Mycelial mass with non-electrical carbon dioxide transfer

Abstract

Carbon dioxide benefits plants in restricted indoor growing areas. Plants will deplete carbon dioxide levels in an indoor environment over time. The present invention provides a process design, system, and apparatus for a controlled, non-electrical, non-heat generating, non-mechanical, production source of CO.sub.2. The source of CO.sub.2 is fungi inoculated into a scientifically sterilized, enclosed growth medium prepared in a laboratory setting. The fungi is provided with an optimum food source from which the fungi may produce CO.sub.2 for at least six months. The CO.sub.2 produced is passively transferred from the fungi growing environment to an indoor plant growing environment under the optimization of the present invention. The transfer is non-electrical and preferably occurs through a gaseous interchange portal system which provides an interface between the fungi's enclosed plastic bag and the surrounding plant-growing environment.

Claims

1. A plant-growth enhancement apparatus comprising: a mycelial mass comprised of mycelia, nutrients, and a cellulose-based substrate blend, the mycelia comprising Trametes versicolor (Turkey Tail), a container having at least one opening and at least one gaseous interchange portal, the mycelial mass disposed within the container, wherein the mycelial mass is prepared from a culture of a pure fungal strain of Trametes versicolor, the culture added to a sterilized water and nutrient mixture to form a combination, the combination further mixed into the container with the cellulose-based substrate blend, the at least one opening of the container with the mycelial mass is closed by a seal, the plant-growth enhancement apparatus optimizes generation of carbon dioxide and passively distributes carbon dioxide from an elevation above plants in an indoor growing environment.

2. The apparatus of claim 1 where the apparatus is recyclable by composting the mycelial mass and recycling the container.

3. The apparatus of claim 1 wherein the at least one gaseous interchange portal further comprises a microbial filter.

4. The apparatus of claim 1 wherein the at least one gaseous interchange portal comprises a biological filtered vent.

5. The apparatus of claim 1 wherein the cellulose-based substrate comprises recycled sawdust.

6. The apparatus of claim 1 wherein the sterilized water and nutrient mixture is treated by a combination of heat and pressure.

7. The apparatus of claim 1 wherein the nutrients comprise cereal grain.

8. The apparatus of claim 1 wherein the container comprises a heat tolerant bag.

9. The apparatus of claim 8 wherein the container comprises a polypropylene bag.

10. A plant-growth improvement apparatus comprising: mycelial mass, a container with an opening and a gaseous exchange portal, wherein the mycelial mass is further comprised of: mycelia comprising Trametes versicolor (Turkey Tail), a water and nutrient mixture, and a blend of cellulose-based substrate, the mycelial mass disposed in the container; and whereafter the apparatus affects the optimal generation and passive distribution of carbon dioxide to a plant-growth environment via a non-electrical transfer of carbon dioxide to plants from an elevation above the plants.

11. The apparatus of claim 10, wherein the mycelial mass is formed by steps comprising the following: selecting a pure strain of Trametes versicolor; growing a pure spawn culture from the pure strain of Trametes versicolor; mixing water and nutrient additives to create the water and nutrient mixture; sterilizing the water and nutrient mixture; combining the pure spawn culture with the water and nutrient mixture in a sterile vessel to form a combination; incubating the combination in the sterile vessel.

12. The apparatus of claim 10, wherein the blend of cellulose-based substrate is formed by steps comprising the following: placing the blend of cellulose-based substrate in the container, sterilizing the blend of cellulose-based substrate in the container; and cooling the blend of cellulose-based substrate in the container in a HEPA chamber; adding the mycelia and the water and nutrient mixture to the container while in the HEPA chamber.

