US20220402833A1

US20220402833A1 - Nutritional Compositions for Plants and Soils

Info

Publication number: US20220402833A1
Application number: US17/689,849
Authority: US
Inventors: Sushil K. Bhalla; Devon K. Hooper; Michael A. Laughlin; Jonathan Casassa
Original assignee: ENVIROKURE Inc
Current assignee: ENVIROKURE Inc
Priority date: 2015-12-20
Filing date: 2022-03-08
Publication date: 2022-12-22
Also published as: US20190194081A1; EP3390324A1; IL259997A; US11299437B2; WO2017112605A1; CA3009174A1

Abstract

Nutritional compositions for application to plants and soils are disclosed. In various aspects the compositions comprise an autothermal thermophilic aerobic bioreaction product from a liquid fraction of poultry manure. The compositions can be produced in a manner that permits their use in organic farming programs. Methods of making the compositions and methods of treating plants and/or soils with the compositions are also disclosed.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 16/063,962, filed Jun. 19, 2019 which is an application filed under 35 U.S.C. Sec. 371 of International Application No. PCT/US2016/067614, filed Dec. 19, 2016 which claims benefit of U.S. Provisional Application No. 62/270,009, filed Dec. 20, 2015, the entire contents of which are incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates generally to fertilizers and compositions useful for promoting plant growth and healthy soil structure. In particular, liquid and solid compositions produced by aqueous bioprocessing of poultry manure are disclosed.

BACKGROUND OF THE INVENTION

Various publications, including patents, published applications and scholarly articles, are cited throughout the specification. Each of these publications is incorporated by reference herein, in its entirety.
Two main categories of crop input products are used in agriculture: fertilizers and pesticides. A fertilizer is typically described as any organic or inorganic material of natural or synthetic origin that is added to supply one or more nutrients essential to the growth of plants. Fertilizers provide, in varying proportions, the macronutrients, secondary nutrients and micronutrients required or beneficial for plant growth.
The Food and Agriculture Organization (FAO) has defined pesticide as any substance or mixture of substances intended for preventing, destroying, or controlling any pest, including vectors of human or animal disease, unwanted species of plant or animals, causing harm during or otherwise interfering with the production, processing, storage, transport, or marketing of food, agricultural commodities, wood and wood products or animal feedstuffs, or substances that can be administered to animals for the control of insects, arachnids, or other pests in or on their bodies. The term includes substances intended for use as a plant growth regulator, defoliant, desiccant, or agent for thinning fruit or preventing the premature fall of fruit, as well as substances applied to crops either before or after harvest to protect the commodity from deterioration during storage and transport.
During the last century, there has been extensive use of synthetic fertilizers and pesticides in agriculture. It is now well recognized that the use of synthetic fertilizers adversely impacts the physical qualities of soil and its ability for sustainable growth. In addition, the adverse impacts of these chemicals on environment and humans are being recognized (see, e.g., Weisenberger, D. D., 1993, “Human Health Effects of Agrichemical Use,” Hum Pathol. 24(6): 571-576).
The recognition of the often detrimental effect of synthetic fertilizers and pesticides on plants, soil ecology and human health has provided impetus for resurgent interest in organic crop production, including the use of fertilizers and pesticides of natural and/or biological origin. Indeed, crops that can be described or advertised as “organic” must be produced in accordance with standards set forth by federal and state law. In the United States, the National Organic Program (NOP) is a regulatory program housed within the United States Department of Agriculture (USDA) Agricultural Marketing Service. The NOP is responsible for developing national standards for organically-produced agricultural products. These standards assure consumers that products with the USDA organic seal meet consistent, uniform standards. To comply with NOP rules, growers must use only approved materials and handling processes in their production programs. The NOP accredits various organizations that test and approve products for use in compliance with NOP rules. One example is the Organic Materials Review Institute (OMRI), an international nonprofit organization that determines which input products are allowed for use in organic production and processing. OMRI Listed® products are allowed for use in certified organic operations under the USDA National Organic Program. Another example is the Washington State Department of Agriculture (WSDA) Organic Program. As a certification agent of the USDA NOP, the WSDA Organic Program's role is to uphold the integrity of NOP organic standards by inspecting and certifying organic operations.
The term “organic fertilizer” typically refers to a soil amendment from natural sources that guarantee, at least the minimum percentage of nitrogen, phosphate and potash. Examples include plant and animal byproducts, rock powder, sea weed, inoculants and conditioners. If such fertilizers meet criteria for use in organic programs, such as the NOP, they also can be referred to as registered, approved or listed for use in such programs.
“Biofertilizer” is another term used in the industry. It refers to a substance that contains living microorganisms which, when applied to seed, plant surfaces, or soil, colonize the rhizosphere or the interior of the plant and promote growth by increasing the supply or availability of primary nutrients to the host plant. Biofertilizers add nutrients through the natural processes of nitrogen fixation, solubilizing phosphorus, and stimulating plant growth through the synthesis of growth-promoting substances. Biofertilizers can be expected to reduce the use of chemical fertilizers and pesticides. The microorganisms in biofertilizers restore the soil's natural nutrient cycle and build soil organic matter. Through the use of bio-fertilizers, healthy plants can be grown, while enhancing the sustainability and the health of the soil.
Plant “biostimulants” are diverse substances and microorganisms used to enhance plant growth. In North America, the Biostimulant Coalition defined biostimulants as “substances, including micro-organisms, that are applied to plant, seed, soil or other growing media that may enhance the plant's ability to assimilate applied nutrients, or provide benefits to plant development” (Biostimulant 2013, see URL biostimulantcoalition.org). “They are derived from natural or biological sources and can i) enhance plant growth and development when applied in small quantities; ii) help improve the efficiency of plant nutrients, as measured by either improved nutrient uptake or reduced nutrient losses to the environment, or both; or [iii)] act as soil amendments to help improve soil structure, function, or performance and thus enhance plant response” (Biostimulant 2013). Biostimulants were defined and described by du Jardin (2015, supra) as including several categories, namely: humic substances, protein hydrolysates and other nitrogen-containing compounds, seaweed extracts and botanicals, chitosan and other biopolymers, certain inorganic compounds, and beneficial bacteria and fungi.
It will be understood that biofertilizers and biostimulants, as well as biopesticides and other biocontrol agents, can be “organic” within the meaning set forth above.
With regard to microorganisms, a preferred scientific term for beneficial bacteria is “Plant Growth Promoting Bacteria (PGPB)”. Therefore, they are extremely advantageous in enriching soil fertility and fulfilling plant nutrient requirements by supplying the organic nutrients through microorganisms and their byproducts. Hence, bio-fertilizers do not contain any chemicals which are harmful to living soil.
PGPBs can influence the plant in a direct or indirect way. For instance, they can increase plant growth directly by supplying nutrients and hormones to the plant. Examples of bacteria which have been found to enhance plant growth, include thermophilic members of genera such as Bacillus, Ureibacillus, Geobacillus, Brevibacillus and Paenibacillus, all known to be prevalent in poultry manure compost.
PGPBs are also able to control the number of pathogenic bacteria through microbial antagonism, which is achieved by competing with the pathogens for nutrients, producing antibiotics, and the production of anti-fungal metabolites. Besides antagonism, certain bacteria-plant interactions can induce mechanisms in which the plant can better defend itself against pathogenic bacteria, fungi and viruses. One mechanism is known as induced systemic resistance (ISR), while another is known as systemic acquired resistance (SAR) (see, e.g., Vallad, G. E. & R. M. Goodman, 2004, Crop Sci. 44:1920-1934). The inducing bacteria triggers a reaction in the roots that creates a signal that spreads throughout the plant, resulting in the activation of defense mechanisms, such as reinforcement of plant cell wall, production of antimicrobial phytoalexins and the synthesis of pathogen related proteins. Some of the components or metabolites of bacteria that can activate ISR or SAR include lipopolysaccharides (LPS), flagella, salicylic acid, and siderophores.
Animal manure, particularly nutrient- and microbe-rich poultry manure, has been a subject of extensive research regarding its suitability as a bio-organic fertilizer. It is well established through academic research and on-farm trials that poultry manure can cost-effectively and safely provide all the macro and micro nutrients required for plant growth, as well as certain plant growth promoting bacteria, if the harmful plant and human pathogens can be destroyed. However, significant concerns from the use of raw manure include increased potential for nutrient run off and leaching of high soil P, as well as transmittal of human pathogens to food. As composting has been shown to reduce total volume of runoff and soil erosion and to reduce the potential for pathogen contamination, many states now require poultry manure to be composted prior to field application, leading to advances in composting processes.
Composting can be described as the biological decomposition and stabilization of organic material. The process produces heat via microbial activity, and produces a final product that is stable, substantially free of pathogens and weed seeds. As the product stabilizes, odors are reduced and pathogens eliminated, assuming the process is carried to completion. Most composting is carried out in the solid phase.
Benefits of composting include: (1) enriching soil with PGPB, (2) reduction of microbial and other pathogens and killing of weed seeds; (3) conditioning the soil, thereby improving availability of nutrients to plants; (4) potentially reducing run-off and soil erosion; (5) stabilizing of volatile nitrogen into large protein particles, reducing losses; and (6) increasing water retention of soil. However, the process is time consuming and labor intensive. Additionally, because nutrients are applied in bulk prior to planting, there is a significant potential for nutrients to be lost. There is also a significant potential for inconsistent decomposition and incomplete pathogen destruction. Furthermore, the unavailability of set application rates can lead to uneven nutrient distribution in field application. Lastly, solid compost cannot be used in hydroponics and drip irrigation.
With regard to this last drawback, organic growers have utilized compost leachate (“compost tea”) as a liquid fertilizer. The leachate is produced by soaking well-composted material in water and then separating the solid from the liquid leachate. While such liquid material can be utilized in drip irrigation or foliar application, its production remains time consuming and labor intensive, and the liquid product suffers from the same drawbacks as solid compost in that it may still contain pathogenic organisms and its nutrient content is inconsistent.
Other organic fertilizers include fish-based and plant protein based fertilizers. Fish emulsion fertilizers are typically produced from whole salt-water fish and carcass products, including bones, scales and skin. The fish are ground into a slurry, then heat processed to remove oils and fish meal. The liquid that remains after processing is referred to as the “fish emulsion.” The product is acidified for stabilization and to prevent microbial growth. Fish hydrolysate fertilizers are typically produced from freshwater fish by a cold enzymatic digestion process. While fish fertilizers can provide organic nutritional supplementation to plants and soil microorganisms, they are difficult to use, in part due to their high acidity and oil-based composition in some instances, which can clog agricultural equipment. Plant protein-based fertilizers are typically produced by hydrolysis of protein-rich plant materials, such as soybean, and are an attractive alternative for growers and gardeners producing strictly vegan products, for instance. However, due to their sourcing, these products can be expensive. Furthermore, none of the above-described fertilizers is naturally biologic: beneficial microorganisms must be added to them.
Thus, there remains a need in the art for biologically-derived products, particularly products that can be used in organic programs, which can provide superior plant nutrition and soil conditioning, while at the same time being safe, easy to use and cost-effective. Such products would provide highly advantageous alternatives to synthetic products currently in use, and would satisfy growers' requirements for standardization and reliability.

