CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of the priority of U.S. Provisional Patent Application No. 63/035,596, filed June 5, 2020, the contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The invention is directed to the field of bioaugmentation and to a method of producing high populations of iteratively adapted microbiology for bioaugmentation.

BACKGROUND OF THE INVENTION

[0003] Traditionally the use of microbes to remediate polluted waters and soils, and improve plant growth and health in agriculture, also known as bioaugmentation, has relied upon selecting microbes that will perform specific functions, growing these microbes in factories and applying them in either dry or liquid format to the water or soil.

[0004] It is not uncommon for the liquid or dry format to also contain food and nutrients specific to the microbes being added to transition the microbes to an active, vegetative state before being added to the target water or soil.

[0005] The problems with these approaches are primarily cost which economically constrains the amounts of microbes that can be deployed, and the inefficiency of the approach because the added microbiology, over time, changes the chemical composition of the target water; or are insufficient in populations by cost constraints when applied to change or sustain change in the microbiome. [0006] Traditional methods used in desludging a wastewater treatment lagoon are highly arduous and expensive. The subsequent disposal of the removed sludge pose a serious health and environmental threat to areas surrounding the disposal site. Municipalities across North America spend millions of dollars to clean their lagoons in a continuous cycle and perpetuate the inevitable risks to the environment. Furthermore, the traditional method hardly solves other operational issues involving odor and inefficiencies caused by sludge build up.

[0007] Therefore, there is a need for a method of creating high populations of active microbiology, on site, inexpensively, on a continual basis that also adapts to chemical changes brought about by their addition to the target water source used as the growth substrate. The continual production and addition of the high population of active microbiology brings about competitive exclusion where these adapted microbes dominate the target water or soil where applied.

[0008] Therefore, there is also a need for a method that solves other operational issues involving odor and inefficiencies caused by sludge build up.

SUMMARY OF THE INVENTION

[0009] In one embodiment, the present disclosure relates to a method of creating high populations of active microbiology, on site, inexpensively, on a continual basis. The active microbiology adapts to chemical changes brought about by their addition to a target water source used as the growth substrate. The continual production and addition of these high populations of active microbiology brings about competitive exclusion where the adapted microbes dominate the target water or soil where applied.

[0010] In one embodiment, the present disclosure relates to a process produces a liquid bioaugmentation microbial consortia that is both adapted to or for the target application and present in high populations from using organic materials from such as those existing in the target water, animal manure or other organic wastes.

[0011] Yet still another embodiment of the present disclosure there is provided a method to iteratively adapt a selected microbial formulation with an approach which relies on the principal that microbes can adapt to different food sources by regulating their enzymes needed to consume available nutrients and use available substrate to grow efficiently to high populations. In this aspect, the method comprises an iterative approach methodology to grow beneficial microbes on site in a process that is continually adapted to the changing chemistry and conditions within the system being treated.

[0012] In one embodiment of the present disclosure there is provided a method reducing contamination at a remediation site, the method comprises: obtaining target liquid material, pasteurizing it and using it to grow a microbiology composition to high, active concentrations, then adding this high concentration of active microbiology back to the target liquid.

[0013] In aspects, the microbiology composition comprises microbes of the genus

Bacillus that are non-pathogenic, facultative, naturally occurring and ubiquitous in nature, non-genetically modified and proven to be plant growth and health promoting.

[0014] In one embodiment there is provided a process to produce a totally organic and natural liquid product, which includes organic waste materials as the growth medium for specific microbiology formulations prepared and delivered in a carefully controlled process that iteratively adapts to the optimum growth rate within the medium, regardless of changes in the growth medium composition.