13. The apparatus of claim 10, wherein the container filled with the mycelial mass is sealed at the opening.

14. The apparatus of claim 10, wherein the container filled with the mycelial mass is incubated for a period of time.

15. The apparatus of claim 10, wherein the blend of cellulose-based substrate comprises recycled sawdust.

16. The apparatus of claim 10, wherein the container comprises a heat tolerant bag.

17. The apparatus of claim 16, wherein the heat tolerant bag comprises a polypropylene bag.

18. The apparatus of claim 10, wherein the gaseous exchange portal further comprises a microbial filter.

19. The apparatus of claim 10, wherein the gaseous exchange portal comprises a biological filtered vent.

20. The apparatus of claim 10, wherein the nutrient mixture comprises cereal grain.

21. A method to improve plant growth, comprising: selecting a pure strain of Trametes versicolor (Turkey Tail); culturing a spawn culture from the pure strain; mixing water and nutrient additives to create a water and nutrient mixture; sterilizing the water and nutrient mixture; combining the spawn culture with the water and nutrient mixture in a sterile vessel to form a combination; incubating the combination in the sterile vessel; placing a blend of cellulose-based substrate in a container with at least one opening and at least one gaseous interchange portal; sterilizing the blend of cellulose-based substrate in the container; cooling the container of the blend of cellulose-based substrate in a HEPA chamber; transferring the combination from the sterile vessel to the container; forming a mycelial mass by mixing the combination with the blend of cellulose-based substrate in the container; sealing the opening of the container; and offering the container for placement above an elevation of the plants in an indoor growing environment; wherein the mycelial mass produces carbon dioxide, which is released from the container to affect non-electrical transfer of the carbon dioxide, improving the plant growth.

22. The method of claim 21, further comprising incubating the mycelial mass mixture in the container for a period of time.

23. The method of claim 21, wherein the at least one gaseous interchange portal further comprises a microbial filter.

24. The method of claim 21, wherein the at least one gaseous interchange portal is a biological filtered vent.

25. The method of claim 21, wherein the blend of cellulose-based substrate comprises recycled sawdust.

26. The method of claim 21, wherein sterilizing is conducted using a combination of heat and pressure.

27. The method of claim 21, wherein the nutrient additives comprise cereal grain.

28. The method of claim 21, wherein the container comprises a polypropylene bag.

29. The method of claim 21, wherein the container comprises a heat tolerant bag.

30. A method to improve plant growth, comprising: selecting a pure fungal strain; culturing a spawn culture from the pure fungal strain; mixing water and nutrient additives to create a water and nutrient mixture; sterilizing the water and nutrient mixture; combining the spawn culture with the water and nutrient mixture in a sterile vessel to form a combination; incubating the combination in the sterile vessel; placing a blend of cellulose-based substrate in a container with at least one opening and at least one gaseous interchange portal; sterilizing the blend of cellulose-based substrate in the container; cooling the container of the blend of cellulose-based substrate in a HEPA chamber; transferring the combination from the sterile vessel to the container; forming a mycelial mass by mixing the combination with the blend of cellulose-based substrate in the container; sealing the opening of the container; and offering the container for placement above an elevation of the plants in an indoor growing environment; wherein the mycelial mass produces carbon dioxide, which is released from the container to affect non-electrical transfer of the carbon dioxide, improving the plant growth.

31. The method of claim 30, wherein the pure fungal strain is a white rot fungus.

32. The method of claim 30, wherein the pure fungal strain is Trametes versicolor (Turkey Tail).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The following drawings further describe by illustration, the methods, advantages, and objects of the present invention. Each drawing is referenced by corresponding figure reference characters within the DETAILED DESCRIPTION OF THE INVENTION section to follow.

(2) FIG. 1 is a schematic depiction of tissue culture petri plate production including the initial steps of the inventive process which results in a finished petri plate inoculated with a pure fungi strain.

(3) FIG. 2 is a schematic depiction of the spawn production, or those steps taken to spawn the petri plate fungi strain of the present invention.

(4) FIG. 3 is a schematic depiction of the carbon dioxide cultivator production or final production steps of inoculating the spawn within a combination of a cellulose-based substrate such as but not limited to a combination of sawdust, nutrient additives, and water.

(5) FIG. 4 is a schematic depiction of CO.sub.2 production with the finished mycelial mass produced in the steps reflected in FIGS. 1-3 and illustrates the non-mechanical, flow of CO.sub.2 produced by the mycelial mass in the polypropylene bag and the CO.sub.2 transferring by diffusion or other natural dispersal to the ambient air.

