FIELD OF THE INVENTION

The present invention relates to producing a desirable wood color with particular fungal species and the lumber treating process with the fungi.

BACKGROUND

Wood color is produced by progressive accumulation of wood cells with a complex of diverse substances called extractives during tree growing. Pigmented extractives determine most of the visual appearance quality of the hardwood species; therefore, they affect wood usefulness and value of the wood products. Many recognizable and commercially desirable qualities of the heartwood such as cherry, walnut and rosewood etc are a result of the presence of pigmented extractives. The presence of the pigmented extractives is mostly distributed in the heartwood of trees. In some species such as maple or spruce the extractives are light color, and the heartwood of these species remains light color similar to the sapwood: these wood species are called light heartwood trees. In some other species such as oak or cedar the extractives presented in heartwood are a dark-color; therefore, the heartwood has various color intensities and can be visually recognized from sapwood. These trees are described as regular heartwood trees.

Fungal infection of wood can cause wood color change (lighter or darker). The well-known fungal discoloration of wood is called blue stain. Blue stain is caused by a particular group of fungi that commonly attack only the sapwood of trees to bluish or greyish discoloration of the wood; therefore, it is also called sapstain. This type of fungi utilizes simple sugars and starches presented in the sapwood as nutrients and produce dark pigment called melanin during their growth. The wood discoloration caused by fungal melanin may cover the whole sapwood or may appear as streaks or patches of bluish to black intensities. However, the bluish black wood color resulted from these fungi is not desirable for wood end users. Most studies on wood blue stain are focused on preventing or controlling color development on wood products. One of such approaches is inoculating wood with a colorless mutant of a sapstain fungus such as Ophiostoma piliferum, and the preoccupation of wood surfaces by the colorless fungus can prevent later invasion of wood by staining fungi and thereafter wood color change. No study has been conducted to artificially inoculate blue stain fungi to produce bluish black wood color for high wood value use.

Another wood color change caused by fungal infection is a green color caused by Chlorociboria species. The wood discoloration is caused by the production of a fungal pigment xylindein, which is classified as a napthaquinone. The naturally green-stained wood had been used as woodcrafts in European countries since 14 ^th - 15 ^th century.

Wood decay can also change wood color. A well known example is called spalted wood that is in high demand in the decorative wood market. Spalted wood is caused by certain decay fungi growing in wood (white-rot). The decay fungal attack can cause random patches of contrasting colors to appear on the surface of some hardwoods such as maple and birch. In addition, when two or more competing fungi are meeting together in wood, it may create brown to black zone lines on wood in the border of each fungal territories. In this way, spalted wood forms map-like figures of different shapes and color contrasts. It may also produce unusual multicoloured streaks on wood caused by reaction between the wood and decay fungi. However, the pattern and color changes produced on spalted wood by these decay fungi are not predictable and repeatable.

Most common methods for coloring wood products are using pigments or dye materials which are carried either in a liquid solution or as a dispersion.

SUMMARY

In accordance with one aspect of the present invention, there is provided a method of coloring and treating wood with a pigmented fungal species, the method comprising: providing the fungal species in an active form; providing the wood to be treated; applying the active form of the fungal species to the wood to produce a treated wood; incubating the treated wood for a period of time; drying the treated wood.

In accordance with another aspect of the method described herein, the fungal species provided is selected from the group consisting of Penicillium variabiles Fusarium culmorum ; Coryne microspore; Diatrypella placenta; Arthrographis cuboidea; Poria aurea; Corticium polosum; Lentinus cyathiformis; Lecythophora hoffmannii; Tyromyces balsameus; Trogia crispa; Polyporus dryophilus; Polyporus dryophilus var. vulpinus; Peniophora piceae; Sporotrichum dimorphosporum; Gliocladium verticilloides; Nectria ochroleuca; Trichoderma atroviride; Trichoderma sp; Verticillium sp; Chlorosplenium aeruginascens; Scytalidium lignicola; Ophiostoma p ceae; Aureobasidium pullulans; Phiaiophora alba; Penicillium expansum; Penicillium implicatum; Fusarium verticillioides; Dactylium dendroides; Phialemonium dimorphosporum, Fusarium oxysporum, Ascocoryne cylichnium; Cephalotheca purpurea and combinations thereof.

