BACKGROUND OF THE INVENTION Microscopic fungi and fungal spores are ubiquitous in nature, unfortunately also in the materials manufactured by humans as consumer items e. g. clothing, shoes, furniture, carpets, wallpaper, and also more basic materials used for construction purposes. Fungal spores are spread in many ways, airborne through the wind, through droplets or running water, and by touch.

A number of different problems are associated with the occurrence of fungal growths (molds) in human habitats. Molds often leave stains which are aesthetically not pleasing, and very often they give off unpleasant earthy mildewy odors. However, more importantly, mold growths may lead to potential health problems for the occupants of a building where they occur, since many molds are allergenic and/or produce toxins. Toxic compounds that have been identified to be produced by various common indoor molds e. g. of the genera Aspergillus, Stachybotrys, Penicillium, Fusarium, or Alternaria, include Aflatoxin, Satratoxin H, Putulin, Vomitoxin, Fumonisin, Zearalone, Patulin, Ochratoxin, and Alternariol. It is thought that these toxins may act synergistically to produce a wide variety of symptoms and disease states. The symptoms of exposure to allergenic or toxic molds may range from allergenic sinusitis, asthma, bronchiopulmonary aspergillosis, and hypersentivity pneumonitis, to lethargy, gastrointestinal disturbances, liver toxicity and varieties of cancers.

In addition to the potential health problems, if the underlying cause of an indoor mold growth is not remediated, then given time the eventual progression of the mold may lead to structural damage within the building where it occurs. Current building construction

practices, while providing less costly construction and greater energy savings, have yielded themselves to the propagation of molds in the indoor environment. The highly insulated modern buildings are prone to high levels of humidity if not sufficiently aerated. Moisture may be trapped within the building structures as a result of accidentally leaking plumbing or due to condensation from air conditioning, also specific construction materials such a vinyl wall "paper"tends to trap moisture at the interstitial space between the vinyl and the wall board.

Unfortunately, the one requirement that is critical to mold growth is moisture; filamentous fungi require water activities above approximately 0.60, and virtually all fungi occurring indoors require water activities above approx. 0.85.

Fungi grow on a wide variety of substrates. In the indoor environment the primary substrate for growth is considered to be cellulose. The most common construction material in the United States for interior walls is gypsum-based wallboard. Gypsum wallboard is composed of a gypsum center covered with a paper coating, an excellent growth substrate for cellulolytic fungi such as Stachbotrys sp. and Chaetomium sp.

Cleaning mold infected materials with detergents, surfactants, and bleach only removes the surface contamination. Standard cleaning agents most likely do not destroy the fungal mycelia below the surface, which can regrow when exposed to conditions which led to fungal growth in the first place, nor do they destroy toxins produced by the fungi nor their allergenic properties. Complete removal of the contaminated material is the recommended strategy for the treatment of mold contaminated surfaces, however this may be extremely costly. In the state of Texas there were an estimated 44,285 insurance claims for mold abatement in the years of 2000 and 2001, with a total estimated cost of $1,007, 038,839.

A spore-forming Bacillus species denoted SB 3086 (ATCC 55406) was described in WO 03/000051 as having antifungal activity, especially in relation to plant fungal diseases.

Clearly, anything, even alternatives, that may provide even the slightest inhibition of mold growth could translate into a substantial economic savings and/or healthier living environments.

SUMMARY OF THE INVENTION The present inventors have identified a number of spore-forming bacterial species as having antifungal properties/. e. the bacterial cells are capable of inhibiting fungal growth most likely through production and release of antifungal substances. The antifungal bacteria of the present invention are capable of producing very resilient spores which can remain dormant for years and can tolerate exposure to very harsh conditions including dessication.