(6) FIG. 5 demonstrates the results of Test 1 of a series of carbon dioxide tests where one bag of the present invention was enclosed in a 20 cubic foot sealed vessel for 12 hours and testing occurred at hourly intervals.

(7) FIG. 6 shows the carbon dioxide testing results of Test 2, when one bag according to the present invention was enclosed in a 100 cubic foot sealed vessel for 72 hours and results were recorded every 24 hours.

(8) FIG. 7 illustrates the result of Test 3 of the series of carbon dioxide levels tests where a bag according to the present invention was enclosed in a 750 cubic foot sealed room for 72 hours and carbon dioxide levels were recorded at 24 hours intervals.

(9) FIG. 8 is a line graph illustrating the respective Block and Retort temperatures reached in optimum sterilization techniques used for the present invention.

DETAILED DESCRIPTION OF THE INVENTION

(10) The present invention generally comprises the processes of creating and using an isolated fungi growing environment inside a larger indoor plant growing environment whereby the invention allows the user to enhance CO.sub.2 exposure of the plants in the larger ambient growing area. In order to create an optimum, isolated, and sterile fungi growing environment which will generate and expel CO.sub.2 into a plant growing environment, the invention provides an apparatus, system, and process comprising: starting a fungus tissue culture; creating a spawn of the cultured fungus; preparing a bulk substrate; filling a heat-tolerant bag with the substrate; sterilizing the substrate and bag; cooling the substrate and bag; inoculating the substrate in the bag with spawn to create a mycelial mass; sealing the bag; incubating the bag's mycelial mass; and utilizing the mycelial mass and bag to produce a CO.sub.2 interface with an indoor plant growing environment.

(11) These steps are set forth below in greater detail. To start the process of mycelial growth a specific strain is introduced to an agar medium to grow from spores or tissue culture. As demonstrated in FIG. 1, the beginning phase of the process is to start a population of fungi from a purified tissue culture. Agar plates with master cultures are prepared by using sterile petri plates that have been filled with Potato Dextrose Agar (PDA) and sterilized. The depiction of FIG. 1 begins at the left with a petri plate, a Potato Dextrose Agar, water, and a tissue culture of the desired mushroom species. The Potato Dextrose Agar and water are mixed together and placed in the petri plate. These agar plates or master cultures are created by using sterile petri plates that have been filled with PDA and sterilized at 250 degrees Fahrenheit for 1 hour. The agar and plate combinations are sterilized such as by autoclave and allowed to cool.

(12) As illustrated in FIG. 1, the cooled plates containing the agar are inoculated with the sterile transfer of spores or tissue by known laboratory procedures and protocols. The preferred protocol calls for first sterilizing the instrument used for the transfer with flame or other sterilizing agent followed by transferring a small amount of spores or tissue into said petri plate and placing spores or tissue so that it comes in contact with agar in petri plate. Once contact is made spores or tissue is left on agar and the instrument is removed and petri plate is covered and sealed. With incubation (at the desired temperature of 70 degrees Fahrenheit), growth of mycelium will be noticeable in 24-72 hours after spore or tissue transfer and will continue until a layer of mycelium covers the entire agar surface.

(13) The diagram in FIG. 2 continues the process and depicts spawn production from the petri plate culture created in FIG. 1. The process begins with a sterile vessel (glass is suggested), nutrient-rich additives, water, and the culture from the petri plate. Ideal nutrient additives may be cereal grains (e.g., oats, rye, milo, or similar grains). The nutrient additives and water are blended together and placed in a sterile vessel for sterilization. The sterilization process should be done with heat and pressure, such as by autoclave, and then allowed to cool. Optimally, the nutrient blend is sterilized at 250 degrees Fahrenheit for at least one hour. The combination is allowed to cool in a HEPA (High Efficiency Particulate Air) filtered chamber. Once cooled to approximately 75 degrees Fahrenheit, the sterile, nutrient rich blend is inoculated with the pure culture which was previously grown on the agar petri plates. The result is the pure culture spawn used in later bulk inoculation (see FIG. 3).