[0015] In one embodiment, the present disclosure relates to a method for iteratively reducing biological contamination at a remediation site. [0016] In one embodiment, the present disclosure relates to a method for reducing contamination at a remediation site microbiologically by iterative microbiological adaptation, the method comprises: pasteurizing an extract obtained from a remediation site to inactivate resident vegetative microbiology in the extract, flowing pasteurized extract into a growth reservoir, growing in the growth reservoir one or more microbes suitable for growth at the remediation site with the pasteurized extract to allow the one or more microbes to adapt to the pasteurized extract, releasing from the growth reservoir, when the one or more microbes is near or at the log growth phase, an amount of growth material comprising the one or more adapted microbes and the pasteurized extract back into the remediation site such that the one or more adapted microbes dominate the remediation site and reduce contamination at the remediation site. [0017] In aspects, the pasteurizing is by elevating the temperature of the extract to about 165F to about 185F.

[0018] In aspects, once the temperature of the extract is from about 165F to about

185F, maintaining the temperature of the extract from about 165F to about 185F for about 20 seconds or more.

[0019] In aspects, once the temperature of the extract is from about 165F to about

185F, maintaining the temperature of the extract from about 165F to about 185F for up to about 60 seconds.

[0020] In aspects, after the pasteurizing and before the flowing, the temperature of the extract is reduced from about 165F to about 185F to about 85F and about 103F.

[0021] In aspects, the temperature of the extract is rapidly reduced from about 165F to about 185F to from about 85F and about 103F. [0022] In aspects, the temperature of the extract is rapidly reduced from about 165F to about 185F to from about 90F and about 103F.

[0023] In aspects, the growing comprises introducing a nutrient rich food source to encourage growth of the one or more microbes.

[0024] In aspects, the releasing of growth material is at a rate of about 3 to 10 times the rate of the pasteurized extract flowed into the growth reservoir.

[0025] In aspects, the releasing of growth material is stopped when the volume of the growth material is from about 10% to about 50% of the volume of the growth reservoir. [0026] In aspects, the method further comprises, after the releasing of growth material is stopped, seeding an amount of the one or more microbes into the growth material comprising the one or more adapted microbes remaining in the growth reservoir so as to allow the seeded amount of the one or more microbes to adapt to conditions in the growth reservoir; obtaining and pasteurizing extracts obtained from the remediation site; flowing the pasteurized extracts into the growth reservoir; growing in the growth reservoir the one or more seeded microbes with the pasteurized extracts to allow the one or more seeded microbes to adapt to the conditions of the pasteurized extracts, releasing from the growth reservoir, when the one or more microbes is near or at the log growth phase, an amount of growth material comprising the one or more adapted microbes and pasteurized extracts back into the remediation site to further reduce contamination at the remediation site.

[0027] In aspects, the amount of the one or more microbes seeded is an amount that does not negatively affect the adaptation of the one or more adapted microbes.

[0028] In aspects, the one or more microbes seeded is in a spore state. [0029] In aspects, the method further comprises, before the growing, selecting the one or more microbes based their ability to grow from within a sample of the extract obtained from the remediation site. In aspects, the one or more microbes is from Bacillus. In aspects, the one or more microbes is Bacillus subtilis.

[0030] In aspects, the extract is contaminated water and/or contaminated soil. In aspects, the contaminated water and/or contaminated soil comprises organic waste. In aspects, the organic waste is fecal matter or manure.

[0031] In aspects, the method further comprises aerating the growth reservoir.

[0032] In one embodiment, the present disclosure relates to an adapted microbial composition for use in treating waste and changing nutrient balances at a remediation site, the microbial composition produced according to a process that comprises: pasteurizing an extract obtained from a remediation site to inactivate resident vegetative microbiology in the extract, flowing a volume of pasteurized extract into a growth reservoir, growing in the growth reservoir one or more microbes suitable for growth at the remediation site with the pasteurized extract to allow the one or more microbes to adapt to the pasteurized extract, retrieving from the growth reservoir, when the one or more microbes is near or at the log growth phase, the one or more adapted microbes.