The conditions for germination and growth of these bacterial spores were found to be very similar to the conditions under which fungal spores are known to germinate. The spores only

require a certain level of moisture and a suitable growth substrate to germinate, and once again form actively metabolizing cells. Suitable substrates for the bacterial cells may be found in a variety of materials, for instance gypsum wall boards include cellulose, a starch binder, and other nutrients. After adding or applying the bacteria or their spores to a number of different manufactured materials, the present inventors were able to demonstrate a resulting increase in the materials'resistance to fungal growths. The present inventors found, that when fungal and bacterial spores are both present in a material, under conditions conducive for spore germination, then the bacterial spores are activated sufficiently to effectively inhibit fungal growth.

Accordingly, in a first aspect, the invention relates to a method of preventing or inhibiting fungal growth on and/or in a manufactured material, the method comprising the addition of at least one bacterial cell and/or at least one spore thereof to the material during the manufacture of the material, and/or the application of at least one bacterial cell and/or at least one spore thereof to one or more surface of the material, wherein the at least one bacterial cell has antifungal activity and is capable of forming spores.

In the present context, the term"manufactured material"is intended to include any material manufactured by man, non-limiting examples of which could be construction materials, gypsum-based wall boards, clothes, shoes, carpets, timber, synthetic materials etc. Essentially biological materials are not included in this term, specifically excluded from this term are plants, seeds, plant shoots, plant roots, vegetative propagules, trees etc.

A bacterial spore in the present context has the meaning generally recognized in the art, non-limiting examples of spores according to the invention are the spores formed by many Bacillus species.

A bacterial cell is defined as having antifungal activity for the purposes of this invention if it produces a zone of inhibition of at least 0.20 millimeters from the edge of a bacterial colony to the edge of the growth of a fungal mycelia, when tested against at least one of the three commercially available fungi as listed in the following assay : In a 100 x 13 mm Petri dish containing 20 ml of solidified standard agar (agar 15 g/l, tryptone 5.0 g/l, yeast extract 2.5 g/l dextrose 1.0 g/I) a circular well with a diameter of 1.0 mm is cut or punched out in the center of the plate using-e. g. a sterile pipette-tip. A suspension of a fungus (Cladosporium herbarum ATCC 76226, Alternaria alternata ATCC 52170, or Stachybotrys chartarum ATCC 16026) is spread evenly on the plate in sufficient concentration to achieve lawn growth, and 10 microliters of a bacterial spore suspension is placed into the well. After 5 days incubation at 25°C, the clear zone of inhibition of fungal growth around each well is measured. The suspensions are prepared as outlined in the examples below. This assay

may be modified and/or scaled to accommodate high throughput screening and use of robotic sampling e. g. a"colony picker", reference is made to WO 01/32834.

In a second aspect, the invention relates to an antifungal mixture or composition, suitable for preventing or inhibiting fungal growth on or in a manufactured material, said mixture or composition comprising at least one bacterial cell and/or spore thereof, wherein the at least one bacterial cell has antifungal activity and comprises one or more Bacillus cell having the identifying characteristics of a Bacillus cell chosen from the group of Bacillus cells consisting of SB 3001 (deposited on 14 March 2003 with ATCC and assigned patent deposit designation PTA-5059), SB 3086 (ATCC 55406), Bacillus SB 3114 (ATCC 14580), Bacillus SB 3115 (ATCC 53757), Bacillus SB 3130 (ATCC 6051A), and Bacillus SB 3131 (ATCC 12713).

And in final aspect, the invention relates to a manufactured material comprising at least one bacterial cell having antifungal activity and/or at least one spore thereof, said material having an increased resistance to fungal growth when compared to an otherwise identical material under conditions conducive to fungal growth.

Whether the manufactured material has an increased resistance to fungal growth when compared to an otherwise identical material under conditions conducive to fungal growth may be tested using the following assay: The two otherwise identical samples of the manufactured material are placed in glass Petri dishes and steam sterilized by autoclaving.

