Developing Your Mushroom Substrate Sterilization Procedures
By: Priorclave North America
Category: Lab Practices
Contamination is the most common source of failure in any mushroom crop. This especially affects slow-growing mushrooms with longer colonization times.
The same characteristics that make mushroom substrate delicious to mushroom mycelium also make it appealing to mold and bacteria. Those mold and bacteria often take hold and flourish much more quickly than your desired fungi, outcompeting them for resources in the substrate. That leads to reduced yields, and even complete crop failure.
Some hearty mushroom varieties (e.g., most popular edible mushrooms, including all the varieties of Oyster and Agaricus mushrooms) can thrive in non-supplemented straw substrates, which don’t tend to support very tenacious microorganisms. But if you’re working with moist, dense, nutrient-rich, highly supplemented substrates like manure, whole grains, soy hulls, and “master’s mix” (a mix of soybean hulls and hardwood sawdust), you’re going to need to sterilize that substrate to give your mushrooms a fighting chance. That means developing a set of validated sterilization procedures for your preferred mushroom substrates.
Mushroom Substrate Pasteurization vs. Sterilization
Clarity about the terms “pasteurization” and “sterilization” is essential.
Pasteurization kills dangerous pathogenic bacteria and weakens microorganisms. It is only effective in less-dense, less biologically active substrates (e.g., non-supplemented straw). Pasteurization does not remove, kill, or deactivate all fungi, bacteria, viruses, spores, endospores, unicellular eukaryotic organisms, etc. Heat-based pasteurization methods rely on holding the substrate between 150 and 180 degrees Fahrenheit for several hours. Because heat-based pasteurization is only weakening invasive microorganisms, there isn’t a practical way to validate this process. That makes it inherently unreliable for many uses. For example, some bacterial spores become more likely to germinate if heated and held at 150 degrees Fahrenheit; therefore, heat pasteurization can be worse than skipping the step altogether.
Sterilization aims for complete, reliable destruction of all microorganisms and spores in your mushroom substrate. Sterilization requires heating your substrate to 250 degrees Fahrenheit under pressure and holding the temperature steady for several hours. Remember that sterilization time varies wildly (and not necessarily linearly) by substrate type, volume, and density. For example, you can sterilize small volumes of hardwood sawdust in less than twenty minutes. That same sawdust in blocks or large bags could take more than 2.5 hours to sterilize. Jars of grain spawn can likewise sterilize in forty-five minutes to two hours, depending on the size of the container, moisture of the mixture, and so on.
With validated sterilization some extra steps will confirm that your sterilization procedure is successfully destroying all spores and other biological contaminants. In general, that means running test sterilization procedures with a high-quality “biological indicator” (usually spore ampoules) to confirm that controlled variables successfully sterilize a specific load size and configuration.
A Note on “Steam Recycling”
We occasionally hear about people “recycling” the steam from their autoclave. Ffor example, they might inject that steam back into the fruiting room as a cost-effective way to heat the space and maintain high humidity. While this might be acceptable in smaller mushroom operations that only handle edible fungus and do no other lab operations, we advise against the practice in any mycological laboratory for three reasons:
- Safety: As a rule, we advise that labs vent their autoclaves outside the building, well above ground level, using a heat-resistant pipe not less than 30mm (~1 in) in diameter. This is because effluent and vented gas can potentially carry and distribute viable spores during some portions of the autoclave cycle, even with non-biohazardous loads. If you’re regularly using your autoclaves to process potentially hazardous, pathogenic, or contaminated waste, you should both vent outside your building and plumb a direct connection to the building sanitary sewer drain.
- Consistency: Many cycles (especially those with freesteaming or vacuum stages) can produce large volumes of waste water and steam (which may, in turn, carry viable spores and endospores). Biohazard risks aside, the volume of steam can be hard to manage, causing unacceptable spikes in heat and humidity.
- Containment: Pumping steam into your fruiting room will create positive pressure, which runs the risk of dispersing mushroom spores and contaminants from the fruiting room elsewhere in your lab (such as the inoculation and incubation spaces), leading to cross contamination.
We applaud every effort to increase efficiency and cut costs. Still, this is a case in which a small win for efficiency can be overshadowed by huge blows to crop consistency and yields.
Beware “Sterilization” That Isn’t Sterilization
A leading culprit of botched mushroom substrate is inaccurate verbiage: Several types of pasteurization are casually referred to as “sterilization,” even though they do not sterilize the substrate. Principal among these are “cold sterilization” and “atmospheric sterilization.”
“Cold sterilization” is a broad term for a range of non-heat pasteurization options that include lime baths, peroxide baths, and cold-water fermentation. “Atmospheric sterilization” is more accurately considered “super pasteurization.” Here, substrate is boiled in an open vat or barrel, with heat somewhat higher (kept as close as possible to the boiling point: 212 degrees Fahrenheit) and the dwell time significantly longer (up to eighteen hours).
Broadly speaking, you need to hold mushroom substrate at 250 degrees Fahrenheit (121 degrees Celsius) or hotter for one to four hours to reliably kill all bacteria and fungal spores in your substrate. This is substantially hotter than boiling water or steam at regular pressure (i.e., in an open container). You simply cannot heat boiling water and steam higher than 212 degrees Fahrenheit at sea level, regardless of how much heat you apply to it. As a rule, if you are not pressurizing a chamber, you are not reliably sterilizing your substrate.
Developing Your Validated Mushroom Substrate Sterilization Procedures
Most people growing anything other than common grocery store mushrooms will need several different procedures for their various substrates and preparations (bagged substrate, blocks, jars or sample containers, etc.). Here’s a good starting point to establish a reliable, validated substrate sterilization procedure:
- Prepare several identical substrate samples (e.g., bags, jars, trays, etc.)
- Load these into an autoclave tray.
- Embed biological indicators (BIs) in the load, making sure to have one at the center of the load’s mass (i.e., in the middlemost jar, in the middle of a bag of hulls, etc.), and several along the length of the load at various depths.
- Process the load for 120 minutes at 121 degrees Celsius or twenty minutes at 135 degrees Celsius
- Incubate your BIs.
- If the BIs show no activity (i.e., they don’t change color/turbidity after 48 hours of incubation), your procedure is valid.
- If the initial sterilization protocol activated the BIs, note which BIs failed. (You may discover a cold spot in your autoclave chamber that can be addressed by simply positioning the entire load farther back, for example.)
- Repeat your procedure with an identical load (same style tray, same substrate type and amount, same number and placement of spore ampoules), but process it twice as long as the first load to ensure BI incubation. If biological activity is still evident, repeat the procedure, processing for three times the original cycle time.
Reliable Tools and Validated Procedures Give Reliable Results
Are you frustrated by an unreliable autoclave or do you need help finding the right sterilization systems for your lab? Let us know; we’d love to help.
Reliable tools are the key to a reliable mushroom substrate preparation. We optimize Priorclaves for long-term reliability and low-energy/low-water/low-maintenance operation. Each unit is backed by our free lifetime technical support—including advice on validating custom cycles and protocols like these.