In accordance with yet another aspect of the method described herein, the step of providing the fungal species in an active form comprises incubating the fungal species to produce a fungal culture, homogenizing the culture to produce a suspension.

In accordance with still another aspect of the method described herein, the suspension produced comprises a concentration of spores/mycelia fragments per ml of suspension of at least about 1 x 10 ⁵ .

In accordance with yet still another aspect of the method described herein, the suspension produced comprises a concentration of spores/mycelia fragments per ml of suspension of about but not limits from 1 x 10 ⁶ to 1 x 10 ⁸ .

In accordance with a further aspect of the method described herein, the wood provided to be treated is sapwood and heartwood of sugar maple, white birch and yellow birch but may extend to all other hardwood and softwood species.

In accordance with yet a further aspect of the method described herein, the step of applying the active form of the fungal species to the wood is by dipping, by spraying or by brushing.

In accordance with still a further aspect of the method described herein, the step of applying the active form of the fungal species to the wood is by dipping.

In accordance with yet still a further aspect of the method described herein, the step of incubating the treated wood for a period of time is for more than 1 week at a temperature from 5°C to 35°C and a relative humidity at least 75% or higher.

In accordance with one embodiment of the method described herein, the treated wood is incubated at 25°C and 75% RH for 1 to 4 weeks.

In accordance with another embodiment of the method described herein, the treated wood is dried at a temperature from 50°C to 105°C. ln accordance with yet another embodiment of the method described herein, the wood color change is evaluated visually or with a colorimeter.

In accordance with still another aspect of the present invention, there is provided a fungal species treated wood product produced by a method comprising: providing the fungal species in an active form; providing the wood to be treated; applying the active form of the fungal species to the wood to produce a treated wood; incubating the treated wood for a period of time; drying the treated wood.

In accordance wit another aspect of the product described herein, the fungal species provided is selected from the group consisting of Penicillium variabile; Fusarium culmorum ; Coryne microspora; Diatrypella placenta; Arthrographis cuboidea; Poria aurea; Corticium polosum; Lentinus cyathiformis; Lecythophora hoffmannii; Tyromyces balsameus; Trogia crispa; Polyporus dryophilus; Polyporus dryophilus var. vulpinus; Peniophora piceae; Sporotrichum dimorphosporum; Gliocladium verticilloides; Nectria ochroleuca; Trichoderma atroviride; Trichoderma sp; Verticillium sp; Chlorosplenium aeruginascens; Scytalidium lignicola; Ophiostoma piceae; Aureobasidium pullulans; Phialophora alba; Penicillium expansum; Penicillium implicatum; Fusarium verticillioides; Dactylium dendroides; Phialemonium dimorphosporum, Fusarium oxysporum, Ascocoryne cylichnium; Cephalotheca purpurea and combinations thereof.

This invention provides methods and manufacturing processes to produce various desirable wood colors with different particular fungal species for high wood value use by 1 ) using selected fungal species to produce a particular desirable wood color, and 2) producing wood color changes predictable, uniform, stable and repeatable.

DESCRIPTION OF THE DRAWINGS

Figure 1 is a histogram illustrating a brown wood color variation between sapwood and heartwood produced by Trogia crispa (FT 473C) according to one embodiment of the present invention;

Figure 2 is a histogram illustrating a grey wood color variation between sapwood and heartwood caused by Penicillium expansum (FTK 828A) according to one embodiment of the present invention; Figure 3 is a histogram illustrating a black wood color variation between sapwood and heartwood caused by Aureobasidium puilulans (FTK 1321) according to one embodiment of the present invention;

Figure 4 is a histogram illustrating a purple wood color variation between sapwood and heartwood caused by Dactylium dendroides (FTK 597A) according to one embodiment of the present invention;

Figure 5 is a histogram illustrating a red wood color variation between sapwood and heartwood Arthrographis cuboidea (FTK 706B) according to one embodiment of the present invention;

Figure 6 is a histogram illustrating a green wood color variation between sapwood and heartwood Chlorosplenium aeruginascens (FTK 401A) according to one embodiment of the present invention;

Figure 7 is a photograph of black coloration of sugar maple sapwood (left) and heartwood caused by Aureobasidium puilulans according to one embodiment of the present invention;