SUMMARY OF THE INVENTION

One aspect of the invention features a liquid composition for application to plants and soils, comprising an autothermal thermophilic aerobic bioreaction product from a liquid fraction of poultry manure. In particular embodiments, the poultry manure is from chickens, such as from chickens raised for meat or egg-laying chickens.
In an embodiment, the composition endogenously comprises at least one biostimulant. More particularly it comprises several biostimulants, e.g., 2, 3, 5, 10, 15, and/or 20 or more biostimulants. The biostimulants can include one or more amino acids, bacteria, fungi and combinations thereof.
In an embodiment, the composition endogenously comprises at least one living species of plant growth promoting bacteria or fungi. More particularly it comprises several such species of bacteria or fungi, e.g., 2, 3, 5, 10, 15, and/or 20 or more species.
In an embodiment, the composition endogenously comprises at least one non-living substance that promotes plant growth and is not a macronutrient or a micronutrient. More particularly, it comprises several such substances, e.g., 2, 3, 5, 10, 15, and/or 20 or more such substances. In certain embodiments, the substance that promotes plant growth is selected from citramalic acid, salicylic acid, pantothenic acid, indole-3-acetic acid, 5-hydroxy-indole-3-acetic acid, galactinol, and any combination thereof.
In an embodiment, the composition endogenously comprises at least one biocontrol agent selected from a living organism, a non-living substance, or a combination thereof, that promotes a plant pathogen resistance response. More particularly, it comprises several such biocontrol agents, e.g., 2, 3, 5, 10, 15, and/or 20 or more such agents. In certain embodiments, the non-living substance is selected from salicylic acid, phenolic compounds, and any combination thereof.
In any embodiment of the composition set forth above, the liquid fraction of poultry manure comprises a liquid fraction of a poultry manure slurry comprising between about 80% and 90% moisture and a pH between about 4 and about 7. In certain embodiments, the poultry manure slurry is heated to between about 60° C. and about 75° C. for between about 1 hour and about 4 hours.
In any embodiment of the composition set forth above, the autothermal thermophilic aerobic bioreaction of which the composition is a product comprises maintaining the liquid fraction at a temperature of about 45° C. to about 80° C. under aerobic conditions for a pre-determined time. In particular embodiments, the pre-determined time is between about 1 day and about 18 days.
In any embodiment of the composition set forth above, the composition endogenously comprises all macronutrients and micronutrients required for plant growth. The composition endogenously comprises less than about 0.5 wt % phosphorus.
Any embodiment of the composition can comprise at least one additive. In certain embodiments, the additive is selected from a macronutrient, a micronutrient, a biostimulant, a biocontrol agent, and any combination thereof.
The compositions described above can be formulated in a variety of ways, such as for application to soil or a medium in which a plant is growing or will be grown. Alternatively, they can be formulated for application to a seed or plant part.
Any of compositions described above can be produced or formulated in a manner suitable for use in an organic program.
Another aspect of the invention features a method of improving health or productivity of a selected plant or crop. The method comprises: (a) selecting a plant or crop for which improved health or productivity is sought; (b) treating the plant or crop with a composition comprising an autothermal thermophilic aerobic bioreaction product from a liquid fraction of poultry manure; (c) measuring at least one parameter of health or productivity in the treated plant or crop, and (d) comparing the at least one measured parameter of health or productivity in the treated plant or crop with an equivalent measurement in an equivalent plant or crop not treated with the composition; wherein an improvement in the at least one measured parameter in the treated, as compared with the untreated, plant or crop is indicative of improving the health or productivity of the selected plant or crop.
In various embodiments, the plant or crop is selected from angiosperms, gymnosperms, ferns, mosses, fungi, algae and cyanobacteria. In certain embodiments, the plant or crop is grown or cultivated in a medium selected from, soil, soil-less solid, hydroponic or aeroponic. In certain embodiments, the treating comprises applying the composition to one or more of: seeds of the plant, a medium in which the plant or crop is growing or will be planted, portions of the plant or crop, and any combination thereof. In particular, the composition is applied in a manner selected from one or a combination of: to the medium pre-planting or pre-inoculation, or pre-emergence; to the medium as a side dressing; in the course of irrigation; and as a direct application to the plant or crop.
In certain embodiments, the at least one parameter of health or productivity in the plant or crop is selected from one or more of: germination rate, germination percentage, robustness of germination, root biomass, root structure, root development, total biomass, stem size, leaf size, flower size, crop yield, structural strength/integrity, photosynthetic capacity, time to crop maturity, yield quality, resistance or tolerance to stress, resistance or tolerance to pests or pathogens, and any combination thereof. Yield quality can include one or more of dry matter content, starch content, sugar content, protein content, appearance, Brix value, and any combination thereof.
In certain embodiments, the at least one measured parameter in the treated plant or crop is compared with an equivalent parameter in an equivalent untreated crop: (a) grown in substantially the same location during the same growing season; or (b) grown in the substantially same location during a different growing season; or (c) grown in a different location during the same growing season; or d) grown in a different location during a different growing season.
In any embodiment of the above-described method, the plant or crop is grown in accordance with an organic program and the composition is approved for use in the program. In particular, the organic program is a United States Department of Agriculture (USDA) National Organic Program or equivalent thereof, such as an equivalent program in a state, or in another country.
Another aspect of the invention features a method of conditioning a selected soil. The method comprises: (a) selecting a soil for which conditioning is sought: (b) treating the soil with a composition comprising an autothermal thermophilic aerobic bioreaction product from a liquid fraction of poultry manure; (c) measuring at least one parameter of conditioning in the treated soil, and (d) comparing the at least one measured parameter of conditioning in the treated soil with an equivalent measurement in an equivalent soil not treated with the composition, or before treatment with the composition; wherein an improvement in the at least one measured parameter in the treated, as compared with the untreated soil, or with the soil prior to treatment, is indicative of conditioning the selected soil.
In one embodiment, the selected soil is one in which plants or crops are or will be planted. In certain embodiments, the selected soil comprises at least one feature selected from compaction, nutrient deficiency, microbial deficiency, organic matter deficiency, and any combination thereof.
In certain embodiments, the at least one parameter of conditioning the soil is selected from one or more of: soil organic matter, microbial diversity, nutrient profile, bulk density, porosity, water permeation, and any combination thereof.
In certain embodiments, the at least one measured parameter in the treated soil is compared with an equivalent parameter prior to treatment of the same soil, or at various time points during a treatment regimen. In other embodiments, the at least one measured parameter in the treated soil is compared with an equivalent parameter in an equivalent untreated soil in substantially the same location or in a different location.
Other features and advantages of the invention will be apparent by references to the drawings, detailed description and examples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block-diagram of an exemplary embodiment of nutritional composition production process where the circled numbers (1-7) indicate samples taken from various stages of the process and collected for analysis. Sample 1 refers to raw manure (e.g., raw chicken manure), sample 2 refers to the slurry taken after the preparation of feedstock material, sample 3 refers to the solid taken after separation by centrifugation, sample 4 refers to the liquid stream taken after separation by centrifugation, sample 5A refers to the sample taken after 24 hours in the bioreactor and prior to primary formulation, sample 5B refers to the sample taken after 72 hours in the bioreactor and prior to primary formulation, sample 6 refers to the formulated sample that has not been subjected to heat pasteurization, and sample 7 refers to the sample taken after the heat pasteurization step, but prior to the filtration step.

FIG. 2 is a graph showing FTIR spectra from samples collected at various stages in the production process of FIG. 1 . Band assignments are based on Filip and Hermann (Eur. J. Soil. Biol., 2001, 37:137-143); Maquelin et al. (J. Microbiol., 2002, 51:255-271), and Rodriquez (Clin. Microbiol. News., 2000, 22:57-61), the contents of each of which are incorporated by reference herein in their entireties. The vertical lines indicate the location of absorption bands characteristic for functional groups contributing to the formation of absorption bands at specific wavenumbers. 1 Raw, raw manure or sample 1; 3 Cake, filter cake or sample 2; 4 Centrate, liquid stream centrate or sample 4; 5A T24, liquid product after 72 hours in aerobic bioreactor or sample 5A; 6 Form, formulated unpasteurized liquid product or sample 6: 7 Post, formulated post-pasteurized liquid product or sample 7.

FIG. 3 is a bar graph showing the concentration of microbial biomarker groups in samples collected at various stages in the fertilizer production process of FIG. 1 . The x-axis identifies the various samples, including the raw chicken manure (Raw), the slurry taken from the slurry stage (Slurry), the solid taken after centrifugation (Cake), the liquid taken after centrifugation (Centrate), the sample taken after 24 hours in the bioreactor (T24), the formulated sample taken after 72 hours in the bioreactor and prior to the pasteurization step (Form), and the formulated sample taken after the final heat pasteurization step (Post). The y-axis indicates the concentration of the microbial markers in pmol g⁻¹.

FIG. 4 depicts the heat map resulting from hierarchical cluster analysis (Ward's minimum variance method) showing the relative peak abundance of the known compounds in the 7 samples (rows) taken from the fertilizer production process of FIG. 1 . Each column represents one of the 254 identified metabolites where the colors from blue, gray to red reflect the relative abundance of the metabolites from lowest to highest. The dendrogram to the right of the heat map indicates similarities between the samples.

FIG. 5A is a bar graph showing the relative peak abundance (y-axis) of several known plant growth promoting compounds in the 7 samples taken from the fertilizer production process of FIG. 1 . The top panel represents the relative peak abundance of 5-hydroxy-3-indoleacetic acid. The second panel from the top represents the relative peak abundance of indole-3-acetate. The third panel from the top represents the relative peak abundance of citramalic acid. The bottom panel represents the relative peak abundance of salicylic acid.

FIG. 5B is a bar graph showing the relative peak abundance (y-axis) of galactinol in the 7 samples taken from the fertilizer production process of FIG. 1 .

FIG. 6 is a heat map resulting from hierarchical cluster analysis (Ward's minimum variance method) showing the relative peak abundance of the unknown compounds in the 7 samples (rows) taken from the fertilizer production process of FIG. 1 . Each column represents one of the unidentified metabolites where the colors from blue, gray to red reflect the relative abundance of the metabolites from lowest to highest. The dendrogram to the right of the heat map indicates similarities between the samples.

FIG. 7A is a photograph of the modified sieve test filtering apparatus.

FIG. 7B is a photograph of the modified sieve test retain fraction collection.

FIG. 7C is a photograph showing retained material on a filtration disc following pressure filtration in an embodiment of the fertilizer production process.

FIG. 8 is a graphical representation of the modified sieve test data. The x-axis represents data from

mesh sizes

230, 200, 170, and 140. The y-axis represents the grams of material retained.

FIG. 9A is a graphical representation of the fertilizer challenge study on Salmonella spp. The x-axis represents minutes after inoculation of the sample with the bacteria. The y-axis represents the viable count of bacteria in log 10 scale. Lines A, B, and C represent samples done in triplicate.

FIG. 9B is a graphical representation of the fertilizer challenge study on Listeria spp. The x-axis represents minutes after inoculation of the sample with the bacteria. The y-axis represents the viable count of bacteria in log 10 scale. Lines A, B, and C represent samples done in triplicate.

FIG. 9C is a graphical representation of the fertilizer challenge study on E. coli O157:H7. The x-axis represents minutes after inoculation of the sample with the bacteria. The y-axis represents the viable count of bacteria in log 10 scale. Lines A, B, and C represent samples done in triplicate.

FIG. 9D is a graphical representation of the fertilizer challenge study on generic E. coli. The x-axis represents minutes after inoculation of the sample with the bacteria. The y-axis represents the viable count of bacteria in log 10 scale. Lines A, B, and C represent samples done in triplicate.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention features nutritional compositions for plants and soils. These compositions include both liquid and solid products produced from animal manure and related waste products as a starting material. In particular embodiments the starting material comprises poultry manure.
Avian manure tends to be very high in nitrogen, phosphorous, and other nutrients, as well as a robust microbial community, that plants require for growth and is therefore suitable for use in embodiments of the present invention. Shown in the table below is a comparison of typical nutrient and microbial content contained in manure from several different poultry species.

