximately 20 cm long and 10 cm in diameter and were bagged and transported to the laboratory. Inoculation of random, unsterilized logs occurred two to three days after cutting.

Fungi used in the laboratory study consisted of cultures of Phanerochaete gigantea strain TE1. To inoculate the logs, cultures were grown at room temperature under normal lighting conditions in 2% malt extract broth for 14 days prior to inoculation in order to allow fungal mat formation. A dewatered fungal mat was used to inoculate each log end To determine the average weight of the mycelia inoculum, mats which were not used in inoculations were dried and weighed. Averaged dry mat weights were O. lOlg/mat +/- 0.009g.

Treatments included inoculation with Phanerochaete gigantea and non-inoculated control logs. A total of 20 logs were used per treatment. Additional logs were placed in a freezer to be used for non-inoculated controls and to determine the characteristics of the wood at time 0.

Log ends were inoculated by placing one fungal mat on each end of the red pine log. Fungal mats were evenly spread over the entire end of the log using a sterile glove pressed firmly enough to ensure adherence. Simultaneous inoculation of two fungi involved

mixing both mats by hand in a beaker, vortexing for 20 seconds, and placing them on the log end.

After inoculation, the fungus was allowed to grow on the logs stored at room temperature under normal lighting conditions in clear plastic bags, filled with air and tied closed with one moist paper towel. The bags were opened at 20 days after inoculation to allow air exchange and remove excess liquid, refilled with air, and tied closed. Sampling and analysis of logs was carried out 16, 32 and 64 days after inoculation.

Analyses were performed to determine pitch content (by extractives) and for Simons' staining.

Wood used for analyses was debarked and the center column of heartwood was removed. The sapwood was chipped into approximately 1 inch by 1 inch chips, and air dried. For pitch analyses the wood chips were ground to pass a 40 mesh screen and extracted with dichloromethane (DCM) using the TAPPI standard Procedure T204 OM-88. The results are presented in Table 5.

Additional chips used for the Simons' stain assay were refined using a mechanical pulp refiner with a setting of 0.04 inches. Coarse fibers obtained after one pass through the refiner were collected and stained with the Simons' stain reagent (see TAPPI Journal 75:121-124 for procedures). The results are presented in Table 6.

Energy savings (according to Wood and Fiber Science, Vol. 27) is:

Aspen wood logs are hand barked, chipped, screened and then homogenized. Three plastic bags are each filled with 475 g of the wood chips (52-53% solids), which are inoculated with 10 ml of CARTAPIP ® 97 (5 X 10 6 cells/ml), 10 ml Phlebia tremellosa strain BRI-1 18 (1 X 10 6 cells/ml) or 10 ml water, respectively.

Each treatment sample is observed in duplicate at 1, 2, 3 and 6 weeks after treatment and is thereafter subjected to conventional chemical pulping 1 during which kraft pulping of treated chips are determined.

liquor: water/ratio is set at about 4:1 (not including treatment) ; Liquor strength is about 16% active alkali and about 25% sulphidity; chips are cooked up to 150-400°C over 83-190 minutes; H-Factor is 800-1400. In this Example, chips are cooked up to 170°C, for 83-90 minutes, at an H-factor of 1400.

The comparative data on pulp yield, Kappa number, pulp brightness and viscosity is set forth in Tables 7-10, respectively below.

'Liquor: wood ratio is set at about 4: 1; Liquor strength is about 16% active alkali and about 25% sulphidity; chips are cooked up to 150 - 400°C over 83 - 190 minutes; H- Factor is 800 - 1400. In this Example, chips are cooked up to 100-250°C for 83 - 240 minutes, at an H-factor of 1400.

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One measurement only

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Values given above are the average of two measurements

The results indicate that pretreatment with P. tremellosa followed by conventional chemical treatment results in a marked decrease in Kappa number, and also produces a pulp of high yield and brightness without concomitant loss in viscosity. In addition, the observed physical properties are consistent with a paper of improved strength, as set forth below in Tables 11-13.

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EXAMPLE 5

Southern Yellow Pine wood logs are barked and chipped. Four plastic bags are each filled with 300 g. (o.d.) of the wood chips, which are inoculated with 10 ml of Phanerochaete gigantea strain TE1 (1 X 10 6 cells/ml), 10 ml Phlebia tremellosa strain BRI-118 (1 X 10 6 cells/ml) or 10 ml water to each of a frozen and aged control, respectively.

Each treatment lasts for fourteen (14) days after which each sample is air dried, screened, homogenized and subjected to conventional chemical pulping 2 during which kraft pulping of treated chips are determined in duplicate (Treatments A and B).