Figure 8 is a photograph of purple coloration of yellow birch sapwood (left) and heartwood caused by Dactylium dendroides according to one embodiment of the present invention;

Figure 9 is a photograph of red coloration of white birch sapwood (left) and heartwood caused by Arthrographis cuboidea according to one embodiment of the present invention;

Figure 10 is a photograph of green coloration of sugar maple sapwood (left) and heartwood caused by Chlorosplenium aeruginascens according to one embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

EXAMPLE 1 - Selection of fungal species for coloring

Selection of fungal species for coloring wood was performed in Petri plates (85 mm in diameter) holding 20 ml of a 2% (w/v) malt extract agar medium in each plate. One mycelia plug (5 mm in diameter) was cut from each fungal colony and transferred to the middle of each plate. The plates were sealed with a Parafilm and incubated at 25°C and 75% RH for 14 days. The colors produced by these fungi on agar were visually evaluated. Based on the principal colors produced by these fungi, 33 fungal species were selected for testing on wood. The principal colors are pink, red, brown, orange, yellow, green, black, blue and purple. 1 to 5 fungal species per color were selected for the test on wood. The selected fungal species and associated colors in agar plates are shown in Table 1.

All these fungal species came from the Culture Collection of Wood-inhabiting Fungi (FTK) holding by FPInnovations, Quebec, Canada. All fungal cultures were maintained in a liquid nitrogen reservoir for cryopreservation at -198°C before use.

EXAMPLE 2 - Preparation of fungal solutions and wood specimens for coloring

The selected fungal species were retrieved from the liquid nitrogen reservoir and grown on a 2% (w/v) malt extract agar medium in Petri plates at 25°C for one week. Mycelia plugs (5 mm in diameter) were cut from each fungal colony and transferred 3 plugs to each 125 ml flask containing 50 ml of a sterile 2% (w/v) Difco malt extract broth (Becton, Dickinson and Company, Sparks, MD, USA) in distilled water. After incubation, the fungal cultures were homogenized 3 times (30 seconds per time) with a homogenizer into a fine mycelia fragments and spore suspension. One drop of the suspension was loaded on a hemacytometer and spores and mycelia fragments in the solution were counted under a microscope. Fungal suspensions having at least 1 x 10 ⁵ spores/mycelia fragments per ml of suspension have been found to be effective. However, concentrations of the present fungal suspensions were determined to be 1 x 10 ⁶ to 1 x 10 ⁸ spores/mycelia fragments per ml of the solution. These fungal suspensions were used immediately to treat wood specimens.

Fresh log sections of sugar maple {Acer saccharum Marshall), white birch (Betula papyrifera Marshall) and yellow birch {Betula alleghaniensis Britton) were provided by a local Quebec company. The sapwood and heartwood of log sections were identified and cut separately into wood specimens at the size of 60 mm x 20 mm x 5 mm. A total of 792 wood samples were prepared from these 3 wood species for testing selected 33 fungal species. EXA PLE 3 - Treatment of wood specimens and evaluation

Wood specimens were placed in containers based on wood species and autoclaved at 121 °C for 10 minutes. After cooling, wood specimens were dipped for 30 seconds in a fungal solution, 4 specimens per treatment. Following the treatment, two pieces of specimens were placed on a W-shaped glass support sitting over 2 layers of wet filter paper in a Petri plate. These plates were incubated in a growth chamber set at 25°C and 75% RH. Wood specimens were visually inspected for wood color change each week up to 4 weeks. At the end of the test, half amount of the wood specimens was dried at 50°C and another half was dried at 105°C. The final wood colors after drying were measured with a colorimeter.

The wood coloring with selected fungal species is shown in Table 1. In most cases, one fungal species colored all three wood species tested into a similar color. In addition to wood species, most of the fungal species colored sapwood and heartwood of a wood species at a similar intensive level. Therefore, wood colors shown in Table 1 represented the major color observed from all wood specimens treated with each fungal species.