TABLE 1A

Poultry manure analysis (source: Biol. & Agric. Eng. Dept. NC State
University, Jan 1994; Agronomic Division, NC Dept of Agriculture &
Consumer Services)

	Unit		Chicken
Parameter	(mean)	Layer	Broiler	Breeder	Turkey	Duck	Range

Total Solids	% wet basis	25	79	69	73	37	25-79
Volatile Solids		74	80	43	73	66	43-80
TKN	lb/ton	27	71	37	55	17	17-71
NH₃N	% TKN	25	17	21	22	22	17-27
P₂O₅	lb/ton	21	69	58	63	21	21-69
K₂O	lb/ton	12	47	35	40	13	12-47
Ca	lb/ton	41	43	83	38	22	22-83
Mg	lb/ton	4.3	8.8	8.2	7.4	3.3	3.3-14
S	lb/ton	4.3	12	7.8	8.5	3	3-12
Na	lb/ton	3.7	13	8.3	7.6	3	3-13
Fe	lb/ton	2	1.2	1.2	1.4	1.3	1.2-2
Mn	lb/ton	0.16	0.79	0.69	0.8	0.37	0.16-8
B	lb/ton	0.055	0.057	0.034	0.052	0.021	0.021-0.057
Mo	lb/ton	0.0092	0.00086	0.00056	0.00093	0.0004	0.0004-0.0092
Zn	lb/ton	0.14	0.71	0.62	0.66	0.32	0.14-0.71
Cu	lb/ton	0.026	0.53	0.23	0.6	0.044	0.026-0.6
Crude Protein	% dry basis	32	26		18		18-32
Total Bacteria	col/100 gm	7.32E+11	1.06E+11		5.6E+11
Aerobic Bacteria	col/100 gm	6.46E+10	1.58E+09

TKN, Total Kjeldahl Nitrogen (organic nitrogen, ammonia, and ammonium)

Thus, manure from domestic fowl, or poultry birds, may be especially suitable for use in the present manufacturing methods as they tend to be kept on farms and the like, making for abundant and convenient sourcing. In particular embodiments, the poultry manure is selected from chickens (including Cornish hens), turkeys, ducks, geese, and guinea fowl.
In preferred embodiments, the raw manure used in the present manufacturing process comprises chicken manure. Chicken farms and other poultry farms may raise poultry as floor-raised birds (e.g., turkeys, broilers, broiler breeder pullets) where manure is comprised of the animal feces or droppings as well as bedding, feathers and the like. Alternatively, poultry farms may raise poultry as caged egg layers that are elevated from the ground and where manure consists mainly of fecal droppings (feces and uric acid) that have dropped through the cage. In particular aspects, the chicken manure is selected from the group consisting of egg layer chickens, broiler chickens, and breeder chickens. In a more particular embodiment, the manure comprises egg layer manure.
A typical composition of chicken manure is shown in the table below (analysis in wt % or ppm). The moisture content can vary from 45% to 70% moisture. In addition to macro and micro nutrients the manure contains a diverse population of microorganism which have a potential of being PGPB and also pathogenic characteristics. The manufacturing process is designed to reduce or eliminate the pathogenic organisms and cultivate beneficial organisms, including PGPB.

TABLE IB

Raw Chicken Manure Nutrients Analysis

Nutrient	Average	Range

Ammonium Nitrogen	0.88%	0.29-1.59
Organic Nitrogen	1.89%	0.66-2.96
TKN	2.78%	1.88-3.66
P₂O₅	2.03%	1.33-2.93
K	1.40%	0.89-3.01
Sulfur	0.39%	0.13-0.88
Calcium	3.56%	1.98-5.95
Magnesium	0.36%	0.22-0.60
Sodium	0.33%	0.10-0.88
Copper	90 ppm	>20 ppm-309 ppm
Iron	490 ppm	314 ppm-911 ppm
Manganese	219 ppm	100 pm-493 ppm
Zinc	288 ppm	97 ppm-553 ppm
Moisture	51.93%	31%-71%
Total Solids	49.04%	69%-29%
pH	7.60	5.5-8.3
Total Carbon	17.07%	9.10%-29.20%
Organic Matter	22.32%	15%-30%
Ash	19.00%	15-25%
Chloride	0.39%	0.19%-0.80%

In certain embodiments, the selected poultry manure comprises between about 17 lb/ton and about 71 lb/ton (i.e., between about 0.85% and about 3.55% by weight) total kjeldahl nitrogen (TKN), which is the total amount of organic nitrogen, ammonia, and ammonium. In particular aspects, the manure comprises about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or 71 lb/ton TKN.

