Method of forming fibrils from a lignocellulosic substrate

Abstract

Method of forming fibrils from a lignocellulosic substrate, comprising: providing (100) a lignocellulosic substrate; growing (101) mushrooms on the lignocellulosic substrate; harvesting (102) said mushrooms; collecting (103) the lignocellulosic substrate on which the mushroom has been grown, which substrate is a spent mushroom substrate; fibrillating (104) the spent mushroom substrate, forming fibrils. The formed fibrils may be further formed into fibrillar films or foams.

Description

[0001] METHOD OF FORMING FIBRILS FROM A LIGNOCELLULOSIC SUBSTRATE

[0002] TECHNICAL FIELD

[0003] The present disclosure relates to a method of forming fibrils from a lignocellulosic substrate, wherein the substrate is collected and fibrillated after mushrooms have been grown on the substrate and harvested therefrom.

[0004] BACKGROUND ART

[0005] In the well-managed mushroom industry, up to 70% dry mass of the initial substrate remains as spent mushroom substrate (SMS) (Zhang et al., Oyster mushroom cultivation with rice and wheat straw, Bioresource Technology 82 (2002), 277-284). The world production of cultivated mushrooms was approximately 39 million tons in 2017 (Chapter 2, Current Overview of Mushroom Production in the World, Daniel J. Royse, Johan Baars, Qi Tan, Book Editor(s): Cunha Zied Diego, Arturo Pardo-Gimenez, 2017, https: / / doi.org / 10.1002 / 9781119149446.ch2). After mushroom cultivation and harvesting (normally after 20 and 150 days (Chen et al., Potential for combined production of food and biofuel: Cultivation of Pleurotus pulmonarius on soft- and hardwood sawdusts”, Journal of Cleaner Production 266 (2020) 122011 ; Shaojun et al., Energy-efficient substrate pasteurization for combined production of shiitake mushroom (Lentinula edodes) and bioethanol, Bioresource Technology 274 (2019) 65- 72), the initial wood substrate remains as spent mushroom material (SMS). SMS is currently considered a waste material, causing environmental concerns and additional expenses for its disposal.

[0006] SUMMARY OF THE INVENTION

[0007] It is an object of the present disclosure to provide a method of forming fibrils from a spent mushroom substrate, i.e. a substrate collected after mushrooms have been grown and harvested therefrom.

[0008] The invention is defined by the appended independent patent claims. Non-limiting embodiments emerge from independent claims, the appended drawings and the following description. According to a first aspect there is provided a method of forming fibrils from a lignocellulosic substrate, comprising: providing a lignocellulosic substrate, growing mushrooms on the lignocellulosic substrate, harvesting the mushrooms, collecting the lignocellulosic substrate on which the mushrooms have been grown, which is a spent mushroom substrate, fibrillating the spent mushroom substrate, forming fibrils, wherein the spent mushroom substrate is collected and thereafter fibrillated without any intermediate chemical or thermal treatment step.

[0009] The mushroom allowed to grow on the lignocellulosic substrate until harvest may be any kind of mushroom. Preferably the mushroom is an edible mushroom, such as: white button, cremini, portobello, shiitake, chanterelle, porcini, enokitake, lion’s mane, morel, shimeji, reishi, maitake, and / or oyster.

[0010] Harvesting takes place normally after 20 and 150 days (Chen et al., Potential for combined production of food and biofuel: Cultivation of Pleurotus pulmonarius on soft- and hardwood saw dusts”, Journal of Cleaner Production 266 (2020) 122011 ).