Because of the interference of wood cells, the colors shown on agar may or may not be the same as the one shown on wood. For example, agar and wood were both colored into green by Verticillium sp. (FTK 164C) and Chlorosplenium aeruginascens (FTK 401A); colored into purple by Dactylium dendroides (FTK 597A) and Phialemonium dimorphosporum (FTK 669A); colored into brown by Trogia crispa (FTK 473C) and Polyporus dryophilus var. vulpinus (FTK 483A); and colored into black by Aureobasidium pullulans (FTK 1321). Some fungal species produced different colors on agar and on wood. For example, Fusarium culmorum (FTK 750A) produced red color on agar, but purple on wood; and Fusarium oxysporum (FTK 31 A) produced dark purple color on agar, but brown on wood. Other fungal species produced a similar color on agar, but different colors on wood. For examples, both Phialophora alba (FTK 772A) and Penicillium expansum (FTK 828A) produced pink pigments on agar, but on wood the former caused light brown and the later caused grayish color. Still some fungal species produced different colors on agar, but a similar color on wood. For example, Arthrographis cuboidea (FTK 706B) produced light brown and Poria aurea (FTK 1 1 OA) produced brown color on agar, but both species produced red color on wood. There were several fungal species that produced pigments on agar but not on wood such as, in agar plate cultures, Penicillium variabile (FTK 659B) produced red pigment, Coryne microspora (FTK 239A) produced light brown pigment, and Sporotrichum dimorphosporum (FTK 306D) produced yellow pigment, while none of them produced any color on wood.

Wood specimens dried at different temperatures, in general, did not significantly change principal wood colors but significantly changed color lightness. Those wood specimens dried at 105°C were significantly darker than those dried at 50°C.

Table 1 - Fungal coloring from agar plate test and on wood specimens

Fungal

Number code Fungal species Color on agar Color on wood

FTK

No coloring

1 659B Penicillium variabile Sopp Red

FTK Fusarium culmorum (W. G. Sm.)

Purple

2 750A Sacc. Red

FTK

No coloring

3 239A Coryne microspora Ellis & Everh. Light brown

FTK

No coloring

4 430A Diatrypella placenta Rehm Light brown

FTK Arthrographis cuboidea (Sacc. et

Red

5 706B Ellis) Sigler Light brown

FTK

Red

6 1 10A Poria aurea Peck Brown

FTK

Brown

7 534A Corticium polosum Burt Brown

FTK

Brown

8 795A Lentinus cyathiformis Bres. Brown

FTK Lecythophora hoffmannii (van

Brown

9 893A Beyma) W. Gams & McGinnis Brown

Tyromyces balsameus (Peck)

Brown

10 FTK 79A Murrill Dark brown

FTK

Brown

1 1 473C Trogia crispa Fr. Dark brown

FTK

Brown

12 482B Polyporus dryophilus Berk. Dark brown

FTK Polyporus dryophilus var. vulpinus

Brown

13 483A (Fr.) Overh. Dark brown

FTK Peniophora piceae (Pers.) J.

Brown

14 840A Erikss. Dark brown

FTK Sporotrichum dimorphosporum v.

No coloring

15 306D Arx. Yellow

FTK Gliocladium verticilloides

Grayish yellow

16 790A Pidoplichko Yellow

FTK Nectria ochroleuca (Schweinitz)

Grayish yellow

17 843C Berkeley Yellow

FTK Yellowish

Grayish brown

18 585E Trichoderma atroviride P. Karst. orange

FTK Yellowish Yellowish

19 872B Trichoderma sp. orange brown

FTK

Green

20 164C Verticillium sp. Green

FTK Chlorosplenium aeruginascens

Green

21 401A (Nyl.) Karst Green

FTK

Grayish blue

22 197P Scytalidium lignicola Pesante Dark blue

FTK Ophiostoma piceae (Munch) Syd.,

Grayish brown

23 387AN H. & P. Syd. Dark blue Fungal

Number code Fungal species Color on agar Color on wood

Aureobasidium pullulans (deBary)

Black

24 FTK 1321 Arnaud Black

FTK

Light Brown

25 772A Phialophora alba von Beyma Pink

FTK

Gray

26 828A Penicillium expansum Link Pink

FTK

Green

27 837A Penicillium implicatum Biourge Pink

FTK Fusarium verticillioides (Sacc.)