A fungal spore suspension of Cladosporium herbarum ATCC 76226, Alternaria alternata ATCC 52170, or Stachybotrys chartarum ATCC 16026, prepared as outline in the examples section below, is then either sprayed or swabbed over the samples to provide a fungal inoculum. To the Petri dishes is then added enough volume of SSC medium (-30 ml) to provide a sufficiently high water activity for spore germination, through the wicking action of the materials. The plates are incubated for at least 5 days and are then assesed visually and compared for the presence of fungal growth.

DETAILED DESCRIPTION The first aspect of the invention relates to a method of preventing or inhibiting fungal growth on and/or in a manufactured material, the method comprising the addition of at least one bacterial cell and/or at least one spore thereof to the material during the manufacture of the material, and/or the application of at least one bacterial cell and/or at least one spore thereof to one or more surface of the material, wherein the at least one bacterial cell has antifungal activity and is capable of forming spores. The second aspect relates to a manufactured material comprising at least one bacterial cell having antifungal activity and/or at least one spore thereof, said material having an increased resistance to fungal growth

when compared to an otherwise identical material under conditions conducive to fungal growth.

As already mentioned above, the inventors envision embodiments relating to the method the first aspect and the material of the second aspect, wherein the manufactured material comprises construction materials, in particular wherein the manufactured material comprises cellulose and/or gypsum. Also materials comprising paper and/or cardboard are envisioned as preferred embodiments.

A particularly preferred embodiment relates to a method of the first aspect or material of the second aspect, wherein the manufactured material comprises gypsum boards, plywood, and ceiling tiles, especially gypsum wall boards.

However, the method of the first aspect or the material of the second aspect of the invention also relates to many other materials that are susceptible to fungal growths, other preferred embodiments relate also to clothes items, garments, footwear, shoes, carpets, wowen fabrics, fibers, synthetic and natural such, cotton, wool.

A preferred bacterial cell for the method of the first aspect or the material of the second aspect is a Gram positive bacterium including, but not limited to, a Bacillus cell, e. g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis. In a preferred embodiment, the bacterial cell is a Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus or Bacillus subtilis cell. In an even more preferred embodiment, the bacterial cell comprises a Bacillus cell having the identifying characteristics of one or more Bacillus cell chosen from the group of Bacillus cells consisting of SB 3001 (deposited on 14 March 2003 with ATCC and assigned patent deposit designation PTA-5059), SB 3086 (ATCC 55406), SB 3114 (ATCC 14580), SB 3115 (ATCC 53757), SB 3130 (ATCC 6051A), and SB 3131 (ATCC 12713).

Many isolates of Bacillus have been found by the present inventors to be effective against a broad array of fungal growths. Because of such broad range efficacy and the availability of relatively large-scale growth and delivery of these organisms, practitioners in the industry can now employ biological control methods against fungal infestation of manufactured materials. Some of the advantageous properties of these species include: efficient and rapid growth under standard large-scale fermentation conditions and media; broad temperature range for optimal growth (18°C-50°C) ; propensity to form stable spores in a high percentage of vegetative bacterial population under known fermentation procedures; ability of the spores to survive for long periods under sub-optimal conditions, such as extremes of temperature and desiccation; inherent resistance of the spores to UV

sunlight; ability to grow under low oxygen conditions typically encountered in the manufactured materials of the invention; and their verified safety (generally recognized as safe; GRAS) to animals and plants based on standard toxicology and pathogenicity studies.

The medium used to culture the cell may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e. g. in catalogues of the American Type Culture Collection). <BR> <BR> <P>The media are prepared using procedures known in the art (see, e. g. , references for bacteria and yeast; Bennett, J. W. and LaSure, L., editors, More Gene Manipulations in Fungi, Academic Press, CA, 1991).

In a preferred embodiment, the invention relates to a method of the first aspect, wherein the at least one bacterial cell is added or applied in the form of a liquid mixture or composition, said mixture or composition comprising one or more active vegetative cell and/or spore thereof, and a carrier liquid such as, but not limited to, water, and mixtures of water and other liquids e. g. surfactants, preservatives, fungicides, biosupplements.