[0011] All mushrooms have mycelium as they are the fruiting bodies of living mushrooms, and mushrooms themselves consist of tightly bunched mycelium. Through the mushroom cultivation, the lignocellulosic substrate undergoes a biological pretreatment. The formed spent mushroom substrate (SMS), in addition to the remaining lignin, hemicellulose and cellulose materials, also contains remnants of the mushroom / fungi such as the mycelium or mycelia and / or parts of the stem. (Shaojun et al., Energyefficient substrate pasteurization for combined production of shiitake mushroom (Lentinula edodes) and bioethanol, Bioresource Technology 274 (2019) 65-72) 2019). Through the mushroom cultivation, the lignocellulosic substrate undergoes a biological pretreatment. In general, this results in a degradation of the lignin of between about 15 to 35 wt.%, meanwhile cellulose and hemicellulose are largely remaining (Chen et al., Potential for combined production of food and biofuel: Cultivation of Pleurotus pulmonarius on soft- and hardwood sawdusts”, Journal of Cleaner Production 266 (2020) 122011 ; Shaojun et al., Energy-efficient substrate pasteurization for combined production of shiitake mushroom (Lentinula edodes) and bioethanol, Bioresource Technology 274 (2019) 65-72). The formed fibrils are composed of a natural combination of cellulose and mycelium, wherein the mycelium act as a binder of the cellulosic fibrils. All mushrooms can, hence, be used to form the spent mushroom substrate. This as all mushrooms have mycelium. The lignocellulosic substrate is biologically pretreated by the mycelium, which uses the lignin as food / nutrient and this allows the fibrillation of the lignocellulosic substrate, the production of spent mushroom substrate, without any pretreatment. The type of mushroom grown on the lignocellulosic substrate is, hence, subordinate.

[0012] In the above-describe method, except for the mushroom cultivation, no other chemical, mechanical or thermal pre-treatment processes are applied to the SMS to be able to fibrillate the substrate.

[0013] In the fibrillation process, the SMS is fibrillated to separate the fibrils, forming a fibrillar gel.

[0014] With fibrils is here meant fibrils with diameters in a range typically between 1 nm and 1 pm. Fibrils are traditionally defined as cylindrical structures with an outer diameter below 1 ,000 nm and an aspect ratio - the ratio between length and width - greater than 50.

[0015] Walaiporn et al. (Use of waste mushroom beds for the production of value-added biodegradable fiber sheet, E3S Web of Conferences 302, 02016 (2021 )) shows the utilization of waste mushroom beds (WMB) after the harvesting of oyster (WMB-0) and lingzhi mushrooms (WMB-L) for the preparation of fiber sheets. The WMB material was treated by steam explosion (a physicochemical pretreatment technology in which the material is heated to 160-260°C in a closed vessel under high pressure (7-50 bar) or with NaOH and thereafter mixed with a binder, tapioca starch. Results indicated that an increase of NaOH concentration in the pretreatment led to an increase in the toughness and water absorption of the fiber sheet. The additional tapioca starch promoted the interaction between cellulose fiber networks. The WMB showed a potential for use as e.g. biodegradable packaging, packing materials etc.

[0016] Ser et al. (Isolation and Optimization of Cellulose Crystals (CCs) from Substrate Mushroom Spent, Proceeding International Multidisciplinary Conference (IMC 2020)) shows that cellulose crystals (CCs) were isolated from SMS and the yield optimized. The SMS material was chemically pre-treated to remove lignin and part of hemicellulose. After the pre-treatment process, the biomass underwent a bleaching process to whiten the raw material in order to produce pure CCs, which is white in colour. After the bleaching isolation was performed, an acid solution was used to synthesize CCs.

[0017] The present method differs from these prior art techniques in that no steam explosion or NaOH treatment is applied to the SMS after the mushroom cultivation and that no chemical of thermal pre-treatment, bleaching or acid hydrolysis is used.

[0018] That the spent mushroom substrate is collected and thereafter fibril lated without any intermediate thermal treatment step is here meant that the collected spent mushroom substrate, before the fibrillation step, does not undergo any specific or deliberate heating process intentionally designed to alter its properties. The temperature of the collected spent mushroom substrate reaches as a maximum a temperature of 80°C, or at maximum 50°C, or at maximum 30°C, before fibrillation. Preferably, the temperature of the collected spent mushroom substrate is at or around room temperature when starting the fibrillation process. The collected spent mushroom substrate could be stored in a freezer and thawed before fibrillation.

[0019] Generally, an intermediate thermal treatment of the collected substrate before fibrillation would comprise increasing the temperature of the collected substrate to a temperature of at least 100°C.

[0020] During the fibrillation step there might be a change of temperature of the substrate at a maximum of ± 10°C as compared to the temperature of the substrate material at the beginning of the fibrillation process.

