KickStart Your Myco-Culture - Open Source Practices from FabTextiles Lab

Kick Start Your Myco-Culture !

An introduction to cultivating mycelium for products, materials or to grow mushrooms!

Curated with love by Jessica Dias for Fabtextiles

Contents 1. What is mycelium?

2. The magic of mushrooms Part 1 - Cultivating mycelium - The basics

a) Sterile experiment protocols

b) Agar preparation

c) Spawn preparation

d) Bulk substrate preparation

Part 2 - Material application

a) 3D forms

b) Myco-textile

c) Post processing

i) Compression

ii) Plasticiser

ii) Dehydration

3. Recommended reading

What is mycelium?

Mycelium is the root-like vegetative part of the fungi, it is formed of a filamentous hyphal network that branches out, forming connections with other hyphae in search for nutrients and moisture from its surroundings, where it feeds on organic matter by releasing enzymes to help it digest the medium it grows on. During this process it is able to fuse together the substrate, making it an excellent and natural binder for organic materials.

Nature’s natural binder, mycoremediation and the magic of mushrooms!

Different species of fungi posses different mycelial behaviours and properties, therefore the species of mushrooms to use very much depends on the overall objective. For example, the Oyster mushroom is a great starter species as it grows fast, consumes almost anything and is delicious! Its mycelium however, is not strong enough to use to make a textile, but can be considered as a good binder for larger objects with coarse substrates such as sawdust and straw.

Like bacteria, certain fungi strains have excellent bio-remediation properties, meaning they are able to digest particular toxins from soils and water and can be considered as a great natural filtering system in polluted areas.

Mushrooms themselves possess powerful healing properties, and have been used as holistic alternatives for energy, as nootropics, immune boosting supplements, for focus and sleep aid.

The Pleurotus Ostreatus, oyster mushroom, digests most matter, making it the perfect strain when starting cultivation. It is also known to break down oils and toxins, alluding it to be useful for cleaning up contaminated waters and soils.

The Magic of Mushrooms!

A few examples of available species and what they can be used for: Oyster (Pleurotus ostreatus)

The pearl oyster mushroom or tree oyster mushroom, is a common edible mushroom. It was first cultivated in Germany as a subsistence measure during World War I and is now grown commercially around the world for food. It is related to the similarly cultivated king oyster mushroom.

Reishi (Ganoderma lucidum)

The lingzhi mushroom is a polypore mushroom belonging to the genus Ganoderma. Its redvarnished, kidney-shaped cap gives it a distinct appearance. When fresh, the lingzhi is soft, corklike, and flat. It lacks gills on its underside, and instead releases its spores via fine pore

Horsehoof fungus (Fomes fomentarius)

A species of fungal plant pathogen found in Europe, Asia, Africa and North America. The species produces very large polypore fruit bodies which are shaped like a horse’s hoof and vary in colour from a silvery grey to almost black, though they are normally brown.

Lions mane (Hericium erinaceus)

An edible and medicinal mushroom belonging to the tooth fungus group. Native to North America, Europe and Asia it can be identified by its long spines, its appearance on hardwoods and its tendency to grow a single clump of dangling spine.

Commercial products Companies such as Ecovative and Mycoworks are taking advantage of mycelial binding properties, making use of agricultural waste or organic residue from another manufacturing process, and are spreading the awareness on Biotechnologies and encouraging designers and makers to design and cultivate their own pieces.

There are different techniques to produce different outcomes, each company and designer has their own protocols, species and nutrients for their desired outcome.

Methods include using agricultural waste as a substrate. Generally a bulk substrate is sterilised and inoculated in sterile conditions, allowed to colonise and then broken up, removed and placed in a designed mould. After several days the mould can be removed and the substrate can be dehydrated, deactivating the mycelium and forming a strong but lightweight solid object.

Pioneers of the field -

Mycoworks Ecovative Mycotech Natura studios Biohme Bolthreads

Part 1: Cultivating mycelium - The basics!

Let’s Begin! a) What you will need...

To begin your mushroom cultivation, you will firstly need to purchase either spores, spawn or a fresh mushroom of your choice. With oyster mushrooms being one of the fastest reproducing hyphae and the most readily available, we recommend purchasing a bulk spawn bag from https://grocycle.com/how-to-grow-oyster-mushrooms or https://www.boletsdesoca.com/. Alternatively you can cultivate your mushroom from the flesh of a freshly picked mushroom.

Spawn, Spores or fresh mushrooms Spores are the ‘seeds’ of the mushroom and are usually dispersed throughout the mushrooms gills. You can purchase a spore syringe online containing spores of the strain you would like to use in a liquid medium, this can then be injected directly into the grain jar or agar plates.

You can also harness the spores of a fresh mushroom by collecting the spores released before the cap fully opens and making a spore print.

Spawn is the nutritional substrate, usually grains like rye or barley, which can be either purchased as a pre-colonoised bag or can be prepared using a healthy mycelium plate. With a spawn jar or bag you can store healthy mycelium in a cool dry place to keep the mycelium alive but de-active. A healthy spawn is the starter point for scaling up mushroom cultivation for fruiting or material preparation.