Light Brown

28 754A Nirenberg Light purple

FTK Dactylium dendroides (Bulliard)

Purple

29 597A Fr. Purple

FTK Phialemonium dimorphosporum

Purple

30 669A W. Gams & W. B. Cooke Purple

Fusarium oxysporum

Brown

31 FTK 31A Schlechtend.:Fr. Dark purple

FTK Brownish

32 392A Ascocoryne cylichnium (Tul.) Korf Dark purple purple

FTK Cephalotheca purpurea (Shear)

Light Brown

33 433A Chesters Dark purple

EXAMPLE 4 - Matching color on sapwood and heartwood

Color evaluation of sapwood and heartwood wood blocks after fungal treatment and drying were performed with a colorimeter Color-guide 45/0 de BYK-Gardner USA.

Colors are perceived as combinations of green and yellow, red and blue, and red and yellow. Based upon the equation of the CIE 1976 from Haegen et al.:

L*a*b* color space system, colors are assigned to a rectangular coordinate system. The color coordinates are L* the lightness coordinate, a* the red/green coordinate (+a* indicating red and -a* indicating green), and b* the yellow*/blue coordinate (+b* indicating yellow and -b* indicating blue). Because the CIE L*a*b* colors space system is three-dimensional, it can often be difficult to relate actual differences in color values to visually perceived differences. One method developed for examining color differences uses the color metric difference (AE* _ab ) where:

AE* _ab = V ((LYL* ₂ ) ² + (aYa ^* ₂ ) ² +(bVb ^* ₂ ) ² ) Mathematically, the color metric difference (AE* _ab ) is the Euclidean distance between two colors, L^a*^*-, and L* ₂ a* ₂ b* ₂ . It is relatively proportional to color differences perceived by human observers (Billmeyer and Saltzman 1981 ). Haeghen et al. (2000) determine that AE* _ab color difference values less than 3 are considered unnoticeable to the human eye.

In a study on white beech looking at color problems with the drying process (Rodolfo et al. 2007), the magnitude of AE* _ab was classified according to the grading rules as follows:

0.2 < AE* _ab = Not visible difference;

0.2 < AE* _ab < 2 = Small difference;

2 < AE*ab < 3 = Colour difference visible with high quality screen;

3 < AE* _ab < 6 = Colour difference visible with medium quality screen;

6 < AE* _ab < 12 = High colour difference; and

AE* _ab > 12 = Different colours.

With this classification, AE* _ab > 6 correspond to a high color difference and if >12 as different colours.

Color variations (AE* _ab ) of all fungal treated wood samples compared with the untreated controls are presented in Table 2. All of the fungal treatments lead to a significant color change of the tree hardwood species, both on sapwood and heartwood, with AE* _a value going from 25.2 and up to 73.6.

We also looked at the wood color variation between sapwood section and heartwood section of a same wood species (Figures 1 to 6). Most of the fungal treated samples had a AE* _ab value below 10 with a least one representative of each wood color below 3, the critical level of what could be seen by the human eye. Pictures of different wood colors obtained by fungal coloration are presented in Figures 7 to 10. Table 2 - Wood color change (AE* _ab ) after fungal treatment

Number Fungal

code Fungal name Sugar maple White birch Yellow birch heart- sap- heart- sap- heart- sap- wood wood wood wood wood wood

6 FTK 1 1 OA Poria aurea 57.1 52.5 56.5 55.1 60.3 46.4

24 Aureobsidium

FTK 1321 pullulans 35 33.5 46.1 44.5 47.6 43

20 FTK 164C Verticillium sp. 59.4 55.8 56.1 59.5 57.4 57.7

22 Scytalidium

FTK 197P lignicola 46.1 52.1 45.8 39.4 51 .5 41 .9

3 FTK 239A Coryne microspora 64.7 65.9 67.3 66.7 63.9 68.1

15 Sporotrichum

FTK 306D dimorphosporum 62.7 66.5 63.2 68.8 58.7 68

31 Fusarium

FTK 31A oxysporum 55.2 61.5 61.9 59.5 58.7 60.5

23 FTK

387AN Ophiostoma piceae 59.7 60.5 61 .1 53.6 54.6 54.1

32 Ascocorune

FTK 392A cylichnium 40.2 25.2 55.7 54.5 54.6 49.3

21 Chlorosplenium

FTK 401 A aeruginascens 57.3 62.8 59 61.5 59.1 57.2

4 FTK 430A Diatrypella placenta 64.8 70.9 69.6 70.1 61.9 70.4

33 Cephalotheca

FTK 433A purpurea 60.3 63.8 60.9 58.8 56.6 62.7

11 FTK 473C Trogia crispa 56.2 60.8 59.2 58.3 57.1 56.6

12 Polyporus

FTK 482B dryophilus 64.6 67.5 66.7 61.8 64.8 66.7

Polyporus

13 dryophilus var.