The compositions of the invention are produced from the animal waste by a process that combines physical (e.g., mechanical, thermal), chemical and biological manipulations that reduce or eliminate pathogens while promoting the growth of a diverse microbial population and generating metabolic products of those microorganisms, all of which act together to promote plant and soil health, as described in detail below. In this regard, the inventors have discovered that manipulation of the time, temperature, oxidation reduction potential value, and/or pH in various stages of the process can alter the microbial and biochemical profile of the compositions.
While not wishing to be bound by theory, the metabolites in the compositions are believed to act as precursor building blocks for plant metabolism and can enhance regulatory function and growth. In one aspect, the bacteria in the compositions can produce allelochemicals that can include, for example, siderophores, antibiotics, and enzymes. In another aspect, precursor molecules for the synthesis of plant secondary metabolites can include flavonoids, allied phenolic and polyphenolic compounds, terpenoids, nitrogen-containing alkaloids, and sulfur-containing compounds.
All percentages referred to herein are percentages by weight (wt %) unless otherwise noted.
Ranges, if used, are used as shorthand to avoid having to list and describe each and every value within the range. Any value within the range can be selected, where appropriate, as the upper value, lower value, or the terminus of the range.
The term “about” refers to the variation in the numerical value of a measurement, e.g., temperature, weight, percentage, length, concentration, and the like, due to typical error rates of the device used to obtain that measure. In one embodiment, the term “about” means within 5% of the reported numerical value.
As used herein, the singular form of a word includes the plural, and vice versa, unless the context clearly dictates otherwise. Thus, the references “a”, “an”, and “the” are generally inclusive of the plurals of the respective terms. Likewise the terms “include”, “including” and “or” should all be construed to be inclusive, unless such a construction is clearly prohibited from the context. Similarly, the term “examples,” particularly when followed by a listing of terms, is merely exemplary and illustrative and should not be deemed to be exclusive or comprehensive.
The term “comprising” is intended to include embodiments encompassed by the terms “consisting essentially of” and “consisting of”. Similarly, the term “consisting essentially of” is intended to include embodiments encompassed by the term “consisting of”.
As used herein, “animal waste” refers to any material that contains animal manure, including litter, bedding or any other milieu in which animal manure is disposed. In one aspect, “animal waste” comprises avian or fowl manure, more particularly poultry manure (e.g., chicken, turkey, duck, goose, guinea fowl). In particular, “animal waste” comprises chicken manure, for example, from broilers or layers. In other aspects, “animal waste” can refer to waste from other animals, such as, for example, hogs, cattle, sheep, goats, or other animals not specifically recited herein. In yet another aspect, “animal waste” can refer to a mixture of waste products from two or more types of animals, for instance, two or more types of poultry.
“Poultry litter” refers to the bed of material on which poultry are raised in poultry rearing facilities. The litter can comprise a filler/bedding material such as sawdust or wood shavings and chips, poultry manure, spilled food, and feathers.
Manure slurry refers to a mixture of manure and any liquid, e.g., urine and/or water. Thus, in one aspect, a manure slurry can be formed when animal manure and urine are contacted, or when manure is mixed with water from an external source. No specific moisture and/or solids content is intended to be implied by the term slurry.
The term “autothermal thermophilic aerobic bioreaction,” or “ATAB,” is used herein to describe the bioreaction to which the substantially liquid component of the animal manure slurry is subjected in order to produce the liquid nutritional compositions of the present invention. As described below, the term refers to an exothermic process in which the separated liquid component of an animal waste slurry is subjected to elevated temperature (generated endogenously at least in part) for a pre-determined period of time. Organic matter is consumed by microorganisms present in the original waste material, and the heat released during the microbial activity maintains thermophilic temperatures.
In this regard, a “bioreaction” is a biological reaction, i.e., a chemical process involving organisms or biochemically active substances derived from such organisms. “Autothermal” means that the bioreaction generates its own heat. In the present disclosure, while heat may be applied from an outside source, the process itself generates heat internally. “Thermophilic” refers to the reaction favoring the survival, growth and/or activity of thermophilic microorganisms. As is known in the art, thermophilic microorganisms are “heat loving,” with a growth range between 45° C. and 80° C., more particularly between 50° C. and 70° C., as described in detail herein. “Aerobic” means that the bioreaction is carried out under aerobic conditions, particularly conditions favoring aerobic microorganisms, i.e., microorganisms that prefer (facultative) or require (obligate) oxygen.
The term “endogenous” as used herein refers to substances or processes arising from within—for instance, from the starting material, i.e., the animal waste, or from within a component of the manufacturing process, i.e., the separated liquid component, or from within a product of the manufacturing process, i.e., a nutritional composition as described herein. A composition may contain both endogenous and exogenous (i.e., added) components. In that regard, the term “endogenously comprising” refers to a component that is endogenous to the composition, rather than having been added.
As used herein, a biostimulant is a substance or microorganism that, when applied to plants or to the soil, stimulates existing biological & chemical processes in the plant and/or associated microbes (e.g., mycorrhizal fungi) to enhance the plant's growth, yield and/or quality through improving nutrient update, nutrient use efficiency and/or tolerance to abiotic stress (e.g., heat, saline soils).
As used herein, biofertilizers are materials of biological origin, e.g., plants, seaweed, fish, land animals, and the like, that contain sufficient levels of plant nutrients (nitrogen, phosphorus, potassium, calcium, magnesium, etc.), in forms that are either directly absorbed by plants, or are sufficiently quickly decomposed to available forms, to cause an increase in plant growth and/or quality.
As used herein, “biocontrol agents” or “biopesticides” are substances of natural or biological origin, or are organisms themselves, that facilitate a plant's inherent disease or pest-resistance mechanisms. These formulations may be very simple mixtures of natural ingredients with specific activities or complex mixtures with multiple effects on the host as well as the target pest or pathogen.
As used herein, a “soil conditioner” is a substance added to soil to improve the soil's physical, chemical or biological qualities, especially its ability to provide nutrition for plants. Soil conditioners can be used to improve poor soils, or to rebuild soils which have been damaged by improper management. Such improvement can include increasing soil organic matter, improving soil nutrient profiles, and/or increasing soil microbial diversity.
Process:
The manufacturing process comprises the following steps: (1) preparation of the starting material (the animal waste, also referred to herein as “feedstock material”); (2) separation of the prepared feedstock material into a substantially solid and a substantially liquid component; (3) drying the substantially solid component to produce a solid nutritional composition of the invention; (4) subjecting the substantially liquid component to an autothermal thermophilic aerobic bioreaction (ATAB); and (5) subjecting the bioreaction liquid product to one or more further processing steps including filtration, pasteurization and formulation via addition of other components. A schematic diagram depicting an exemplary embodiment of the manufacturing process applied to layer chicken manure is shown in FIG. 1 and described in Example 1. If manure is supplied as poultry litter, e.g., from broiler chickens, the bedding is removed prior to initiation of the above-summarized process.
In the preparation step, the feedstock material is first adjusted for moisture content and pH. The moisture content is adjusted by adding a liquid to form an aqueous slurry that is sufficiently liquid to be flowable from one container to another, e.g., via pumping through a hose or pipe. In certain embodiments, the aqueous slurry has a moisture content of at least about 80%. More particularly, the slurry has a moisture content of at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, with the understanding that about 99% moisture is an upper limit. In particular embodiments, the slurry has a moisture content of between about 80% to about 95%, even more particularly between about 84% and about 87%, or between about 85% and about 90%.
The pH of the slurry is adjusted to neutral or acidic through the addition of a pH adjusting agent. Typically, the slurry will need to be acidified. In particular embodiments, the slurry is adjusted to a pH of between about 4 and about 7, or more particularly to between about 5 and about 7, or even more particularly to between about 5.5 and about 7, or even more particularly to between about 6 and about 7. In preferred embodiments, the pH of the slurry is between about 6.0, or about 6.1, or about 6.2, or about 6.3, or about 6.4, or about 6.5, or about 6.6, or about 6.7 or about 6.9 and about 7.0. Acidification of an otherwise non-acidic (i.e., basic) feedstock is important to stabilize the natural ammonia in the manure into non-volatile compounds, e.g., ammonium citrate.
An acid is typically used to adjust the pH of the slurry. In certain embodiments, the acid is an organic acid, though an inorganic acid may be used or combined with an organic acid. Suitable organic acids include but are not limited to formic acid (methanoic acid) acetic acid (ethanoic acid) propionic acid (propanoic acid) butyric acid (butanoic acid) valeric acid (pentanoic acid), caproic acid (hexanoic acid), oxalic acid (ethanedioic acid), lactic acid (2-hydroxypropanoic acid), malic acid (2-hydroxybutanedioic acid), citric acid (2-hydroxypropane-1,2,3-tricarboxylic acid) and benzoic acid (benzenecarboxylic acid). Preferably, the acid is one typically used to adjust the pH of food or feed. A preferred acid is citric acid.
The slurry preparation system is designed to prepare a homogeneous slurry in an aqueous medium at a pH of 4 to 7, preferably 6 to 7 and at an elevated temperature. The temperature is elevated at this stage for several purposes, including (1) to promote mixing and flowability of the slurry, (2) to kill pathogens and/or weed seeds, and/or (3) to facilitate growth of thermophilic bacteria present in the feedstock. The temperature can be elevated by any means known in the art, including but not limited to conductive heating of the mixing tank, use of hot water to adjust moisture content, or injection of steam, to name a few. In certain embodiments, the slurry is heated to at least about 60° C., or at least about 61° C., or at least about 62° C., or at least about 63° C., or at least about 64° C., or at least about 65° C., or at least about 66° C., or at least about 67° C., or at least about 68° C., or at least about 69° C., or at least about 70° C. Typically, the temperature does not exceed about 80° C., or more particularly it is less than about 75° C., or less than about 70° C. In certain embodiments, the temperature of the slurry is maintained at between about 65° C. and about 75° C.
The pH-adjusted aqueous slurry is maintained at the elevated temperature for a time sufficient to break the manure down into fine particles, fully homogenizing the slurry for further processing, killing pathogens and weed seeds, and/or activating the native thermophilic bacteria. In certain embodiments, the slurry is held at the elevated temperature for at least about one hour and up to about 4 hours. Typically, the slurry is subjected to chopping and/or mixing during this phase. In certain embodiments, the preparation step as outlined above is segregated from subsequent steps of the process to reduce the likelihood that downstream process steps could be contaminated with raw manure.
In an exemplary embodiment, the slurry system consists of a stainless steel tank, equipped with a chopper pump, nozzle mixing and an aeration system, pH and temperature controls and a biofiltration system for off-gases.
An exemplary process consists of charging the tank with water, heating it to 65′C or higher, lowering the pH to 7 or lower, preferably to a range of 6-7, with citric acid. The chopper pump, nozzle mixers, aeration and off gas biofiltration systems are turned on before introducing the feedstock to ensure a moisture content of, e.g., 85 to 90%. It is a batch operation and, in various aspects, can take one to four hours to make a homogeneous slurry. The operation ensures that each particle of the manure is subjected to temperatures of 65° C. or higher for a period of at least one hour to kill substantially all the pathogens and weeds.
In certain embodiments, the animal waste slurry prepared as described above is transferred from a slurry tank by pumping, e.g., using a progressive cavity pump. Progressive cavity pumps are particularly suitable devices for moving slurries that can contain extraneous materials such as stones, feathers, wood chips, and the like. The transfer line can be directed into a vibratory screen where the screens can be either vibrating in a vertical axial mode or in a horizontal cross mode. The selected vibratory screen will have appropriate sized holes to ensure that larger materials are excluded from the slurry stream. In one embodiment, the screens exclude materials larger than about ⅛ inch in any dimension.
In particular embodiments, the slurry stream is directed into storage tanks, which may be equipped with pH and temperature controls and/or an agitation system. The slurry can be aerated to remove odiferous compounds that have been formed while the manure was in transit or storage. Optionally, the off-gases are subjected to bio-filtration or other means of disposal. The slurry stream leaving the storage tank is sent to the centrifuge for the next step of the process.
In the separation step, the slurry stream from the storage tank is pumped into a solid-liquid separation system, which can include but not limited to mechanical screening or clarification. The purpose of this step is to reduce solids, such as cellulosic and hemicellulosic material (e.g., feathers, bones) that is unsuitable for the subsequent ATAB. It is noteworthy that a substantial fraction of phosphorus and calcium present in the feedstock tends to separate with the solids in this step.
A preferred separation method employs a decanter centrifuge that provides a continuous mechanical separation. The operating principle of a decanter centrifuge is based on gravitational separation. A decanter centrifuge increases the rate of settling through the use of continuous rotation, producing a gravitational force between 1000 to 4000 times that of a normal gravitational force. When subjected to such forces, the denser solid particles are pressed outwards against the rotating bowl wall, while the less dense liquid phase forms a concentric inner layer. Different dam plates are used to vary the depth of the liquid as required. The sediment formed by the solid particles is continuously removed by the screw conveyor, which rotates at different speed than the bowl. As a result, the solids are gradually “ploughed” out of the pond and up the conical “beach”. The centrifugal force compacts the solids and expels the surplus liquid. The compacted solids then discharge from the bowl. The clarified liquid phase or phases overflow the dam plates situated at the opposite end of the bowl. Baffles within the centrifuge casing direct the separated phases into the correct flow path and prevent any risk of cross-contamination. The speed of the screw conveyor can be automatically adjusted by use of the variable frequency drive (VFD) in order to adjust to variation in the solids load.
Thus, the separation process results in formation of a substantially solid component and a substantially liquid component of the prepared animal waste slurry. The term “substantially solid” will be understood by the skilled artisan to mean a solid that has an amount of liquid in it. In particular embodiments, the substantially solid component may contain, e.g., from about 40% to about 64% moisture, often between about 48% and about 58% moisture, and is sometimes referred to herein as “solid,” “cake,” or “wet cake.” Likewise, the term “substantially liquid” will be understood to mean a liquid that has an amount of solids in it. In particular embodiments, the substantially liquid component may contain between about 2% and about 15% solids (i.e., between about 85% and about 98% moisture), often between about 4% and about 7% solids, and is sometimes referred to herein as “liquid,” “liquid component,” or “centrate” (the latter if the separation utilizes centrifugation). Approximately 30% of the raw feedstock is retained in the substantially liquid component, with about 70% being retained in the cake.
The solids from the separation step are dried to a moisture content suitable for subsequent handling and packaging of the material. In certain embodiments, the solid component is dried to less than about 20% moisture. In particular embodiments, the solids are dried to less than about 19%, or less than about 18%, or less than about 17%, or less than about 16%, or less than about 15%, or less than about 14%, or less than about 13%, or less than about 12%, or less than about 11% b, or less than about 10% moisture. In a preferred embodiment, the solid component is dried to less than 12% moisture. The dried solid is sometimes referred to herein as “dried cake.”
In certain embodiments, the manufacturing process is a closed-loop system in which off-gases and water vapors from any or all stages of the system, including the dryer, are captured and condensed into a nutrient-rich liquid form. This liquid can be re-integrated into the liquid manufacturing processes described below, e.g., into the feedstock slurry, the bioreactor or into the base product exiting the bioreactor.
The next step involves subjecting the substantially liquid component to an autothermal thermophilic aerobic bioreaction (ATAB). ATAB is an exothermic process in which the separated liquid component with finely suspended solids is subjected to elevated temperature for a pre-determined period of time. Organic matter is consumed by microorganisms present in the original waste material, and the heat released during the microbial activity maintains thermophilic temperatures. Autothermal thermophilic aerobic bioreaction produces a biologically stable product which contains macro and micro nutrients and PGPB.
In certain embodiments, the elevated temperature conditions are between about 45° C. and about 80° C. More particularly, the elevated temperature conditions are at least about 46° C., or 47° C., or 48° C., or 49° C., or 50° C., or 51° C., or 52° C., or 53° C., or 54° C., or 55° C., or 56° C., or 57° C., or 58° C., or 59° C., or 60° C., or 61° C., or 62° C., or 63° C., or 64° C., or 65° C., or 66° C., or 67° C., or 68° C., or 69° C., or 70° C., or 71° C., or 72° C., or 73° C., or 74° C., or 75° C., or 76° C., or 77° C., or 78° C., or 79° C. In particular embodiments, the elevated temperature conditions are between about 45° C. and about 75° C., more particularly between about 45° C. and about 70° C., more particularly between about 50° C. and about 70° C., more particularly between about 50° C. and about 65° C., and most particularly between about 55° C. and about 60° C.
In certain embodiments, the liquid component is maintained at the elevated temperature for a period of several hours to several days. A range of between 1 day and 18 days is often used. In certain embodiments, the conditions can be maintained for 1, 2, 3, 4, 5, 6, 7, 8 or more days. For purposes of guidance only, the bioreaction is maintained at the elevated temperature for a longer period, e.g., three or more days, to ensure suitable reduction of pathogenic organisms, for instance to meet guidelines for use on food portions of crops. However, inasmuch as the length of the bioreaction affects the biological and biochemical content of the bio-reacted product, other times may be selected, e.g., several hours to one day or two days.
One challenge in operating under aerobic thermophilic conditions is to keep the process sufficiently aerobic by meeting or exceeding the oxygen demand while operating at the elevated temperature conditions. One reason this is challenging is that as the process temperature increases, the saturation value of the residual dissolved oxygen decreases. Another challenge is that the activity of the thermophilic micro-organisms increases within increasing temperature, resulting in increased oxygen consumption by the microorganisms. Because of these factors, greater amounts of oxygen, in various aspects, should be imparted into the biomass containing solutions.
In certain embodiments, oxygen is delivered to the bioreactor by using a jet aeration device. Jet aerators utilize two-phase jet nozzles to supply atmospheric oxygen to chemical and biological treatment processes. Process benefits of jet aeration include high oxygen transfer efficiency, independent control of oxygen transfer and mixing, superior mixing, capital and energy savings, and reduced off-gas. In addition to the efficiency inherent with a fine bubble dispersion of gas into liquid, the turbulent nature of jet aeration produces constant renewal of the gas/liquid interface, further facilitating oxygen transfer. Suitable jet aeration devices are commercially available, e.g., from Fluidyne Corp. (Cedar Falls Iowa), Kla Systems, Inc., (Assonet, Mass.) and Mass Transfer Systems, Inc. (Walpole, Mass.), to name a few.
In certain embodiments, oxygenation of the bioreaction is measured in terms of oxidation-reduction potential (ORP). Typically, the ORP of the bioreaction is maintained between about −480 mV to about +10 mV. More particularly, it is maintained within a range of between −250 mV and −50 mV.
To monitor the temperature, pH and oxygenation parameters of the ATAB, the bioreactor can be equipped with automated controllers to control such parameters. In some embodiments, the bioreactor is equipped with a programmable logic controller (PLC) that effectively controls pH, ORP, and other parameters by adjusting the air supply and feed rate of a pH adjuster to the bio-reactor.
The off-gases from the slurry preparation tank and slurry storage tank contain carbon dioxide, air, ammonia, and water vapors; whereas the off-gases from the bioreactor contain oxygen depleted air, carbon dioxide and water vapors. In certain embodiments, these off-gases are directed to a biofilter. When applied to air filtration and purification, biofilters use microorganisms to remove undesired elements. The air flows through a packed bed and the pollutant transfers into a thin biofilm on the surface of the packing material. Microorganisms, including bacteria and fungi are immobilized in the biofilm and degrade the pollutant.
The product stream from the bioreactor is directed into a receiving container and can be used as a final product at that stage or subjected to further processing. This composition is sometimes referred to herein as “base composition” or “base product.” In certain embodiments, the receiving vessel for the base composition is equipped with an agitation system that maintains the colloidal components of the liquid stream in the homogeneous suspension.
The initial heat step and the heat and other conditions applied in the ATAB are effective to substantially or completely eliminate human pathogenic organisms, as well as weeds and seeds (see, e.g., Examples 1 and 7), leaving beneficial aerobic thermophiles and mesophiles. However, in certain embodiments, the base composition is subjected to a second heat treatment for the purpose of further reducing the microbial load so that the composition can be supplemented with exogenous microorganisms as desired, e.g., in a customized product. This step, referred to herein as “pasteurization” or “flash pasteurization,” depending on the time and temperature of treatment, comprises heating the liquid composition to between about 65° C. and about 100° C. for between about 5 minutes and about 60 minutes. In certain embodiments, the base composition is heated to at least 70° C., or at least 75° C., or at least 80° C., or at least 85° C., or at least 90° C., or at least 95° C. In certain embodiments, the base composition is heated for at least 10 minutes, or at least 15 minutes, or at least 20 minutes or at least 25 minutes, or at least 30 minutes, or at least 35 minutes, or at least 40 minutes, or at least 45 minutes, or at least 50 minutes, or at least 55 minutes, noting that the heating time typically is inversely proportional to the heating temperature. In certain embodiments, the composition is heated to about 95° C. for about 30 to 45 minutes.
The liquid composition can also be subjected to one or more filtration steps to remove suspended solids. The solids retained by such filtration processes can be returned to the manufacturing process system, e.g., to the aerobic bioreactor.
Filtration can involve various filter sizes. In certain embodiments, the filter size is 100 mesh (149 microns) or smaller. More particularly, the filter size is 120 mesh (125 microns) or smaller, or 140 mesh (105 microns) or smaller, or 170 mesh (88 microns) or smaller, or 200 mesh (74 microns) or smaller, or 230 mesh (63 microns) or smaller, or 270 mesh (53 microns) or smaller, or 325 mesh (44 microns) or smaller, or 400 mesh (37 microns) or smaller. In particular embodiments, the filter size is 170 mesh (88 microns), or 200 mesh (74 microns), or 230 mesh (63 microns), or 270 mesh (53 microns). In certain embodiments, a combination of filtration steps can be used. e.g., 170 mesh, followed by 200 mesh, or 200 mesh followed by 270 mesh filtrations.
Filtration is typically carried out using a vibratory screen, e.g., a stainless mesh screen, or a pressure filter vessel, or a combination thereof. Filtration typically is carried out on products cooled to ambient air temperature, i.e., below about 28° C.-30° C.
The base product can also be further formulated to produce products, sometimes referred to herein as “formulated products,” “formulated compositions,” and the like, for particular uses. In certain embodiments, additives include macronutrients, such as nitrogen and potassium. Products formulated by the addition of macronutrients such as nitrogen and potassium are sometimes referred to as “formulated to grade,” as would be appreciated by the person skilled in the art. In exemplary embodiments comprising a liquid nutritional composition prepared from chicken manure, the base composition is formulated to contain about 1.5% to about 3% nitrogen and about 1% potassium.
In other embodiments, additives include one or more micronutrients as needed or desired. Though the base composition already contains a wide range of micronutrients and other beneficial substances as described in detail below, it is sometimes beneficial to formulate the composition with such additives. Suitable additives include, but are not limited to, blood meal, seed meal (e.g., soy isolate), bone meal, feather meal, humic substances (humic acid, fulvic acid, humin), microbial inoculants, sugars, micronized rock phosphate and magnesium sulfate, to name a few. Other materials that are suitable to add to the base product will be apparent to the person of skill in the art.
In certain embodiments, the materials added to the base composition are themselves approved for use in an organic farming program, such as the USDA NOP. In particular embodiments, nitrogen is added in the form of sodium nitrate, particularly Chilean sodium nitrate approved for use in organic farming programs. In particular embodiments, potassium is added as potassium sulfate.
The base composition can be formulated any time after it exits the bioreactor and before it is finished for packaging. In one embodiment, the product is formulated with macronutrients prior to any subsequent processing steps. In this embodiment, the product stream is directed into a formulation product receiving vessel where the macronutrients are added. Other materials can be added at this time, as desired. The formulated product receiver can be equipped with an agitation system to ensure that the formulation maintains the appropriate homogeneity.
It will be apparent to the skilled person that the above-described subsequent processing steps, i.e., pasteurization, filtration and formulation, may be performed either singly or in combination, and in any order. Thus, for instance, one embodiment comprises formulation to grade, pasteurization, two levels of filtration and a secondary formulation step. Another embodiment comprises no pasteurization and one or two levels of filtration. Other combinations are also suitable, depending on the desired properties of the finished composition.
Prior to packaging and/or storage, it can be beneficial to adjust the final pH of the liquid composition to enhance stability. Thus, in certain embodiments, the final products can be adjusted to a pH between about 4 and about 7, or between about 4.5 and about 7, or between about 5 and about 7 or between about 5.5 and about 7 or between about 6 and about 7, using a suitable pH adjusting agent as described above. In particular embodiments, the pH adjusting agent is an organic acid, such as citric acid.
In specific embodiments, post-ATAB processing includes one or more of the following steps. The base composition is formulated to grade either as 1.5-0-3 or 3-0-3 (N-P-K) by adding sodium nitrate and potassium sulfate. The pH of the composition is adjusted to 5.5 with citric acid. The composition is flushed through a vibratory screener at about 40 gallons per minute. The vibratory screener is fitted with a 200 mesh stainless steel screen. The filtered product is then pumped through a cartridge filter. Typical operating parameters of the cartridge filter include one or more of the following: (1) differential pressure up to 40 PSI; (2) inlet temperature 29.5° C. (85° F.) or less; and (3) vessel housing pressure up to 40 PSI.
Packaging of the finished product can include dispensing the product into containers from which the material can be poured. In certain embodiments, filled containers may be sealed with a membrane cap (“vent cap,” e.g. from W. L. Gore, Elkton, Md.) to permit air circulation in the headspace of the containers. These membranes can be hydrophobic and have pores small enough that material cannot leak even in the event the containers are completely inverted. Additionally, the pores can be suitably small (e.g., 0.2 micron) to eliminate the risk of microbial contamination of the container contents.
Compositions:
The process described above produces two useful compositions from animal waste. The solid composition is produced from one of the two process streams of the separation step. Once dried to an appropriate moisture content, it may be packaged, stored and shipped for use as a solid nutritional composition for plants. In certain embodiments, the dried solid fertilizer is in a free flowing granular form. In particular embodiments, it comprises a bulk density of 50 lbs/ft³.
In a particular embodiment, the process utilizes chicken manure. Table 2 provides a typical analysis for the dr product produced from chicken manure as the starting material.