Accordingly, in a preferred embodiment, the mixture or composition further comprises one or more surfactant, one or more preservative, one or more fungicide, one or more biosupplement, and/or one or more bacterial substrate.

The term"biosupplement"as used herein is defined as commonly recognized inorganic nutrients and micronutrients. Examples of such biosupplements include : i) sea plant extracts, such as that obtained from the Norwegian kelp plant Ascophyllum nodosum ; ii) animal manures and processed sewage sludge ; iii) animal-derived products, such as bone, feather, hair, and fish meal ; iv) humic and fulvic acid materials, such as from mined leornardite, peat; v) paper processing by-products (e. g. lignin sulfonate) ; vi) compost material obtained by microbial metabolism and partial degradation of plant and animal waste substances; and the like.

A mixture or composition in accordance with the present invention is a concentrated liquid formulation which may include micronutrients such as organic nitrogen, cheated zinc, cheated iron, as well as natural organic growth enhancing agents, such as sea plant extract, in addition to the active ingredient of the antifungal bacterial cell and/or spore. Of course, as it may be suggested to those skilled in the art, other levels and types of nutrients, organic materials and the like may be substituted or added in the formulation.

In a preferred embodiment, the mixture or composition is concentrated, and the concentrate is then diluted with about 2 to 300 volumes of liquid before application.

Alternatively, a dry formulation of the mixture or composition of the invention may be prepared by spray-drying the antifungal bacterial cell and/or spore into a flowable powder.

This may then be blended with dry nutrients, micronutrients, and organic growth agents. For application, the dry formulation containing the spray-dried spores may be suspended in water e. g. to the preferred spore concentration of about 8.0 x 107 CFU/ml. It will be appreciated that these examples are merely illustrative and not limiting of the invention.

Many variations and modifications will be apparent to those skilled in the art and all such variations and modifications are included within the purview and scope of the claims.

Accordingly, a preferred embodiment relates to a method of the first aspect, wherein the at least one bacterial cell is added or applied in the form of a dust, powder, spray, or granule comprising one or more spore of the at least one bacterial cell, preferably said dust, powder, spray, or granule further comprises one or more surfactant, one or more preservative, one or more fungicide, one or more biosupplement, and/or one or more bacterial substrate.

If a material is susceptible to fungal infections, but does not in itself comprise suitable substrates for the bacterial spores, then suitable substrate (s) for the bacterial spores may be added to the spore-containing formulation of the invention. Additional antifungal substances may also be added to the formulation to act in concert with the antifungal activity of the bacterial cells, e. g. the growth rate of the fungi could be slowed by the additions of carbonates to the final formulations.

The second aspect of the invention relates to an antifungal mixture or composition, said mixture or composition comprising at least one bacterial cell and/or spore thereof, wherein the at least one bacterial cell has antifungal activity and comprises one or more Bacillus cell having the identifying characteristics of a Bacillus cell chosen from the group of Bacillus cells consisting of SB 3001 (deposited on 14 March 2003 with ATCC patent deposit designation PTA-5059), SB 3086 (ATCC 55406), Bacillus SB 3114 (ATCC 14580), Bacillus SB 3115 (ATCC 53757), Bacillus SB 3130 (ATCC 6051A), and Bacillus SB 3131 (ATCC 12713).

Preferably, the mixture or composition of the second aspect is a liquid, a dust, a powder, a spray, or a granule, and preferably it further comprises one or more surfactant, one or more preservative, one or more fungicide, one or more biosupplement, and/or one or more bacterial substrate.

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the methods and materials described herein are preferred. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are only exemplary and not limiting.

EXAMPLES The test bacteria were grown on standard agar plates (agar 15 g/l, tryptone 5.0 g/l, yeast extract 2.5 g/l dextrose 1.0 g/I) for 1-2 weeks at room temperature. At the end of the incubation period, the plates were scraped of their surface growth and the bacterial paste was suspended in 0.3 mM potassium-phosphate buffer pH 6.3 with 0.9 mM MgCI2 added.