[0033] In one embodiment, the present disclosure relates to use of an adapted microbial composition to treat waste and change nutrient balances at a remediation site.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is a flow diagram of the process according to an embodiment of the present invention; [0035] Figure 2 is a photo showing biofilm and spore of the microbial composition according to one embodiment;

[0036] Figure 3 is a photo showing a side-by-side comparison of the ability of the microbial composition to reduce total solids (TS) on an approximately 9% (by weight) manure mixture (raw manure + lagoon water): 3:1, and 20:1;

[0037] Figure 4 is a photo showing a close up view of the test sample having a clearly visible layer of bubbles on surface indicating respiration (off-gassing of CO2) and nitrification-denitrification (off-gassing of N2);

[0038] Figure 5 is a photo showing a before treatment of starting raw manure of 19 oz and an after treatment with microbiology of residual wet “solids” of 3 oz;

[0039] Figure 6 is a table showing the % TS reduction over time;

[0040] Figure 7 is a graph showing the amounts of E-Coli (EC) and Coliforms (CO) from lagoon water after treatment;

[0041] Figure 8 is a graph showing the amounts of TS from 2018 to the final day of the testing on June 30, 2020;

[0042] Figure 9 is a graph showing the amounts of TS from June 22, 2020 to the final day of the testing on June 30, 2020;

[0043] Figure 10 is a graph showing the amounts of Microcystin (MI) was reduced to zero or at least less than 0.1 ug/L;

[0044] Figure 11 is a photo showing the clear liquid produced after incubation with the active microbiology of the present disclosure; and

[0045] Figure 12 is a photo showing the results of the biosolid destruction in a relatively short time. DETAILED DESCRIPTION OF THE INVENTION

[0046] Definitions.

[0047] The terminology used herein is for the purpose of describing embodiments only and is not intended to be limiting of the invention.

[0048] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0049] Furthermore, to the extent that the terms “including”, “includes”, “having”,

“has”, “with”, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner like the term “comprising.”

[0050] As used herein, the terms “comprising,” “comprise” or “comprised,” and variations thereof, in reference to defined or described elements of an item, composition, apparatus, method, process, system, etc. are meant to be inclusive or open ended, permitting additional elements, thereby indicating that the defined or described item, composition, apparatus, method, process, system, etc. includes those specified elements— or, as appropriate, equivalents thereof— and that other elements can be included and still fall within the scope/definition of the defined item, composition, apparatus, method, process, system, etc.

[0051] The term “about” or “approximately” means within an acceptable error range for the value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the value should be assumed.

[0052] “Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0053] “Media” is meant to include any medium used in the germination, growth, maintenance, and general health of the microbiology.

[0054] “Material” and “platform material” are used interchangeably herein and applies to the material produced by the systems and methods herein. Materials are liquid form necessary for the production, processing, storage, distribution, delivery to applications. The materials can be used as a platform for addition of any one or more, for example: nutrients, vitamins, minerals, essential and non-essential amino acids, etc. Accordingly, the composition of the platform material can be varied based on the desired end use.

[0055] Detailed embodiments of the instant invention are disclosed herein, however, it is to be understood that the disclosed embodiment is merely exemplary of the invention, which may be embodied in various forms and is in no way intended to limit the invention, its application or uses. Therefore, specific composition ranges disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representation basis for teaching one skilled in the art to variously employ the present invention in virtually any appropriately detailed composition. The embodiments of the invention may be practiced without the theoretical aspects presented. Moreover, the theoretical aspects are presented with the understanding that the Applicant does not seek to be bound by the theory presented. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. One having ordinary skill in the relevant art, however, will readily recognize that the invention can be practiced without one or more of the specific details or with other methods. The present invention is not limited by the illustrated ordering of acts or events, as some acts may occur in different orders and/or concurrently with other acts or events. Furthermore, not all illustrated acts or events are required to implement a methodology in accordance with the present invention. Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0056] With reference to figure 1, there is disclosed generally and without limitation, a process 100 for producing an adapted microbial composition 10 suitable for use in treating waste and changing nutrient balances at a remediation site that includes pathogens or otherwise undesirable/harmful bacteria (not shown), the process comprises: extracting, from a source 2 (e.g. from a remediation site such as a lagoon in need of remediation), raw water 12 comprising organic materials that is the substrate for growing the microbiology by way of the raw water pump 4 which pumps the raw water 12 into a raw water tank 20 which, according to one embodiment, also maximizes thermal efficiency. [0057] In the raw water tank 20, the raw water 12 undergoes some partial heating via heat exchange (as described below). This preheated raw water 12 is pumped out of the raw water tank 20 by a process pump 22 through to a heating section 30 where the temperature of the raw water 12 is elevated to between 165F and 185F as indicated by a temperature sensor TS-1.

