WO2019170355A1

WO2019170355A1 - Substrate mixture for fungal material, fungal culture and use thereof

Info

Publication number: WO2019170355A1
Application number: PCT/EP2019/052989
Authority: WO
Inventors: Francis W.M.R. Schwarze; Javier RIBERA REGAL; Tanja Zimmermann
Original assignee: Empa Eidgenössische Materialprüfungs- Und Forschungsanstalt
Priority date: 2018-03-08
Filing date: 2019-02-07
Publication date: 2019-09-12
Also published as: DE102018105329A1; EP3606330A1; ECSP20052411A

Abstract

A substrate mixture for fungal material contains water, a binder and nanocellulose. The substrate mixture is suitable for producing a fungal culture or for storing, transporting or applying fungal material, such as fungal spores, mycelium and hyphae, and thus makes possible the treatment of materials based on cellulose, hemicellulose and/or lignin, soil and cultivated land, waste from agriculture and forestry, plants and wood, bioaugmentation, bioremediation or biological pest control.

Description

Substrate mixture for mushroom material, mushroom culture and their use

The invention relates to a substrate mixture for fungal material, a fungal culture, the use of the substrate mixture and the fungal culture and a method for the treatment of cellulosic, hemicellulose and / or lignin-based materials, soil and culture soil, agricultural and forestry waste, from

Plants and wood, for bioaugmentation, for bioremediation or for biological control of cholera.

The vegetative, long-lived body of a fungus is an extensive network of microscopic filaments (known as mycelium) that completely penetrate the soil, tree trunks, or other substrates in which the organism grows. Under

Ecologists recognize that the health of the soil is directly related to the presence, abundance and diversity of the fungal community. Healthy ecosystems include a variety of fungal communities. For example, mycorrhizal fungi (including many fungal fruit bodies) form a mutually dependent, beneficial relationship with the roots of host plants derived from

Trees over grasses to agricultural crops range. Saprophytic fungi (decomposers of wood and organic matter) are the primary decomposers in nature and work with a range of microorganisms and plants to degrade and recycle organic and inorganic compounds and materials. It has also been found that saprophytic fungi form a symbiotic, mutually beneficial relationship with a number of agricultural crops. For example, it is known that corn in the presence of straw bales inoculated with Stropharia rugosoannulata provides greater yields compared to unvaccinated straw bales. The no-till method of agriculture also benefits from the growth of

Basidiomycetes enclosing fungi that break down plant stoppers into nutrients. Parasitic fungi have their own role in a healthy ecosystem, although they can become overly destructive in unhealthy systems. A other broad class of decomposers are the more primitive, non-fruiting fungi imperfecti, which also include molds and yeasts.

Without the presence of fungi, few organisms can effectively break down the complex biopolymers cellulose and lignin, the two major components of woody plants. Cellulose and especially lignin are generally resistant to microbial attack and degradation. The fungi, especially white rot fungi that can degrade lignin, and brown rot fungi, the main decomposers of cellulose, produce a complex series of enzymes that completely oxidize the polymer structures to water and carbon dioxide. Both liquid substrate and solid substrate cultures of white rot fungi were

The subject of years of research into bioremediation, as shown by the large number of publications and patents in this field. Such saprophytic wood decaying white rot fungi have demonstrated the ability to treat contaminated sites such as polyhedral aromatic hydrocarbons (PAH), alkanes, creosote, dichlorodiphenyltrichloroethane (DDT), polychlorinated biphenyls (PCBs),

Pentachlorophenol (PCP) and trinitrotoluene (TNT), dioxin, nitrogenous compounds such as ammonium nitrate, urea, purines and putrescine, as well as agricultural waste and agricultural wastewater. These bioremediation processes, however, have significant limitations that hinder the transition from laboratory to large field applications and therefore in the

Generally not be used commercially. A particular problem is that there are no economical and effective systems for the large-scale application of white rot fungi available.

The saprophytic fungi have also proved to be efficient digestors for potentially harmful organisms such as coliform bacteria and nematodes. The voracious Austempilze (Pleurotus ostreatus) have proven to be parasitic to nematodes. Extracellular enzymes act like an anesthetic and numb the nematodes, allowing for the penetration of the mycelium directly into their immobilized bodies. However, the fungal material may also be derived from parasitic and / or saprophytic and / or symbiotic (endo- or ectomycorrhizal) fungi.