[0021] That the spent mushroom substrate is collected and thereafter fibril lated without any intermediate chemical treatment step is here meant that there are no deliberate chemical treatments applied to modify the collected spent mushroom substrate before the fibrillation starts. It remains in its natural state without any chemical alterations. The collected lignocellulosic substrate is not subject to any enzymatic treatment, oxidative treatment, alkaline treatment or acidic treatment.

[0022] In summary, the collected spent mushroom substrate is fibrillated directly from its raw form without any intermediate chemical of thermal steps. Hence, the above-described method of forming fibrils from SMS is a simpler (it requires no pre-fibrillation chemical or thermal treatment) and more environmentally friendly (requires no harsh chemicals) method of taking care of the SMS and forming fibrils therefrom as compared to known methods.

[0023] The spent mushroom substrate may be fibrillated using mechanical separation, grinding, homogenization, and / or shear extrusion.

[0024] The mechanical separation may comprise a step of mechanical disintegration of the spent mushroom substrate into smaller pieces ranging between 1 mm and 5 cm before fibrillation takes place.

[0025] The formed spent mushroom substrate may be dispersed in a suspension to a concentration of 1 wt.% to 50 wt.% before fibrillation.

[0026] The spent mushroom substrate may be dispersed in a suspension to a concentration of 1 -50 wt.%, 1 -45 wt.%, 1 -40 wt.%, 1 -35 wt.%, 1 -30 wt.%, 1 -25 wt.%, 1 -20 wt.%, 1 -15 wt.%, 1 -10 wt.%, 1 -8 wt.%, 1 -6 wt.%, 1 -4 wt.%, 2-50 wt.%, 4-50 wt.%, 6-50 wt.%, 8-50 wt.%, 10-50 wt.%, 15-50 wt.%, 20-50 wt.%, 25-50 wt.%, 30-50 wt.%, 35-50 wt.%, 40- 50 wt.%, 45-50 wt.%, 2-5 wt.%, 10-20 wt.%, 20-30 wt.%, or 30-40 wt.%.

[0027] The suspension may for example be water, alcohol or glycol.

[0028] When shear extrusion is used for fibrillation the spent mushroom substrate may be dispersed in a suspension to a concentration of up to 50 wt.% before fibrillation. When mechanical separation, grinding and / or homogenization is used, a suspension having a somewhat lower concentration may be used.

[0029] The lignocellulosic substrate may be selected from straw, hardwood, softwood, or any mixture thereof.

[0030] A hardwood substrate may be birch, oak, beech, hickory, maple, poplar etc. A softwood substrate may be pine, spruce, etc. The substrate may be in the form of branches, logs, wood chips, sawdust, or mixtures thereof, etc. Preferably the lignocellulosic substrate has a size range: pm to cm size.

[0031] The lignocellulosic substrate chosen may depend on the mushroom choice. Before mushroom culturing, the lignocellulosic substrate may be pre-treated by adding water and / or nutrients. The nutrients may be cereal: wheat, oats, rice, rye, corn etc., bran or grain. The lignocellulosic substrate may possibly also be pre-processed by sterilization or pasteurization to remove contaminants.

[0032] The formed fibrils, a fibril gel, may be vacuum filtrated, or casted, thereby forming a fiber network.

[0033] Before vacuum filtering, the fibrils may be diluted to a concentration between 0.2-2.0 wt.%.

[0034] The fiber network mat be dried, forming a film.

[0035] Drying of the fiber network may comprise a step of pressing the fiber network.

[0036] The pressing may take place during at least a portion of the drying step. By pressing is here meant to apply a force on at least a major surface of the film in a direction parallel with a normal of the major surface. Through the drying and pressing, flat, densely packed films may be generated. The pressing / drying may take place at an elevated temperature of at least 30°C, or at least 50°C or at 70-110°C.

[0037] The formed film may be applied onto at least a surface of a packaging material or paper.

[0038] There are different types of packaging materials used in the packaging industry to wrap and pack items. Some examples onto which the formed film may be applied includes corrugated boxes, cardboard boxes, etc.

[0039] The film may be applied onto at least a surface of a packaging material after the film has been formed or during the film is formed.

[0040] The formed fibrils may be freeze-dried, thereby forming a foam.

[0041] The formed gel may be frozen in liquid nitrogen and then freeze-dried to form foams

[0042] According to a second aspect there is provided a fibrillar film formed by the method described above.