[0058] Heated raw water 12 flows into a residence section 32 where the heated raw water 12 is held for a period of time needed to achieve destruction of all vegetative microbiology. The temperature in the residence section 32 is determined by a temperature sensor TS-2.

[0059] Leaving the residence section 32 is pasteurized water 16 which is flowed into a cooling coil 18 in the raw tank 20 where temperature of the pasteurized water 16 is reduced to between 85F and 103F as monitored by a cooling temperature sensor 38. As shown, in the raw tank 20 there is a countercurrent flow to exchange heat between the pasteurized water 16 and incoming raw water 12 from the source 2. This countercurrent exchange preheats the incoming raw water 12. Fluid level in the raw tank 20 is monitored by a level control LC-A.

[0060] The cooled pasteurized raw water 16 is flowed out from the raw tank 20 into a growth reservoir 40. In aspects the flow out of the raw tank 20 is continuous and without interruption.

[0061] The growth reservoir 40 is operated as a continuous batch process which initially starts with a separately prepared volume of a microbial formulation 50 that has been activated and comprising microbes determined to be useful in remediation.

[0062] The pasteurized flow 16 enters the growth reservoir 40 where it serves as the growth media for active microbes 50 as the tank slowly fills over a time duration determined by the division rate of the microbiology under the composition and temperature within the growth reservoir 40 such that the microbiology is just reaching the peak of the log growth phase before the growth material 10 comprising adapted microbes are released from the growth reservoir 40.

[0063] The active microbes 50 suitable for use as a starting point can comprise a consortium of beneficial microbes is selected for their ability to utilize nutrients/substrate in a sample of the target extract. In some aspects, selection of the active microbes can be determined in a laboratory setting. It is well established that non-pathogenic, facultative anaerobes from the genus Bacillus will remediate organic materials in both water and soil. Also, different species and strains within the genus are known to perform specific functions as well as work in symbiotic relationships. Additionally, formulations comprised of species selected for specific performance have been proven to be beneficial to plant health and growth.

[0064] The inflow of the cooled pasteurized mixture 16 into the growth reservoir 40 continues while outflow of growth material 10 comprising adapted microbiology in pasteurized water 16 and growth media from the growth reservoir 40 is released using a pump 42 at a rate that is greater than the inflow. In some respects the rate of the outflow is between 3 to 10 times the rate of the inflow. The outflow stops at a predetermined volume which can vary from as little as approximately 10 percent to as much as 50 percent of the growth reservoir volume, leaving a volume of the active microbiology as a residual “heel” inside the tank 40. The heel contains a portion of the continuing iterative adaption adapted microbiology. The amount of residual heel can be determined, for example, based on parameters of the speed of replication of the microbiology in the pasteurized substrate and the growth temperature. Therefore, both the speed of filling the growth tank 40 and the retained heel can be adjusted to optimize the final output concentration.

[0065] When the outflow of growth material 10 comprising adapted microbiology ceases, a small amount of the original microbial concentrate 50, in a spore state, is added to the heel as the tank continues to fill for another cycle to refresh the genetic composition and prevent outside microbial contamination that may survive the pasteurization.