There is therefore a great deal of interest in fungi for applications such as the introduction of mycorrhizal fungi, bioaugmentation of soils, bioremediation, biological pest control and the production of fungi.

Typical methods for applying fungal spores and hyphae to the soil include carriers such as cereals, sawdust and wood shavings, alginate hydrogels with and without additional nutrient sources, vermiculite and peat optionally saturated with nutrient broths, vermiculite and rice flour, or cereal flour, straw or other agricultural waste products. which are overgrown with mushroom mycelium pelleted

Fungal inoculants and the like.

The usual methods of inoculation with fungi are typically expensive, labor-intensive and / or ineffective. Various techniques have been used to inoculate growing substrates with mushrooms. These include vaccination procedures for wood chips, compost, turf and soils. There are also methods of seeding soils with

Mushrooms known for bioremediation purposes.

Wood chips are typically inoculated by spreading mycelium impregnated sawdust and / or wood chips over the wood chips. Compost bunkers are typically similarly inoculated with cereal impregnated with mycelium, although in some cases also impregnated with mycelium

Sawdust can be used. The use of expensive inoculum with limited shelf life produced by labor and equipment intensive sterile culture techniques is one of the disadvantages of this approach.

Other methods are vaccination with spores or vaccination with mycelium that has been fermented under sterile conditions. In the first method can

Spores are collected and scattered. Preferably, however, mushrooms are dipped in water to produce a spore slurry, molasses, sugar and / or sawdust added to stimulate spore germination, aerated, incubated and the aqueous spore slurry applied. This approach and the similar approach to liquid mycelium grown under sterile conditions can be used successfully. However, these approaches require either Irish spore-producing fungi or sterile culture techniques, and must be applied during the time frame of vigorous spike growth after germination or inoculation, otherwise the mycelial fragments will not grow together into a contiguous mycelial mat. There is still a need for more convenient products and methods for extended application of biologically active spores and / or mycelial vaccine material.

Trees, lawns and seedbeds were inoculated with mycorrhiza species using tablets or gels made from spores or mycelium. Trees may also be vaccinated with mycorrhizal fungi by dusting the roots of seedlings with spores or fungal mycelium or by dipping the exposed roots of seedlings in water enriched with spores of the mycorrhizal species. Another method of vaccinating mycorrhizae requires the planting of young seedlings near the root zones of proven fungus-producing trees, whereby the seedlings can become infected with the mycorrhiza of an adjacent tree. After a few years, the new trees are dug up and transplanted. Another method involves sending spore mass to the root zones of trees. Such approaches are labor intensive, expensive, unsafe, and / or unsuitable for widespread use.

The fungus is often in an encapsulated form for such applications in a matrix consisting of polymers (alginate salts, carrageenan salts, iota-carrageenan salts, maltodextrin, corn starch, modified starch, whey protein concentrate, skimmed milk powder, dry yeast autolysate,

Dry yeast extract and polyvinyl alcohol), which do not allow high germination rates and long storage times. In addition, these carriers are often not in the correct application form and contain substances that are questionable in terms of environmental friendliness. The object of the present invention is therefore to enable a simple, sterile, cost-effective and rapid production, storage and use of fungal inoculum.

According to the invention, this object is achieved by a substrate mixture according to claim 1. The substrate mixture according to the invention is suitable for the production of a fungal culture or for the storage, transport or application of fungal material, such as fungal spores, fungal mycelium and hyphae, thus enabling the treatment of cellulosic, hemicellulose and / or lignin-based materials, terrestrial and Cultivated land, of agricultural and forestry waste, of plants and of wood, bioaugmentation, bioremediation or biological

Pest control.

Advantageous embodiments of the invention are the subject of the dependent claims.

According to the invention, the substrate mixture for fungal material contains water, a binder and nanocellulose. Cellulose with structures in the nanometer range is called nanocellulose.