[0043] According to a third aspect there is provided a fibrillar foam formed by the method described above. According to fourth aspect there is provided a fibrillar film having an oxygen permeability coefficient below 1 x w16cm3cm / s cm2Pa, a contact angle of 63° to 91 °, a mechanical strength of 20 to 100 MPa, an elastic modulus of 3000 MPa to 7000 MPa, and an elongation at break between 1 % and 10%.

[0044] A plasticizer may be added to the formed fibrils before forming the fibril network and the film in order to adjust the mechanical properties of the fibrillary film.

[0045] The formed films are composed of a natural combination of cellulose and mycelium, wherein the mycelium act as a binder of the cellulosic fibrils. This is not found in other films for packaging or paper.

[0046] In one example, the fibrillar film has an oxygen permeability coefficient below 1 x w16cm3cm / s cm2Pa, a contact angle of 63° to 91 °, a mechanical strength of 40 to 70 MPa, an elastic modulus of 3800 MPa to 5300 MPa, and an elongation at break between 1.5% and 2.5%.

[0047] The oxygen permeability coefficient (PO2) is defined as the thickness and pressure normalized rate at which O2 passes through a material and may be expressed in cm3pm / (m2day atm).

[0048] BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Fig. 1 shows A) spent mushroom substrate (SMS) as received after mushroom cultivation, B) SMS after mechanical disintegration of the SMS by hand, C) SMS after fibrillation, i.e. an SMS fibrillar gel product, D) an SMS film, E) an SMS foam.

[0050] Fig. 2 shows viscosity as a function of energy demand during fibrillation of SMS, dispersed to different concentrations in a solution, into fibrils.

[0051] Fig. 3. shows images of the SMS samples of Fig. 2, dispersed in a solution to different concentrations, before (upper row) and after fibrillation (lower row).

[0052] Fig. 4 shows representative stress and strain curves for films formed through fibrillation of SMS dispersed in a solution to different concentrations.

[0053] Fig. 5 schematically shows a method of producing fibrills from lignocellulosic substrates. Fig. 6 shows antioxidant activity of fibrillated and freeze-dried SMS and reference material as a function of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical after 30 min (upper graph) and 20 h (lower graph) of incubation. Figs 7a-7c show different properties of fibrillated SMS material formed into a foam product. Fig. 7a) shows stress versus strain curves for a compression test of the foam. Fig. 7b shows water and oil absorption capability after five minutes immersed in the fluid. Fig. 7c shows absorption capability of the foam to remove the oil from the water, and the picture of the foam after removal.

[0054] DETAILED DESCRIPTION

[0055] Below is described an example of a method of forming fibrils from a lignocellulosic substrate. Mushrooms, below exemplified with shiitake mushrooms, are grown on the substrate and harvested. The thus formed spent mushroom substrate (SMS) is collected and fibrillated, forming a fibrillary gel. The gel is then formed into films and foams.

[0056] Preparation of the material

[0057] SMS material produced by growing shiitake mushrooms on lignocellulosic material from hard wood and possible a small amount of wheat bran was stored in a freezer at - 23°C until use. The SMS was removed from the freezer to defrost and was disintegrated by hand (Fig.1a). In order to further fibrillate the material in a ultra-fine grinder, a low solid content was required. The solid content was first determined based on the raw material by using a moisture analyzer (HR83 Halogen, Mettler Toledo, Columbus, Ohio, USA) and then reduced to a concentration of 2.5 wt. %, 4.5 wt.%, 6.5 wt.% and 8.5 wt.%, respectively, (Fig. 1 b) 1 h before the grinding process. Prior to fibrillation, the suspension was pre-dispersed using an Ultra Turrax (T25 digital ULTRA TURRAX, IKA, Steifen im Breisgau, Germany) which rotated with 9000 rpm for 15 min (Fig. 1c). The suspension was then stirred until the start of the grinding process using a magnetic stirrer (VMS-C7, VWR, Radnor, USA) to prevent segmentation of the particles (Fig. 1d).