The comparative data on %DCM Extractives reduction, pulp yield (screened and unscreened), Kappa number and % rejects is set forth in Tables 14-18, respectively below.

2 Liquor:wood ratio is set at about 4: 1 ; Liquor strength is set at about 1 1, 13, 15 or 17% active alkali and about 25% sulphidity in the respective Examples; chips are cooked for 98 - 135 minutes; H-Factor is in the 780 - 830 range.

2 Liquor: water/ratio is set at about 4:1 (not including treatment); Liquor strength is set at about 11, 13, 15 or 17% active alkali and about 25% sulphidity in the respective Examples; chips are cooked up to 170 C C in 98-135 minutes; H- Factor is in the 780-830 range.

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EXAMPLE 6

Pine wood logs (2 and 8 feet in length) are inoculated with 10 ml of CARTAPIP ® 97 (5 X 10 6 cells/ml), 10 ml Phanerochaete gigantea TEl (1 X 10 6 cells/ml) or 10 ml water to each of a frozen and aged control, respectively, over twenty weeks, and are thereafter hand barked, chipped, screened and then homogenized. Each treatment sample (300 g chips, o.d.) is subjected to conventional chemical pulping, (Liquor: wood ratio is set at about 4: 1 ; Liquor strength is set at about 14 or 16% active alkali and about 25% sulphidity; chips are cooked for 101 - 180 minutes; H-Factor is about 800), during which chips made from treated logs are evaluated in duplicate at 10 and 20 weeks after treatment.

The comparative data on % extractive reduction, pulp yield % (screened and unscreened), Kappa number, and % rejects is set forth in Tables 21-24, respectively below. DCM Extractives at 10 and 20 weeks, and Simons' stain results at twenty weeks after treatment are presented in Tables 19-20.

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large chunk could not be refined

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EXAMPLE 7

Red pine wood logs (8 feet in length) are inoculated with 10 ml Phanerochaete gigantea TEl (1 X 10 6 cells/ml) or 10 ml water, respectively. The logs are then hand barked, chipped, screened and then homogenized.

The properties of chips made from treated logs are determined in duplicate at 16, 32, and 64 days after treatment. Thereafter, the 64 day treated sample (300 g chips, o.d.) is subjected to conventional chemical pulping (Liquor: wood ratio is set at about 4: 1 , Liquor strength is set at about 14 and 16% active alkali and about 25% sulphidity; chips are cooked for 90 - 180 minutes; H-Factor is about 800.).

The comparative data on % Extractive reduction and Simons' stain is set forth in Tables 25-26, respectively below.

EXAMPLE 8

The cooking conditions of the previous example (Example 7) were modified in three subsequent chemical cooks of wood chips (300 g (o.d.)) comparing P. gigantea to aged control. The first cook (A) was set at 16% active alkali and lasted 190 minutes (H- factor=830). The second cook (B) was set at 14% active alkali, preheated to 385°C, and lasted 128 minutes (H-factor=856). The third cook (C) was set at 14% active alkali, preheated to 385°C, and lasted 114 minutes (H factor=816).

The results of each cook on % yield (screened), % rejects and kappa number are set forth below in Table 27:

In addition, the observed physical properties are consistent with pulp/paper of improved strength, as demonstrated below in Table 28 for the pulp produced by cook A.

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EXAMPLE 9

Sterile pine wood logs are inoculated with 10 ml of Phanerochaete gigantea TEl ( 1 X 10 6 cells/ml) or 10 ml water, respectively and six days later are hand barked, chipped. screened and then, homogenized. 300 g (o.d.) of the wood chips are subjected to conventional chemical pulping, (Liquor: wood ratio is set at about 4: 1 ; Liquor strength is set at about 16% active alkali and about 25% sulphidity; chips are cooked up to 170°C for 83 - 190 minutes; H-Factor is about 800), during which chips made from treated logs are evaluated in duplicate.

The comparative data on % extractive reduction, % Klason lignin, the carbohydrate analysis, pulp yield (screened and unscreened), Kappa number, and % rejects is set forth in Tables 29-31, respectively below.

EXAMPLE 10

The cooking conditions of the previous example (Example 9) were modified in a subsequent conventional chemical pulping lasting 160 minutes (H-factor=790) of P. gigantea TEl -treated pine wood chips (350 g (o.d.)). The results of four separate cooks at various levels of active alkali are provided below in Table 32.

EXAMPLE 1 1

The cooking conditions of the previous Example (Example 10) were again modified in two subsequent conventional chemical pulping of control and P. gigantea TEl- treated wood chips (300 g (o.d.)). The first cook (A) was set at 16% active alkali and lasted 1 15 minutes (H-factor - 909) * and the second cook (B) was set at 15.5% active alkali and lasted 148 minutes (H-factor = 846).

