EP4677061A1

EP4677061A1 - A method for preparing a product comprising water repellent low-density fiber-based material, a product comprising water repellent cellulose fiber foam and use of cellulose fiber foam for preparing the product comprising water repellent cellulose fiber foam

Info

Publication number: EP4677061A1
Application number: EP24725920.3A
Authority: EP
Inventors: Geza SZILVAY; Tiina PÖHLER; Anniina VALTONEN; Petri Jetsu; Ali Harlin; Philippe AMSTISLAVSKI
Original assignee: VTT Technical Research Centre of Finland Ltd
Current assignee: VTT Technical Research Centre of Finland Ltd
Priority date: 2023-04-13
Filing date: 2024-04-10
Publication date: 2026-01-14
Also published as: FI20235420A1; WO2024213832A1

Abstract

A method for preparing a product comprising water repellent low-density material, the method comprising providing a product comprising dewatered foamed cellulose fiber foam, providing an aqueous dispersion of mycelium, applying the aqueous dispersion of mycelium on the surface of the dewatered foamed cellulose fiber foam to obtain a treated foamed cellulose fiber foam, incubating the treated foamed cellulose fiber foam for a time period enabling the mycelium to grow into the treated foamed cellulose fiber foam to obtain a composite product comprising mycelium filament network incorporated in the cellulose fiber foam, and drying the composite product at elevated temperature to obtain the product comprising water repellent low-density material. A product comprising water repellent cellulose fiber foam comprising mycelium filament network incorporated in the cellulose fiber foam, wherein the concentration and/or density of the mycelium filament network in the cellulose fiber foam is higher near the surface of the cellulose fiber foam compared to the interior of the cellulose fiber foam. Use of cellulose fiber foam for preparing a product comprising water repellent cellulose fiber foam.

Description

A method for preparing a product comprising water repellent low-density fiber-based material, a product comprising water repellent cellulose fiber foam and use of cellulose fiber foam for preparing the product comprising water repellent cellulose fiber foam

Field of the application

The present application relates to a method for preparing a product comprising water repellent insulation material and to a product comprising water repellent cellulose fiber foam. The present application also relates to use of cellulose fiber foam, or a product comprising cellulose fiber foam, for preparing the product comprising water repellent cellulose fiber foam and/or insulation material.

Background

The need for environmentally sustainable insulation is steadily growing and driving the demand for alternatives to petroleum-based plastics and other carbon-positive materials. Common synthetic thermal insulation materials, such as extruded polystyrene, have the second highest CO2 emissions and environmental impact of all building materials behind steel.

Cellulose fiber based materials can achieve carbon negativity and are recyclable, compostable, affordable and light-weight. Their carbon storing capacity provides a marked environmental benefit over synthetic incumbents used in thermal insulation and packaging. Foamed cellulose and its bio-based composites also provide an opportunity to reduce carbon emissions and plastic pollution associated with thermal and acoustic insulation and product packaging, however, some difficult challenges remain to be solved. Foamed cellulose materials are hygroscopic, like most bio-based foams, and their thermal and mechanical properties are sensitive to moisture content and do not inhibit microbial growth.

Concerns over the impact of plastic pollution on human health and ocean, greenhouse gas (GHG) emissions, and the mounting clean-up costs of plastics provide an impetus for the current exploration of biologically produced and biodegradable composite materials. These efforts are synergetic with the climate action policies aimed at the reduction of single-use plastics from all sources in the consumer products and in the industries where plastics have long been a mainstay. Plastics comprise 90% of all marine debris, with single-use food and beverage containers being one of the most common items found in ocean and coastal waters. As most plastics resist degradation the environmental pollution accumulates over time. These concerns provided the motivation for research into bio-based alternatives to plastics. Such alternatives are actively sought by a wide range of industries including the food packaging, cold-chain shipping and construction industry.

There is a need to find improved biobased materials for insulation, packaging, nonwoven fabrics, interior design objects and similar uses. Such materials should tolerate challenging conditions, such as moisture, grease, microbes as well as thermal and mechanical stress. At the same time the materials shall be environmentally friendly, such as recyclable, biodegradable, carbon negative and microplastic free.

There is also a need to find improved methods for preparing the materials and products. The methods shall be simple, fast and enable forming products with high accuracy.

Summary

In the solutions presented herein it was found out how to overcome drawbacks of prior art and to prepare products which fulfil discussed needs. The present method may be applied to existing foamed cellulose products and to methods for preparing thereof to produce cellulose-mycelium composite products and also enable creating new types of products and operations. The preparation can be implemented as an industrial scale process, wherein the forming of the product is simple and fast. Products with desired shape, accuracy and other desired properties can be prepared efficiently.

The present methods enable providing soft water repellent foamed products tolerating mechanical forces, and having high quality and structural accuracy. The product can be obtained with simple methods which can be optimized specifically in terms of the formation of the product. This is achieved when a product comprising dewatered foamed cellulose fiber foam is formed before applying the mycelium to the formed dewatered product, which has a fixed shape which does not change during and/or after application of the mycelium. The present method was found to produce a different product compared to methods wherein the cellulose foam is not dewatered before contacting with mycelium. If the mycelium was applied to a moist substrate, which is in a mouldable state and does not exhibit a fixed shape, the mycelium can penetrate the moisture-containing substrate mass and the shape of the mass continues changing. Further, as the growth of mycelium takes a relatively long time, the nonfixed foam is prone to collapse.

Foam formed cellulose fiber materials potentially have many advantages over synthetic plastic foams. They originate from renewable sources, are able to store carbon, and provide recyclability and biodegradability. The foam forming process is well understood and enables the production of low-density cellulose fiber materials and is achieved by mechanically mixing water, cellulose fibers, and a foaming agent to create a wet, fibrous foam, where the air bubbles keep the fibers apart until dewatering and drying. Foam formed cellulose materials provide a sustainable alternative to synthetic polymeric foams such as polystyrene and polyurethane because they are environmentally benign, and have comparable mechanical, thermal and acoustic insulation properties. Foam formed cellulose and other natural fiber materials have less embodied energy than synthetic polymer materials. In addition, these natural materials have low or no toxicity compared to many synthetic polymers.

High humidity environment and direct contact with water are common in temperature-controlled packaging and in building insulation, for example within external wall cavities. Prior art foam formed cellulose materials are not suitable for those applications as they are hygroscopic and the mechanical strength is affected by moisture. For example, tensile strength parallel to faces of foam formed cellulose decreases significantly when the relative humidity (RH) reaches above 50%. Therefore, there are limitations in prior art foam formed cellulose fiber materials, which may be overcome with the present solutions.

In the present disclosure it is shown how fungal mycelium influences the properties of light-weight cellulose-based materials. Different types of fungal composites were prepared and compared. To achieve mycelium-cellulose composite foams new biofabrication methods were found which combine mycological and foam forming techniques. The use of porous cellulosic substrates for the mycelium increases the efficiency of the biofabrication process and therefore afford cost-competitiveness in manufacturing. Properties such as compression strength, water contact angle, and thermal conductivity properties of the fabricated materials were tested and found suitable for a range of applications where plastic foams are currently being used.

The present application provides a method for preparing a product comprising water repellent low-density fiber-based material, the method comprising -providing a product comprising cellulose fiber foam, -providing an aqueous dispersion of mycelium,

-applying the aqueous dispersion of mycelium on the surface of the cellulose fiber foam to obtain a treated cellulose fiber foam,

-incubating the treated cellulose fiber foam for a time period enabling the mycelium to grow into the cellulose fiber foam to obtain a composite product comprising mycelium filament network incorporated in the cellulose fiber foam, and

-drying the composite product at elevated temperature, such as at 70°C or more, preferably without pressing, to obtain the product comprising water repellent low- density fiber-based material.

The present application also provides a product comprising water repellent cellulose fiber foam comprising mycelium filament network incorporated in the cellulose fiber foam, wherein the concentration and/or density of the mycelium filament network in the cellulose fiber foam is higher near the surface of the cellulose fiber foam compared to the interior of the cellulose fiber foam.

The present application also provides use of cellulose fiber foam, or a product comprising cellulose fiber foam, for preparing the product comprising water repellent cellulose fiber foam.

The main embodiments are characterized in the independent claims. Various embodiments are disclosed in the dependent claims. The embodiments and examples disclosed herein are mutually freely combinable unless otherwise explicitly stated.

