Pitch and Lignin Degradation With White Rot Fungi

The present invention relates to the use of certain fungi in the reduction of the pitch and/or lignin content of cellulosic materials. In particular, the white rot fungi Schizophyllum commune, Trichaptum biforme and Phanerochaete gigantea are useful in reducing pitch and/or lignin, or both, which saves electrical energy during the mechanical reining of pulps and pulpwoods used in making cellulosic products.

Wood is a complex material composed of cellulose, hemicellulose, 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 are known in the art, e.g. waste stream pollution.

In Nilsson, et al., U.S. Pat. No. 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 discoloring 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 (based on U.S. patent application Ser. No. 310,814, filed 13 Feb. 1989) 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 (based on U.S. patent applications having Ser. No. 560,521, filed Jul. 31, 1990 and copending Ser. No. 657,581, filed Feb. 19, 1991).

In copending U.S. patent application Ser. No. 889,796, filed Jun. 17, 1992, there are described other strain derivatives of a preferred wood-penetrating fungus 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, particularly for example when used on substrates for chemical pulping, 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 including Basidiomytes 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 are also particularly useful since the action of the fungi in degrading pitch avoids metabolic states in which cellulose, hemicellulose and lignin may be attached, hence allowing such Basidiomycete fungi to protect against staining fungi over adequate periods of time without adversely affecting the quality of wood as structural wood. White rot fungi which degrade pitch and grow very well on non-sterile wood are Schizophyllum commune, Trichaptum biforme, Phanerochaete gigantea and Phlebia tremellosa.

Staining fungi protected against by the invention involve those which typically penetrate deeply into the wood and which themselves involve the fungal classes Ascomycetes and Deuteromycetes, which staining fungi are typically represented by those also known as blue stains. Such fungi reduce pitch as is now known. While we do not wish to be bound by any theory concerning the invention, the beneficial results provided by the invention are probably due at least in part to the ability of the pitch-degrading white/colorless growing fungi to deprive the staining fungi of their primary food source.

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 also been of interest for their ability to degrade lignin and produce lignin degrading enzymes. The original "biopulping" concept 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. Such biopulping processes have been thought to improve efficiency, reduce energy consumption and reduce environmental problems in the pulp and paper industry.

A white rot fungi judged particularly suitable for such purpose is Ceriporiopsis subvermispora as described in U.S. Pat. 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.

An objective of the present invention is to expand the field of fungi useful in degrading pitch and/or lignin in pulps and pulpwoods, and particularly in non-sterile substrates such as wood chip piles.

Another object is to provide pitch degrading fungi having or combining desired properties such as color effects, pitch-degrading ability, good growth on non-sterile substrates, flexibility in temperature of operation, greater action or flexibility of action on different wood species and the like.

The present invention relates to the use of certain fungi in the reduction of the pitch and/or lignin content of cellulosic materials. In particular, the white rot fungi Schizophyllum commune, Trichaptum biforme and Phanerochaete gigantea are useful in reducing pitch and/or lignin, or both, which saves electrical energy during the mechanical refining of pulps and pulpwoods used in making cellulosic products.

In accord with the present invention, it has been found that white rot fungi of the species Schizophyllum commune, Trichaptum biforme and Phanerochaete gigantea are desirably effective in reducing the pitch and/or lignin content of wood substrates, including particularly pitch in non-sterilized wood substrates. Use of such fungi in pulping have been discovered to reduce electrical energy consumption, improve paper strength and reduce undesirable environmental contamination.

Accordingly, the invention provides a method of reducing the pitch and/or lignin content of wood, particularly pulpwoods and pulps, said method comprising applying to pulpwood or pulp an inoculum of the fungus Schizophyllum commune or of the fungus Trichaptum biforme or of the fungus Phanerochaete gigantea, or a mixture of inoculums for such fungi, and thereafter maintaining the inoculated pulpwood or pulp under conditions allowing growth of the fungus for a time sufficient to reduce the pitch and/or lignin content of the pulpwood or pulp.

It has also been found in accord with the present invention that the color staining of structural wood by color-staining fungi may be surprisingly suppressed to a great degree merely by inoculating both the cut end of timber logs with a fungus which grows white and/or colorless and which acts to reduce the pitch content of the wood.

Despite the fact that the pitch degrading fungi will deeply penetrate and leave voids where pitch has been removed, it has been found that such voids have substantially no adverse affect on the quality of structural lumber produced from logs treated with such fungi in accord with this invention. Because such previously disclosed pitch-degrading fungi are among the more virulent growing known to us, they generally constitute preferred fungi for use in the invention.