[0066] The amount of microbial concentrate 50 can be determined from the volume of the growth reservoir and the heel and is added as a known genetic composition primary source to the adapting consortia and maintain the direction of the adaptation with each batch. Without being limited to any particular theory, it is believed that iterative adaptation process of the present disclosure alters the expression of enzyme of the individual microorganisms in the formulation. The addition of an amount of the original microbial formulation triggers the restart of the adaption process in the added microbes, ensuring a robust process that is constantly refreshed. The fluid level in the growth reservoir 40 can be determined using low level sensor LS 1 and high level sensor LS 2.

[0067] Growth material 10 comprising adapted microbiology can be flowed back into the remediation site 2 and/or can be harvested for later applications.

[0068] Therefore, according to one embodiment, the process broadly comprises three features.

[0069] The first is controlling of all vegetative indigenous microbiology using heat pasteurization. The first feature involves the control of the indigenous microbiology, which, in most cases, is harmless to the environment and may, in some cases, be beneficial. To reduce the indigenous, vegetative bacteria in the system, the raw water 12 is sent to a pasteurization process where it is heated from to 165 to 185 degrees Fahrenheit (F) and held for about one minute or more within the purification system.

[0070] In the case of milk, for example, flash pasteurization does not kill all the microbiology, but suppresses the viable population because it is done at lower temperatures and holding times. It is well known that higher temperatures and hold times causes the denaturation of the proteins and affects taste and flavor in milk. There are no real concerns for taste or flavor in the instant invention, so some degradation of the proteins is very acceptable and even desirable.

[0071] In one embodiment, the higher temperatures effectively reduce the number of psychrophilic and mesophilic indigenous, vegetative microbiology. The resulting liquid is an ideal environment to grow a specific microbiology to a desired concentration and thereby transform the liquid into a controlled platform substance, which may be applied in a repeatable, specific manner to achieve desired results. It will be understood some indigenous microbiology may exist as heat resistant spores, such as other species or strains of Bacillus , in which case the pasteurization heat will activate them to become vegetative in the growth reservoir 40. Since this microbiology is indigenous, they may actually be beneficial in the environment.

[0072] In one embodiment, the mixture 16 heated to a temperature between 165-185 degrees Fahrenheit and held for a sufficient time to inactivate indigenous microbiology. In aspects, the hold time is between 20-60 seconds or more before being immediately cooled in heat recovery equipment 18, in the present invention using the cooling to preheat the raw water 12, within the purification system to a temperature around or below 103 degrees Fahrenheit where it is then enters the growth reservoir 40.

[0073] The second feature is controlling the rate of flow of the pasteurized liquid 16 into the growth reservoir 40 to ensure growth inside the tank 40 is at or near the top of the log growth phase when the tank 40 is full.

[0074] The third feature is managing the residual heel volume of the adapting (or adapted) microbiology and addition of spore state microbial concentrate with a constant genetic composition to maintain the desired genetic composition of the active, adapted microcosm.

[0075] Additional steps, which may be applied if desired, of controlling the indigenous microbiology and may be necessary to produce a viable, sustainable product. Thus, according to one embodiment of the present invention, the method includes the addition of a microbial composition of naturally occurring bacteria from the genus Bacillus selected specifically for their ability to work synergistically with each other and specific applications where it will be applied to the pasteurized mixture in the growth reservoir. In this embodiment, the flow of pasteurized mixture 16 can held for a period time to allow the added microbial composition 50 to achieve a desired concentration at or before flow of the pasteurized mixture such that the microbial composition 50 is at a suitable concentration and in a vegetative state so it will work immediately upon application.

[0076] The specific volume and microbial concentration 50 to be added to the pasteurized mixture 16 can be determined using predetermined known growth rates of the one or more microbes in the formula in pasteurized mixtures tested to achieve a desired concentration of the specific microbial formulation mentioned.

[0077] Aeration may also be used in the growth reservoir if required by the nature of the organic substrate and the growth profile of the microbiological formulation in the substrate by various means.

[0078] Additionally, while it is desirable to have a purely natural organic environment without any added artificial chemicals, this is not a limiting factor if the use of the liquid does not preclude such added chemistry.