Biological fabrication is a novel avenue for the manufacture of bio-based materials by employing the growth of living cells for the fabrication process, rather than chemical and or physical manufacturing processes. The biological fabrication has potential in the future carbon-neutral society. Mycelium-based materials provide a prominent example of biological fabrication where the fungal cellular mass propagates through hyphal tip extension and branching, resulting in a bottom-up assembly of a microscopic fibrous network. The mycelium comprises a three- dimensional network of hyphae having a diameter of less than 10 micrometers. This growth process can be utilized in the assembly of fibrous networks in composites. Such biologically-bound fiber materials can find applications in various everyday applications, ranging from bulk insulation, leather-like fabrics, and packaging materials, to biomedical and hygienic products and filter materials.

Mycelium growth requires a nutritive media, containing for example lignocellulosics. As the cellulose fibers are gradually valorized by the fungus, they are completely or partially consumed and replaced by the growing mycelium biomass. The growing mycelium breaks down the nutritive medium through mechanical force and by enzyme secretion. The resulting three-dimensional mycelium architecture acts as a biological binder or a scaffold. As the mycelium converts the nutritive media or substrate into living cells it cements the substrate, and a multi-scale bio-composite material is produced. The robust growth of the fungi, their ability to bind and valorize various feedstocks, including by-products of forestry and agriculture as nutritive media make them an attractive candidate for novel biobased materials. In nature, most filamentous fungi, including many members of phyla basidiomycota and ascomycota, have evolved to decompose wood and other lignocellulosics. Cellulose is the most abundant naturally polymer on the Earth, accounting for 30-50% of the plants dry weight. It represents a massive source of energy for the fungi and for other organisms. Wood pulp, the principal source of industrial cellulose, is widely available with a global production of 176 million tons in 2015.

Currently, the limiting factor for the fabrication of the mycelium-based materials with prior art methods is the low efficiency of the process, which involves mechanical mixing mycelium with nutritive solid media or substrate (i.e., agricultural waste or chopped wood), and water up to 70% of water by weight before incubation. Agricultural waste or chopped wood fibers are not universally available, is bulky, challenging to transport and store at the fabrication site. This substrate is heavy, wet and perishable due to its contamination by other organisms it needs to be sterilized, which requires a large autoclave for at scale production, and specialized facilities and the costly autoclaving equipment. This need for sterilization may negate the low-carbon emissions potential of such mycology-based materials. These operations are not in the common general knowledge of a cellulose foam product manufacturer. The present method can benefit the whole production chain and any operators operating in the chain. For example initial cellulose foam products can be manufactured without involving the mycelium, which enables many different operators to carry out the manufacturing with their existing equipment without need to invest new equipment, reagents and/or without specific knowhow, such as in the field of biology. Another operator can then prepare the composite product, which enables each operator to use their best resources and operate in economical manner. Different method steps may be carried out at different locations and/or at different facilities, and/or at different time and/or by different operators. The composite product may be further processed by another operator and/or at different location and/or facilities. Two or more, or all, process steps can be also carried out by one operator and/or at the same facilities.

The present approach provides improved energy efficiency of production of mycelium-based materials and lesser energy inputs and results in mycelium- cellulose composites that are competitive with the synthetic polymeric foams on compression strength, thermal conductivity, and water contact angle.

Antimicrobial properties of the mycelium-cellulose composites also deserve attention. Many viruses remain active on common surfaces for extended period of time, increasing the risk of fomite transmission of viral illnesses. For example, active SARS-CoV-2 and SARS-CoV-1 viruses have been shown to be viable for up to 72 hours on the common plastic, cardboard and stainless steel surfaces. Present initial screening for the antiviral and antimicrobial properties of the mycelium-cellulose composites produced with the 4 polypore species shows that extracts of all 4 species caused a statistically significant decrease in active MS-2 virus and in Staphylococcus aureus over a 24 h contact period. The present materials provide a decrease in active viruses and microbe pathogens, which can be utilized in pathogen reduction on packaging, face masks, PPE, and high-touch surfaces (HITES).

The present findings are relevant for the scaling of the production of myceliumbased materials. For example, the feedstock for the tested materials can be either thermo-mechanically or chemically derived cellulose from the locally harvested biomass resulting from forestry operations, such as small-dimensional timber, or formed into compact sheets for easy transport and storage. The mycelium-based materials provide many advantages over the synthetics in creating a carbon-negative manufacturing. From the vantage point of circular economy and environmental sustainability such materials are advantageous because they can be produced with low energy inputs, are bio-degradable, and can potentially be recycled in the existing paper and cardboard stream. The final product can be post-processed, pressed, and shaped to produce insulating panels, packaging materials, and other objects. At the end of the lifecycle these biodegradable composites can be recycled, composted or re-used, for example as animal feed, soil fertilizers and substrate plant seedlings.

The present mycelium-cellulose composites can be applied in several applications where plastics or extruded polystyrene foams, for example known as Styrofoam, and the like materials are currently used, because mycelium-based materials are benign to humans and the environment and consist of natural polymers and are biodegradable. In the jurisdictions with established recycling systems cellulose- mycelium composites with high cellulose content can be recycled in the paper or cardboard stream.

Brief description of the figures

Figure 1 shows, on the left, the mixer and mixing vessel for generating fiber foam, on the right top, a sheet mould, and on the right bottom, a schematic picture of the pouring of fiber foam via a funnel into a sheet mould comprising a forming wire.

Figure 2 shows a schematic structure of the kraft honeycomb board used to produce samples of honeycomb-mycelium composite. 1. Kraft paper cover. 2. Webbing of wells by created by strips of kraft paper glued at predetermined distance to form a honeycomb pattern. 3. Depth of the wells, 10 mm. 4. Thickness of the kraft webbing, approximately 0.5 mm. 5. Width of the wells, 12 mm. 6. Second kraft paper cover was removed to expose the ridged wells to enable the mycelium growth throughout the entire board.

Figure 3 shows a mixing vessel with a disk-type stirrer used for making of the Co-foam material.

Figure 4 shows a process of inoculation of a single honeycomb-mycelium board composite. Semi-liquid fungal inoculum (1) poured into the bottom of into a covered tray (2) (lid removed on this illustration). Then kraft honeycomb board with kraft paper removed on the bottom side of the board (3) is placed in the tray so that the ridges of the honeycomb wells (4) are pressed into the semi-liquid fungal inoculum to ensure contact between mycelium inoculant and the board. The ridges of the honeycomb wells are partially submerged into the inoculum as the bottom of the board touches the bottom of the tray. Additional amount of the inoculum (5) is poured and spread over the top of the honeycomb board to facilitate the growing mycelium filling the inside of the honeycomb wells (6).

Figure 5 shows a composite made from three kraft honeycomb-mycelium boards. 1 . Several boards were pressed and held together from the top and the bottom (shown by the arrows). 2. A sheet of kraft paper that has been added to cover the honeycomb wells of the middle board. 3. Enlarged view of the mycelium growth on the surface of the kraft paper cements the boards together. 4. A schematic view of the mycelium propagation inside the hexagonal honeycomb wells.

Figure 6 shows Table 2 presenting photos of the three different composite materials without and with mycelium.

Figure 7 shows a microscopic image of mycelium (stained blue) growing inside the highly porous cellulose substrate. Foamed pulp with T. versicolor was stained with cotton blue in lactic acid.

Figure 8 shows SEM images of the mycelium-cellulose materials showing the colonization of the medium by T. versicolor.

Figure 9 shows SEM images of sample cross-sections. Images a) and c) of the surface of the samples with a layer of pure mycelium. Images b) and d) are of the inside of the samples and show mycelium interspersed among the cellulose fibres.

Figure 10 shows compression strength at 50% deformation. N=1 .

Figure 11 shows recovery from compression. N=1 .

Figure 12 shows thermal conductivity for the mycelium-cellulose materials and controls without mycelium at 10°C and -7.5°C. Materials 1 and 2 have consistently lower thermal conductivity than the honeycomb-based materials. Figure 13 shows tests of antimicrobial activity of homogenized mycelium- cellulose composites produced with strains of Irpex lacteus, Fomes fomentarius, Trametes versicolor and Fomitopsis pinicola. Antimicrobial activity was determined using the modified standard method EN 1276: 2019 “Ell Chemical disinfectants and antiseptics ". Tested microbes were Escherichia coli, Staphylococcus aureus, Bacillus atrophaeus, Candida albicans, and Aspergillus niger, as well as the virus MS2.