(14) The bulk substrate of mycelial mass is produced as may be better understood by viewing FIG. 3. To begin, a cellulose-based substrate such as but not limited to sawdust, more nutrient additives such as cereal grains, and water are blended to achieve a substrate with a optimal moisture content of approximately 65%. While this is indicated to be optimal moisture content, it is typical to have ranges between 60%-75%. Other ranges (e.g., about 50%-80%) are known to maintain functionality, but are not ideal. This substrate is placed in a container with a gaseous interchange portal. The container is desirably a autoclave-able bag, preferably having a single air-vent with a microbial filter. After the substrate is placed in the bag, it is autoclaved. The process of sterilizing the bulk substrate involves utilizing steam generated from a steam boiler that is piped into an autoclave and allowed to be put under pressure at a temperature of 250 degrees Fahrenheit. Sterilizing the substrate under these conditions for at least one hour is required. Preferred sterilization time is up to 2 hours. The bag and the substrate are allowed to cool to approximately 75 degrees Fahrenheit or cooler. The cooling of the substrate is a vital step in this process. Cooling must take place in a HEPA filtered room that is positively charged with air. If this is not done the substrate will become contaminated and will not be suitable for inoculation. Once the substrate is properly cooled to approximately 75 degrees Fahrenheit, it is inoculated with pure culture spawn. The bulk substrate is suitable for spawn growth and because the media has been sterilized at every juncture, bacteria, undesired fungi, and other contaminants will be minimized. In the preferred embodiment, bags are filled with substrateapproximately to the half-way point or up to the gaseous interchange portal means. The bag and substrate are inoculated with spawn forming the mycelial mass 1 of the present invention. In the preferred embodiment, the combination weighs approximately 6 pounds.

(15) The substrate as inoculated creates the mycelial mass 1, as schematically shown in FIG. 4, inside the transparent or translucent polypropylene bag 2 with a gaseous interchange portal 3. The bag or container 2 may be opaque and still function according to the objectives of this invention. As has been described, the inoculation of the substrate is done by adding pure spawn under sterile conditions. Preferably, about of a cup of pure culture spawn will be added from the sterile vessel to each 6 pounds of bagged and sterilized substrate in order to optimize good mycelial growth and available food and nutrient consumption over a six month period. A heat impulse sealer is preferably used to seal the bag. In this case, the seal is approximately 1.5 inches from the top of the bag. However, any air-tight sealing means may be employed. The sealing of the bag closes the sterile environment and the mycelium can produce CO.sub.2 using the food in the mycelial mass. The bag should not be opened again except for disposal and recycling. Opening the bag would interrupt the flow of CO.sub.2 and could possibly contaminate the mycelial mass. The use of a filter such as the Unicorn filter bag or biological breather patch allows the most ideal environment for the mycelial mass to create and transfer CO.sub.2 to the surrounding environment.

(16) After sealing the bag containing the mycelial mass, the combination must be incubated. In the preferred method, incubation should occur at a temperature of 70 degrees Fahrenheit for 3-4 days. This allows mycelial growth to begin and to reach the ideal environmental conditions for the proliferation of the mycelial mass. For the preferred species, once mycelial growth is appreciable to the human eye, the product is ready for commercial distribution or use in any environment where increased CO.sub.2 is desired. The use of the mycelial mass 4 in the bag 2 is demonstrated in schematic in FIG. 4. The polypropylene bag 2 schematically demonstrated in FIG. 4 contains the substrate inoculated with the spawn and incubated to the prescribed level of production. In the preferred embodiment, the necessary level of production will have been reached once the preferred fungus (see infra) white rot begins to be visible to the naked human eye. CO.sub.2 is constantly being expired or expelled by the saprobes or fungi in the mycelial mass. That CO.sub.2 is passed from the interior of the bag to the ambient air surrounding it by natural dispersal by air-exchange chemical processes. Contrary to prior belief, it is not necessary to actuate this expulsion with any agitation or mechanical or electrical means but the transfer will occur naturally to a beneficial level if the growth and containment is controlled according the present invention disclosure.

