Leather production by cultivating Reishi.pdf

Instructions for leather substitute production by cultivating Reishi(Ganoderma lucidum)



Fungal-based leather substitutes offer an alternative fabrication materialto animal-based leather as well as synthetic leather. Not only is it animal friendly, more importantly it is environmentally friendly. In times of environmental uncertainties, such sustainable solutions are in  high demand.
This article explains how to make leather substitutes from fungal material.
For all experiments and productions the mushroom Reishi (Ganoderma lucidum) was used, but other genera are suitable as well.For the production of the fungal substitutes two media are used; solid and liquid cultures.The two procedures allow different growth possibilities for the fungal mycelium. In liquid culture, which is a nutrient solution, the fungus can grow in three dimensions. In the solid medium, the so-called petri culture, the fungus can grow two-dimensionally. Both processes can be used for the production of leather substitutes.Which method is used for depends on the desired end product.

1. Preparation of an agar plate as a nutrient medium in Petri dishes

In these instructions, a suggested recipe for 1 L "MEA" (malt extract agar medium) is presented, which is suitable for most fungal genera. For working with spores, antibacterial agar mixtures are also available, which only need to be mixed with water.

1.1 Materials

Recommended quantities for petri culture

-         24 g agar agar

-         20 g malt extract

-         2 g dry yeast

-         1 g peptone

-         1 L (drinking) water

Additional materials and recommendations

-         Sterile disposable petri dishes or glass petri dishes

-         Measuring beaker

-         Scale

-         Parafilm

-         Latex gloves

-         Face mask and hair net

-         Hand and surface sanitizer

-         Sterile work area; sterile air flow (HEPA, laminar flow)

1.2 Method

1.2.1 Mixing substrate

Mix the ingredients together with warm water in an Erlenmeyer flask (closefirmly andshake). We recommend filling the mediumflask only up to 2/3. Thenplace the Erlenmeyer flask (without the lid) into the pressure cooker. In order to avoid condensation water getting into the solution,the neck of the flask is wrapped in aluminium foil (see picture right).

1.2.2 Sterilise substrate

The agar is sterilised for 45 minutes, we use a pressure cooker for this part. Add about 2-3 cm of water into the pot and a layer (please note the product information by the manufacturer). Place the flask upright into the pot, it shouldn’t float! If necessary pour off some water.
Close the lid tightly and place the cooker on a hotplate. Starting from the time where the pressure cooker reaches the highest level (for household pressure cookers) or 121°C/1.05 bar for professional pressure cookers, the sterilisation time starts (45 minutes).Make sure that the temperature does not exceed the temperature mentioned (121°C), especially not for a longer time period.This can lead to thedestruction of the nutrients contained in the solution.
The liquid should have a yellowish colour after sterilisation. If the liquid is rather brownish, the temperature was too high and the medium is unusable (note: the colour of the medium may differ depending on the recipe).
After the sterilisation, the pot should be placed somewhere sterile to cool down. Preferably in front of a HEPA filter (sterile air flow). During cooling, a damp (clean) cloth can be placed over the pot, this additionally filters the air that is sucked into the pot.

1.2.3 Pour agar mixture into petri dish

After the pressure cooker has cooled down and the pressure indicator is at zero, the pot can be opened.The agar mixture should be poured into the petri dishes as quickly as possible, as it hardens during cooling.If glass petri dishes are used, they should be sterilised at the beginning.We recommend 25-30 ml agar per dish, which corresponds to about 90 mm.A 1 L mixture thus corresponds to about 35-40 filled petri dishes.
The ready-cast petri dishes should be placed in front of an air filter or in an inoculation box during cooling to avoid contamination.As soon as the culture mediums are firm and have cooled down to room temperature, they can be processed.Unused petri dishes can be stored in the refrigerator (2 - 4°C) for up to 4 weeks, to avoid contaminationwe recommend that the trays are sealed with parafilm and packed in a plastic bag.

2. Preparation of liquid nutrient medium

2.1 Materials

Recommended quantities forliquid cultures

-         20 g malt extract

-         2 g dry yeast

-         1 g peptone

-         1 L (drinking) water

Additional materials and recommendations (see 1.1 Materials)

2.2 Method

The method used for liquid cultures is equivalent to solid cultures, with the exception of agar agar.

3. Strains/fungal cultures

For the production of the leather substitute, we have cultivated a Reishiculture (Ganoderma lucidum)suitable for both growth media applications.

Liquid culture: firm growth with horizontal, two-dimensional fruiting body formation

Petri culture: “fluffy” growth with three-dimensional mycelium growth

3.1 Fluffy MyceliumDualuse

Fluffy Mycelium Dualuse is a process by which insulation panels (Dämmstoffplatten) for building materials industry and leather substitutes are grown together in one form. The aim is to produce both end products (insulation panels and “leather”) at the same time.

3.1.1 Materials

In these instructions, a suggested recipe for 15 L form medium is presented, which issuitable for most fungal genera.

Recommended quantities for form culture

-         5 kg wood pellets/cellulose

-         1 kg vermiculite

-         9 L (drinking) water

Additional materials and recommendations

-         Compulsion mixer

-         Breeding bag

-         Measuring beaker

-         Scale

-         Latex gloves

-         Face mask and hair net

-         Hand and surface sanitizer

-         Autoclave

-         Sterile working area; sterile air flow (HEPA, laminar flow)

3.1.2 Method

1.       Place the ingredients in the compulsory mixer and mix for about 5 minutes until a homogeneous base substrate is obtained.

2.       Fill the mixture into culture breeding bags and autoclave (for approx.80 to 90 minutes).

3.       Let the substrate bag cool down to 26°C in the autoclave.

4.       Inoculate substrate bags with fungal culturein laminar flow and seal them airtight.

5.       Allow the bags to grow for 1-3 days at 24°C (without light) at 1200 ppm CO2.

6.       Fill substrate into moulds and cover with a porous membrane, then incubate again. (note: Basic substrate can also be placed directly in moulds and then inoculated with liquid culture, this saves working steps. However, the work must take place in a sterile room.)

7.       Fill the moulds with CO2 to achieve a CO2 value of 900-1200 ppm. Open moulds after 3-14 days, leather substitute has formed on the top of the membrane and can be peeled off and used for further processing. A moulding plate has formed under the membrane, which can be demoulded and dried at a minimum of 38°C.

4. Physical Properties

4.1 Elasticity (tear resistance)

Elasticity (tear resistance) of the mycelium-based leather compared to the reference for leather (8-25% elongation).

4.2Thermogravimetric analysis

The measuring device used is a Q500 TGA from TA Instruments. This allows thermogravimetric analyses to be carried out in the temperature range between room temperature and 1000 °C.
For all measurements in this report, about 5 mg of the material was heated from room temperature to 1000 °C at 10 K/min. Artificial air was chosen as the atmosphere.For the material samples, the temperature at which 90% and 50% of the material is still present was measured in each case.


Figure 1: Measurement results of the samples of the B/P series.


Table 1: Summary of temperatures (in °C) at which 90% or 50% of the starting mass is still present

The samples from the B/P* series (compared to P/L* and A/P/F**) show the best thermal resistance. This could play a role in further processing methods where the material has to be heated.


























* B/P and P/L are a mycelium-based leather substitute

** A/P/F is a fruitbody-based leather substitute


5. Application

Fungal-based leather substitutes offer an alternative fabrication material to animal-based leather. Not only is it animal friendly, more importantly it is environmentally friendly. In times of environmental uncertainties, such sustainable solutions are in high demand.