Figure 14 shows different dried sheets prepared with the present method with different thicknesses (14A), sheets cut according to the size of carboard sheets (14B) and sheets with different thicknesses applied inside cardboard boxes (14C, 14D).

Figure 15 shows water durance test with T. versicolor Mycelium-CFF blocks compared to the control sample CFF. The mycelium containing Mycelium-CFF material survived shearing in water well as seen in the image after 2 min mixing, while the control sample of CFF without mycelium already disintegrated after 0.5 min mixing.

Figure 16 shows microcombustion calorimeter measurements of mycelium from T. versicolor (16A), Trichoderma reesei (16B) and bleached softwood kraft pulp (16C). The results show that both fungal mycelium have much lower total heat release and peak heat release rates compared to cellulose pulp.

Detailed description

In this specification, percentage values, unless specifically indicated otherwise, are based on weight (w/w, by weight, or wt%). If any numerical ranges are provided, the ranges include also the upper and lower values. In specific examples the open term “comprise” used herein can be limited with a closed term “consisting of’. The sizes or diameters disclosed herein, unless specifically indicated otherwise, refer to the smallest diameter, and may be presented as average or number-average diameter and in the case of small objects may be determined microscopically, such as by using optical or electron microscope, which may be equipped with a dedicated software for analyzing and/or outputting results. The present disclosure provides a method for preparing a product comprising or consisting of water repellent fiber-based material, such as water repellent low- density fiber-based material or water repellent lightweight fiber-based material. The fiber-based material may be also called as fibrous material or fiber-derived material. The fiber-based material may be or may be obtained from cellulosic material, for example it may be cellulose fiber based material. The present disclosure also provides a method for preparing a product comprising or consisting of water repellent insulation material. The present disclosure also provides a method for preparing a product comprising or consisting of water repellent cellulose fiber foam, more particularly comprising mycelium filament network incorporated in the cellulose fiber foam. The method may be a method for preparing a product having barrier properties, an antimicrobial surface and/or a product having fire retardant properties.

The methods may refer to the same method steps, and can be presented as a combination of two or more methods, and to production of products or materials with same features and/or properties. The material or the product can be used for a variety of applications, such as ones disclosed herein, including the applications benefiting from the water repellent properties, but also applications such as filtering and/or fire retarding.

The fiber-based material may be natural fiber-based material, such as comprise or consist of natural fibers. The low-density or lightweight as used herein may refer to material which has a lower density or weight than a corresponding material, which is not foamed. The lightweight may refer to foamed and/or to low-density. The low- density fiber-based material may refer to foamed fiber-based material. The low- density or lightweight may be specified as having a density, such as a bulk density, of 0.1 g/cm³ or less, such as 0.090 g/cm³ or less, 0.080 g/cm³ or less, or 0.070 g/cm³ or less.

The method comprises providing a product comprising or consisting of cellulose fiber foam. The product is formed beforehand, i.e. it is already formed or readymade, so that the product already exists before applying the mycelium. The cellulose fiber foam in the product is fixed, dried, cured, stabilized and/or sized. A hard foam product is obtained, which can be further handled, stored, transported, processed and treated, for example with industrial equipment and/or in an industrial process. The product has a shape, which may be the shape of the final product, or which may be modified to obtain the final product, such as cut and/or refined into the form of the final product. This product, which does not contain mycelium, may be called as a first product, and in the method a second product is obtained, which may be the final product or from which the final product may be obtained. The second product contains the mycelium incorporated in the cellulose fiber foam. During the method the shape of the product does not preferably change, or preferably it does not substantially change. Preparing the first product may be part of the method, or alternatively the first product is obtained as already prepared and/or as formed. The first product may be prepared by a different operator, and/or it may be a commercially available product.

The method may comprise providing cellulose fibers and forming the cellulose fibers into the cellulose fiber foam. The cellulose fiber foam may be in the form of a product or a part of a product, such as the first product, or it may be further processed to obtain the first product. The cellulose fibers may be formed into the cellulose fiber foam by using known methods.

Hjelt et al. Foam forming of fiber products: a review, Journal of Dispersion Science and Technology, 2002, Vol 43, No 10, 1462-1497 discloses aqueous foams for preparing lightweight materials from different raw materials. The review discloses structural and rheological properties of wet foams, forming methods, and various product applications. The disclosed cellulosic foams and methods for preparing thereof, as well as characterizing methods, can be applied to the present invention. Any other suitable methods for preparing a cellulose foam can be also used.

The cellulose fibers may be any suitable cellulose fibers that can be foamed, and they can be obtained from cellulosic or lignocellulosic material. The cellulose, or the cellulose fibers, may comprise or consist of pulp. One example of pulp is chemical pulp, which lacks lignin and therefore represents cellulosic material. The chemical pulp may be bleached chemical pulp, such as bleached Kraft pulp, for example bleached chemical softwood Kraft pulp (BSKP). The source of cellulose fibers may therefore substantially comprise only cellulose or substantially consist of cellulose, but in some cases also hemicellulose and/or lignin may be present. However preferably the source of cellulose fibers, or the cellulose fibers used for foaming, do not contain other types of fibers or fibrils, such as organic fibers, fibrils and/or polymers, especially synthetic, for example ones comprising thermoplastic or thermosetting polymers, and/or inorganic fibers, fibrils and/or polymers. The cellulose fibers may be obtained in a desired form, such as in the form of suitable pulp, for example from a cellulose pulp manufacturer or provider. The cellulose fibers may be provided as a commercial product. The provided cellulose fibers or the foamed product do not preferably contain fungi or other microbes.

The foamed cellulose may be prepared by providing cellulose or a source of cellulose, such as pulp, and providing one or more additional agents, such as one or more surfactant(s), one or more sizing agent(s) and/or one or more wet strength resin(s). The cellulose, for example in the form of dry pulp, may be disintegrated and dispersed in water or aqueous solution to obtain a suitable concentration/consistency.

For example a pulp concentration in the range of 1-3% by weight may be used, such as 1 .5-2.5% by weight. The sizing agent and the wet strength agent may be provided as 1-3% by fiber weight, such as about 2% by fiber weight. The surfactant may be provided with a dosage in the range of 1-3 g/l, such as 1 .5-2.0 g/l. The cellulose dispersion may be foamed by using a mechanical mixer, such as a stirrer. The mixing may be carried out in the presence of gas, such as air, so that the gas can be incorporated in the mixed cellulose dispersion. The mixing may be carried out until a desired foaming degree is obtained, which may be determined by percentage of gas content. The gas content may be for example in the range of 40-60% by volume.

The obtained cellulose fiber foam may be formed into a desired shape by using a mould, a wire and/or other suitable means. In one embodiment the forming into the cellulose fiber foam and/or to the first product is carried out in a mould and/or on a wire, such as in a mould with a wire bottom.

After the foam is settled, water may be filtered through the wire or other suitable permeable means to obtain a dewatered foam. The dewatering and/or forming of the product may be facilitated by applying pressure. However too high pressure shall be avoided to maintain the foamed form. The obtained product may be further dewatered and dried, such as at increased temperature, for example in an oven or by using other suitable heating means. For example the foamed product may be dried at less than 100°C, such as at 70-80°C, for example for at least 12 hours, such as for 12-24 hours, or less if applicable. After drying the products may be further heat treated at a higher temperature, such as over 100°C, for example at about 105°C, for a short period of time, to activate the sizing agent and to finalize the foamed product.

The cellulose fiber foam or the product comprising cellulose fiber foam, especially when dewatered and preferably finalized, may have the shape of the final product, which may comprise water repellent insulation material or other type of material discussed herein. The cellulose fiber foam may have a dry basis weight in the range of 250-800 g/m², such as 300-500 g/m², 250-450 g/m² or 300-450 g/m². Preferably the cellulose fiber foam is homogenous or substantially homogenous.

The product comprising or consisting of cellulose fiber foam, such as the first (foamed) product, may be provided as dewatered and preferably finalized. The product, or the cellulose fiber foam, may have a moisture content in the range of 5-40% by weight or lower, such as 5-20% by weight. This first product or the cellulose fiber foam may be used in the method in this moisture content, or the moisture content may be adjusted to obtain a moisture content which facilitates the growth and penetration of the mycelium into the first product. It was found out that when the mycelium is applied onto the surface of the foam, a suitable moisture content is in the range of 15-35% by weight or 20-35% by weight. The product may be provided in this moisture content.

In one embodiment the cellulose fiber foam has a moisture content in the range of 15-35% by weight prior to applying the aqueous dispersion of mycelium on the surface of the cellulose fiber foam.

