Choosing a Sterilizer aka the Retort or Autoclave

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Although home-style pressure cookers are i leal for sterilizing agar media and for small-to-medium batches of grain, they have insufficient capacity for the sterilization of bulk substrates. The problems faced by the mushroom cultivator in Thailand or the U~:ted States are the essentially the same. In developing countries, the sterilizer is often a make-shift, vertical drum, heated by fire or gas. A heavy lid is placed on top to keep the contents contained. The boiling of water generates

Figure 135. Commercial double door steam sterilizer contamina'' or If too much water is added, exceeding the carrying capacity of the media, the excess collects at the bottoms of the bags, dis couraging mycelium and stimulating bacterial blooms and anaerobic activities. Ideally, sawdust is wetted to 60-65% water. If the wetted mix can be squeezed with force by hand and water droplets fall out as a stream, then the r ,;x is probably too wet.

The easiest way to determine moisture content is by gathering a wet sample of the mixture, weighing it, and then drying the same sample in an oven for 1 hour at 350° F. (180° C.) o in a microwave for 10-15 minutes. If, for instance, your sample w( .ghed 100 grams before drying, and only 40 grams after dr; "Tig-then obviously 60 grams of water were lost The moisture content was ^0%.

Once the person making the substrate obtains experience with making up a properly steam, which over many hours, sufficiently sterilizes the substrate. This method works well within the model of many rural agricultural communities.

The pressurized steam autoclave is far better suited for commercial production. The most useful autoclaves for sterilizing bulk substrates are horizontal and have two doors. (See Figure 134) Since the autoclave is the centerpiece upon which the entire production process is dependent, many factors must be considered in its acquisition: size; configuration and placement. Another important feature is its ability to hold a vacuum subsequent to the sterilization cycle. [f the autoclave can not hold a vacuum as it coo's, a valve should be installed for the controlled intake of filtered air. If the influx of air is not filtered, the contents can contaminate after sterilization. (See Figure 137.)

Hospital autoclaves are typically made of stainless steel and equipped with a pressurized steam jacket. These types of autoclaves are usually smaller than those needed by commercial mushroom cultivators, measuring only 2 x 3 ft. or 3 x 4 ft. by 3-6 ft. deep. Furthermore, they usually have only one door, and their pressure ratings have been engineered to operate at 100 psi, far exceeding the needs of most mushroom growers. Unless obtained on the surplus market for a fraction of their original cost, most knowledgeable spawn producers avoid these types of autoclaves. The most cost-effective vessels are those developed for the canning industries. These are commonly called "retorts" and are constructed of steel pipe, 1/4 to 3/8 inch thick, and ideally fitted with doors at both ends. The doors come in a variety of configurations. Quick-opening, spider doors are popular and durable. Wing-nut knock-off doors are slower to open and close but are less expensive to have fitted onto steel pipe. With autoclaves longer than 6 feet, steam spreader pipes are needed so that the entire mass heats up evenly. More suggestions follow for choosing an autoclave:

Recommendations for equipping an autoclave:

B Double-doors (i.e. doors at both ends) E Redundant pressure/temperature gauges (at least two)

E Pressure/Vacuum Gauge (+ 50 psi to - 50

psi) w/valves E Electrical safety interlocks with warning lights

E I.and-operated vent valve on top of autoclave for venting cold air E 25 psi and 50 psi excess-pressure relief, safety blow-out valves E Hand-operated drain valve for drawing off condensate

E Coated with heat-resistant, anti-corrosive paint

3 At least four 1 inch, and/or two 2 inch ports for inputs, exhausts, and sensors. U One-way gate valve in series with a vacuum gauge that allows the drawing in of clean-room air post autoclaving, (See Figure 137.)

Recommendations for the placement of the autoclave:

E Recessed "wells" (2 ft. x 3 ft. x 2 in.) underneath each door, with sealable drains, for removing excess condensate from the autoclave after opening. C Length of autoclave framed in its own insulated room (R=18 to R=32 with active exhaust (500+ CFM.)) □ One door of autoclave opens into clean room.

C Escape doors located remote from the door seals of the autoclave should metal fatigue suddenly release an impassable curtain of steam.