For the purposes of the present invention, this means cellulose whose fibrils / fibers have a mean diameter in the range from 5 to 100 nm. The mean length of these cellulose particles is from 100 nm to several micrometers. Nanocellulose is widely differentiated on the basis of size and manufacturing method in nano- and microfibrillated cellulose (NFC and MFC), nanocrystalline cellulose (NCC), and bacterial nanocellulose (BNC). Nano- and microfibrillated cellulose (NFC and MFC), sometimes referred to as nano- or microfibrils or simply referred to as fibrillated cellulose, is obtained by high pressure homogenization of pulp at high temperatures. Nanocrystalline cellulose (NCC), sometimes called cellulose nano or microcrystals, cellulose crystallites, cellulose whiskers, rod-shaped cellulose, is obtained by partial hydrolysis of classical cellulose. In contrast, bacterial nanocellulose (BNC), also referred to as bacterial cellulose, microbial cellulose or biocellulose, by the biochemical polymerization of low molecular weight building blocks (eg, alcohols and sugars, such as glucose). The sources of nanocellulose are wood, sugar beet, potato tuber, hemp, flax, wood, cotton, wheat straw, mulberry bark, ramie, avicel, tunicin, cellulose from algae and bacteria, and generally wood pulp, paper pulp, plant pulp or any other biological pulp

Cellulose source in question. For an overview of nanocellulose, see the article by D. Klem et al, Nanocellulose: A New Family of Nature-Based Materials, Angew. Chem. Int. Ed., 2011, 50, 5438-5466.

Nanocellulose serves as an energy or carbon source for the fungi. In addition, the nanocellulose influences the theological properties of the substrate mixture and acts as a gelling agent. According to the invention, the nanocellulose is selected from the group consisting of nano- and micro-fibrillated cellulose (NFC and MFC), nanocrystalline cellulose (NCC), and bacterial nanocellulose (BNC) or mixtures thereof. The content of nanocellulose in the substrate mixture is preferably 0.5 to 20 wt .-% and particularly preferably 1 to 15 wt .-%.

The binder serves to adjust the rheological properties of the substrate mixture (gelling agent, thickener). The binder is not particularly specified and can be selected by the user according to the requirements of the substrate mixture. However, the binder should preferably be ecologically safe, i. biodegradable and in particular compatible with the fungi to be used.

Suitable binders are alginate, agar-agar, carrageenan, locust bean gum, guar gum, tragacanth, gum arabic, xanthan, gellan, pectin, modified starch, alkylcellulose, hydroxyalkylcellulose, carboxyalkylcellulose and their alkali, alkaline earth and ammonium salts, poly (meth) acrylic acid and theirs Alkali,

Alkaline earth and ammonium salts, polyvinyl alcohol, polyacrylamide, polyvinylpyrrolidone, polyethylene glycol and mixtures thereof. Particularly preferred binders are alkali, alkaline earth and ammonium salts of Polyacrylic acid and most preferably the binder is sodium polyacrylate.

The content of binder may be selected according to the wishes of the user or the requirements of the substrate mixture in use, for example to adjust a desired viscosity.

Suitable contents of binder in the substrate mixture are from 0.1 to 1.0% by weight, preferably from 0.2 to 0.8% by weight or particularly preferably from 0.3 to 0.5% by weight.

%.

The substrate mixture may further contain other conventional additives to promote the growth of the fungi, such as nutrients such as organic or inorganic

Carbon, nitrogen, sulfur and phosphate sources, inorganic salts, trace elements, inhibitors, buffer substances and growth factors. Corresponding additives are well known in the art.

Suitable nitrogen sources are, for example, ammonium salts, nitrates or urea, it being possible for the contents to be from 0.05 to 0.2% by weight. In addition to the nanocellulose discussed above, for example, glucose and glycerol may be used as the further carbon source, with preferred contents being from 0.2 to 0.4% by weight. Further, as nutrients, malt extract (preferably 0.1 to 4% by weight), yeast extract (preferably 0.1 to 0.5% by weight), peptone (preferably 0.1 to 1% by weight), TBS ( tryptone soya broth) (preferably 1 to 4

Wt .-%) and tyrosine (preferably 1 to 2.5 wt .-%) into consideration.

A fungal culture according to the invention contains the substrate mixture according to the invention and fungal material. The fungal material may be fungal spores, fungal mycelia or hyphae. The fungal material may be derived from saprophytic fungi and, in particular, wood decomposing fungi. In particular, the fungi are species of the department Ascomycota and

Basidiomycota. Particularly suitable candidates are, for example, Physisporinus vitreus and Kretzschmaria deusta. The substrate mixture according to the invention can be used for producing a fungal culture or for storage, transport or application of fungal material (fungal spores, fungal mycelia or hyphae).