One embodiment provides a method for preparing a product comprising water repellent low-density fiber-based material, the method comprising -providing a product comprising dewatered foamed cellulose fiber foam, -providing an aqueous dispersion of mycelium,

-applying the aqueous dispersion of mycelium on the surface of the dewatered foamed cellulose fiber foam to obtain a treated foamed cellulose fiber foam, -incubating the treated foamed cellulose fiber foam for a time period enabling the mycelium to grow into the treated foamed cellulose fiber foam to obtain a composite product comprising mycelium filament network incorporated in the cellulose fiber foam, and

-drying the composite product at elevated temperature, such as at 70°C or more, to obtain a product comprising water repellent foamed cellulose fiber foam comprising mycelium filament network incorporated in the foamed cellulose fiber foam, preferably wherein the concentration and/or density of the mycelium filament network in the foamed cellulose fiber foam is higher near the surface of the foamed cellulose fiber foam compared to the interior of the foamed cellulose fiber foam.

The method may comprise providing the product comprising or consisting of cellulose fiber foam in a form of a product or a part of the product selected from one or more of a sheet, a container, packaging material, an insulation product such as thermal insulation product, acoustic insulation product, moisture insulation product and/or grease insulation product, such as a product having barrier properties, i.e. a barrier product or a product comprising a barrier, a nonwoven product, a fire retardant product, a construction product, an interior design product, an automotive product, and a filter. The product comprising water repellent insulation material may be one or more of said products, or part of one or more of said products. The product may comprise the foam as material providing one or more of the discussed functionalities, such as insulation material, for example thermal insulation material, acoustic insulation material, moisture insulation material and/or grease insulation material, such as a material having barrier properties, nonwoven material, fire retardant material, construction material, interior design material, automotive material, and/or filter material.

As the first product is formed in the absence of mycelium, the process is simplified and can be optimized specifically in terms of the formation of the product, not in terms of the mycelium. In the formation, which includes forming the cellulose foam into a product with a specific shape and form, such conditions can be used, that could be harmful to the mycelium, such as high temperatures, use of chemicals and/or the like. This enables forming the product in substantially shorter time using efficient methods and equipment. For example the products may be dried at higher temperatures, which shortens the drying time and/or sizing time, and which may facilitate forming a more accurate shape of the product. This also lowers the risk of collapsing of the foam. Further, there is no need to specifically provide conditions optimal for the mycelium growth or survive during forming, such as sterile conditions, nutrients and/or the like. The cellulose used as raw material and the forming equipment do not need to be sterilized, but the formed product itself may be sterilized before applying the mycelium. This enables using desired cellulose raw material as such in the process of preparing the first product, so there is no need to provide sterilized or sterilizing conditions or equipment for the cellulose raw material, which enables carrying out the forming of the products in a variety of locations and by different operators, which are not specialized in working at sterilized conditions. No fungal waste is formed, which should be treated by using specific methods and/or at specific conditions. The formed product may be postprocessed, such as cut or otherwise modified to obtain a desired shape, form and/or size, before applying the mycelium. The formed products, i.e. the first products, may be fed with industrial equipment automatically and/or with high speed to further method step(s), which include the application of the mycelium on the formed products. These further steps may be carried out by an operator specialized in microbiology. On the other hand this operator does not need to prepare the foamed product.

The method comprises providing an aqueous dispersion of mycelium. This may be called as inoculum and it may be obtained from a culture of a desired mycelium spawn, which may be mixed with distilled deionized water (DDIW), for example in a ratio of 1 :1 to 1 :5, such as about 1 :3 (w/w), to obtain a semi-liquid aqueous slurry, i.e. a dispersion. The inoculum may be obtained from a liquid culture. The concentration of the slurry may be in the range of 1-10% by weight, such as in the range of 1-5% by weight, for example in the range of 1-3% by weight. The dispersion is spreadable so it can be applied by using any suitable application means, such as spraying means, for example comprising one or more nozzles and a source of the dispersion, for example a tank or other container, and a source of pressure, for example source of pressurized air; one or more brushes or the like applicators. The application of the mycelium dispersion may be automatized. For example products, or surfaces thereof, proceeding in a system, such as by a conveyor, can be treated with the dispersion as they pass the application means. Such arrangement enables treating a large number of products in a short time at an industrial scale.

The total amount of the used aqueous dispersion of mycelium may be 10-30% of the total moist foam material weight, for example in the case of sheets, or the like forms having two large/main surfaces, 5-15% per surface.

The mycelium may be obtained from Polyporaceae family. In the tests it was found out that not all species from the division of Basidiomycetes nor all the genera from the Polyporaceae family were optimal for the tested purposes. Two most promising ones were Fomes genus and Trametes genus, for example Fomes fomentarius or Trametes versicolor. The method comprises applying the aqueous dispersion of mycelium on the surface of the cellulose fiber foam to obtain a treated cellulose fiber foam. The cellulose fiber foam may be the product, form the product or be a part of the product. There is no need to impregnate the foam with the dispersion, which simplifies the application as no vacuum, pressure and/or specific applying means are required. Treating only the surface(s) of the foam is enough as the mycelium can penetrate into the foam in the present conditions, especially the mycelium can usually penetrate through the smallest thickness of the products, in most cases several centimeters. For example a sheet-like foamed product or other foam product comprising large surfaces may be treated by applying the dispersion only to the largest surfaces. This enables implementing industrial processes, wherein the treatment of the foamed product is fast and can be automatized.

Next the method comprises incubating the treated cellulose fiber foam, or the treated first product, for a time period enabling the mycelium to grow into the cellulose fiber foam to obtain a composite product comprising mycelium filament network incorporated in the cellulose fiber foam. The treated cellulose fiber foam, or more particularly one or more products comprising the treated foam, may be transported and/or applied into an incubator, wherein the subsequent incubating is carried out. The incubator may be a closed space, such as a room, a cabinet or the like, which can provide suitable conditions for the growth of the mycelium, such as suitable temperature, humidity, atmosphere, light, sterility and/or the like. Providing suitable conditions may include controlling and/or adjusting one or more of the conditions, such as changing the said conditions gradually or stepwise during the cultivation.

The incubating may be carried out for 1-14 days, such as for 1-7 days, 1-10 days, 3-10 days, or 7-14 days, or for any other time period suitable to obtain desired growth and/or penetration of the mycelium into the cellulose foam. The incubating may be carried out at a temperature in the range of 20-37°C, such as already in the range of 22-28°C, which saves energy and does not require complicated incubating facilities and/or equipment. The incubating may be carried out for a time period and/or at conditions enabling obtaining cellulose fiber foam comprising mycelium filament network incorporated in the cellulose fiber foam, wherein the concentration of the mycelium filament network in the cellulose fiber foam is higher near the surface of the cellulose fiber foam compared to the interior of the cellulose fiber foam. This is an indication of the present method, wherein the mycelium grows from the surfaces of the foam towards the interior of the foam. The density and/or concentration of the mycelium is therefore higher at or near the surface where the mycelium dispersion was applied, and a concentration gradient and/or a density gradient is formed as the mycelium grows. The concentration or density differences would eventually be decreased if the incubation would be continued for a prolonged time. This is however usually not desired as it would slow down the process and the desired properties can be obtained already with a shorter incubation time. The incubating may be also carried out for a time period and/or at conditions enabling obtaining a product comprising a layer comprising mycelium on the surface of the cellulose fiber foam, as disclosed herein. After a desired mycelium growth and distribution is obtained, the incubating may be ended and mycelium may be inactivated, for example by elevating temperature.

A concentration gradient is a measurement of how the concentration of something changes from one place to another. The concentration and/or density of the mycelium decreases gradually from the surface towards the inner area/interior part of the foamed cellulose foam, thus forming the gradient.

When dewatered foamed cellulose foam is used as a substrate and incubated with the mycelium, the foam, or a product formed from the foam, does not change its shape, for example collapse, during incubation and the characterizing concentration gradient is formed. On the contrary, if water is present in the substrate, it would allow the mycelium to freely proceed inside the substrate, and the characterizing concentration and/or density difference is not formed. This rather results in thorough impregnation of the product with the mycelium. Thus the result is similar to the tested cofoaming process (“Co-Foam”), which produced hard and brittle products.