[0058] Fibrillation process

[0059] A super mass colloider (MKZA6-3, Masuko Sangyo Co., LTD., Kawaguchi, Japan) with coarse silica carbide (SiC) grinding stones was used for the grinding (fibrillation) process, with the aim to fibrillate (separate) the cellulosic biomass into nanofibers. The fibrillation was conducted in contact mode, with the gap of the two discs set to contact and then gradually adjusted to -90 pm, at 1500 rpm. During the grinding process, samples were collected for viscosity measurements and optical microscopy (OM) analysis, which were used as an assessment during the grinding process. For the viscosity measurements, a Vibro Viscometer SV-10 from A&D Company, Ltd. (Tokyo, Japan) at a constant shear rate and periodical circulation of the sensor plates from zero to peak (sine-wave vibration) was used at a frequency of 30 Hz. Because the temperature increased during the fibrillation owing to the compression and abrasive shearing forces, the viscosity measurements were repeated at a stabilized temperature of 22.3 ± 1 .0 °C to confirm that a viscosity plateau had been reached during the process. The presented values are an average of three measurements for each sample. The aim is to visually observe a reduction of size in the OM together with an increased viscosity, which is an indication of more separated fibers that forms a stronger network formation. The process is terminated when there are no longer visually intact larger structures in the OM combined with a reached viscosity plateau. The optical microscope used was a Nikon Eclipse LV100N POL (Kanagawa, Japan), equipped with imaging software NIS-Elements D 4.30. The energy consumption of the fibrillation process was established by the direct measurement of the power with an energy analyzer, EM24 DIN, Carlo Gavazzi (Belluno, Italy) and from the monitored processing time. The energy demand was calculated from the product of the power and the time, and the energy consumption is expressed as kilowatt-hour per kilogram of dry weight of nanofiber.

[0060] Results of viscosity and energy demand measurements - fibrillation process

[0061] The viscosity as measured during the fibrillation as a function of the energy demand is presented in Fig. 2 for concentrations ranging between 2.5 wt.% and 8.5 wt.%. The viscosity as measured during the fibrillation gradually increases during the fibrillation process at all concentrations, thus indicating a reduced particle size despite the increase of temperature upon fibrillation. Although comparable behavior was observed upon fibrillation, the different concentrations resulted in a large difference in viscosity value as seen from the different scale on the y-axis. Also, the energy demand for the fibrillation process was affected by the fibrillation concentration, where a higher concentration appear to be beneficial for an energy-efficient fibrillation process with lower energy consumption. Results of optical microscopy - before and after fibrillation

[0062] As described in the Ultrafine grinding section, samples were taken during the grinding process for optical microscopic analysis. Fig. 3 shows representative samples from the different batches before and after fibrillation process. The fibrillated samples display no larger intact structure, indicating that the fibrillation has successfully separated the material into nano sizes.

[0063] Antioxidant activity of the SMS material

[0064] Radicals scavenging activity of the SMS material and a chemically pretreated reference material, a commercial Kraft pulp (a process for conversion of wood into wood pulp, which consists of mainly cellulose fibres, after fibrillation were analysed. Adequate amount of freeze-dried material was weighted in a tube and 0.4 mL distilled water was added. Then, 0.4 mL of 0.2 mM 2,2-diphenyl-1-picrylhydrazyl (DPPH) ethanol solution were added. The final concentration of freeze-dried material varied between 0.0625-12.5 mg mL-1. The final concentration of DPPH was 0.1 mM. A blank sample was prepared containing 0.4 mL distilled water and 0.4 mL of 0.2 mM DPPH ethanol solution. Moreover, a series of blank samples were prepared containing the materials without DPPH adding 0.4 mL distilled water and 0.4 mL ethanol. The final mixtures were allowed to shake at 1000 rpm and room temperature in the dark. At 30 min and 20 h, an homogeneous aliquot was withdrawn, and the sample mixture was centrifuged for 2 min at 5,000 rpm to remove the hydrogel solids. The supernatant was pipetted into a 96-well plate and its absorbance at 517 nm was measured via a microplate reader. The DPPH radicals scavenging activity was calculated as following: DPPH radicals scavenging activity (%) = [1 - (As - A1 ) / A0] x 100 where As is the absorbance of the sample and DPPH, A1 is the absorbance of the sample (hydrogel in water: ethanol 50:50 v / v), and A0 is the absorbance of the control (DPPH 0.1 mM in water: ethanol 50: 50 v / v).