It is also within the scope of the invention to protect the length of logs between the treated ends against the infestation or natural inoculation of staining fungi which could stain the wood portions between the ends. For example, in areas where bark beetles are present which can bore into logs and carry with them, as is known, spores or other inoculum of the staining fungus, the log lengths may be treated with an amount of an insecticide effective to suppress the bark beetles. Lengthwise areas which have been debarked in tree-falling or handling may also be inoculated with a white/colorless growing fungus. As an alternative or where inoculum of staining fungi are particularly high, the logs may be scored lengthwise, preferably at intervals of 8 to 20 inches around the log circumference, and the white/colorless growing fungus inoculated into the scoring which generally will be carried out to a depth sufficient to substantially reach the under-the-bark wood. If and when logs are to be debarked, and then stored, it is within the scope of the invention to treat the entire debarked surface with the white/colorless growing fungi to protect against staining fungi.

Moreover, a considerable expense is encountered in the structural wood industry in protecting structural wood after cutting from logs against the color staining fungi which could infect the wood before or after cutting to form the structural wood, typically by spraying with an environmentally unsound fungicide such as pentachlorophenol. It is further within the scope of this invention to protect such structural wood against staining fungi by inoculating at least the lengthwise surfaces, or at least 60% of the surface area, preferably 80% and more, preferably all surfaces, of such wood with a pitch degrading fungus which grows white/colorless. The fungus is then allowed to grow on the structural wood which is maintained under environmental conditions sufficient to permit growth for at least about 14 days. Such inoculation desirably takes place no more than two weeks after the structural wood is cut from its log source, preferably in no more than one week, more preferably in no more than 4 days and most preferably in no more than 2 days. Such treatments are particularly useful to inhibit staining when the structural wood is stored and/or shipped for long periods in environments where staining fungi may be present, such as in ships or trucks which had previously carried infected wood forms such as logs, wood chips and the like.

By the terms "resin" or "pitch" (which are used interchangeably) is meant that complex mixture of hydrophobic substances in wood, commonly known as pitch, which are soluble in neutral organic solvents, such as methylene chloride, diethyl ether, benzyl alcohol and the like. These include the terpenes, the diterpene ("resin") acids, fatty acids and 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 methylene chloride will suffice for measuring the reduction in resins which is an object of the invention.

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 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 he 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 invention may be generally applied to reduce the pitch content of pulpwoods and pulps used in the manufacture of cellulosic products. 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.

White-rot 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 basidiomycetes 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. 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 carrying out the biopulping aspect of the invention it is important to inoculate the log ends soon after cutting down off the tree, the timing being influenced in part by the potential for infestation of staining fungi in the area. Desirably, the log ends will be inoculated in no more than two weeks after falling off the tree, preferably in no more than one week, more preferably in no more than 4 days and most preferably in no more than 2 days after cutting down of the tree. The particular fungus to be used will be selected in accord with guidelines given herein including growth ability on the particular wood type being treated. As is known, fungi grow on differing extents on different wood types, particularly when the wood is non-sterile. Hence, generally preferred fungi are those which grow well on the wood type of the substrate to be treated. Fungi more suitable for particular wood types are generally known from their history of natural growth habit on particular woods.

Any of the wide variety of wood types or genera processed by industry for structural woods may be treated in accord with the invention. These include both Gymnosperms and Angiosperms, and in particular both hardwoods and softwoods. Particular classes or types of wood therefore include without limitation conifers, pines, cedars, oak, maple, aspen, firs and birch. Softwoods such as pines generally have high pitch content and are readily colonized by pitch degrading fungi. Hence, they are more susceptible to invasion by pitch degrading staining fungi, but equally more easily treated in accord with the invention. Hardwoods, particularly those with low pitch contents, may in some instances require more thorough or high dose inoculum of the white/colorless growing fungi in order to ensure optimum germination.

The invention may therefore be utilized in one aspect thereof to at least partially reduce the resin and/or lignin component of refined pulpwood and incompletely refined pulps by applying to the pulpwood or pulp an inoculum of at least one of the indicated fungi, 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 resin and/or lignin component of the pulpwood or pulp by the fungus. The invention may be applied to unrefined pulpwoods such as cut timber in debark 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 and effect a reduction in the resin component thereof.

Such "biopulping" processes appear 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 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 content of both of which are incorporated herein by reference. 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), the content of which is incorporated herein by reference.