The present characterizing features indicate water repellent foamed products having high quality and structural accuracy. The structure of the foam is homogenous as it has not collapsed during the preparation, which could deteriorate the properties of the foamed product. As the preparation of the foamed product can be optimized in terms of the formation of the product, not in terms of survival of the mycelium, the foamed product can be prepared with high accuracy.

Finally the method comprises drying the composite product, preferably without pressing, at elevated temperature, such as at 70°C or more, such as 70-80°C, to obtain the product, such as the product comprising or consisting of water repellent insulation material. The drying of the composite product at elevated temperature may be carried out in an oven or at any other suitable conditions and/or in a suitable system. The drying may be continued until a dry matter content of 80% by weight or more, such as 85% by weight or more, for example 90% or more, is obtained. The final product may be provided at this dry matter content.

From the present method specific products were obtained with distinguishing properties. The properties relate for example to the distribution of the mycelium in the foam as well as formation of a specific layer on the surface of the foam. The products also exhibit specific measurable properties.

The present disclosure provides a product comprising or consisting of water repellent cellulose fiber foam comprising mycelium filament network incorporated in the cellulose fiber foam. The product may be obtained by the method disclosed herein.

The concentration and/or density of the mycelium filament network in the cellulose fiber foam may be higher near the surface of the cellulose fiber foam compared to the interior of the cellulose fiber foam. This may refer to the concentration/density gradient and/or to the formed denser layer at the surface.

It was found out that the present products contained a denser film-like area or areas on the surface of the foam. Such film or layer is formed by the mycelium applied onto the surface, and can be seen and identified from microscopic images, such as shown in Figure 9c. The layer is distinct from the cellulosic material, and these may not be substantially mixed, i.e. there may be a recognizable border between the layer and the cellulose. Proteins such as hydrophobins provide water repellency to mycelium and these proteins can be detected from the product, for example by antibody based methods, mass spectrometry or other methods. The mycelium also contains other water-repellent compounds, such as waxes and lipids. All these compounds may be detected and used for characterizing the product, for example for identifying the mycelium and distribution thereof.

The present product may comprise a layer comprising, such as substantially consisting of, mycelium on the surface of the cellulose fiber foam. The layer may have a specific thickness, which is remarkably high, such as a thickness of 500 pm or more, such as 700 pm or more or 800 pm or more, for example 500-1500 pm, such as 700-1500 pm or 800-1200 pm. This specific thickness can contribute to specific properties in the product, such as increased water repellency, insulation properties, fire retardant properties, antiviral and/or antimicrobial properties, and/or other properties discussed herein. The layer may have a substantially uniform density, concentration, distribution and/or thickness, and it is preferably distinct from the concentration gradient formed inside the foam. The layer may contribute to barrier properties and/or it may act as a barrier.

One example provides a product comprising or consisting of water repellent cellulose fiber foam comprising mycelium filament network incorporated in the cellulose fiber foam, the product comprising a layer comprising mycelium on the surface of the cellulose fiber foam.

When the already formed products comprising cellulose foam were treated with the present method, the obtained final products were soft under compression, and thus they tolerate mechanical forces. In the present products also recovery after compression was high, which indicates elasticity. In comparison, products obtained for example by cofoaming with included mycelium were hard and more brittle, which makes them less suitable for many applications requiring mechanical durability and/or elasticity. Also forming products other than simple sheets was more challenging with the cofoaming process. In the present products the thermal conductivity of the product was not affected when compared to corresponding cellulose foam products without the mycelium, which is another difference to cofoamed products, wherein the thermal isolation was worse when compared to corresponding cellulose foam products without the mycelium.

In one embodiment the product is part of, comprises or consists of one or more of a sheet, a container, packaging material, an insulation product, fire retardant material, construction material, interior design material, automotive material and a filter.

The insulation material or product may provide one or more types of insulation properties. The insulation properties may comprise barrier properties, such as water, grease and/or odor barrier properties. The insulation product may be for example thermal insulation product, acoustic insulation product, moisture insulation product and/or grease insulation product. The product may comprise for example thermal insulation material, acoustic insulation material, moisture insulation material and/or grease insulation material. Such products or materials may be in a form of sheets, or may comprise a sheet, and/or may comprise another form, such as designed to fit into a target. Packaging material may comprise materials and/or products, which have been conventionally prepared from foamed plastics, such as polystyrene foams, for example expanded polystyrene (EPS), or the like. Such packaging material may be material fitted inside boxes, such as carboard boxes, wherein the material usually covers the inner surfaces of the boxes and provides insulating properties. The packaging material may be configured to protect an object or material packed in the package. Packaging material may be also configured to fit to the net shape of the object being packed and/or protected. Packaging materials may comprise one or more sheets, but also more complex forms moulded to fit into a box may be provided. Such forms may have other surfaces formed according to the interior of the box and inner surfaces formed according to the product(s) intended to be packed.

The present materials are especially suitable for packing food products, such as fresh food, for example fish, meat and vegetables, which could easily contaminate such packing material which would absorb water, grease or fat and other substances derived from the food products, which may cause odors and act as a basis for microbial growth. The present material can provide barrier properties against such substances. The present materials tolerate moisture and grease/fat and do not absorb or allow the substances to permeate, which makes the material especially suitable for food product use, such as for packing food products. The present materials can also protect the cardboard boxes from contamination. Even if the packaging materials would be contaminated by the food products, they can be recycled and/or decomposed by using environmentally friendly methods. For example the materials may be biologically decomposed by using anaerobic and/or aerobic methods, such as by composting.

It was found out that the present materials can provide a positive influence on reaction-to-fire properties. Micro-combustion calorimeter tests of pure mycelium showed that mycelium has much lower total heat release and peak heat release rate compared to kraft pulp cellulose alone, thus the mycelium being significantly more heat and fire resistant. The properties of mycelium can help protecting the pulp from fire in the present products, and therefore enable forming products with heat resistant properties. The product may be a fire safe product, such as a fire retardant product, which may be provided for example as a sheet, such as a sheet of construction material or product, or interior design material or product, or as a part of such material or product. The product may have a bulk density in the range of 0.040-0.100 g/cm³, such as in the range of 0.050-0.070 g/cm³, for example in the range of 0.050-0.060 g/cm³. Bulk density is defined as the mass of the material divided by the total volume it occupies. To determine the bulk density of the present products and materials, the sample area, basis weight and thickness may be measured, and the density is calculated. A ruler, a balance and a laser measuring device may be used for the measurements.

The product may have a water contact angle in the range of 105-150°, such as in the range of 110-145°, for example in the range of 115-142° determined by sessile drop method, such as with t=0.3 s, t=1 s, t=60 s and/or t=600 s intervals for recording images. The sessile drop contact angle may be measured by a contact angle goniometer using an optical subsystem to capture the profile of a pure liquid on a solid substrate. The angle formed between the liquid-solid interface and the liquid-vapor interface is the contact angle. This may be detected by systems employing high resolution cameras, a light source and software to capture and analyze the contact angle, such as by using an optical tensiometer preferably equipped with dedicated software to determine and output results. The sessile drop method is used to define the surface free energy of solid surfaces. As the surface energy of the solid cannot be directly measured, the sessile drop method is utilized to measure the contact angles of a probe liquid on the surface. The contact angle values can be used to model the surface free energy of the surface. The sessile drop method is also used to determine the wettability of the surface. The water contact angle can be used to determine the hydrophilicity of the surface.

The product may exhibit a compression stress at 50% deformation in the range of 20 - 200 kPa, such as in the range of 20 - 100 kPa, preferably 30-50 kPa, and/or a specific compression strength in the range of 0.4-0.6 Nm/g. The compression strength is measured by a mechanical tester for example a Lloyd LR10K universal tester with a suitable load cell (Lloyd Instruments Ltd, Bognor Regis, West Sussex, UK) using the method EN 826 (2013) or similar. A compressive force is applied perpendicular to the major faces of a sheet-like material and the maximum stress is recorded at 50% deformation. Measurements may be performed at RH50%. Recovery after compression is measured from the material dimensions before and after a compression/release cycle. The product may have a thermal conductivity in the range of 0.020-0.040 W/mK, preferably in the range of 0.028-0.033 W/mK at 10°C and/or in the range of 0.020-0.035 W/mK, preferably in the range of 0.025-0.030 W/mK at -7.5°C, determined according to ISO 8301 , for example by using a Heat Flow Meter HFM, Fox314 (TA instruments, U.S.A.).