[0065] From Fig. 1 , the fibrillated SMS material, at the highest tested concentration of 12.5 mg mL-1, 91 .25% scavenging activity was observed after 30 min of incubation (upper graph), and after 20 h of incubation (lower graph), a 50% scavenging of the free radical was observed at only 0.31 mg mL’1, while the reference material had negligible antioxidant activity even at the highest loading. Furthermore, for SMS, a 100% scavenging activity was observed at only 2.5 mg mL’1, which signifies that biological pretreatment, i.e. the growing of mushrooms on the lignocellulosic substrate, could be beneficial to preserve functional properties such as antioxidant activity compared to chemically pretreated and fibrillated materials.

[0066] Film manufacturing process

[0067] The production of films from the grinded material can be divided into two steps. First, vacuum filtration is used to generate a fiber network and then various drying processes are used to dry the material.

[0068] Vacuum filtration

[0069] The films produced have a grammage of 100 and 150 grams per meter square. The suspension was diluted to 0.3 wt. % and then stirred with a magnetic stirrer (Variomag Multipoint 15, Thermo Scientific, Waltham, Massachusetts, USA) for 30 to 40 minutes directly before vacuum filtration has started. To perform vacuum filtration, a Buchner funnel, a Buchner flask, a vacuum pump (VCP 80, VWR, Radnor, USA) and a filter membrane (Durapore PVDF from Millipore, Darmstadt, Germany (Diameter: 90mm, Filter-Type: 0.1 pm)) were used. The Buchner funnel was placed in the Buchner flask and the output of the Buchner flask was connected to the vacuum pump using a rubber hose. The filter membrane was then placed as centrally and flat as possible in the Buchner funnel and a spirit level was used to measure whether the construct was standing straight. Then the vacuum pump was switched on and the suspension was carefully poured onto the center of the filter membrane. Therefore, the time for filtering also depends on the thickness of the film. Depending on the thickness of the film and the strength of the pump, this process takes 4 to 15 hours for the films manufactured so far. The process was stopped as soon as a coherent network was formed. There was no longer any visible liquid in the Buchner funnel, but the film was not completely dry, in order to facilitate the removal of the filter membrane. The pump was switched off and the Buchner Funnel was removed. The filter membrane was removed from the Buchner funnel together with the film. Then the filter membrane was carefully removed from the film. If the film has already dried too much, it can be moistened again by spraying distilled water to be able to remove it better from the filter membrane. After removing the membrane, the drying process continues. Drying

[0070] The drying of the films can also be divided into two steps. The first is to dry the film as much as possible and the second is mainly responsible for generating flat, densely packed films.

[0071] For the first step, a vacuum oven (NSV 9000, LABEX, Helsingborg, Sweden, vacuum pump: MZ 2C, Vacuubrand GmbH & Co KG, Wertheim, Germany), 2 aluminum plates (300 mm x 300 mm x 1.8 mm, 0,36 kg), paper, two thin-meshed metal nets (Pore size: 50pm) and a weight (5,3 kg) were used. To dry the film, a layer system of different materials was used. The film was placed between the two metal nets immediately after the filter membrane was removed after the vacuum filtration. The paper was then placed around the metal nets, and then covered by the two metal plates. The weight was then placed on the top of it. This setting was then placed in the vacuum oven at 80°C and -950 mbar for 30 min. After 30 minutes, the oven was brought to normal pressure, and everything was removed. Then the film was released from the setting.

[0072] The second step required a hot press (LabEcon 300, Fontijne Press, Vlaardingen, Netherlands), 2 metal plates (300 mm x300 mm x 1.8 mm, 0,36 kg) and two Mylar films (100 MICRON MYLAR, Lohmann Technologies, Milton Keynes, UK). The film was placed between the Mylar foils directly after the first step and then surrounded by the metal plates. Then this setting was put into the hot press preheated to 100°C and pressed for 10 min at 2.5 MPa. After 10 min, the press was cooled down to 20°C and the film was removed.

[0073] Finished films

[0074] The resulting films were 150 grams per meter square films and had a heterogeneous particle distribution. However, the films are flexible and have a very smooth surface that resembles a plastic film.