Once the bags are filled, the supplemented (sawdust) substrate must be heat treated for an extended period of time before inoculations can proceed. In a small pressure cooker, two to three hours of sterilization at 15 psi or 250°F. usually suffices for supplemented sawdust substrates. When sterilizing more than 100 bags in a large pressure vessel, however, the thermodynamics of the entire mass must be carefully considered in choosing a successful sterilization protocol. Hundreds of bags tightly packed in an autoclave achieve different degrees of "sterilization." When bags are stacked against one another, the entire mass heats up unevenly. Even so, this practice is common with those whose autoclaves must be packed to capacity in order to meet production requirements. Bear in mind that sawdust has high insulating properties, making heat penetration through it difficult.

Other factors affect the minimum duration of sterilization. The substrate mixture should be wetted just prior to filling. If water is added to the formula and allowed to sit for more than 6 hours, legions of contaminants spur to 'ife. The more contaminants at make-up, the more that are likely to survive the sterilization cycle.

Fresh hardwood sawdust needs 2-3 hours of sterilization at 15 psi or 250°F. The same mass of sawdust supplemented with rice bran needs 4-5 hours of sterilization. Hence, one of the cardinal rules of mushroom culture: as the percentage of nitrogen-supplements increases relative to the base substrate, the greater the likelihood of contamination, and thus the greater the need for full and thorough sterilization.

I prefer the aforementioned formula using alder sawdust, alder chips, and rice bran. An autoclave filled tightly 5 bags high, 6 bags wide, and 8 bags deep (240 bags) requires exposure to steam pressure for 5 hours at 18 psi to assure full sterilization. The lower, central core is the slowest to heat up. (See Figure 136.) By placing, heat-sensitive sterilization indicator strips throughout the mass of sawdust filled bags, a profile of sterilization can be outlined. Each cultivator must learn the intricacies of their system. Since the combination of variables is too complex to allow universal judgments, each cultivator must fine tune his techniques. Even the type of wood being used can influence the duration of the sterilization cycle. Woods of higher density, such as oak, have greater thermal inertia per scoop than, say, alder. Each run through the autoclave is uniquely affected by changes in the substrate formulation.

Those with ample space in their autoclaves separate the layers so thermal penetration is u^;form. This is ideal. The sterilization cycle can be shortened, again best affirmed by sterilization-sensitive markers. However, few individuals find themselves in the luxurious position of having an autoclave capable of running several hundred bags with one or two inches of separation between the layers of bags. These one or two inches could be used to increase the capacity of the run by approximately 20%. Many small scale cultivators are soon forced to maximum capacity as their production expands with market demand. In the long run, dense packing is generally more cost-efficient compared to loose packing. Hence, dense packing, although not the best method, is usually the norm not the exception with the small to mid-size cultivator. Thus, the manager of the autoclave cycle operates from a precarious decision-making position, constantly juxta-

Autoclave Wood Cycle

Poorest

Good

Completely Sterilized Bag

Autoclave Cross-Sections Showing Density of Fill

Incompletely Sterilized Bag

Separation Rack to Provide Even Steam Penetration

Figure 136. Four profiles depicting the degrees of sterilization as affected by the method of packing the bags into the autoclave. _>?ote cen*. al, lower core is most resistant to deep steam penetration.

Swing Gauge

Diagram Steam Steriliser

Figure 137. A microporous filter canister is attached to a pipe equipped with a gate valve which in turn is connected to a one-way check valve leading directly into the autoclave. Located on the clean-room side, this design allows pressure to be equalized in the autoclave after the sterilization cycle without introducing contaminants.

Breaking the vacuum to equalize pressure in the autoclave without introducing contamination.

Swing Gauge

Figure 137. A microporous filter canister is attached to a pipe equipped with a gate valve which in turn is connected to a one-way check valve leading directly into the autoclave. Located on the clean-room side, this design allows pressure to be equalized in the autoclave after the sterilization cycle without introducing contaminants.

posing the needs of production and the dangers of uneven sterilization due to heavy loading.