The present disclosure provides use of cellulose fiber foam, or a product comprising cellulose fiber foam, for preparing a product or material for the product, such as a product comprising or consisting of water repellent cellulose fiber foam or the product comprising water repellent insulation material, or any other product or material disclosed herein. The product or material may be obtained with the method disclosed herein.

Examples

Example 1

Different types of composite products of cellulose material with mycelium from order Polyporales were prepared by using different methods. Composite products comprising foamed cellulose were prepared by a co-foaming method and by the present method, and composite product obtained from a honeycomb board was prepared by applying mycelium suspension on the surfaces of the honeycomb board. The methods differed in respect of methods steps, and also the properties of the obtained products were different.

Materials and methods strains

In total, seven cosmopolitan polypores (basidiomycota) species were selected for this study and were obtained from the culture collection of VTT (http://culturecollection.vtt.fi/) and from the USDA Forest Service Forest Products Laboratory. The selected strains were Irpex lacteus (VTT D-79108), Irpex lacteus (USFS Mad 517 strain), Fomes fomentarius (VTT D-061139), Trametes versicolor (VTT D-99747), Ganoderma lucidum (VTT D-06390), Fomitopsis pinicola (environmental isolate), Schizophyllum commune (VTT D-88362) and Pleurotus ostreatus (VTT D-90415). The strains were routinely cultivated on MEA (Malt Extract Agar) plates at +25°C. Preparation of the mycelium spawn culture and semi-liquid inocula

The spawn cultures were prepared in filtered polypropylene bags (SacO2). The media bags for each strain contained 250 g dry rye grain (Secale cereale), 10 g calcium sulphate dihydrate and 250 g of DDI water. The media was soaked at + 4°C overnight followed by autoclavation for 2 h at 121 °C. The work was carried out aseptically under the laminar flow hood. After cooling to the ambient room temperature, each bag was inoculated with actively growing mycelium from agar plates and sealed with an impulse sealer. The bags were incubated at + 25°C for 3 weeks. After the mycelium growth was clearly visible on grain kernels, a semiliquid mycelium inocula was prepared by mixing the spawn culture with sterile DDIW in the ratio of 1 :3 (w/w) by using a Waring blender resulting in a semi-liquid aqueous slurry.

Preparation of inoculum from liquid culture

Inocula from liquid cultures were prepared from fungal cultures grown in shake flasks with Standard Nutritional Liquid (SNL) medium containing 30 g/L D-glucose monohydrate, 4.5 g/l L-asparagine monohydrate, 3 g/l yeast extract, 1.5 g/l KH2PO4, 0.89 g/l MgSO4 x 7 H2O, 1 ml/l trace element solution in H2O, and having the pH adjusted to 6.0 with KOH. The work was performed under sterile conditions using sterile labware. Pre-cultures were prepared from fungal cultures grown on agar plates by cutting 3 pieces (5 x 5 mm) from a plate and homogenizing them in 5 ml of SNL liquid media using a sterile Ultraturrax homogenizer and successively adding 0.5 ml of this suspension to 50 ml of SNL media. The cultures were grown at 25°C shaking with 150-180 rpm for several days until visible growth. After cultivation the cultures were centrifuged (4000 rpm, 5 min in 50 ml centrifuge tubes), the supernatants were discarded, 5 ml of fresh SNL media was added, the pellets resuspended and the samples homogenized with a sterile Ultraturrax homogenizer. 3 ml of these homogenized samples were then used to inoculate 300 ml liquid cultures (SNL media) and cultivated at 25°C shaking with 150-180 rpm for five or more days. After the cultivation, the dry matter contents of the cultures were determined by filtering a known volume of the produced mycelium using a GF/A filter (Whatman), washing the retentate with water, drying the retentate in an oven and weighing the remaining dry matter. From the well grown shake flask cultures, 250 ml were divided into 50 ml aliquots, centrifuged for 5 min at 4000 rpm followed by removal of the supernatant, addition of 5 ml of fresh SNL media, homogenization with an Ultraturrax homogenizer for 20 seconds, and combining the aliquots for use as an inoculum.

Cellulose and Lignocellulose Media

Foam Formed Cellulose Sheets

Bleached chemical softwood kraft pulp (BSKP) sheets were obtained from a Finnish pulp mill (Metsa Fibre Oy, Aanekoski Bioproduct Mill). The dry pulp sheet was soaked in water at least 24 h and disintegrated into single fibers in pulper. Schopper-Riegler value of the pulp was 13 (ISO 5267-2:2001 ) and the average fibre length was 2.2 mm (L&W Fiber Tester Plus). The consistency of BSKP pulp after disintegration was 4.3%. Cellulose fibre foam (CFF) material with target basis weight of 400 g/m² was produced as follows: BSKP pulp was diluted to 1 .5% and pH of pulp was adjusted to 7. BSKP pulp (initial volume 2 I), surfactant (Simulsol SL10) with the dosage of 1.7 g/l, sizing agent (Fennosize KD364M) with the dosage of 2% from fibre weight, and wet strength resin (Fennostrength PA21 ) with the dosage of 2% from fibre weight were added to the foaming vessel. The vessel diameter was 160 mm and the volume was 8.8 I. Fibre foam was generated with the help of mechanical mixer (power 1.5 kW) fitted with a disk-type stirrer (Figure 2). A circular disc (0 87 mm) with two opposing 25 degree bends with their fulcrum positioned at a distance of 25 mm from the centre was used as a mixing blade. Mixing speed was 3800 rpm. Mixing time was adjusted so that the target 60% air content for fibre foam was achieved. Generated aqueous cellulose fibre foam was poured into a mould with wire bottom, with mould size of 220 mm x 350 mm with the help of funnel (Figure 1). After the fibre foam was settled, the water was partly filtered through a wire by gravity (no vacuum). The filtration time was 20 min. During the filtration the fibre foam was pressed by hand to 12 mm thickness. The wet sheets were removed on the wire from the mould and the sheets were dried in oven (80°C). The drying time of sheets was 24 h. After drying, the sheets were treated for 5 min in 105°C temperature in the oven to activate the sizing agent.

Honeycomb Board Media

Honeycomb board (Eltete TPM Ltd, Finland) was split in half resulting in a 10 mm thick honeycomb material covered from one side by a cardboard (the other side was open). The honeycomb material had a well width of 12 mm (Figure 2). Fabrication of the cellulose-mycelium composites

Fabrication of the cellulose-mycelium composites was carried out under aseptic conditions in the laminar flow hood. Three types of cellulose-mycelium composites were produced using the mycelium from all selected strains. The materials were Co-Foam, Mycelium-CFF, Mycelium-Honeycomb, and the respective control samples without mycelium. The material properties tests were performed with the samples produced with T. versicolor. The water contact angle tests were performed for material samples produced with T. versicolor, F fomentarius, I. lacteus, G. lucidum, S. commune and P. ostreatus. All material samples were prepared by placing the mycelium-cellulose composites in sealed sterile square culture dishes in order to maintain a consistent micro-environment for fungal propagation. The plates were incubated at +25°C and monitored for colonization of the media by fungal mycelium.

Preparation of Co-Foam material

For the preparation of the first material, termed Co-Foam, an aqueous foam of mycelium and cellulose pulp fibers was generated. The BSKP pulp was concentrated to 8% and sterilized by autoclaving at +121°C. Simulsol SL10 at 1.7 g/l was added to the pulp and an aqueous foam was generated by a mechanical mixer with a disk type stirrer (Figure 3). The air content was -50%, meaning that the original volume was doubled.

Subsequently a 10% (w/w) semi-liquid fungal inocula was added to the prefoamed cellulose and mixed until homogenous. The aqueous foam was poured into a mould with a wire bottom, with mould size of 165 mm x 165 mm. After the fibre foam was settled, the water was partly filtered through a wire by gravity (no vacuum). The filtration time was 15 min. Subsequently the fibre foam was pressing by hand to 15 mm thickness with a 90 micron mesh plunger. The resulting wet sample was transferred on to a sterile culture dish and incubated at + 25°C for 14 days. Additionally, a control sample without mycelium was produced (Co-Foam control).

Preparation of Mycelium-CFF

For the preparation of the second material, termed Mycelium-CFF or Mycelium- Cellulose Fibre Foam, which represents the presently claimed material, semi-liquid fungal inoculum or liquid inoculum was used to introduce the mycelium to foam formed cellulose sheets, which were prepared as described in previous. The foam formed cellulose sheets were first moistened by spraying sterile water on the foam material until and the water addition was 20% (w/w) of the foam material weight. Then semi-liquid or liquid inoculum was evenly spread on top and bottom surfaces of the sheets. The total amount of used inoculum was 20% of the total moist foam material weight (10% per sheet surface). The inoculated samples were cultivated on sterile culture dishes at +25°C for 14 days.