When purchasing a spawn bag, the substrate should come colonised, you can then break this up in separate sterile bag and store them in a

Equipment and Tools • • • • • • • • • •

Pressure cooker Sterile workspace Agar agar Malt extract Petri plates Scalpel Open flame / Bunsen burner Glove box (optional) Gloves Alcohol

b) Sterile experiment protocols The sterilisation process is imperative to working with mycelium as it removes any traces of bacteria or moulds that could otherwise contaminate the substrate and prevent mycelium from fully colonising it.

When working at a small scale like preparing agar plates or spawn jars, working within a 10cm radius of an open flame generally provides a sufficient level of sterility, especially if the room is clean, the work surface is disinfected with alcohol and there is no airflow during the time of the transfer.

Alternatively, working within a glove box or a laminar flow hood is an even better strategy for avoiding the risk of contaminants entering the substrates.

For the sake of this introduction, we will work with an open Bunsen flame.

Phase 1: Cloning or transferring healthy mycelium!

Preparing Agar Plates Malt Extract Agar is a good all rounder when it comes to agar recipes for most mushroom strains. Malt extract can either be purchased in powder form or bought in health stores as a kind of syrup. In any case, the recipe is the same.

1. Mix all ingredients in a jar or blue capped bottle and autoclave for 15 minutes, ensuring the lid is loose 2. Allow agar to cool just enough to be able to hold the bottle without burning your hand, and carefully pour into Petri dishes working within the 10cm radius of the open flame.

Working within 10cm radius of open flame

10cm

MEA (4 Petri Plates) 200ml Distilled water 4g Malt Extract powder 4g Agar 0.5g Yeast

Culture transfer 3. Once the plates are solid (approx 10-15 minutes) continuing to work in the sterilised space, sterilise a scalpel with the flame until

the blade is red hot, allow to cool within the sterilized perimeter and then slice either a pre-colonised agar plate or the inside of a fresh mushroom - a triangular shape is often the most effective way to slice. Carefully transfer this to the fresh plate.

4. Seal plates with either micro-pore or parafilm and mark each plate with the date, mushroom strain and type of media.

MEA plates

Cloning species using a fresh mushroom

Phase 2: Spawning Mycelium!

Spawning mycelium Mycelium spawn is a nutritious substrate that has been fully colonised by mycelium, it usually comes in the form of grains or bird seeds, as the smaller particles work better when inoculating a larger substrate.

Rye grain is a good go-to substrate for spawn preparation. The following technique makes 4 jars of grain spawn when using half a cup of dried rye grain.

1. Thoroughly rinse grain in warm tap water until water runs clean 2. Soak grain ½ cup of grain 1 cup distilled water for 12-24 hours adding a ¼ tsp of Gypsum for calcium carbonate

3. Boil and simmer grain in pot for 10 minutes 4. Strain grain and shake to help grain dry out 5. Fill ¾ of glass jars with semi dry grain and place lids loosely on top - lids should have air exchange holes using either micropore tape or filter wool to cover

6. Place foil over lids to ensure no water droplets fall on lids 7. Place jars in pressure cooker and pour in enough water for 1.5 hours of sterilisation at 15 psi 8. Remove pressure cooker lid and tighten jar lids when cool enough 9. Remove jars and allow to cool before inoculating 10. In a sterile space, either glove box or open flame, transfer healthy mycelium from a colonised plate or healthy spawn jar, adding a few cuts to each jar 11. Seal jars and place in incubator, after a few days hyphae growth should be visible

Phase 3: Bulk Substrate

Bulk substrate Once the spawn is fully colonised and healthy, it is time to transfer to a larger substrate. Generally bulk substrate are less nutritious but larger in size then substrates used for spawning. Good substrates are sawdust, straw, wood chippings, coffee grains, cow manure and luckily most organic matter which can be sourced as a by product of other processes such as spent grain from beer industry, or agricultural waste.

Unlike spawn, the bulk substrate, which is generally less nutritious, only needs pasteurising, which means it is heated at a high enough temperature for a period of time to kill any nasty microbes, but leaving those that help protect the substrate whilst the mycelium colonises it.

There are a number of techniques for pasteurising your substrate. The process generally occurs between temperatures of 70ยบC - 80ยบC for an hour and can be done using water in a boiling pot, in the oven or with steam. Below is a step by step process for pasteurising substrate in a pot.

1. Using a large cooking pot (enough to fit the amount of substrate you wish to pasteurise), fill halfway with tap water and bring to boil.

2. Place substrate in a mesh bag, canvas of nylon work well, and place in pot of pre boiled water Transferring spawn to sterilised substrate

3. Turn down the heat and allow to simmer for an hour 4. Ideally maintain a temperature of 75ยบC 5. Turn heat off, and remove the bag when cool enough 6. Rinse excess water and place bag on drying tray to allow water to evaporate 7. Place pasteurised substrate into the sterilised container or bag you wish to use.