[0075] Foam manufacturing process

[0076] Fibrillated materials were also used for foam production. The material was frozen in liquid nitrogen and then freeze-dried in two steps. An Alpha 1-4 LSCpIus freeze dryer (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany.) was used for freeze drying. In the first step, the samples were dried at 10°C and a pressure of 1 mbar for 40 hours. In the second step, the pressure was further reduced to 0.1 mbar for one hour. The fibrillated materials form porous lightweight foam structures after the process.

[0077] Properties of the prepared films

[0078] Oxygen permeability

[0079] Barrier properties are important for packaging application, where for example a low permeability is of importance. Oxygen permeability was measured by measuring the flow rate calculated by the distance of a bubble traveling a volume / time at 25°C. a sample film disc (diameter = 25 mm) was clamped between one o-ring and an AI2O2 membrane, and a constant oxygen gas flow at a pressure of 2.7 bar was applied to the film, having a thickness of 62 pm. Taking into consideration the formula presented by Stem, the permeability coefficient can be calculated by

[0080] Q x d P = — -

[0081] A x AP where P is the coefficient of permeability (expressed in 1 barrer = 1O’10cm3(STP) cm / sec cm2cmHg), Q is the mass flux of gas through the film of area A and thickness d under a partial pressure gradient AP. It is important to note that the values expressed in STP, are related to standard temperature and pressure (273 K and 1 bar).

[0082] No flow could be detected through the SMS films at 1 bar. Common polymers used in packaging applications such as PLA or polyethylene (PE) displayed an oxygen permeability coefficient between 5.8 x 10’15and 1.0 x 10’9cm3cm / s cm2Pa.

[0083] Displaying that the formed SMS films displayed a good barrier against oxygen.

[0084] Contact angle

[0085] The wettability of the nanofiber networks was characterized using water contact angle measurements with an EASYDROP measuring system, drop shape analysis control (DSA1), and evaluation software (Kruss GmbH, Hamburg, Germany). A 4 pl drop of water was placed onto the sample and the software of the instrument calculated the contact angle over time with a sessile drop technique. The contact angle was determined immediately after a stable drop was formed on the sample surface. The reported values are the average based on three measurements. The contact angle analysis was first carried out before film production was developed further, where the filter membrane was removed from the film using distilled water and placed on a Mylar foil. After this, the film was covered with a thin-pored metal net, paper and 1 kg of weight. This created a film that was smooth on one side of the Mylar film and a little rougher on the other side.

[0086] The contact angle was measured to evaluate the hydrophilic or wettability behavior of the material. It was measured to be between 63° to 91° directly after the drop had stabilized on the surface. The material displayed hydrophilic behavior, but also demonstrated that the surface wettability could be altered with pressing to achieve hydrophobic behavior.

[0087] Mechanical properties

[0088] Samples were prepared for the tensile test. A 150 g / m2film was used, from which samples with a width of 5.9 mm were cut out. Then the samples were measured three times each in thickness with an ABS digital thickness gauge (Series 547, Mitutoyo, Kanagawa, Japan) to calculate the average thickness of the samples. The samples were then glued into paper frames which were made with a laser cutting machine (CMA0604-B-A, Han's Yueming Laser, Guangdong, China), and had a window size of 2 cm in height and 1.4 cm in width. Afterwards the samples were conditioned for 115 hours. For the tensile test the universal testing machine Shimadzu AG-X (Shimadzu, Japan) equipped with a 1 kN load cell (SLBL-1 KN, Shimadzu, Japan) was used. To operate the machine and record the measurements, the software Trapezium 1.1.1 (Shimadzu, Japan) was used. The test speed was 2 mm / min. The results of the tensile test and representative stress-strain curves is shown in Fig. 4.

[0089] The SMS films displayed mechanical properties that were comparable to for example polylactic acid (PLA), which is a commonly used material for packaging applications. The strength was measured to about 40 to 70 MPa, compared to PLA with a strength of 53 MPa. Elastic modulus or stiffness of the SMS films was between 3800 MPa to 5300 MPa, and elongation at break between 1 .5% and 2.5%, compared to PLA having elastic modulus of 3600 MPa and elongation at break of 2.6%, respectively.

[0090] Properties of the prepared foams

[0091] Mechanical characterization for SMS foams

[0092] Foams in a cylindrical shape with 15 mm in height were subjected to a compression test using a Instron 4411 Series (Instron) at a strain rate of 10% / min with a 500 N load cell. In terms of compression properties (Fig. 7a), at 50% strain, the foam showed a compression strength of 51 kPa, while the modulus was found to be 0.08 MPa.