[0079] Yet still another embodiment of the disclose is a method to iteratively adapt a selected microbial formulation with an approach which relies on the principal that microbes can adapt to different food sources by regulating their enzymes needed to consume available nutrients and use available substrate to grow efficiently to high populations. [0080] For example, Bacillus subtilis does not make the enzymes for tryptophan biosynthesis if tryptophan is present in the environment. If both glucose and lactose are present in a media, E. coll will use the glucose first because it takes two less enzymes to use glucose than it does to use lactose. For example, the bacterium Neisseria gonorrhoeae will begin transcription of an iron gathering siderophore and transport system when iron is in short supply in its environment.

[0081] It is known that moving microbiology from one media to another can create what is termed a "shock" (lag phase) condition where reproduction is hindered or suspended until the microbes adapt to the new media. Once in the application target, introduced bacteria face challenges such as reduction in population from predation and competition as well as stress from abiotic factors such as pH, osmotic pressure, temperature, and availability of suitable nutrients. Bacteria introduced into natural environments tend to rapidly decline in numbers and rarely grow after being introduced. As a result, lasting change only occurs with repeated applications of the microbes to the target, and it returns to its original state once the applications stop. The present disclosure producing high populations of active microbiology iteratively adapted to a changing substrate addresses these barriers.

[0082] Changes in nutrients/chemistry in a source substrate also impacts the composition of a consortia of microbes. Microbes within a consortium that are best adapted for a nutrient/substrate source will increase in population while less fit microbes will decrease. The ability of a consortium to adapt to changing chemistry is what makes microbial consortia an ideal tool to treat waste and change nutrient balances (for example, to decrease nitrogen and phosphorus levels in lagoons, or increase nitrogen content in soil, depending upon the microbial consortium selected).

[0083] Pasteurized flow of water 16 continues into the growth reservoir 40 at an initial rate calculated so the population in the growth reservoir 40 is near the peak of the log growth rate curve based upon the initial laboratory testing at which time a calculated percentage of the grow tank volume (comprising the adapted microbiology) is released back into the initial target wastewater, “treated system” (for example, dairy lagoon water or chemically contaminated water or soil).

[0084] The microbiology in the growth reservoir 40 adapt to the nutrients/substrate/chemistry in the target wastewater is then released back into the treated system. This would be a population of bacteria 10 that are adapted to the treatment system at T=0. As the adapted, active bacteria 10 is released into the treated system consume nutrients/substrate and biochemically change the water chemistry, the nutrient concentrations and ratios will also change.

[0085] As discussed above, periodically, pasteurized source water/soil extracts from the treated system is used to grow a fresh consortium that will be adapted to the new chemistry and again released back into the treated system. Microbes left over from the T=0 chemistry would be grown on the new chemistry from the treated system to be T=1 population, and so on.

[0086] In one embodiment, the method is designed to iteratively adapt the enzyme regulation within the individual microbes as well as the fitness of the consortium with successive cycles in the growth reservoir with microbes adapted in the previous cycle. This iterative approach leads to successful adaptation to changing chemistry, even if the source chemistry is affected by more than just changes catalyzed by the microbes (such as additional nutrient loading).

[0087] As the adapted, active bacteria 10 released into the treated system consume nutrients/substrate and biochemically change the water chemistry, the nutrient concentrations and ratios will also change. . In particular, the method reduces contamination at a remediation site microbiologically by taking advantage of the microbiological adaptation when the adapted microbial composition 10 is iteratively added to the remediation site according to the methods, and uses of the present disclosure. The site is considered remediated biologically because contamination of remediation site is reduced or eliminated when the adapted microbes dominate and/or begin to out compete undesirable microorganisms at the remediation site.