Preparation of Mycelium-Honeycomb composites

For the preparation of the third material, termed Honeycomb-Mycelium composite, the sterilized honeycomb board, having its top side covered by kraft paper and the bottom side with exposed honeycomb webbing, was pushed into the semi-liquid fungal inoculum which was evenly spread (5 mm thick) at the bottom of a 22 x 22 cm plastic tray. Samples were prepared either as a single layer or as several stacked layers of inoculum and honeycomb material. The tray was then covered with a lid and incubated at +25°C for 14 days. See Figures 4 and 5 for the visual description of the process.

Tests used to characterize material properties

The testing methods used to characterize the material properties are shown in

Table 1 .

Table 1 . Testing methods used to characterize the produced materials.

Tests of antiviral and antimicrobial properties

To screen for the potential antimicrobial properties of materials a modified standard EN 1276: 2019 “Ell Chemical disinfectants and antiseptics" was used. The mycelium-cellulose samples were homogenized and provided 24 hours contact time.

Results and Discussion

Growth performance of the fungi on prepared mycelium-based materials

All samples containing mycelium were removed from the plates and dried. After drying, the samples were stored in the laboratory at approximately 20°C and 30% RH until testing.

Initially several fungal species were used and ranked by visual inspection for mycelium growth and the strength of the filamentous network. T. versicolor showed the best growth robustness, followed by F. fomentarius and I. lacteus with medium growth, and F. pinicola with poor growth. Therefore, the tests were continued only with T. versicolor for the fabrication of three composites materials shown in the Table 2 of Figure 6. Photos of the three different composite materials without and with mycelium are presented in the Table 2.

Basic material properties

Three types of composite materials with mycelium and cellulosics/lignocellulosics were prepared and compared to control samples without mycelium. The basic material properties of the three composite material types with and without mycelium are shown in Table 3. The bulk density of the Co-Foam material with mycelium was higher and the thickness lower compared to the Co-Foam control material (without mycelium) because of the partial wet fibre foam collapse during the incubation time. Similar collapse of structure was not observed with the Mycelium-CFF material. The densities of the Mycelium-Honeycomb samples and honeycomb control without the mycelium were higher than the density of the CFF and mycelium-CFF materials but lower than the density of Co-Foam sample with mycelium. The bulk density for EPS (expanded polystyrene) has been reported to be 0.96-0.28 g/cm³.

Table 3. Bulk density, thickness and basis weight of the three materials each produced with T. versicolor mycelium. Control samples are prepared without mycelium.

Morphology and microstructure of mycelium-cellulose composites

The microstructure of mycelium composites was investigated using light microscopy and SEM. Light microscopy showed that the scale of the fibrillar structure of mycelium was clearly smaller in diameter compared to the scale of the cellulose fibers (Figure 7). SEM analysis showed that a continuous mycelium layer was growing on top of all samples incubated with the mycelium (Figure 8). The surface of the Mycelium-Honeycomb composite material and Mycelium-CFF material had patches of denser film-like areas. As expected, in the control samples only the cellulose fibres were observed on the sample surface.

The cross-sectional SEM images revealed that the mycelium had colonized the bulk interior of the studied cellulose materials. The pure mycelium surface thickness was around 0.4 mm and 0.9 mm in Co-Foam and Mycelium-CFF samples, respectively. Cross-sectional images from the material core showed that mycelium was well spread out inside the material. In Figure 9 images a) and c) of the surface of the samples show a layer of pure mycelium without cellulose fibres. Images b) and d) show mycelium interspersed among the cellulose fibres inside the material and away from the surface.

Compression strength properties

To study the mechanical performance of the biofabricated materials compression tests were measured for maximum stress at 50% deformation using a Lloyd LR10K universal tester (Lloyd Instruments Ltd, Bognor Regis, West Sussex, UK) at 50% RH. The measurement method is essentially as in standard EN 826 (2013). Measured properties are presented in Table 4. The compression strength of the Co-Foam material was significantly higher compared to the Co-Foam control material (Figure 10). However, this is at least partly due to the higher, almost doubled density level caused by the foam collapse during incubation. The foam formed cellulose material with (Mycelium-CFF) or without mycelium (CFF control) had similar compression strengths. The mycelium colonization in the honeycomb materials significantly lowered the compression strength. The recovery after compression was recorded and for all cases the recovery was not affected by the mycelium addition and varied between 70% and 85%. The highest recovery after compression was observed for the Mycelium-CFF material (Figure 11).

The bending strength and tensile strength of Co-foam control material and Cofoam cellulose-mycelium composite material were very different when evaluated by handling the material. The Co-foam control was brittle but the Co-foam cellulose mycelium composite was very hard and strong.

Table 4: Measured bulk density, compression stress and specific compression strength from different samples prepared by using T. versicolor.

Surface hydrophobicity was tested by water contact angle (WCA) measurements and recorded for 100 seconds or more (Table 5). The Co-Foam control material was hydrophilic and had a WCA of -40° and the absorption of the water droplet was very fast. In contrast, the WCA of the Co-Foam material was -140° and remained unchanged after 600 s. The foam-formed cellulose control material (containing hydrophobic agent) showed a WCA of -115° being also stable after 600 s. The Mycelium-CFF material had an increased WCA of -135° and remained similar during the measurement. The Mycelium-Honeycomb composite material showed an unexpectedly low WCA, lower than the control material without mycelium. This could be due to uneven coverage of the cellulose scaffold by the mycelium, as seen in the SEM images. Overall, the results show that the mycelium was able to provide hydrophobicity to the Co-Foam and the Mycelium-CFF materials. The WCA value for expanded polystyrene (EPS) has been reported to be 75-90°.

Table 5. Hydrophobicity characteristics as measured by WCA of the material surface after 0.3 seconds and 600 seconds. aContains a hydrophobic sizing agent (Fennosize KD364M). ^b WCA not measurable as the water droplet was absorbed into the material.

Thermal conductivity

To assess the thermal insulation properties of the materials, thermal conductivities of the composite materials at +10°C (left bar) and -7.5°C (right bar) (Figure 12) were determined with method ISO 8301 using a Heat Flow Meter HFM, Fox314 (TA instruments, U.S.A.). Thermal conductivity of the Honeycomb control material was the highest, and the addition of mycelium was able to decrease the conductivity. The Co-Foam and OFF materials had clearly lower thermal conductivity level compared to the Honeycomb materials. The addition of mycelium into the Co-Foam material increased the thermal conductivity slightly whereas with the CFF material, the thermal conductivity remained unchanged with mycelium addition. The higher thermal conductivity level of Co-Foam materials in relation to CFF materials may be explained by the possible different fibre orientation in the materials: the CFF materials have more laterally oriented fibers compared to the Co-Foam samples that were prepared at high fibre consistency resulting in more random fibre orientation.

Antiviral and antimicrobial properties

The effect of the mycelium-cellulose materials produced on active virus and pathogenic microbes over a 24 h contact period was tested using a method that is essentially the same as standard EN 1276: 2019 “EU Chemical disinfectants and antiseptics". Mycelium-cellulose materials were prepared with four different polypore species T. versicolor, I. lacteus, F. fomentarius, and F. pinicola. A statistically significant decrease (over 1 .0 log cfu/sample) on the active MS-2 virus during 24 h contact time in all mycelium-cellulose materials was observed. F. pinicola derived extract also had a statistically significant decrease in viable Staphylococcus aureus during 24 h (Figure 13). All mycelium-cellulose composite samples made with the different species had a slight growth promoting effect on the tested fungal species (C. albicans, and A. niger). Conclusions

Mycelium composites are complex materials with at least two scales, mesoscale of the cellulose fibers and the microscale of the fungal hyphae. Use of the foamed cellulose as the nutritive substrate and scaffolding for mycelium-based materials may be advantageous in terms of total life-cycle energy required to achieve reduction in carbon emissions during fabrication and ease of production.

Different strains produced materials with different thermal and antimicrobial properties (Figures 12 and 13). T. versicolor strain was ultimately chosen as the most suitable inoculant for the present purposes because the highest speed of its growth and the properties of the resultant composites approximate thermal and other physical properties of EPS, a major non-biodegradable synthetic competitor used in thermal insulation and packaging materials for the cold chain shipping, storage and other building insulation. Mycelium-CFF materials showed good thermal insulation properties, low stiffness, good recovery from compression and high water contact angles.