The results of each cook are provided in Table 33 below:

"Thermocouple failure for 50 minutes renders the data only an approximation.

EXAMPLE 12

The cooking conditions of the previous Example (Example 1 1) were again modified in two subsequent conventional chemical pulping of control and P. gigantea TE1 - treated wood chips (300 g (o.d.)). Both cooks were set at 15% active alkali. The first cook (A) lasted 103 minutes (H-factor = 808) and the second cook (B) lasted 1 13 minutes (H-factor = 823). The results of each cook are provided in Table 34 below. In addition, the observed physical properties are consistent with pulp/paper of improved strength, as demonstrated below in Table 35.

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F.X AMPLE 13

Growth on sterile and non-irtcrile Snnthern Yellow Pine:

An evaluation of fungal growth on Southern Yellow Pine was performed on both sterile wood chip samples and non-steπle wood chip samples, the wood chips having been aged about 5 days Each of tne samples contained 100 g of wood chips, prepared as descπbed above An inoculant of each of the fungi was prepared as descπbed e. and 5 g of mycelial mat (wet weight) were used to inoculate the 100 g of chips in the manner descπbed above The bags were then stored at room temperature for a peπod of 12 days Evaluation of the growth of the fungi was periormed at tne second, fifth and twelfth da> after the inoculation of the samples The results of this growth on steπle and non-steπle southern pine is reported in Tables 36 and 37 below With regard to the results on non-steπle substrates (Table 37) a minor background growth was observed on some wood chips after 12 days with some of the background appeaπng under the otherwise white growing lest fungus

TABLE 36 - GROWTH OF FUNGI ON STERILE SOUTHERN YELLOW PINE

Species 2 days growth 5 days growth 12 days growth

Schizophvllum commune 100% 1009.

Trichaptum hiforme 100% 100. 100%

EXAMPLE 14

Growth and Pitch Reduction on Non-Sterile Hardwoods:

Following the procedure of the preceding Examples, Phlebia tremellosa strain BRI-1 18 was evaluated together with

CARTAPIP ® 97 for growth and pitch reduction on 500 g samples of non-sterile mixed hardwood wood chips which were inoculated one day after chipping and which showed no background growth at time of inoculation. The hardwood mixture involved 75% maple, 20% yellow birch and 5% oak. The BRI-1 18 sample was harvested from an 8 day shaking flask culture and each inoculum involved 3 g of mycelial mat with an estimate CFU count of 7.1 X 10 5 /g of mat. Treatment time was 14 days. Growth results are reported in Table 38 and pitch reduction in Table 39 (against the ambient control).

Table 38 indicates good detectable growth of the fungus of the invention on hardwoods and Table 39 indicates a supeπor pitch reduction for Phlebia tremellosa over CARTAPIP ® 97.

EXAMPLE A

G ROWTH CH A R A CTER OR FUNG I I N LI QUID SH A KE FLASK CULTURE

Phlebia tremellosa (BRI-1 18) was grown in shake flask liquid culture using 500 ml of a YNPD medium prepared as above described. The medium was inoculated with a small plug of mycelia from an actively growing malt/yeast extract agar plate. The flask was shaken at 200 rpm at 23-25 C for 1 1 days and a 1 ml sterile sample from each culture was removed for microscopic analysis. The culture showed a dense growth of mycelial balls and the culture masses were also indicated to include from about 0.5 to 1.5% blastospores. This product can be used as inoculum or processed in vaπous ways to produce inoculum forms, e.g., by homogenizing and freezing for later use. Inoculum based essentially on the spore content of the cultures may also be prepared by freeze drying.

Schizophyllum commune and Trichaptum biforme were each separately grown in shake flask liquid culture using 50 ml of a malt extract/yeast extract medium prepared by dissolving 20 g malt extract and 2 g yeast extract in distilled water to a total volume of 1 liter. The medium was inoculated with a small plug of mycelia from an actively growing

malt/yeast extract agar plate. The flask was shaken at 200 rpm at 23-25 °C for 5 days and a 1 ml sterile sample from each culture was removed for microscopic analysis. Both cultures showed a dense growth of mycelial balls and the culture masses were also indicated to include from about 40 to 60% blastospores (about 40% for T. biforme and 50-60% for £_ commune). Both products can be used as inoculum or processed in various ways to produce inoculum forms, e.g., by homogenizing and freezing for later use. Inoculum based essentially on the spore content of the cultures may also be prepared by freeze drying.