[0088] To preserve the integrity of the adapted consortium, a small volume and concentration of the starting formulation (amount and concentration to be determined from testing) is added with each cycle of the growth reservoir. The added amount is controlled to not negatively impact the ongoing adaptation within the growth reservoir microcosm. [0089] Example 1

[0090] Figures 2 to 6 show the results of a study of facultative digestion with starting culture grown on pasteurized cow manure. In this study, microbiology was grown using about 15 ml of concentrate. Shown in figure 2 is biofilm and spores that form as the initial (synthetic) food source is depleted. When in this spore state, the microbiology moves into a cannibalistic state so that the microbiology will be ready to work as soon as the pasteurized manure is introduced in the growth reservoir.

[0091] Figure 3 shows a side-by-side test was undertaken to compare the efficacy of the biology to reduce total solids (TS) on an approximately 9% (by weight) manure mixture (raw manure + lagoon water): 3:1 Test: 1.25 gallons of manure mixture + 3.75 gallons of microbiology = 5 gallons total (on the left); and 20: 1 Test: 0.25 gallons of manure mixture + 4.75 gallons of microbiology = 5 gallons total. It is notable that a sedimentation layer was visible within 30 minutes of initial mixing.

[0092] Figure 4 shows a close-up view of a sample of the mixture having a clearly visible layer of bubbles on surface indicating respiration (off-gassing of CO2) and nitrification-denitrification (off-gassing of N2).

[0093] Figure 5 shows a before treatment of starting raw manure of 19 oz and an after treatment with microbiology of residual wet “solids” of 3 oz (from a total of 2 buckets). [0094] Figure 6 is a table showing the % TS reduction over time. The microbial composition of the present disclosure reduced TS by 25% within 2 hours without aeration, heating (buckets at room temperature) or constant mixing, 40% within 4 hours and 50% by 16 hours. As shown, the growth process slows as food depletes. The power of the microbiology is demonstrated by the fact that the rate of TS destruction was nearly identical as between the 3:1 and 20:1 tests. A decant sample taken at the conclusion of the test showed Total Suspended Solids (TSS) of 50 ppm and 100 ppm in the 20:1 and 3:1 buckets, respectively.

[0095] From the example, it has been demonstrated that all digesters have a population of active microbiology existing between filling and emptying to make room for introduction of more material. If active microbiology is adapted to the material entering the digester, the conversion of TS will be rapid as compared to if the microbiology has not been adapted or iteratively adapted according the present invention. .

[0096] Taking a portion of the digester material, pasteurizing, using that to grow new microbiology and continually re-introducing them into the digester will keep the resident microbiology at high concentration and aggressive towards new raw material inflow.

[0097] Since aerobic digesters are usually designed at 20: 1 in volume versus inflow, the present example clearly demonstrates that using the microbial composition of the present disclosure that digester size of 3 : 1 is just as effective as 20: 1. This translates into all aspects of the process where the goal is to keep the microbiology concentration far in excess of raw inflow to ensure rapid degradation and, because the microbiology is facultative, the process can proceed with no energy input (zero aeration) .

[0098] Example 2 - wastewater lagoon

[0099] A demonstration project was placed at a wastewater lagoon in Kindersley,

Saskatchewan to determine the effectiveness of the disclosed methods and microbial compositions and the viability of a less intrusive alternative to traditional lagoon remediation. A portion of the lagoon containing the effluent and sludge to be treated was sectioned in an 8’ x 40’ container in the lagoon at the north end. When placed, the removed floor allowed the container to settle to the clay liner of the lagoon floor, while the top remained open for equipment access and observations. It is estimated that 32,000 liters of effluent and sludge were contained within this test portion.

[00100] The sample testing was to measure 4 key metrics to determine success. The first three, Total Solids (TS), E-coli (EC), and Coliforms (CO) are conventional metrics used in these types of projects. Total Solids (or sludge) in particular, is important for measuring the success of the process because sludge build up is a problem for wastewater treatment plants.

[00101] Figure 7 shows the process decreased the amounts of TS, MI, EC, and CO. The process had a major effect on EC with a 99.18% total decrease and Similar results are seen in the total decrease of CO with 93.15%.