[0093] Absorption capacity

[0094] Water and oil absorption capability of foams are investigated by immersed the foams in distilled water, and mineral oil, respectively, for 5 min at room temperature. Excess water / oil on the bottom was removed with filter paper, and the foams were weighed. A total of tree replicants per sample were obtained. The absorption capacity (CAB) was calculated by the following equation:

[0095] CAB = (ms ~ wi0) / m0where m0and msare the weight of the foam before and after immersion, respectively.

[0096] The water and oil absorption capacities are shown in Fig. 7b, the foam showed absorption capacity for water of 21 w / w, whereas a higher value was found for the absorption in oil (28 w / w). After 5 min of immersion, no disintegration of the foams was found in both fluids, suggesting good network formation and stability of both foams. For oil fluid, the foams were very stable, and no deformation was found. In addition, the absorption of oil in water medium was also performed, as showing in the sequential pictures in Fig. 7c, the foam could successfully absorb the oil in the water medium, without coalescence of the foam.

[0097] Density and porosity for SMS foams

[0098] The skeletal density (pS) of the foam samples was determined using helium pycnometry (Accupyc 1340, micromeritrics) using of crushed foam samples (approx. 0.05 g) in a 1 cm3 cell, with 10 purges per measurement. To determine the foam density (pF), five samples (around 0.5 x 0.5 x 0.5 cm3) per foam were analyzed using an envelope density analyzer (Geopyc 1360, micromeritrics). The porosity of the foams was calculated as described in equation:

[0099] P(%) = (1 - PF / PS ^ IOO

[0100] The skeletal density was found to be 1 .67 g / cm3, while the foam density was 0.087 g / cm3, the calculated porosity was 94.8%. Summary

[0101] The approach starts with residual wood from Swedish sawmills and leftovers from forest thinning as substrates for edible mushroom production, as one step of cascade uses of resources. In the next step, spent mushroom substrates (SMS), after mushroom harvest, was directly without any use of chemicals, processed for nanofiber production, using fibrillation. This approach can considerably reduce environmental impact and energy consumption due to no need of chemical pretreatment that is conventionally used to separate cellulose from lignin for nanofiber production. During cultivation the substrate undergo a biological pretreatment, where the edible fungi can selectively degrade lignin during growth and leave SMS (70% of initial substrate dry mass) as a cellulose rich feedstock that can result in an easier and more cost-effective process. The nanofibers form very good networks and were further assembled into films and foams and evaluated for their use in packaging applications. The films display comparable or better properties (mechanical and barrier) to those of commonly used packaging materials such as plastic or paper. Foams could be formed into lightweight porous structures.

Claims

CLAIMS1 . Method of forming fibrils from a lignocellulosic substrate, comprising: providing (100) a lignocellulosic substrate, growing (101 ) mushrooms on the lignocellulosic substrate, harvesting (102) said mushrooms, collecting (103) the lignocellulosic substrate on which the mushrooms have been grown, which substrate is a spent mushroom substrate, fibrillating (104) the spent mushroom substrate, forming fibrils, wherein the spent mushroom substrate is collected and thereafter fibrillated without any intermediate chemical or thermal treatment step.

2. The method of claim 1 , wherein the spent mushroom substrate is fibrillated using mechanical separation, grinding, homogenization, and / or shear extrusion.

3. The method of any of the preceding claims, wherein the formed spent mushroom substrate is dispersed in a suspension to a concentration of 1 wt.% to 50 wt.% before fibrillation.

4. The method of any of the preceding claims, wherein the lignocellulosic substrate is selected from straw, hardwood, softwood, or any mixture thereof.

5. The method of any of the preceding claims, wherein the formed fibrils are vacuum filtrated or casted, thereby forming a fiber network (105).

6. The method of claim 5, wherein the formed fiber network is dried (106), forming a film.

7. The method of claim 6, wherein drying (106) the fiber network comprises a step of pressing the fiber network.

8. The method of any of claims 5 to 7, wherein the formed film is applied (107) onto at least a surface of a packaging material or paper.

9. The method of any of claims 1 to 4, wherein the formed fibrils (108) are freeze- dried, thereby forming a foam.

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