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 inocula include fungal cultures or preparation 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 "mycelium". Fungi typically reproduce asexually by means of spores called conidia which are given off by the mycelia or produce 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 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) 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.degree. C. to -80.degree. C., more usually -10.degree. C. to -75.degree. 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 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. 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 has been incorporated in vegetable and/or mineral oil 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 pulp, 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 inoculation containing from 0.5 to 10 grams of mycelia (wet weight of dewatered mycelia, see Example 1) per 100 grams of pulp or pulpwood, preferably from 1 to 5 grams of mycelia per 100 grams of substrate to be treated. 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 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 or suppress the background growth of fungi which naturally infected the wood prior to inoculation.

The fungus to be used in the biopulping aspect of the invention 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. When the fungus to be inoculated forms spores, a suitable inoculum involves, for example, relatively concentrated aqueous spore suspensions having from 10.sup.5 to 10.sup.10 CFU (colony forming units per milliliter, more usually 10.sup.6 to 10.sup.9 CFU/ml., although more or less concentrated forms may also be used. Similarly, the specific activity of mycelia in colony forming units (CFUs) may be determined by homogenizing the mycelia, e.g. for 5-10 minutes, and approximating the number of colonies resulting therefrom in a conventional manner when the fragments are grown on a nutrient substrate to determine the specific activity in CFUs for a given volume. Mycelia expressed as CFU will be used in similar activity concentrations to those of spores as given above. However, mycelia mats may also be simply dewatered and used as such as inoculum as demonstrated herein.

The fungal inoculum may be admixed with or applied concurrently with various 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. Carboxymethylcellulose is preferred for these purposes, although a variety of materials may also be used.

In another embodiment, chips which have been previously inoculated and incubated according to 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 40%, 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.degree. C. it will generally be more suitable to have a temperature of at least 10.degree. C. such as a temperature of from 10.degree. C. to 40.degree. C. more preferably of from 15.degree. C. to 33.degree. C., most preferably of from 22.degree. C. to 28.degree. 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 pipes or a plurality of openings for releasing warm air or steam. 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.

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 lignin removal, the temperature and moisture conditions, the extent of inoculation and the like. However, satisfactory results may generally be obtained after a period of time extending from 3 to 40 days, 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 fungi generally should be conducted for periods which effect desired pitch reduction 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. by isolation from wood sources on which they grow in nature. While some variation among strains can be expected depending on factors such as the wood source from which they may be isolated, our fungi demonstrated remarkable growth on unsterilized Southern Yellow Pine, Red Pine and Aspen and can be expected to grow well on other wood types commonly used in making cellulosic products. Naturally occurring isolates of our fungi can be modified by various known means of strain selection, mating and mutation without losing their identifying species characteristics. Hence, 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 fungi will include not only such isolates but also all other isolates and modifications which substantially possess at least the pitch degrading and growth properties on unsterilized Southern Yellow Pine, Red Pine and Aspen that are possessed by 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 fungi which naturally infect unsterilized substrates, the fungi of the invention may be used to produce a product requiring less bleaching to obtain the final paper product.

We have under the Budapest Treaty deposited with the Northern Regional Research Center (NRRL) at Peoria, Ill. U.S.A. the following fungi refined to herein, which deposits were assigned the Accession Numbers given below along with their date of deposit. These deposits are irrevocable and all restrictions on availability to the public will be removed upon grant of a patent herefrom.

The above deposited fungi were all obtained as natural isolates from fallen timber in the State of Minnesota, U.S.A., but all can be obtained from a variety of other global locations. The S. commune and T. biforme were isolated from a hardwood and the P. 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).

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, sterile and non-sterile aspen wood chips were used. Wood chips are stored at 5.degree. C. prior to evaluation. Each evaluation was performed on substrates of the same wood species and upon wood chips 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.degree. 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 deionized distilled water, and subsequently autoclaved at 121.degree. 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.degree. 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.

Evaluation of the pitch content of substrates was determined according to standard TAPPI Procedure T204 OS-76 which provides results expressible as milligrams of pitch content per gram of substrate extracted with "DCM" which is methylene chloride. In accordance with the TAPPI Procedure, as used on a substrate such as wood chips, the treated chips are splintered with pruning shears to a width of about 1 cm., then dried overnight at 60.degree. C. and then ground into sawdust using a Thomas-Wiley intermediate Mill with 10-mesh screen (10 gauge wire screen). Sawdust is extracted with DCM or other solvents as described in TAPPI Procedure T 206 OS-76. The dish residue is then heated in an air-circulation oven at 60.degree. 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 milligrams (mg.) and correlated to the amount of the original sample being evaluated so to provide an expression of mg. of pitch per gram 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.

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 infested 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 fungi, 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.