Example 2

Package material was prepared by treating sheets of cellulose fiber foam with different thicknesses by applying as aqueous dispersion of mycelium from T. versicolor on the surface of the cellulose fiber, and incubating at 25°C until the mycelium was grown inside the cellulose fiber foam, which was in most cases 4-7 days. The treated sheets were dried in an oven at 70°C without pressing until dried sheets with 92% dry substance matter by weight were obtained. The dried sheets were hardened while maintaining their original size and shape, and they exhibited water repellent properties. The dried sheets also had a dense layer of mycelium on their surface, which exhibits antimicrobial properties.

Dried sheets with different thicknesses were prepared and applied inside cardboard boxes (Figures 14A-D) to obtain an insulated package, for example having dimensions of 16.5 x 16.5 x 10.5 cm, which packages are suitable for example as a shipping box and/or for food product use. If necessary, the dried sheets were cut to obtain desired sheet sizes to fit into the cardboard box. Example 3

Water contact angle of Mycelium-CFF materials prepared with various fungal species.

Mycelium slurries were prepared from spawn cultures using the fungal strains T. versicolor, F fomentarius, I. lacteus, G. lucidum , Schizophyllum commune and Pleurotus ostreatus as described in Example 1 . CFF materials (30 x 30 mm) were prepared and inoculated with the mycelium slurry, incubated and dried as described in Example 1 to obtain Mycelium-CFF materials. The water contact angle (WCA) of the Mycelium-CFF material surfaces were determined using an optical tensiometer CAM200 (KSV Instruments) and sessile drop method with axisymmetric drop shape analysis.

The measurements showed that Mycelium-CFF materials prepared with the species T. versicolor and F. fomentarius had a clearly hydrophobic surface as observed by the WCA values of 119° and 117°, respectively (Table 6). The other fungal species produced materials that had surfaces with low WCA values or surfaces that quickly absorbed water.

Table 6. Water contact angle for Mycelium-CFF materials prepared with different fungal species and comparison to CFF material without mycelium.

³ Measurements where the water droplet was absorbed into the material were marked as hydrophilic. Example 4

Water durance of Mycelium-CFF materials

The properties of the Mycelium-CFF and CFF (control) samples (prepared as described in Example 1 ) were further tested for water durance. CFF-mycelium sample prepared with T. versicolor and a CFF sample (control) were placed in beakers with DDI water. A magnetic stir bar was used to vigorously mix both samples and a video was recorded to follow erosion and disintegration qualitatively. The CFF control sample disintegrated already within 30 sec, while the CFF-mycelium sample showed minimal erosion for at least 2 min and remained floating.

Water durance test with T. versicolor Mycelium-CFF blocks compared to the control sample CFF are shown in Figures 15A and 15B. Figure 15A shows foam formed cellulose + mycelium after 2 minutes mixing in water. There is no disintegration for at least several minutes and the sample floats due to water repellency. Figure 15B shows foam formed cellulose control after 0.5 minutes mixing in water. The sample disintegrates easily.

Example 5

Reaction-to-fire tests

Micro-combustion calorimeter tests were done with pure mycelium from T. versicolor, residue 20% (Figure 16A) and T. reesei, residue 32%, (Figure 16B) as well as bleached softwood kraft pulp, residue 6.5% (Figure 16C). The results showed that mycelium has much lower total heat release and peak heat release rate compared to cellulose pulp, thus the mycelium being significantly more heat and fire resistant. It was found out that the properties of mycelium could help protecting the pulp from fire in the present products.

Claims

1. A method for preparing a product comprising water repellent low- density fiber-based material, the method comprising

-providing a product comprising dewatered foamed cellulose fiber foam,

-providing an aqueous dispersion of mycelium,

-drying the composite product at elevated temperature, such as at 70°C or more , to obtain the product comprising water repellent low-density fiber-based material, which is a product comprising water repellent foamed cellulose fiber foam comprising mycelium filament network incorporated in the foamed cellulose fiber foam, preferably wherein the concentration and/or density of the mycelium filament network in the foamed cellulose fiber foam is higher near the surface of the foamed cellulose fiber foam compared to the interior of the foamed cellulose fiber foam.

2. The method of claim 1 , wherein the incubating is carried out for 1-14 days, preferably at a temperature in the range of 20-37°C, such as at a temperature in the range of 22-28°C.

3. The method of claim 1 or 2, comprising providing cellulose fibers and forming the cellulose fibers into the foamed cellulose fiber foam, such as in a mould and/or on a wire.

4. The method of any of the preceding claims, wherein the product comprising the dewatered foamed cellulose fiber foam has the shape of the final product comprising water repellent low-density fiber-based material.

5. The method of any of the preceding claims, comprising providing the product comprising dewatered foamed cellulose fiber foam in a form of a product or a part of the product selected from one or more of a sheet, a container, packaging material, an insulation product such as a product comprising thermal insulation material, acoustic insulation material and/or moisture insulation material, nonwoven material, fire retardant material, construction material, interior design material, automotive material, and filter, and/or wherein the product comprising water repellent low-density fiber-based material is one or more of said products.

6. The method of any of the preceding claims, wherein the product comprising dewatered foamed cellulose fiber foam has a moisture content in the range of 5-40% by weight, preferably having a moisture content in the range of 15-35% by weight.

7. The method of any of the preceding claims, comprising incubating for a time period and/or at conditions enabling obtaining cellulose fiber foam comprising mycelium filament network incorporated in the cellulose fiber foam, wherein the concentration of the mycelium filament network in the cellulose fiber foam is higher near the surface of the cellulose fiber foam compared to the interior of the cellulose fiber foam.

8. The method of any of the preceding claims, wherein the drying is carried out without pressing.

9. The method of any of the preceding claims, wherein the method is a method for preparing a product having an antimicrobial surface and/or a product having fire retardant properties.

10. A product comprising water repellent foamed cellulose fiber foam comprising mycelium filament network incorporated in the foamed cellulose fiber foam, wherein the concentration and/or density of the mycelium filament network in the foamed cellulose fiber foam is higher near the surface of the foamed cellulose fiber foam compared to the interior of the foamed cellulose fiber foam.

11 . The method of any of claims 1-9 or the product of claim 10, wherein the product comprising water repellent foamed cellulose fiber foam comprising mycelium filament network incorporated in the foamed cellulose fiber foam has a dry matter content of 80% by weight or more, such as 85% by weight or more, for example 90% or more.

12. The product of claim 10 or 11 comprising a layer comprising mycelium on the surface of the cellulose fiber foam, such as a layer having a thickness of 500 pm or more, such as 500-1500 pm.

13. The method of any of claims 1-9 or the product of any of claims IQ-

12, wherein the cellulose comprises pulp, such as chemical pulp, for example bleached chemical pulp.

14. The method of any of claims 1-9 or the product of any of claims IQ-

13, wherein the mycelium is obtained from Polyporaceae family, such as from Fames or Trametes genus, for example Fames fomentarius or Trametes versicolor.

15. The product of any of claims 10-14, wherein the product is part of, comprises or consists of one or more of a sheet, a container, packaging material, an insulation product such as a thermal insulation product, acoustic insulation product and/or moisture insulation product, a nonwoven product, a fire retardant product, a construction product, an interior design product, an automotive product and a filter.

16. The product of any of claims 10-15, having a bulk density in the range of 0.040-0.100 g/cm³, such as in the range of 0.050-0.070 g/cm³, for example in the range of 0.050-0.060 g/cm³.

17. The product of any of claims 10-16, having a water contact angle in the range of 105-150° determined by sessile drop method, such as in the range of 110-145°, for example in the range of 115-142°.

18. The product of any of claims 10-17, having thermal conductivity in the range of 0.020-0.040 W/mK, such as in the range of 0.028-0.033 W/mK at 10°C and/or in the range of 0.020-0.035 W/mK, preferably in the range of 0.025-0.030 W/mK at -7.5°C, determined according to ISO 8301 .

19. The product of any of claims 10-18 obtained with the method of any of claims 1-9.

20. Use of foamed cellulose fiber foam, or a product comprising foamed cellulose fiber foam, for preparing a product comprising water repellent foamed cellulose fiber foam with the method of any of claims 1-9.

21. The use of claim 20, wherein the product comprising water repellent foamed cellulose fiber foam is the product of any of claims 10-19.