	TABLE 2

	Nutrients/Info	Value AVG

	Ammonium Nitrogen	0.58%
	Organic Nitrogen	3.25%
	TKN	3.82%
	P2O5	3.45%
	K	1.24%
	Sulfur	0.32%
	Calcium	10.90%
	Magnesium	0.64%
	Sodium	0.16%
	Copper	61 ppm
	Iron	880 ppm
	Manganese	502 ppm
	Zinc	508 ppm
	Moisture	8.15%
	Total Solids	91.90%
	Total Salts	n/a
	pH	6.4
	Total Carbon	31.40%
	Organic Matter	56.65%
	Ash	34.50%
	Chloride	0.16%

The liquid nutritional compositions are produced from the other of the two process streams of the separation step. The base product exiting the bioreactor may advantageously be qualified to meet all requirements for use in government-regulated organic programs, such as the USDA NOP, and further may be approved for listing with various testing agencies, such as OMRI. This will depend in part on certain of the process parameters designed to ensure product safety, e.g., segregation of the process steps from raw manure, reduction in pathogen load by (1) initial heating during feedstock preparation, and/or (2) sufficient time at elevated temperature during the ATAB. Thus, in a preferred embodiment, the nutritional composition are able to qualify as a bio-organic liquid fertilizer, which meets all USDA and OMRI requirements.
The liquid compositions (also referred to as “liquid product”) are referred to herein as “nutritional compositions;” however, the liquid compositions comprise numerous components, both biological and biochemical, that have been classified as biostimulants, biofertilizers, fertilizers, biocontrol agents and/or soil conditioners in agriculture. Therefore, the liquid compositions may be referred to interchangeably herein as “fertilizers,” “biofertilizers” and “bio-organic fertilizers” or “organic biofertilizers,” the latter terms applying to compositions that meet requirements for use in an organic farming program.
A typical but non-limiting example of the chemical composition of the liquid product when produced from chicken manure is: Macronutrients: nitrogen 1-3%; phosphorus<0.5%; potassium 1-3%; calcium 1-2%; magnesium 1-2%; sulfur>0.2%. Micronutrients: zinc>100 ppm; iron>300 ppm; manganese>100 ppm; copper<20 ppm; boron<20 ppm. Advantageously, the liquid nutritional compositions contain very little phosphorus (i.e., less than about 0.5%), which is helpful in instances where phosphate excess in soil or phosphate runoff is of concern.
More detailed analysis of these nutritional compositions are set out in Examples 2 and 5, which provide a snapshot of components at various stages of the above-described process. As can be seen from the examples, the chemical, biochemical and biological profiles of the liquid compositions are different at different stages of the process, e.g., raw feedstock, feedstock slurry, substantially liquid component from the separation step (centrate in embodiments utilizing centrifugation for separating the liquid and solid components), substantially solid component from the separation step (cake), sample taken after 24 hours of ATAB (T24), samples taken after 3 days of ATAB (T72), and after formulation to grade, either before (“Form”) and after (“Post”) pasteurization.
The liquid composition as produced from chicken manure exists as a suspension, inasmuch as it contains suspended solids that migrate with the liquid through the separation and ATAB steps. Larger solids can be filtered out. In certain embodiments, the compositions are flowable and sprayable, e.g., through 200 mesh nozzles. However, filtered material still can comprise a colloidal suspension with an average particle size of suspended solids between about 2 and about 5 microns. The small particles tend to agglomerate with one another. Even so, the product is filterable through a suitably-sized mesh filter as described above.
The nutritional compositions are of varying tan to brown color, with low odor as compared with the starting material. In certain embodiments, the pH of the compositions has been adjusted from the process pH to a final pH between about 4 and about 7, as described above, and may be adjusted as needed, e.g., to between about 4, or about 4.5, or about 5, or about 5.5, or about 6 and about 7.
The liquid compositions contain a variety of viable microorganisms, as shown, for instance, in Examples 3, 8, 9 and 10 herein for chicken manure. Typically, no exogenous microorganisms are added during the process (though they may be added after the ATAB); therefore, all microorganisms present in the liquid compositions are endogenous to the starting material. The liquid compositions comprise at least about 10⁸colony-forming units per milliliter (CFU/mL) as measured in the base composition from the ATAB. However, the actual viable bacterial count is likely several orders of magnitude higher, given that raw poultry manure can contain 10¹¹or more bacteria per mL and the ATAB enriches the material in thermophiles and some mesophiles.
The liquid compositions include the following classes of organisms as assess by phospholipid fatty acid analysis (PFLA) (see, e.g., Examples 3 and 9): Actinobacteria (Actinomycetes), Gram negative bacteria, Gram positive bacteria, fungi, arbuscular mycorrhizal fungi, and protists. The comparative abundance of these classes of organisms is different in samples taken from stage to stage of the manufacturing process (see, e.g., Example 3), which may be of advantage in cases where a particular class of organism, or a particular organism itself, is deemed to be more desirable for a purpose than another.
The microbial communities of the compositions after subjection to ATAB tend to be dominated by Gram positive bacteria, which were also seen to be a substantial component of raw layer manure (see, e.g., Examples 3 and 9). A noteworthy subset of Gram positive bacteria are the Actinobacteria (Actinomycetes), which are consistently present in the liquid compositions that are not subjected to pasteurization (see, e.g., Examples 3, 9 and 10). Certain Actinobacteria are known to produce antibiotics, and they play a significant role in soil nutrient cycling. In addition, several Actinobacteria have been found to produce growth promoting compounds (Strap, J. L., 2012, “Actinobacteria-Plant Interactions: a Boon to Agriculture,” in D. K. Maheshwari (ed.), Bacteria in Agrobiology: Plant Growth Responses, Springer-Verlag Berlin Heidelberg). Other small but noteworthy members of the compositions' microbial community include Rhizobia and arbuscular/mycorrhizal fungi, which have been observed in the base composition prior to pasteurization (see Examples 9 and 10). These organisms are known for their roles in nitrogen fixation and improving plant uptake of nutrients from soil, among other advantages.
Enrichment of thermophilic microorganisms from the starting layer manure is inherent in the manufacturing process described herein. Based on the microbial content of chicken manure, the large fraction of bacteria found in the liquid compositions of the invention will fall into classes of thermophilic bacteria with known advantageous properties (though mesophiles may also be present, particularly those that are spore forming).
Among other advantages, thermophiles are important for the mineralization of nitrogen, phosphorus and sulfur, increasing the availability of those nutrients to plants. Additionally, some of the bacteria cultivated in the products are also known for their nitrogen fixation (e.g., Rhizobium, as mentioned above) and probiotic properties, while others are known as natural pesticides, including but not limited to Bacillus firmus (nemnaticidal), Bacillus pumilus (fungicidal) and Paenibacillus popilliae (effective against Japanese Beetle larvae).
The following thermophiles are the dominant species found in composted manure of layer chickens: Ureibacillus spp. (including U. thermosphaericus), Bacillus spp., Geobacillus spp. (including G. stearothermophilus), Brevibacillus spp., and Paenibacillus spp. They are described in more detail below.
Geobacillus species are known generally to degrade hydrocarbons and are therefore useful in environmental remediation; they are known to degrade nitrogen compounds as well. More specifically, (1) G. stearothermophilus can improve waste treatment of metal-polluted water and soil, and can facilitate cellulose breakdown; (2) G. thermoleovorans is known for denitrification; (3) G. thermocaternuiatus can facilitate cadmium ion biosorption; and G. thermodenitrificans is a denitrification organism that reduces NO₃to NO₂.
Within the genus Bacillus, (1) B. licheniformis can degrade feathers; (2) B. subtilis possesses several beneficial attributes, including biocontrol, plant growth promotion, sulphur (S) oxidation, phosphorus (P) solubilization and production of industrially important enzymes (amylase and cellulose). Strains of B. subtilis have been shown to inhibit the in vitro growth of the fungi Fusarium oxysporum (25-34%) and Botryodiplodia theobromae (100%), isolated from the postharvest rots of yam (Dioscorea rotundata) tubers. Other than biocontrol, B. subtilis is known to promote root elongation in seedlings up to 70-74% as compared to untreated seeds. B. subtilis is also known to oxidize elemental S to sulfate and has shown distinct P-solubilization activity in vitro. (3) B. pumilus has been shown to be an agricultural fungicide in that of the bacterium on plant roots prevents Rhizoctonia and Fusarium spores from germinating; (4) B. arnyloliquelaciens synthesizes a natural antibiotic protein, barnase, a widely studied ribonuclease that forms a tight complex with its intracellular inhibitor barstar, and plantazolicin, an antibiotic with selective activity against Bacillus anthracis: (5) B. firmus—possesses nematicidal activity and is used to protect roots from nematode infestation when applied directly to the soil, foliar treatment to turf, and as seed treatments (for these uses, B. firmus 1-1582 is classified as a biological nematode suppressant); and (6) B. azotoformans can reduce nitrite to molecular nitrogen.
Members of the genus Ureibacillus are known for their ability to break down soil organic matter and other cellulosic and ligneous material, and to mineralize crop residues. Various isolates of U. thermosphaericus have been used in biological detoxification.
Species of Brevibacillus are known for their antibiotic properties, with certain species having additional functionality, e.g., (1) some strains of Br. agri are capable of oxidizing carbon monoxide aerobically; (2) Br. Borsteinensis degrades polyethylene; (3) Br. levickii—metabolizes specific amino acids; and (4) Br. thermoruber is involved in reduction of nitrates to nitrites and then to molecular nitrogen.
Various Paenibacillus spp. also produce antimicrobial substances that affect a wide spectrum of micro-organisms such as fungi, soil bacteria, plant pathogenic bacteria and even important anaerobic pathogens as Clostridium botulinum. More specifically, several Paenibacillus species serve as efficient plant growth promoting rhizobacteria (PGPR). PGPR competitively colonize plant roots and can simultaneously act as biofertilizers and as antagonists (biopesticides) of recognized root pathogens, such as bacteria, fungi and nematodes. They enhance plant growth by several direct and indirect mechanisms. Direct mechanisms include phosphate solubilization, nitrogen fixation, degradation of environmental pollutants and hormone production. Indirect mechanisms include controlling phytopathogens by competing for resources such as iron, amino acids and sugars, as well as by producing antibiotics or lytic enzymes. With respect to particular species, (1) P. granivorans dissolves native soil starches; (2) P. cookii is a P solubizer; (3) P. borealis is a nitrogen fixing organism and suppresses soil-borne pathogens; (4) P. popilliae is a bio pesticide effective against Japanese Beetle larvae; and (5) P. chinjuensis is an exopolysaccharide-producing bacterium.
The liquid compositions also contain a vast assortment of biochemical metabolites (see, e.g., Example 4 herein). These include sugars and sugar acids, polyols and sugar alcohols, growth factors, lipids and fatty acids, amines (including amino acids), phenolics, carboxylic and organic acids, and nucleosides, among the compounds that have been identified.
Non-limiting examples of sugars and sugar acids that can be present in the liquid compositions include: 3,6-anhydro-D-galactose, beta-gentiobiose, cellobiose, glucose, glucose-1-phosphate, glyceric acid, fructose, fucose, galactose, isomaltose, isoribose, isothreonic acid, lactobionic acid, lyxose, maltose, maltotriose, ribose, sucrose, tagatose, threonic acid, trehalose, UDP-glucuronic acid, xylonic acid, xylonic acid isomer, xylose, and/or xylulose.
Non-limiting examples of polyols and sugar alcohols that can be present in the liquid compositions include: 1-deoxyerythritol, 1-hexadecanol, 2-deoxyerythritol, deoxypentitol, diglycerol, erythritol, glycerol, hexitol, lyxitol, mannitol, pinitol, threitol, and/or xylitol.
Non-limiting examples of plant hormones and other growth factors that can be present in the liquid compositions include: citramalic acid, indole-3-acetic acid (IAA), 5-hydroxy-3-indoleacetic acid, 6-hydroxynicotinic acid, galactinol, pantothenic acid, and/or salicylic acid.
Non-limiting examples of lipids and fatty acids that can be present in the liquid compositions include: 1-monoolein, 1-monopalmitin, 1-monostearin, 2-monoolein, arachidic acid, arachidonic acid, beta-hydroxymyristic acid, beta-sitosterol, capric acid, caprylic acid, cerotinic acid, cholesterol, cis-gondoic acid, dihydrocholesterol, D-erythro-sphingosine, glycerol-alpha-phosphate, heptadecanoic acid, hexadecylglycerol, isoheptadecanoic acid, lauric acid, lignoceric acid, linoleic acid, myristic acid, nonadecanoic acid, octadecanol, oleamide, oleic acid, palmitic acid, palmitoleic acid, pelargonic acid, pentadecanoic acid, squalene, stearic acid, and/or stigmasterol.
Non-limiting examples of amines that can be present in the liquid compositions include: valine, aminomalonate, 1,3-diaminopropane, 2,4-diaminobutyric acid, 3-aminoisobutyric acid, 5-aminovaleric acid, 5-methoxytryptamine, alanine, alpha-aminoadipic acid, asparagine, aspartic acid, beta-alanine, beta-glutamic acid, citrulline, cyclohexylamine, cysteine, ethanolamine, glutamic acid, glutamine, glycine, glycyl proline, homoserine, hydroxylamine, isoleucine, leucine, lysine, maleimide, methionine, methionine sulfoxide, N-acetyl-D-galactosamine, n-acetyl-d-hexosamine, N-acetylaspartic acid, N-acetylglutamate, N-acetylputrescine, N-carbamylglutamate, N-methylalanine, N-methylglutamic acid, norvaline, 0-acetylserine, oxoproline, phenylalanine, phenylethylamine, putrescine, serine, spermidine, taurine, threonine, thymine, trans-4-hydroxyproline, tryptophan, tyramine, and/or tyrosine.
Non-limiting examples of phenolics that can be present in the liquid compositions include: 3,4-dihydroxybenzoic acid, 4-hydroxybenzoate, catechol, cis-caffeic acid, ferulic acid, hydroquinone, phenol, tyrosol, and/or vanillic acid.
Non-limiting examples of carboxylic acids and organic acids that can be present in the liquid compositions include: 2-hydroxy-2-nethylbutanoic acid, 2-hydroxyadipic acid, 2-hydroxybutanoic acid, 2-hydroxyglutaric acid, 2-hydroxyhexanoic acid, 2-hydroxyvaleric acid, 2-isopropylmalic acid, 2-ketoadipic acid, 2-methylglyceric acid, 2-picolinic acid, 3-(3-hydroxyphenyl) propionic acid, 3-(4-hydroxyphenyl) propionic acid, 3-hydroxy-3-methylglutaric acid, 3-hydroxybenzoic acid, 3-hydroxybutyric acid, 3-hydroxypalmitic acid, 3-hydroxyphenylacetic acid, 3-hydroxypropionic acid, 3-phenyllactic acid, 3,4-dihydroxycinnanic acid, 3,4-dihydroxy-hydrocinnamic acid, 3,4-dihydroxy-phenylacetic acid, 4-aminobutyric acid, 4-hydroxybutyric acid, 4-hydroxymandelic acid, 4-hydroxyphenylacetic acid, 4-pyridoxic acid, aconitic acid, adipic acid, alpha-ketoglutarate, behenic acid, benzoic acid, chenodeoxycholic acid, citric acid, digalacturonic acid, fumaric acid, gluconic acid, gluconic acid lactone, glutaric acid, glycolic acid, hexuronic acid, hydrocinnamic acid, isocitric acid, isohexonic acid, isopentadecanoic acid, kynurenic acid, lactic acid, malic acid, malonic acid, methylmalcic acid, oxalic acid, oxamic acid, phenylacetic acid, pimelic acid, pipecolinic acid, pyrrole-2-carboxylic acid, pyruvic acid, quinolinic acid, ribonic acid, succinic acid, sulfuric acid, tartaric acid, uric acid, and/or urocanic acid.
Non-limiting examples of nucleosides that can be present in the liquid compositions include: thymidine, 5,6-dihydrouracil, 7-methylguanine, adenine, adenosine, cytosine, guanine, pseudo uridine, and/or uracil.
Non-limiting examples of metabolites that were not placed into the above-mentioned categories but that can be present in the liquid compositions include: zymosterol, 1-methylhydantoin, 1,2-cyclohexanedione, 2-deoxypentitol, 2-deoxytetronic acid, 2-ketoisocaproic acid, 2,3-dihydroxybutanoic acid, 2,8-dihydroxyquinoline, 3-(3-hydroxyphenyl)-3-hydroxypropionic acid, 3-ureidopropionate, 4-methylcatechol, 5-hydroxymethyl-2-furoic acid, butane-2,3-diol, butyrolactam, conduritol-beta-epoxide, creatinine, daidzein, glycerol-3-galactoside, hypoxanthine, isothreitol, lanosterol, methanolphosphate, myo-inositol, nicotinic acid, octadecylglycerol, ononitol, parabanic acid, phosphate, piperidone, propane-1,3-diol, pyrogallol, pyrophosphate, tocopherol acetate, tocopherol alpha-, tocopherol gamma-, urea, xanthine, and/or xanthurenic acid.