Packing an autoclave deeper than 4 bags (32 inches deep) runs other contradictory risks. In the attempt to achieve full colonization, sterilization time is typically extended, potentially causing other problems: over-sterilization of the outer zones and bag-fatigue. Over-sterilization usually occurs when wood substrates are subjected to steam pressure (15-18 psi) for more than five hours. The sawdust takes on a dark brown color, has a distinctly different odor signature and, most importantly, resists decomposition by mushroom mycelium. Prolonged steam sterilization results in complex chemical transformations. (I have yet to find a chemist who can adequately explain what happens from prolonged exposure). Suffice it to say that turpentines, changes in volatile oils, and toxic by-products are responsible for this radical shift in sawdust's myco-receptivity. In the end, the substrate is rendered entirely inhospitable to mushroom myceiium.

After the autoclave has been packed, the displacement of the cold air by introducing steam and top-venting is absolutely critical. The cold air, if not vented, gives a false temperature/ pressure reading. At 15 psi, the temperature within the autoclave should be 252° F. (121° C.). This arithmetic relationship between temperature and pressure is known as Boyle's Law. When a cold mass is introduced into an autoclave or pressure cooker, Boyle's Law does not come into play until the thermal inertia of the affected mass is overcome. In other words, as hot steam is being forcibly injected into the vessel, there is a lag time as the heat is absorbed by the cold mass. (This causes considerable condensation within the autoclave.) Thermal inertia is soon overcome, and Boyle's Uwbecomes operative.

Many autoclaves not only have a combined pressure/temperature gauge but also sport a separate, remote bulb sensor that records temperature Jeep within the autoclaved mass. Th|s combination enables the laboratory personnel», to compare readings between the two gaug"\ The duration of the autoclave run should not be timed until these differentials have been largely eliminated. (A differential of 10° F. should be considered negligible.) In real terms, the differential is normally eliminated within two hours of start-up. Obviously smaller vessels have reduced differentials while the most massive autoclaves have substantial contradictions between pressure and temperature readings. Since the duration of "sterilization" is critical, careful consideration of these temperature trends can not be underemphasized. Cultivators often mistakenly believe that the mass has been autoclaved sufficiently when only paitia sterilization has been effected. Discarding several hundred bags due to insufficient sterilization is a strong incentive for cultivators to understand the nuances of autoclave cycling. Redundant gauges are recommended since devices fail over time.

spheric pressure is attained. Commonly, an autoclave ran swing in pressure from 20 psi to -20 psi within several hours after steam injection has stopped. This radical fluctuation in pressure further enhances the quality of the sterilization cycle. A 40-psi pressure swing is devastating at the cellular level, disabling any g|urviving endospores of bacteria or conidia of 1jjj contaminating molds.

When the steam supply to the autoclave is cut off, pressure and temperature precipitously decline. Ideally, your autoclave should achieve a vacuum as it cools. If your autoclave or steam box does not have a tight seal, and can not form a vacuum, provisions must be made so that the air drawn in is free of airborne contamination. This usually means the timely opening of the autoclave into the clean room air just as atmo

Once the autoclave has achieved a vacuum, the pressure must be returned to atmospheric before the door can be opened. Ideally, a gate valve has been installed on the clean room side, on a pipe connected to the combination pressure/vacuum gauge. A microporous filter canister can be attached for further insurance that the rush of air into the autoclave does not introduce contaminant spores. (See Figure 137). When the pressure has equalized, the next step is to open the drain valve to draw off excess condensate. Several gallons of condensate is common. After a few minutes, the autoclave door on the clean room side can be opened.

If the mass has just been autoclaved, the containers will be too hot to unload by hand unless protective gloves are worn. With the door ajar, several hours of cooling are necessary before the bags can be handled freely. Bear in mind that, as the mass cools, air is being drawn in. If that air is full of dust, contamination is likely. I like to thoroughly clean my laboratory while the autoclave is running. I remove any suspicious cultures, vacuum and mop the floors, and wipe the countertops with alcohol. In a separate pressure cooker, I autoclave towels, extra water, and other equipment essential to the impending inoculation cycle. Selected personnel for laboratory work wear laboratory garments

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Responses

  • Ines
    How to build a autoclave for mushrooms?
    4 years ago
  • kodi
    How much is a commercial steamer for mushroom sterilization?
    1 year ago
  • merimac
    Does an autoclave work the same a retort vessel?
    5 months ago

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