To prepare the substrate mixture according to the invention, the binder and the nanocellulose are simply dissolved or suspended in water. The substrate mixture thus obtained is then added with the fungal material (fungal spores, fungal mycelium or hyphae) to obtain the fungal culture of the present invention. The fungal culture thus obtained can be further incubated to grow the fungi. Alternatively, a slurry of nanocellulose, optionally with the other additives described above, may be added to the fungal material and this slurry incubated to grow the fungi first and then the binder is added.

This means that the nanocellulose in the fungal culture is at least partially digested by the fungus.

The fungal culture according to the invention can be used for the treatment of cellulose, hemicellulose and / or lignin-based materials, soil and culture soil, agricultural and forestry waste, plants and wood, for bioaugmentation, bioremediation or biological pest control.

The method of treating cellulose, home cellulose and / or lignin based materials, soil and crop, agricultural and forestry wastes, plants and wood, for bioaugmentation, bioremediation or biological pest control includes application a fungal culture of the invention to the object to be treated.

The invention will be further explained below with reference to an example.

Kretzschmaria deusta was cultured for 2 weeks in Petri dishes on 2% MEA. Mycelium from actively growing cultures was then used to become a medium which contained 100 g of nanocellulose, 1.5 g of malt extract and 150 ml of sterilized water. It was incubated for 2 weeks at 24 ° C and 65% relative humidity. Thereafter, the colonized nanocellulose was mixed with a blender at 5000 rpm for 30 seconds. Then, 250 g of the mycelial dispersed nanocellulose were mixed under sterile conditions with 1 g of sodium polyacrylate and 500 ml of sterile water. Different amounts (1.5, 2.0 and 2.5 km ² ) of the mycelium / substrate mixture were applied to the surface of beech, ash and ahomwood with dimensions of 35 x 25 x 5 cm. The wood was incubated for four weeks in a chamber over water at 24 ° C and 65% relative humidity. After 7 days the wood surface was partially colonized (extension about 5 cm) and after four weeks the wood was completely colonized by Kretzschmaria deusta (the wood surface was completely covered).

Claims

Claims:

1. Fungus material substrate mixture containing water, a binder and nanocellulose.

2. Substrate mixture according to claim 1, characterized in that the nanocellulose is selected from the group consisting of nano- and micro-fibrillated cellulose (NFC and MFC), nanocrystalline cellulose (NCC), and bacterial nanocellulose (BNC) or mixtures thereof.

3. Substrate mixture according to one of the preceding claims, characterized in that the binder is selected from the group consisting of alginate, agar-agar, carrageenan, locust bean meal, guar gum, tragacanth, gum arabic, xanthan, gellan, pectin, modified starch, alkylcellulose , Hydroxyalkylcellulose, carboxyalkylcellulose and their alkali, alkaline earth and ammonium salts, poly (meth) acrylic acid and its alkali, alkaline earth and ammonium salts, polyvinyl alcohol, polyacrylamide, polyvinylpyrrolidone, polyethylene glycol and mixtures thereof, and is preferably sodium polyacrylate.

4. Substrate mixture according to one of the preceding claims, characterized in that the content of nanocellulose 0.5 to 20 wt .-%, and / or the content of binder is 0.1 to 1 wt .-%.

5. Substrate mixture according to one of the preceding claims, characterized in that the substrate mixture further contains nutrients such as organic or inorganic carbon, nitrogen, sulfur and phosphate sources, inorganic salts, trace elements, inhibitors, buffer substances and growth factors.

A fungal culture containing a substrate mixture according to any one of the preceding claims and fungal material selected from the group consisting of fungal spores, fungal mycelium and hyphae.

A fungal culture according to claim 6, wherein the fungal material is derived from parasitic and / or saprophytic and / or symbiotic fungi and, in particular, wood decomposing fungi.

The fungal culture according to any one of claims 6 or 7, wherein the nanocellulose is at least partially digested by the fungus.

9. Use of a substrate mixture according to any one of claims 1 to 5 for the production of a fungal culture or for storage, transport or application of fungal material selected from the group consisting of fungal spores, fungal mycelium and hyphae.

10. Use of a fungal culture according to one of claims 6 to 8 for the treatment of cellulosic, hemicellulose and / or lignin-based materials, soil and culture soil, of agricultural and forestry waste, of plants and of wood, for bioaugmentation, for bioremediation or for biological control of cholera.

11. A method for the treatment of cellulose, home cellulose and / or lignin based materials, soil and culture soil, agricultural and forestry waste, plants and wood, for bioaugmentation, bioremediation or biological pest control by application or application A fungal culture according to any one of claims 6 to 8.