FTK 483A vulpinus 41.3 65.8 42.6 44.1 47.9 58.2

7 FTK 534A Corticium polosum 65.6 61.3 65.1 66 54.1 60.3

18 Trichoderma

FTK 585E atroviride 62.7 68.6 57.3 63.8 55.6 64.6

29 Dactylium

FTK 597A dendroides 64.2 65.6 55.9 52.3 54.6 51.1

1 FTK 659B Penicillium variabile 65.2 69.3 61 66.1 64.4 63.6

30 Phialemonium

FTK 669A dimorphosporum 49.7 44.9 48.4 65 51 59.3

5 Arthrographis

FTK 706B cuboidea 42 48 37.1 39.9 42.2 40.4

2 FTK 750A Fusarium culmorum 61.4 52.1 61 .4 62 59.3 58.9

28 Fusarium

FTK 754A verticillioides 54.8 60.6 65.3 63.3 57.2 60.6

25 FTK 772A Phialophora alba 68 73.6 65.9 67.3 60.8 69.6

16 Gliocladium

FTK 790A verticilloides 66 68 65.1 71.8 67.4 69.3

8 Lentinus

FTK 795A cyathiformis 61.4 59.3 57.4 63.1 54.4 57.2 Number Fungal

code Fungal name Sugar maple White birch Yellow birch heart- sap- heart- sap- heart- sap-

_L wood wood wood wood [ wood wood

10 Tyromoces

FTK 79A balsameus 64.6 66 71 70 64.3 66.3

26 Penicillium

FTK 828A expansum 60.5 59.3 63.8 61 65.5 62.1

27 Penicillium

FTK 837A implicatum 52.3 52.1 53.2 51 .3 48.1 58.9

14 FTK 840A Peniophora piceae 54.4 60.3 59.4 62.9 56.2 68.7

17 FTK 843C Nectria ochroleuca 63.2 71.9 61.7 72.4 64.6 68.6

19 FTK 872B Trichoderna sp. 58.5 71 .3 61.7 65.7 57.7 66.2

Lecythophora

9 FTK 893A hoffmannii 65.3 69.5 64.6 69.7 65.2 66.4

Using biological method for coloring wood with fungi is a new innovative approach and has a potential to produce preferable wood colors and patterns. The resultant product could be sold as a water based stain substitute in the form of fungal spore suspension. One litre of such suspension is relatively inexpensive, and can be further diluted into 100 L with water as application solution. In an industrial factory application situation, the product can be applied to lumber either by a spraying line or by a dipping tank, which will consume 20 L or 50 L of application solution per thousand board feet measure (Mfbm) of lumber, respectively. Applying the product to lumber at an industrial scale will lead to a cost effective product. After application of the fungal suspension onto lumber, the lumber must be stored in a yard for more than 1 week to allow fungus changing wood color. Of course, this process will take longer time than standard water-based staining methods; however, the process allows color change in depth of wood, whereas the standard staining method can not. Such technology will increase wood market value and enhance the utilization of wood products in competitive marketing of lumber and furniture manufacturing. References:

- Billmeyer, F.W. and M. Saltzmann, 1981 , Principles of color Technology, 2 ^nd Ed. John Wiley and Sons Inc., NY, 240 pp.

- Haeghen, Y.V., J.M. Naeyaert, I. Lemahieu and W. Phillips, 2000, An imaging system with calibrated color image acquisition for use in dermatology, IEEE Transactions on Medical Imaging, 19(7)722-30.

- Rodolfo, C. T. Livio, A. Ottaviano, 2007, White beech: a tricky problem in the drying process, ISCHPO7, p.135-140.

- H. Sugawara et al. (Editor), Bacteria, Fungi and Yeasts, 4th Edition, 1993, World Discovery of Collection of Cultures of Microorganism, WFCC World Data Center on Microorganisms.