(17) The present invention begins producing CO.sub.2 immediately without further action. The invention is designed to be used to increase levels of CO.sub.2 in an indoor gardening setting. Placement in the growing area is important. The mycelial mass bag should be placed above the height of the plants in the growing space. CO.sub.2 will precipitate downward in atmospheric air and thus should be placed at a level higher than growing plants.

(18) The bag is preferably made of recycled polypropylene or other plastic which may be further recycled. The bag material must be heat-tolerant for sterilization purposes. The preferred bags should be designed to withstand temperatures up to 250 degrees Fahrenheit. There are a number of different types of vented bags available which have been developed for the purpose of creating an environment suitable for mycelial growth and production. All of these bags are suitable to use for the present invention's process, apparatus, and application. Ideally, the preferred vented bag will contain a microbiological filter that acts as a gaseous interchange portal that will allow gas exchange without allowing contaminants to enter the bags. The use of a vented bag allows CO.sub.2 to be released without the need for any other dispersal method or any additional human action of any kind other than placing the bag in a location where an increase of CO.sub.2 is desired. In the preferred embodiment, a Unicorn bag or the functional filter-bag equivalent is used as the plastic bag container. While this bag is optimal for the purposes of the invention, it is but one bag which will accomplish the objectives of CO.sub.2 production of the present invention.

(19) As used herein, spawn is actively growing mycelium. In the present invention, spawn is placed on a growth substrate to seed or introduce mycelia to grow on the substrate. This is also known as inoculation, spawning or adding spawn. The primary advantages of using spawn is the reduction of contamination while giving the mycelia a firm beginning Spores are another inoculation option, but are less developed than established mycelia. Either spores or myclia used in the present inventive process are only manipulated in laboratory conditions within a laminar flow cabinet. The process of making the present invention utilizes sterile laboratory protocols and pure, sterile mycelial culture.

(20) While all strains of mycelium from the kingdom Fungi including Basidiomycetes and Ascomycetes are suitable for this application, strains that exhibit little or no fruiting characteristics are preferred. When producing CO.sub.2 it is desirable to avoid primordial production and to have only mycelial growth occur. This is because primordial formation diminishes CO.sub.2 production by fungi. The process disclosed in the present invention will also create an ideal environment for the controlled and non-flowering growth of mycelium.

(21) For the preferred embodiments of this invention, the fungal strain utilized is Trametes versicolor which is a white-rot fungus known by the common name, Turkey Tail. Trametes versicolor causes a general delignifying decay of cellulose-based substrates such as but not limited to hardwoods. The appearance of this fungi is whitish in color which may be aesthetically pleasing when the bag is placed for CO.sub.2 production. This visual appearance of this strain is helpful during the incubation phase of the process when trying to achieve optimum incubation periods. Furthermore, the Trametes versicolor mycelium is very active and aggressive and grows very quickly resulting in good CO.sub.2 production. The use of the polypropylene bag and the naturally occurring strain in organic materials make every aspect of the present invention readily recyclable. Furthermore, while pre-consumer materials my be used, the preferred materials are made of previously used and recycled materials.

(22) An analysis of test performed on dry matter of substrate mixtures reveals that recycled materials actually optimize nutrient conditions. The testing parameters incorporated mixtures of new sawdust, recycled sawdust, and a combination of the two. These several substrate mixtures were compared with and without supplement, and without cooking Where used, the supplement comprised a mixture of ingredients known to benefit fungal growth. The data collected assessed percentages of total dry matter, nitrogen, non-detergent fiber, acid detergent fiber, lignin, hemicellulose, insoluble ash, and cellulose. For informational purposes, neutral-detergent fiber (of substrate) is basically cell wall components (cellulose, hemicellulose) of substrate. This indicates something about the fungus's ability to degrade structural carbohydrates. Acid detergent fiber is the lignocellulose component of the substrate which relates to the fungus's ability to degrade lignocellulase with lignocellulytic enzymes. Lignin is a complex chemical compound (non-carbohydrate aromatic polymer) present in wood and is an integral part of the secondary cell walls of plants and some algae. Cellulose is a polysaccharide (complex carbohydrate) that is the main constituent of the cell wall in most plants. Hemicellulose is another polysaccharide but less complex than cellulose and more easily hydrated. Soluble ash is the soluble carbohydrates in the ash portion and reflects the ability of the fungus to solubilize a portion of the ash content (making it into a soluble form). Insoluble ash is the remaining byproduct. The following is data collected from an ANALYSIS OF SUBSTRATE MIXTURES:

(23) TABLE-US-00001 Row 1: New Sawdust, no supplement Row 2: New Sawdust, with supplement Row 3: Recycled sawdust, no supplement Row 4: Recycled mix: 60% new sawdust, 40% old sawdust, with supplement 20% 100C Total % Insol Row DM % DM % N % NDF % ADF % Lignin % Hemicellulose ash % Cellulose 1 92.63 25.96 0.299 101.48 80.12 17.31 21.36 0.94 61.87 2 90.56 26.45 0.927 82.90 62.49 14.17 20.41 0.84 47.48 3 92.71 52.71 2.297 51.78 37.36 6.50 14.42 6.53 24.33 4 93.41 31.17 1.763 56.03 39.33 7.21 16.70 4.96 27.16 DM = Dry matter; Total % DM = dry matter percent; % N = percent Nitrogen; % NDF = percent non-detergent fiber; % ADF = acid detergent fiber; % Lignin = percent lignin; % Hemicellulose = percent Hemicellulose; % Insol. Ash = percent insoluble ash; % Cellulose = percent cellulose

(24) The data indicates that the Nitrogen percentages are significantly increased with the use of recycled sawdust or a mixture of new and recycled sawdust and further improved with supplementation. The percent of cellulose, lignin, and hemicellulose decreases in the recycled material tests suggest that the fungus has decomposed more of these. White rot fungus strains are known to decompose both lignin and complex carbohydrate compounds like cellulose. Meanwhile the percentage increases of Insoluble Ash are significant for recycled and mixed recycled sawdust versus new sawdust. These results indicate a high nitrogen to carbon ratio and that the decomposition by the fungus is significant; however, all of the parameters measured will depend on the enzymatic ability of the particular fungus involved.

(25) Methods of passive CO.sub.2 transfer were previously believed to be insufficient resulting in the prominent use and development in prior inventions of electrical transfer techniques. The initial tests for CO.sub.2 transfer by the present invention have been very successful. In particular, the production of CO.sub.2 after the inoculation of the spawn and incubation of the mycelial mass has proven to achieve CO.sub.2 production levels which can enhance growth of plants. For the majority of greenhouse crops, net photosynthesis increases as CO.sub.2 levels increase from 340-1,500 parts per million (ppm). Most crops show that for any given level of photosynthetically active radiation (PAR), increasing the CO.sub.2 level to 1,500 ppm will increase the photosynthesis by about 50% over ambient CO.sub.2 levels. Ambient CO.sub.2 levels in outside air are typically about 340 ppm by volume. All plants grow well at this level but as CO.sub.2 levels are raised by 1,000 ppm photosynthesis increases proportionately resulting in more sugars and carbohydrates available for plant growth. Any actively growing crop in a tightly clad greenhouse with little or no ventilation can readily reduce the CO.sub.2 level during the day to as low as 200 ppm. Plants cannot thrive under these conditions.

(26) During particular times of the year, in new greenhouses, and especially in double-glazed structures that have reduced air exchange rates, the carbon dioxide levels commonly drop below outside air CO.sub.2 levels of 340 ppm. This nutrient deficit has a significant negative effect on the crop. Ventilation during the day can raise the CO.sub.2 levels closer to ambient levels, but rarely reach levels equivalent to 340 ppm. Supplementation of CO.sub.2 is seen as the only method to overcome this deficiency and increase the level above 340 ppm which is beneficial for most crops. The level to which the CO.sub.2 concentration should be raised depends on the crop, light intensity, temperature, ventilation, stage of the crop growth and the economics of the crop. For most crops, the CO.sub.2 saturation point will be reached at about 1,300-2,000 ppm under ideal circumstances. Increased CO.sub.2 levels will shorten the growing period by 5%-10%, improve crop quality and yield, as well as increase leaf size and leaf thickness. The present invention will allow growers to optimize and utilize desired CO.sub.2 levels for their respective crops. The process is an organic system to produce, retain, and non-mechanically siphon carbon dioxide from a mycelium mass to a photosynthesizing plant.