As with the microorganisms discussed above, the comparative abundance of these classes of biochemicals is different in samples taken from stage to stage of the manufacturing process (see Example 4), which may be of advantage in cases where a particular class of substances, or a particular substance itself, is deemed to be more desirable for a purpose than is another. A particularly noteworthy class of metabolites present in the liquid compositions is the class of growth factors, including but not limited to, citramalic acid, salicylic acid, galactinol, indole-3-acetic acid (IAA) and 5-hydroxy-IAA. Among other functions, citramalic acid and salicylic acid have been found to solubilize soil phosphorus to facilitate its uptake into plants (Khorassani, R. et al., 2011, BMC Plant Biol. 11: 121). Salicylic acid is a known signaling molecule in host defense reactions such as ISR and SAR. Galactinol has been found to act in concert with other sugars (e.g., raffinose) as osmoprotectants and stabilizers of cellular membranes, and also as scavengers of reactive oxygen species (ROS). As such, galactinol can play a role in the protection of cellular metabolism (particularly photosynthesis in chloroplasts) from oxidative damage (Nishizawa, A., et al., 2008, Plant Physiol. 147(3): 1251-1263). Indole-3-acetic acid (IAA) is the most common, naturally occurring, plant hormone of the auxin class. As do all auxins, IAA has many different effects, such as inducing cell elongation and cell division with all subsequent results for plant growth and development. On a larger scale, IAA serves as signaling molecule necessary for development of plant organs and coordination of growth (Zhao, Y., 2010, Ann. Rev. Plant Biol. 61: 49-64). The IAA derivative 5-hydroxy-3-indoleacetic acid possesses related, though typically less potent, plant hormone properties and is also known as a metabolite of serotonin.
Certain embodiments of the invention can utilize the distinctive metabolite profiles exhibited at certain points of the manufacturing process to enrich for particularly useful compounds, or groups of compounds, such as known plant growth factors. In this regard, it is noteworthy that relatively larger fractions of growth factors can be obtained from an ATAB after 24 hours (e.g., “T24” in Example 4) than later in the process. Among the growth factors identified in the compositions, citramalic acid and/or galactinol may be more readily isolated directly from the separation step (in the centrate), while indole-3-acetic acid and 5-hydroxy-3-indoleacetic acid may be more readily isolated from the ATAB at 24 hours. Such selection of liquid products from different stages may be used to advantage to produce products with different modes of action, e.g., improving P acquisition, favoring induced systemic resistance, promoting overall plant growth, among others.
Other noteworthy metabolite classes that are significantly represented in the liquid compositions include phenolics, amino acids, fatty acids and organic acids.
Phenolics and organic acids are building blocks for complex organic acids such as humic acid and fulvic acid, among other biologically relevant compounds. Fatty acids and lipids are essential, not only as membrane constituents but also for plant growth and development.
Phenolics are additionally significant in plant defenses mechanisms (see Daayf, F. et al, 2012, Chapter 8 in Recent Advances in Polyphenol Research. Volume 3, 1^stEd., (Eds Cheynier, V. et al., John Wiley & Sons Ltd.) Phenolic-based plant defense mechanisms include physical changes such as lignification and suberization of the plant cell walls, as well as metabolic changes such as de novo synthesis of pathogenesis-related (PR) proteins, and biosynthesis and accumulation of phenylpropanoid secondary metabolites. Many phytoalexins are produced through the phenylpropanoid pathway. In addition, this pathway contributes not only to the pool of free metabolites but also to the group of compounds that are integrated into cell wall reinforcement.
Amino acids and other nitrogen-containing breakdown products of proteins, and their derivatives, have been shown to have a variety of biostimulatory effects on plants. For example, there is considerable evidence that exogenous application of a number of structural and non-protein amino acids can provide protection from environmental stresses or are active in metabolic signaling (see Calvo, P. et al., 2014. Plant Soil 383: 3-41; du Jardin, P., 2015, Scientia Horticulturae 196: 3-14). Several non-protein amino acids have also been shown to have roles in plant defense (see Huang T. et al. 2011, Phytochemistry 72: 1531-1537; Vranova et al. 2011, Plant Soil 342: 31-48).
Uses:
The dried solid product resulting from the above-described reaction scheme contains all macronutrients and micronutrients required for plant growth. It is dried to an appropriate moisture content and is used as a soil amendment and/or additive for other fertilizer products. The solid composition can be applied prior to planting, or as a side dressing, in accordance with known practices.
The liquid compositions can be formulated in a variety of ways known in the industry, as described above and exemplified herein. For instance, they can be formulated for application to dryland crop systems, field irrigation, drip irrigation, hydroponic and/or other soil-free systems, and turf, among others. They can also be formulated for hydroponic, aeroponic and foliar spray application. They are also formulated for use in various soil-less media, including organic media such as peat moss, composted pine bark, coir and the like, and inorganic media such as sand, vermiculite, perlite, rock wool and the like.
The liquid compositions are used to advantage on any plant or crop, including but not limited to angiosperms, gymnosperms, ferns and mosses. These include, but are not limited to: cereals, such as wheat, barley, rye, oats, rice, maize and sorghum: legumes, such as beans, lentils, peas, soybeans, clover and alfalfa: oil plants, such as canola, mustard, poppy, olives, sunflowers, coconut, castor beans, cocoa beans and groundnuts; beet including sugar beet and fodder beet: cucurbits, such as zucchini, cucumbers, melons, pumpkins, squash and gourds; fiber plants, such as cotton, flax, hemp and jute; fruit, such as stone fruit and soft fruit, such as apples, pears, plums, peaches, almonds, cherries, grapes (for direct consumption or for wine production) and berries, e.g. strawberries, raspberries and blackberries; citrus fruit, such as oranges, lemons, grapefruit and mandarins; vegetables, such as spinach, lettuce, asparagus, cabbages, carrots, onions, tomatoes, potatoes and paprika: trees for lumber or forestation, such as oak, maple, pine and cedar; and also tobacco, nuts, coffee, eggplant, sugar cane, tea, pepper, hops, bananas, natural rubber plants, Cannabis, turfgrasses and ornamentals (e.g., woody perennial, foliage and flower ornamentals, and ornamental grasses).
The compositions also will find utility in non-plant crops, for instance in mushroom culture, wherein they are advantageously applied to substrates such as straw (e.g., cereal straw), enriched sawdust, compost, paper and paper products (e.g., shredded cardboard), plant debris and other organic materials such as seed shells, corncobs, and banana fronds. The compositions can also be formulated for use in culture of algae, including cyanobacteria, which are produced commercially for a variety of purposes. For instance, algae are often cultivated for use as nutritional supplements. Additionally, they are used in photobioreactor systems to recycle flue gas emissions (e.g., carbon dioxide) from operations such as power generating plants.
It is noteworthy that the liquid compositions are aqueous and easy to mix with other aqueous materials and to formulate for drip or spray applications. They have been noted in particular for their ease of use for applications involving spraying or liquid injection, because they tend not to clog machinery like certain oil-based compositions.
In some embodiments, the liquid compositions are formulated to grade, e.g., to provide standardized amounts of macronutrients such as nitrogen and potassium. However, due to their biostimulant content, they have been demonstrated to have a beneficial effect on plants and soils even in the absence of added macronutrients. What is more, the beneficial biostimulants in the compositions enhance the effect of the macronutrients, such that less is needed to produce an equivalent plant growth effect observed with traditional fertilizers (see Example 11). As such, these compositions provide numerous advantages when applied to plants and/or soils, to promote plant growth and health, to deter pests and pathogens, and/or to condition the soil.
Typical application rates for a liquid composition of substantially the content shown in Table 2, formulated to grade at 1.5-0-3 (8.6 lbs/gal) or 3-0-3 (with 1% sulfur, 9.6 lbs/gal), will be understood by the skilled person. Examples are as follows, in gallons per acre. (1) Brassica: starter—5-10; side dress, 3-8; (2) cucurbits: starter or side dress, 8-10; (3) leafy greens: Starter, 8; side dress, 8-10; (4) peppers: starter, 5-8; side dress, 8-10; (5) tomatoes: starter or side dress, 5-8; (6) cane fruit: annual/plant ½-1 gallon (7) strawberries: side dress, 10-15; (8) grapevine: side dress, 8-10; (9) corn: starter, 5-8; side dress or foliar, 8-15; (10) soybeans: foliar, 10; (11) small grains: starter, 5-10; side dress, 8-10; (12) hay: starter, 8; side dress or foliar, 5-10. Thus, another aspect of the invention features a method of improving plant health or productivity through the application of the above-described liquid nutritional compositions to plants, plant parts, seeds and/or soils or other media in which plants are grown. The plant or crop selected for such treatment can be any of those listed above, or any other plant or crop known to the skilled person. Depending on the medium in which the plant is grown, the composition may be applied directly to the plant or indirectly through the growth medium, as described above.
The effect of the composition on the health or productivity of the plant can be observed or measured by any means know in the art. For example, plant health or productivity can be observed or measured by one or more of: germination rate, germination percentage, robustness of germination (e.g., hypocotyl, epicotyl, radicle or cotyledon development), root biomass, root structure and development, total biomass, stem, leaf or flower size, crop yield, structural strength/integrity, photosynthetic capacity, time to crop maturity, yield quality (e.g., dry matter, starch and sugar content, protein content, appearance, Brix value), resistance or tolerance to stress (e.g., heat, cold, drought, hypoxia, salinity); and resistance or tolerance to pests or pathogens, (e.g., insects, nematodes, weeds, fungi, bacteria and/or viruses). In certain embodiments, plants treated with the compositions of the invention are compared with untreated plants. “Untreated” plants can include plants treated with a “control,” such as water, or plants treated with one or more other compositions, or plants not treated with any compositions. In other embodiments, various parameters of treated plants can be compared with historical measurements for that type of plant in other locations or at other times (e.g., past seasons). Thus, in various embodiments, one or more parameters of growth and/or productivity can be measured between or among the same or an equivalent crop: (a) grown in substantially the same location during the same growing season; or (b) grown in the substantially same location during a different growing season; or (c) grown in a different location during the same growing season; or d) grown in a different location during a different growing season. “The same or equivalent crop” is intended to mean the same plant genus or the same plant species or the same plant subspecies or variety. “Substantially the same location” is intended to mean, for instance, in an adjacent or nearby plot, or in an adjacent or nearby field, or within a defined geographical distance, e.g., closer than one mile apart.
For purposes of such comparison, observations or measurements of parameters of plant health and/or productivity can be made by any convenient or available method, or any combination of methods. These can include, but are not limited to, visual observations, field measurements and laboratory measurements, all of which are familiar to the person skilled in the art.
Another aspect of the invention features a method of conditioning soil, i.e., building and/or improving the quality of soil. This method is particularly applicable to soil in which crops are grown, but alternatively can be applied as a remediation to damaged or polluted soils in which crops are not grown presently.
The condition or quality of soil is composed of inherent and dynamic soil properties. Inherent properties, such as texture, type of clay, depth of bedrock, drainage class and the like, are not affected to a great extent by management efforts. In contrast, dynamic properties or use-dependent properties can change over the course of months and years in response to land use or management practice changes. Dynamic properties include organic matter, soil structure, infiltration rate, bulk density, and water and nutrient holding capacity. Changes in dynamic properties depend both on land management practices and the inherent properties of the soil. Some properties, such as bulk density, may be considered inherent properties below 20-50 cm, but are dynamic properties near the surface.
Thus, deficiencies in dynamic properties of soil can be addressed by management efforts and the compositions of the invention may be used to advantage in this regard. Such deficiencies include, but are not limited to, deficiencies in organic matter, chemical/nutrient deficiencies, microbial content and structural parameters such as lack of porosity (compaction). Measurable soil quality indicators and their functions in agricultural settings include, but are not limited to: aggregate stability, available water capacity, bulk density, infiltration capacity, respiration, slaking and soil crusts, soil structure and macropores, presence and/or quantity of macronutrients and/or micronutrients, and biological content, i.e., total biomass and breakdown of biological communities, e.g., quantity and type of bacteria, fungi, protists and other soil dwellers (insects, nematodes, earthworms and the like).
The compositions can be applied to soil before planting a crop, or they can be applied to soil containing crops or other growths of plants, or they can be applied to soil between plantings, i.e., between growing seasons. In certain embodiments, application rates can be the same as those exemplified above for treatment of plants. Indeed, in this regard, treatment of plants via application to soils also comprises a treatment of the soil itself. In other embodiments, application rates are different from those selected for treatment of plants.
In certain embodiments, soil treated with the compositions of the invention is compared with untreated soil. “Untreated” soil can include soil treated with a “control,” such as water, or soil treated with one or more other compositions, or soil not treated with any compositions. In one embodiment, such comparison comprises “before and after” measurements, or sequential periodic measurements of the soil being treat over a selected time period. In other embodiments, various parameters of treated soils can be compared with historical measurements for that type of soil in other locations or at other times. Thus, in various embodiments, one or more parameters of soil conditioning can be measured between or among the same or an equivalent soil type in substantially the same location or in a different location. “The same or equivalent soil” is intended to mean the same or similar soil type, and/or a different soil type with a similar deficiency. “Substantially the same location” is intended to mean, for instance, in an adjacent or nearby plot, or in an adjacent or nearby field, or within a defined geographical distance, e.g., closer than one mile apart.
For purposes of such comparison, observations or measurements of parameters of soil condition or quality can be made by any convenient or available method, or any combination of methods. These can include, but are not limited to, visual observations, field measurements and laboratory measurements, all of which are familiar to the person skilled in the art.
The following examples are provided to describe the invention in greater detail. They are intended to illustrate, not to limit, the invention.