The resulting spore suspension had concentration of from about 104 to about 1012 CFU/ml.

All fungi were grown and maintained on potato dextrose agar (PDA) at room temperature. Unless otherwise specified the fungal species used were as follows : Stachybottys chattarum ATCC 16026, Cheatomium globosum ATCC 6205, Cladosporium herbarum ATCC 76226, and Alternaria alternata ATCC 52170. The, Aspergillus oryzae, Cladosporium sp. stud-1, Cladosporium sp. stud-2, Phoma sp. JT, Phoma sp. MT, Penicillium sp. JT, Trichophyton rubium and Penicillium sp. TR were isolated at Novozymes Biologicals Inc. Spores and mycelia from the various fungi were harvested by scraping the plate and material into potassium-phosphate buffer pH 6.3 with 0.9 mM MgCI2. Further maceration of the mycelia and release of the spores was accomplished by vigorous agitation assisted by a vortex mixer. This material was then passed over sterile glass wool to remove the larger fragments. The resulting suspension consisting of fungal spores and mycelia fragments was either sprayed or swabbed onto the surfaces to be tested.

Example 1 Screening of the bacterial species consisted of placing an agar plug containing the test fungus in the center of a 100 mm Agar plate containing PDA. On either side of the fungal plug was inoculated (-3 cm from fungal plug) the bacillus suspected of containing anti-fungal activity. The plates were incubated at room temperature for 7-10 days. The amount of fungal inhibition was assessed visually by the inhibition of the mycelia extension across the surface of the plate as indicated by a clear zone absent of mycelia growth. These were scored from 0-3, where 0 indicates no inhibition, and 3 indicates the greatest relative inhibition; results are shown in Table 1 below. Species 3001 3081 3086 3104 3105 3106 3116 3129 3130 3133 3137 A. alternata 3 2 3 2 1 1 2 0 3 1 3 ATCC 52170 C. herbarum 3 3 3 3 3 3 0 2 3 3 3 ATCC 76226 Cladosporium 3 3 3 0 0 0 0 0 3 3 3 sp. stud-1 Cladosporium 3 2 3 3 2 2 2 2 2 2 2 sp. stud-2 Phoma sp. jt 3 2 3 3 3 2 0 1 0 1 0 Phoma sp. mt 3 3 3 3 2 1 0 1 1 1 1 Penicillium 3 3 3 3 3 3 3 3 3 0 3 sp. Jt Penicillium 1 2 1 2 2 2 1 2 2 0 1 sp. Tr

Table 1. Relative Inhibition of Fungal Species by Bacterial Species Bacillus licheniformis SB 3001 (deposited on 14 March 2003 with ATCC and assigned patent deposit designation PTA-5059), B. subtilis SB 3081, B. licheniformis SB 3086 (ATCC 55406), B. licheniformis SB 3104, B. subtilis SB 3105 (ATCC 55405), B. subtilis SB 3106, B. licheniformis SB 3116 (ATCC 21552), B. polymyxa SB 3129, B. subtilis SB 3130 (ATCC 6051A), B. polymyxa SB 3133 (ATCC 55407), and B. licheniformis SB 3137 (ATCC 21552) Example 2 Selected bacterial strains were tested for their antifungal activity on specific fungal species. This test was carried out for each strain in a 100 x 13 mm Petri dish that contained 20 ml of solidified standard agar (see above), wherein a circular well with a diameter of 1.0 mm had been cut or punched out in the center of the plate using a sterile pipette-tip. A suspension of one of the fungi was spread onto the plate in sufficient concentration to achieve lawn growth, and 10 microliters of a bacterial spore suspension was placed into the well. After 5 days incubation at 25°C, the zone of inhibition of fungal growth around each well was measured. The results are summarized in Table 2. Species C/. herbarum A. altemata S. chartarum Trichophyton Phoma sp. mt ATCC 76226 ATCC 52170 ATCC 16026 rubium SB 3001 5. 0 3. 0 3. 0 >10. 0 5. 0 SB 3086 5. 0 3. 0 3. 0 >10. 0 5. 0 Table 2. Antifungal activity of select bacterial species; the zone of inhibition is indicated in mm. All were done in triplicate with a maximum zone variation of 0.1 mm.