(27) The following tests and corresponding results were measured and tabulated for the present invention. CO.sub.2 levels were tested and recorded using a CO.sub.2 meter inside sealed vessels for specific periods of time. Increased CO.sub.2 levels were demonstrated in each test as demonstrated in FIGS. 5-7.

(28) FIG. 5 demonstrates the results of Test 1 where one bag was enclosed in a 20 cubic foot sealed vessel with a carbon dioxide meter inside. The tested CO.sub.2 level before introducing CO.sub.2 was measured at 380 ppm. After 12 hours of monitoring, the CO.sub.2 level was 8560 ppm. FIG. 5 specifically shows the individual CO.sub.2 levels at hourly intervals beginning at 7 a.m. when the CO.sub.2 level was 380 ppm and ending at 7 p.m. with a CO.sub.2 level of 8560 ppm. The test results are illustrated in FIG. 5.

(29) FIG. 6 demonstrates the results of Test 2 where one bag was enclosed in a 100 cubic foot sealed vessel with a carbon dioxide meter inside. The tested CO.sub.2 level before introducing CO.sub.2 was measured at 465 ppm. After 72 hours of monitoring, the CO.sub.2 level was 3850 ppm. FIG. 6 specifically shows the individual CO.sub.2 levels at 24 hour intervals beginning on December 20, 2009 when the CO.sub.2 level was 465 ppm and ending on Dec. 23, 2009 with a CO.sub.2 level of 3850 ppm. The test results are illustrated in FIG. 6.

(30) FIG. 7 demonstrates the results of Test 3 where one bag was enclosed in a 750 cubic foot sealed room with a carbon dioxide meter inside. The tested CO.sub.2 level before introducing CO.sub.2 was measured at 405 ppm. After 72 hours of monitoring, the CO.sub.2 level was 2150 ppm. FIG. 7 specifically shows the individual CO.sub.2 levels at 24 hour intervals beginning on Jan. 20, 2010 when the CO.sub.2 level was 405 ppm and ending on Jan. 23, 2010 with a CO.sub.2 level of 2150 ppm. The test results are illustrated in FIG. 6.

(31) Sterilization of materials described herein is preferably done utilizing an autoclave or as it is also known, a retort. Autoclaving is the most effective and most efficient means of sterilization. All autoclaves operate on a time/temperature relationship. These two variables are extremely important. Sterilization with steam heat under pressure is used. As steam enters the autoclave chamber the air temperature rises in the chamber. It rises more quickly than the substrate temperature because the substrate has density. The steam pressure also begins to rise and after 7 hours the air temperature and the substrate temperature equalize at 250 degrees Fahrenheit or 15 pounds per square inch (psi). At this point, sterilization begins and is allowed to continue for 2 hours. One hour at 250 degrees Fahrenheit or 15 psi is sufficient, but allowing for 2 hours of sterilization is a preferred in order to ensure sterility.

(32) As can be seen in the following table, after 9 hours the steam pressure inside the autoclave is reduced back down to atmospheric pressure (0 psi), which equates to 212 degrees Fahrenheit. Optimum sterilization of the substrate according to the present invention is demonstrated in FIG. 8.

(33) The substrate retains heat longer than the air inside the autoclave. This causes the bag in which the substrate is enclosed to draw in air from the vessel in an attempt to equalize the pressure inside the bag to be equal to the pressure inside the autoclave. The air that is drawn into the bag must be HEPA filtered, sterile air. Without the use of HEPA filtration the substrate could become contaminated with airborne spores or other airborne contaminants. Once substrate bags have cooled to at least 80 degrees Fahrenheit the inoculation process can begin.

(34) It is further intended that any other variations and embodiments of the present invention which result from any changes in application or method of use or operation, method of manufacture, shape, size, or material which are not specified within the detailed written description or illustrations contained herein, yet are considered apparent or obvious to one skilled in the art, are within the scope of the present invention.