[00102] Figures 8 and 9 show the process also performed its primary function by decreasing the sludge totals in the test area. Comparing the TS from 2018 to the final day of the testing on June 30, 2020, there was a 67.5% total drop (figure 8) and a short term drop of 46.51% by the end of the test period (figure 9).

[00103] Figure 10 shows that Microcystin (MI) was reduced to zero or at least less than 0.1 ug/L (the reliable detection limit) by the end of the period.

[00104] Example 3 - Solution for Confined Animal Feeding Operation (CAFQ) wastewater

[00105] Municipal wastewater treatment consumes large amounts of energy generating greenhouse gases and this new process with microbiology greatly reduces the amount of energy input and associated greenhouse gases.

[00106] The products of traditional wastewater treatment are microbes, deemed biosolids, and effluent water of some quality level, usually containing nitrogen, phosphorous and potassium released into the environment as contributors to eutrophication of receiving waters. Biosolids containing a variety of microbial content are not beneficial to plant growth or health. However, these biosolids are valued for nutrient content, despite concerns over potential problems, and the high cost of alternative methods of dealing with them. These biosolids rely upon indigenous soil microbes to make the nutrients available to plants, and intensification in agriculture practices have depleted the indigenous microbes needed for this to happen quickly.

[00107] The inability of the soil microbiology to process these solids so they can be absorbed into the soil and utilized by plants is well documented. Additionally, applications of chemical fertilizers over time has resulted in an accumulation of phosphates in the soil while reduction in soil microbiology capable of facilitating plant uptake of these phosphates. As a result, these chemical move into runoff water from rainfall and find their way into streams and rivers, contributing to eutrophication.

[00108] In terms of agricultural wastewater, traditionally, animal manure, especially from Confined Animal Feeding Operations (CAFO) have been stored on site in dry stacks, liquid lagoons or pits for long periods of time because they can only be applied to the soil at certain times.

[00109] Overall, the storage requirement creates numerous problems including foul and offensive odors which can impact neighbors while attracting vectors (insects, vermin, etc.) and excessive rainfall often causes problems with runoff and lagoon overflows. [00110] The present example at Alcorn State University Swine Development Farm was designed to determine whether the storage lagoon could be eliminated, water conserved, and energy reduced while producing a liquid byproduct that contained recovered nutrients. [00111] Liquid manure was collected from the barn and pumped into a facultative digester tank where it was combined with high concentrations of the active microbiology and separated quickly with solids settling to the bottom for ongoing digestion. The clear liquid on top was piped off into a smaller tank for recycle to as flush water and byproduct with nutrients. The clear liquid is shown in figure 11.

[00112] Some of the water from the recycle was sent to the small system for pasteurization and used to grow the high concentrations of active microbes to go into the digester tank. Because this recycle water was used to flush the manure in the barn into the collection trough, no fresh water was used, saving fresh water that would otherwise be needed to flush the barn.

[00113] The active bacteria in the recycled flush water helped to solubilize the fresh manure in the troughs before it goes into the digester tank. As well, the recycle water had no noticeable odor.

[00114] Approximately after 2 months of continuous operation, it was noticed that the entire farm outside the barn was odor free. Inside the barn, the only odors were that associated with fresh manure.

[00115] Figures 12 shows the results of the biosolid destruction in a relatively short time carrying out the process and using the microbial compositions of the present disclosure. [00116] While a detailed embodiment of the instant invention is disclosed herein, it is to be understood that the disclosed embodiments are merely exemplary of the invention, which may be embodied in various forms and include most any radius form. Therefore, specific functional and structural details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representation basis for teaching one skilled in the art to variously employ the present invention in virtually any appropriately detailed structure. One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objectives and obtain the ends and advantages mentioned, as well as those inherent therein. The embodiments, methods, procedures, and techniques described herein are presently representative of the preferred embodiments, are intended to be exemplary and are not intended as limitations on the scope. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention and are defined by the scope of the appended claims. Although the invention has been described in connection with specific preferred embodiments, the invention, as claimed, should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention, which are obvious to those skilled in the art, are intended to be within the scope of the following claims.