Example 3 Samples of common construction materials such as gypsum wall board, ceiling tile, wood fiber particle board, and plywood, were steam sterilized by autoclaving in glass Petri dishes. These samples were then inoculated in the Petri dishes with bacterial spores to a concentration of between 1x105 to 1x101° spores/cm2 (100 microliters of buffer) of the bacterial strain designated as SB 3086. Fungal spores were then either sprayed or swabbed over the sample surfaces to provide a fungal inoculum. To the Petri dishes was then added enough volume of SSC medium (-30 ml) to provide a sufficiently high water activity for spore germination, through the wicking action of the materials. The composition of the SSC medium was (all in g/l) : NH4CI (0.8), MgS04 (0.2), CaCI2*H2O (0.01), NaP04 (4.2), KH2PO4 (1.5), Fecal3 (0.005), FeS04*7H40 (0.00028), ZnS04*7H20 (0, 0014), MnS04*H20 (0.00084), CoCl2*6H20 (0.00024), CuS04*5H20 (0.00025), and NaMo04*2H20 (0.00024). The only carbon source for growth is provided in the construction material as such.

The plates were incubated for at least 5 days and the inhibition of fungal growth was assesed visually. In all cases, where the SB 3086 bacterial spores had been inoculated, there was an absence of fungal growth. This was true for wall board, ceiling tiles, wood fiber particle board, and plywood. The fungal strains tested were S. chartarum ATCC 16026, C. globosum ATCC 6205, C. herbarum ATCC 76226, A. alternaria ATCC 52170, and Aspergillus oryzae.

Example 4 To evaluate the ability of the different bacterial spores to inhibit three different fungal growths on wall board, three 10 x 10 cm pieces of gypsum wall board were each partitioned into 49 (7x7) squares of 1 x 1 cm by cutting grooves through the paper surface of the board.

The grooves were sufficiently deep to render each square surface of paper-coating completely severed from the adjacent squares. In the center of each individual square was placed 100 microliters of bacterial spore suspension (in buffer), a number of squares were left blank on each piece as positive controls. Then the entire surface of the each wall board piece was sprayed with a fungal spore/mycelia suspension of Stachybotrys chartarum, Cheatomium globosum and Cladosporium herbarium. The pieces were then placed in a tightly covered dish with SSC media added as described above. The results were visually evaluated after a minimum of 5 days of incubation, and the following bacterial strains were effective in inhibiting the growth of all three fungal species: SB 3001 (deposited on 14 March 2003 with ATCC and assigned patent deposit designation PTA-5059) SB 3086 (ATCC 55406)

SB 3114 (ATCC 14580)<BR> SB 3115 (ATCC 53757)<BR> SB 3130 (ATCC 6051A) SB 3131 (ATCC 12713) The results from Examples 1-4 demonstrate that several Bacillus species are effective in inhibiting fungal growth on cellulose containing construction materials, such as wall board, ceiling tiles, particle board, and plywood. All of the bacteria listed are capable of forming spores resistant to environmental factors such as desiccation, UV-irradiation, and heat. The spores are extremely stable when dried.

Bacillus licheniformis SB 3001 has been deposited with the American Type Culture Collection, 10801 University Blvd. , Manassas, VA 20110, USA, on 14 March 2003, and assigned patent deposit designation PTA-5059. Bacillus licheniformis SB 3086 has been deposited with the American Type Culture Collection, 10801 University Blvd., Manassas, VA 20110, U. S. A. , under accession number ATCC 55406. The deposits shall be maintained in viable condition at the ATCC during the entire term of the issued patent and shall be made available to any person or entity for non-commercial use without restriction, but in accordance with the law governing the deposits.