Example 1. Process for Producing Fertilizer/Nutritional Composition from Chicken Manure

Depicted in FIG. 1 is an embodiment of the production process described herein for producing liquid and solid fertilizers from chicken manure. The production process depicted in FIG. 1 produced pathogen-free products that retained the primary and secondary nutrients, as well as micro-nutrients, present in layer manure. In addition, the process described herein removed potentially problematic phosphorus from the products.
As shown in FIG. 1 , the process began 10 when raw chicken manure was transported to the location directly from the farm(s) in covered live bottom trailers. The trucks were unloaded into mix tanks at the location and combined with citric acid 15 and water to form a homogeneous slurry. The citric acid bound the natural organic ammonia in raw manure.
The next step in the process involved the preparation of feedstock material 20. In this step, the stored slurry was mixed with water 25 adequate to elevate the moisture level of the slurry to a moisture range from about 84% to about 87% moisture. The slurry was then heated with steam 30 to 65° C. for a minimum of 1 hour to break down the manure into fine particles and was fully homogenized into a slurry for further processing. Additionally, the step included both the killing of any pathogens that were found in raw manure as well as the activation of native thermophilic bacteria. In a particular embodiments, this part of the manufacturing process was segregated from the rest of the system to reduce the risk that processed fertilizer material would be contaminated by raw manure. The mixing tank process parameters for the preparation of feedstock material 20 are shown in Table 3.

TABLE 3

Mixing Tank Process Parameters.