An evaluation of fungal growth on Southern Yellow Pine was performed on both sterile wood chip samples and non-sterile wood chip samples, the wood chips having been aged about 5 days. Each of the samples contained 100 grams of wood chips, prepared as described above. An inoculant of each of the fungi was prepared as described above, and 5 grams of mycelial mat (wet weight) were used to inoculate the 100 grams of chips in the manner described above. The bags were then stored at room temperature for a period of 12 days. Evaluation of the growth of the fungi was performed at the second, fifth and twelfth day after the inoculation of the samples. The results of this growth on sterile and non-sterile southern pine is reported in Tables 1 and 2 below. With regard to the results on non-sterile substrates (Table 2) a minor background growth was observed on some wood chips after 12 days with some of the background appearing under the otherwise white growing test fungus.

Evaluations of the growth of the fungal species on aspen were performed on both aged sterile and aged non-sterile wood chip samples. Each of the samples contained 100 grams of wood ships, prepared as described above. An inoculant of each of the fungi was prepared as described above, and 3 grams of mycelial mat (wet weight) were used to inoculate the 100 grams of chips. The bags were then stored at room temperature for a period of 12 days. The growth evaluation was performed at the second, fifth, and twelfth day after the inoculation of the samples. The results on sterile and non-sterile aspen are reported in Tables 3 and Tables 4, below. With regard to the results on non-sterile substrates (Table 4), a minor background growth was observed on some wood chips after 12 days growth.

Following essentially the procedure of Examples 1 and 2, the fungus Phanerochaete gigantea was evaluated for growth on both aged non-sterile Southern Yellow Pine and aged non-sterile Aspen, except that the inoculum in each test was a low dose of 2 grams wet weight of mycelia per 500 grams of wood chips and the evaluation was carried out over 27 days with an intermediate evaluation at 14 days. No background growth was observed either at 14 days or at 27 days. The results are below in Table 5.

The fungal strains 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.degree. 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 gram 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 the fungal species Ophiostoma piliferum in the form of the product available under the registered trademark CARTAPIP.RTM. 97.

Each of the samples were evaluated for the amount of DCM extractable 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; 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. This Klason lignin 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" (1984; TAPPI). In summary, Klason lignin analysis according to the TAPPI T249 cm-85 protocol is as follows; 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. Further, for selected aspen wood chip samples an analysis of the carbohydrates was performed so to evaluate the extent of cellulose and hemicellulose degradation.

Results of the samples being evaluated, % DCM extractives and % Klason lignin are reported on Table 6, and the carbohydrate analysis of selected samples are reported on Table 7, both below.

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

As may be seen from the results of Table 7, 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.

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.degree.-25.degree. 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 S. 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.

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). As used on a substrate such as wood chips, the treated chips are dried overnight at 60.degree. 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 gram 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.

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

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. 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 0.101 g/mat +/-0.009 g.

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 and for Simons' staining (see page 10).

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 using the TAPPI standard Procedure T204 OM-88. The results are presented in Table 8.

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 9.

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

Wood chips of Southern Yellow Pine were inoculated with a spore suspension of Phanerochaete gigantea and incubated at room temperature for 41 days. The inoculum was prepared by growing P. gigantea in a yeast malt media (2 g yeast extract and 20 g malt extract in 1 liter of water). The inoculated media was shaken at room temperature for 13 days and the spore suspension concentrated by spinning down the inoculum in a centrifuge and removing the excess media. The spore pellets were blended in a Vitir Shear homogenizer and diluted with sterile water to a concentration of 4.2.times.10.sup.7 colony forming units per 10 ml. Ten ml of inoculum was added to 500 g of wood chips. The wood chips were incubated at room temperature for 41 days.

Growth on the wood chips was visually determined at various time periods. After 5 days approximately 25% of the chips had white fungal growth present on their surface. At 10 days, 75% of the chips had white growth.

41 days after inoculation, wood chips were refined using a Sprout Waldron 12" laboratory refiner. The refined wood pulp fibers were then assayed for changes using the Simons' stain reagent. Refined pulp from inoculated wood chips showed a diffuse yellowing in over 50% of the fibers observed indicating substantial changes had occurred in the wood cell walls. This intermediate to advanced stage of color change correlates to energy savings (using previous biopulping data) of approximately 12-30%.

Control wood chips that were frozen (time 0) when the study started and allowed to thaw after 41 days were refined. Simons' staining on these pulp fibers showed no change and remained blue.

Control wood chips that were not inoculated but were aged for 41 days in plastic bags were also refined. Simons' staining of these pulps also did not change and fibers remained blue. Blue coloration of pulp fibers correlated with no change in energy use for mechanical pulping.