8. When cool enough and working in sterile conditions, distribute pre-colonised spawn into sterilie bag and mix thoroughly.

Fully colonised substrate bag

Incubation

Incubation Incubation is the process of controlling the conditions in which the mycelium is growing. These conditions vary depending on the growth phase and desired outcome of the cultivation. For example, the incubating conditions for spawning are generally humidity of 70%, temperatures between 21ºC - 27ºC and darkness. This varies slightly when transferred to a bulk substrate as the environmental controls is less important. Maintaining humidity within the substrate is important however, and can be done by minimising air exchange.

Laboratory standard incubators can be purchased and generally act as fridge, controlling the internal temperatures.

In the case of large scale production, for example when using jars or bags of substrate, it is more cost effective and efficient to control a larger space then many small ones. plastic grate to allow heat and moisture to circulate aquarium water heater set to 30ºC water heated by aquarium heater

One easy solution is to use a large plastic or glass container and a heating filament, which can be purchased from an aquarium shop or online. The filament must reach a temperature of 25ºC and be submerged in water in the container. Depending on external conditions, the filament temperature may need to be altered to meet an internal temperature of 21ºC-24ºC, which should be indicated by thermostat within the container.

At the incubation phase air exchange is still important to avoid overheating and growth of unwanted contaminates. Therefore ensuring airholes, and regular air circulation is important.

The inoculated substrates can then be placed in the container, above or away from the water, and left to colonise for 2 weeks, depending on the size of the bags. Regular circulation within the jars or bags is also important, therefore breaking up the material by shaking or squishing the bags is advised.

Part 2: Material Application

3D Forming Once the bulk substrate bags are fully colonised, the material can be transferred into a mould, incubated again and then dehydrated.

It is safer to pre-colonise the substrate before depositing into a mould as it is harder to control the growth conditions of the mould, especially if it is somewhat complex, this way the risk of contamination is less as the mycelium is healthy and strong.

Even though the substrate bags are full colonised, there is still risk of contamination whilst applying the material to the mould therefore this should be done with caution and in sterile conditions, using gloves and alcohol to disinfect the space and any tools.

Once the material has been deposited into the mould, it should be covered with little ventilation and further incubated for 2 more weeks, this may vary depending on size of mould.

For more complex and larger moulds it is recommended to design a space where the substrate can be applied to minimise airflow and the spread of contaminants.

The space should be sterilised inside using bleach and clean protective gear should be worn when inside or close to the mould.

Mycotextile Fungal Skin The mycelium secretes enzymes that convert polymers in the substrate into breakdown products that can be taken up to serve as nutrients.

As a result, the organic material is being degraded in time, while being replaced by fungal biomass on and within substrate particles. At a certain moment, hyphae grow out of the substrate into the air creating a fluffy or compact layer covering the substrate.

Substrate Testing Mycelium feeds on cellulose organic matter, however certain strains are also able to produce special enzymes to break down harmful toxins and synthetic materials.

Variables The strain, substrate, humidity and temperature during the growth process will vary the outcome of the results. Furthermore the post processing of the grown material will determine the flexibility and texture.

Materials for testing - Straw - Sawdust - newspaper - cardboard - coffee - oats - hemp fibres - cotton - silk - coconut fibres - paper waste

Post processing

Compression Hot or Cold compression When creating a sheet with mycelium, the sample can be compressed with a heat press or pressure using a flat smooth surface. The sample can then be left to dehydrate in low temperatures or a plasticiser can be added before this step.

The properties of this sheet will vary depending on the substrate used, the strain of mycelium and the height and amount of substrate.

Adding a plasticiser Once the samples have been dehydrated adding a plasticiser can help maintain flexibility of the material. Depending on the thickness and the desired flexibility, soaking the sample in glycerine for a few hours, or in the case of just

Final Dehydration Remove any remnants of the plasticizer and dehydrate the sample for a final time. You may want to compress the sample again depending on the outcome preferred outcome.

Dehydration Once the material has colonised the mould it is time to dehydrate it and deactivate the mycelium. This is when the magic of the mycelium’s binding properties shine!

For smaller samples a food dehydrator or an oven on a heat of 50ÂşC is suffice, but make sure the mould material can tolerate the heat and be careful of burning!

For larger moulds the object can be places in the sun (protected from insects and birds) or placed near a fan on low power.

Futher Reading Weblinks https://biofabforum.org/t/method-of-making-mycelium-leather/218 https://www.kickstarter.com/projects/mycotech/the-worlds-firstmushroom-wooden-watch?ref=project_build&fbclid=IwAR1B58APXvsIIU4ji7PoDM6aiQNZwEtqrcYc0YyKZnBkPSWxYZufxC0eQDs Research papers https://biofabforum.org/t/method-of-making-mycelium-leather/218 Projects https://biofabforum.org/t/method-of-making-mycelium-leather/218 Tutorials https://biofabforum.org/t/method-of-making-mycelium-leather/218 Books https://biofabforum.org/t/method-of-making-mycelium-leather/218