	Range of Operational
Process Parameter	Parameters		Notes

Mixing Tank	3,000 to 4,000 gallons		Tank Size 5,000 gallons
Axial Turbine Mixer	45 to 60 HZ	75 to 100%	Spins clockwise, forces
			material down turns tank
			over 1 to 3 times per minute
Macerator
	45 to 60 HZ	75 to 100%	Reduces particle size,
			homogenizes mix
Pump
	45 to 60 HZ	75 to 100%	Pump Size	3 HP, Positive
			Displacement
Mixing Tank pH	6.5 to 7.0		Citric acid addition varies
			from patch to patch typically
			1 to 2% by weight addition
Mixing Tank
	65° C. to 75° C.		Measured by thermowell via
Temperature	60 minutes		tank penetration
Moisture %	84 to 87%		Measured by loss of drying
Viscosity	2000 to 3000 CPS
Heating Method	Direct Steam Injection 3		Direct steam injection to
	to 8 PSI		heat the material

HZ, hertz; HP, horsepower; CPS, centipoise; PSI, pounds per square inch

The slurry was then sent to the centrifuge 35, whereas debris, oyster shells, and other grit from chicken feed were removed 40. In preferred embodiments, centrifuge 35 is a decanting centrifuge. Suitable centrifuge parameters for the separation of the solid and liquid fractions are shown in Table 4. The centrifuge 35 separated the slurry into two streams—a liquid stream and a solid stream. The solid stream 42 was dried to about 12% (or less) moisture and used to produce a dry fertilizer product (“dry formulation”). The liquid stream 45 was sent to the aerobic bioreactor 50.

TABLE 4

Centrifuge parameters

	Range of Operational
Process Parameter	Parameters	Notes

Decanting Centrifuge	3250 RPM Max
Influent volume	25-30 gallons per minute	Slurry from mixer being
		pumped into centrifuge
Effluent volume
	25% of input manure by	Liquid fraction exiting the
	weight is extracted as	centrifuge
	finely suspended solids
Solids separation	75% of input manure by	Solids fraction discharge
	weight
Differential
	7 to 12%
Bowl Speed	2900 to 3250 RPM
Torque Scroll
	10% or less

RPM, revolutions per minute

Once the liquid stream 45 was fed to the to the aerobic bioreactor 50, native microorganisms were cultivated. These metabolized the organic components of the feedstock into primary and secondary metabolomic byproducts including, but not limited to, plant growth factors, lipids and fatty acids, phenolics, carboxylic acids/organic acids, nucleosides, amines, sugars, polyols and sugar alcohol, and other compounds. Depending on its age, the liquid feedstock remained in the aerobic bioreactor 50 under gentle agitation (e.g., full turnover occurs 6 times per hour) for a minimum of 1 days to a maximum of about 8 days, and at a uniform minimum temperature of 55° C. The aerobic bioreactor process parameters are provided in Table 5.

TABLE 5

Bioreactor process parameters

	Range of
	Operational
Process Parameter	Parameters			Notes

Data collection Record	1 minute to			How frequent the PLC records data
	30 minutes
Hydraulic Retention time/	1 to 8 days			How long the material resides
Residence time of material in				in the bioreactor
reactors
Bioreactor #
1 Foam Level (feet)	8 to 13 feet			8,000 gallon tank
Bioreactor #
2 Foam Level (feet)	8 to 13 feet.			8,000 gallon tank
Bioreactor Blower (Hz)	0 to 28 HZ	0-46%	0-46% SCFM	0-100 SCFM 6 PSI
Bioreactor Foam Pump (Hz)	0 to 60 HZ	0-100%	0-200 GPM	0-200 GPM pump
				7.5 HP pump
Bioreactor Mixing Pump (Hz)	0 to 60 HZ	0-100%	0-750 GPM	0-750 GPM pump
				15 HP pump
Bioreactor ORP (mV)	−480 to +10			Analytical tool
	mV
Bioreactor pH	6.5 to 7.0			Analytical tool
Bioreactor Temperature (° C.)	45 to 70° C.			Analytical tool
pH peristaltic pump	0-8 GPH			pH adjustment tool ON/OFF
				signal processed via 4-20 ma
				signal from Bioreactor pH
				probe
influent to Bioreactor Pump PSI	3 to 5 PSI			Pressure into the Pump
Discharge Foam Cutting Pump	8 to 10 PSI			Pressure exiting the foam
				cutting spray nozzle at the top
				of the tank.

GPH, gallons per hour; PSI, pounds per square inch; Hz, hertz; ORP, oxidation reduction potential: PLC, programmable logic controller

The liquid product from the aerobic bioreactor 50 was managed in either of two ways. The first was a standard product process, while the second was a specialty product process. Both products were formulated 62 (primary formulation) with supplemental nitrogen (e.g., sodium nitrate, blood meal or hydrolyzed oilseeds) and potassium (e.g., sulfate of potash), and filtered directly into storage or packaging 70. For standard product process, the formulated liquid product was filtered 63 and transferred into a storage tank 70. The formulated standard product was stored under mildly aerobic conditions at a temperature ranging from about 45° C. (i.e., the temperature at which the product enters into the mesophilic state) to about 15-20° C. (i.e., room temperature). For the specialty product, formulated liquid product was flash pasteurized 55, filtered 60, and then further formulated (secondary formulation) 65 for special use, e.g., with custom microbes. The specialty product is then transferred into a storage or packaged 70.
Liquid products were filtered using a vibratory stainless mesh followed by a cartridge filter vessel unit with operating parameters that include a 27 gallons per minute (GPM) inlet flow at 84 pounds per square inch (PSI) with 0 differential pressure at 27° C. In such embodiments, the cartridge filters are rated at 100 mesh with 99.9% absolute rating. For the particular embodiment depicted in FIG. 1 , the formulated liquid product (the standard or the specialty following the pasteurization step 55) was completely homogenized with necessary amendments and cooled to ambient temperature (i.e., about 15-20° C.). For example, the amendments included sodium nitrate and potassium sulfate. The pH of the homogenized product was titrated to 5.50 with citric acid and then flushed through a vibratory stainless mesh screener at about 40 gallons per minute. The vibratory screener was fitted with a 200 mesh stainless steel screen. The filtered product was then pumped through a cartridge filter to a receiving vessel having an approximate 275 gallon tote or a 6,500 gallon storage tank. The operating parameters of the cartridge vessel included a differential pressure up to about 40 pounds per square inch (PSI), an inlet temperature up to about 85° F. (about 29.5° C.), and a vessel housing pressure up to about 40 PSI. The parameters for the pasteurization 55 and filtration 63, 60 are summarized in Table 6.

TABLE 6

Downstream processing after bioreactor

	Range of Operational
Process Parameter	Parameters	Notes

Pasteurization.	65 to 100° C.	Steam injection
	5 to 60 minutes
Filtration Step l	88 to 74 micron	vibratory stainless mesh
Filtration Step
2	50 to 74 micron	pressure filter vessel

For storage 70, storage vessels were maintained under mild aerobic conditions at a pH from about 6.5 to about 7.0. The headspaces of the storage tanks were purged with sterile air and agitated to ensure thorough mixing of the air. While the product was indefinitely stable under these conditions, the storage also served as a maturation stage with mesophilic bacteria converting ligand and cellulosic material into plant-useful compounds. Prior to bulk shipment or packaging 70, a third filtration step was applied. Bottles were sealed with a membrane cap to permit air circulation in the headspace of the containers. The membranes were hydrophobic with pores having a very small size (less than about 0.2 microns) such that material would not leak even when the containers were inverted. The small size of the pores also significantly reduced the potential for microbial contamination from the environment. The storage parameters for certain aspects of the storage and filtration are shown in Table 7.

TABLE 7

Storage parameters

	Range of Operational
Process Parameter	Parameters	Notes

Axial Turbine mixing	50-68 RPM	42 inch axial turbine shaft, 1.5 HP
		1 to 1.6 turnovers per minute
		Interval programmed mixing cycle
Filtration Step
3	50-74 micron	Final filtration prior to shipment
		QC step hybrid sieve test.

HP, horsepower; RPM, revolutions per minute; QC, quality control

In certain embodiments, quality control measures were included to reduce the risk of pathogen reemergence. For instance, the above-described fertilizer production process is a closed system to safeguard against accidental contamination with raw manure. In such aspects, quality control included three major quality assurance steps: 1) raw manure storage was segregated in closed tanks away from the rest of the manufacturing process; 2) the product was transported from formulation/flash pasteurization step directly to storage without exposure; and 3) bulk packages (totes/tankers) and bottles were loaded in an area distant from manure storage.
Samples taken from various stages of the process described above and depicted in FIG. 1 (circles labeled 1-7) were subjected to several analyses, including metabolite profiling, macronutrient composition, micronutrient composition, total carbon content, total nitrogen content, and microbial community characterization. These samples included the raw manure (sample 1 or Raw) taken from the initial process step 10, the slurry taken after the preparation of feedstock material 25 (sample 2 or Slurry), the solid taken after the separation by centrifugation 35 (sample 3 or Cake), the liquid stream 45 taken after the separation by centrifugation 35 (sample 4 or Centrate), the sample taken after 24 hours in the aerobic bioreactor 50 (sample 5A or T24), the sample taken after 72 hours in the aerobic bioreactor 50 and prior to primary formulation 52 (sample 5B or T72), the sample taken after primary formulation without heat pasteurization (sample 6 or Formulated Unpasteurized), and the sample taken after the final heat pasteurization step 55, but prior to filtration 60 (sample 7 or Formulated Post-Pasteurized). In some examples, formulated liquid fertilizer samples were also taken from the product material that had been filtered 60. The samples discussed above were evaluated according to the methods described herein to produce the results described in the following Examples.

Example 2. Chemical Composition of Samples 1-7

The production process was carried out as described in Example 1, and samples 1-7 were taken from various stages of the process as indicated in FIG. 1 . In the particular examples, samples 0.1-4 and 5B were submitted for chemical composition analysis to determine the macro and micronutrient content as well as the fertilizer equivalents within each sample. Each sample was submitted to the University of Kentucky Soil and Plant Testing Laboratory (Lexington, Ky., USA) and analyzed to determine the macronutrient content, micronutrient content, and the fertilizer equivalents. The results for each sample are summarized in Table 8.

TABLE 8

Chemical composition of samples taken from various process stages.

				3-Cake*	3-Cake**
Nutrients/Info	1-Raw	2-Slurry	4-Centrate	WET	DRY	5B-T72

Ammonium Nitrogen	0.88%	0.53%	0.33%	0.72%	0.58%	0.40%
Organic Nitrogen	1.89%	0.35%	0.31%	0.86%	3.25%	0.21%
TKN	2.78%	0.88%	0.64%	1.79%	3.82%	0.61%
P2O5	2.03%	0.72%	0.28%	1.67%	3.45%	0.34%
K	1.40%	0.50%	0.31%	0.57%	1.24%	0.43%
Sulfur	0.39%	0.10%	0.07%	0.46%	0.32%	0.07%
Calcium	3.56%	1.16%	0.27%	5.48%	10.90%	0.30%
Magnesium	0.36%	0.11%	0.05%	0.25%	0.64%	0.06%
Sodium	0.33%	0.06%	0.05%	0.25%	0.16%	0.09%
Copper (ppm)	90	13	>25	>25	61	5
iron (ppm)	490	244	50	934	889	50
Manganese (ppm)	219	78	75	210	502	20
Zinc (ppm)	288	82	42	197	508	25
Moisture	51.93%	88.04%	95.58'%	55.43%	8.15%	94.90%
Total Solids	49.04%	11.96%	4.43%	44.57%	91.90%	5.10%
Total Salts	n/a	2.67%	1.35%	6.29%	n/a	1.31%
pH	7.6	7.0	6.8	7.4	6.4	7.4
Total Carbon	17.07%	3.34%	1.23%	14.28%	31.40%	n/a
Organic Matter	22.32%	5.73%	2.34%	27.12%	56.65%	2.39%
Ash	19.00%	2.00%	1.33%	9.65%	34.50%	1.45%
Chloride	0.39%	0.06%	0.10%	0.04%	0.16%	0.12%

*Sample 3-Cake WET was taken directly following centrifugation.
**Sample 3-Cake DRY was dried to less than 12% moisture content.

In addition, FTIR was performed on samples 1-7 (except sample 5B) to examine how the different steps in the production process transform the raw product. In this example, the samples were evaluated for structural and/or biochemical changes that may have occurred throughout the production process. Fourier Transform Infrared Spectroscopy (FTIR) is a tool suitable for collecting infrared (IR) spectra resulting from the adsorption of molecules within a solid, liquid, or gas sample. IR spectroscopy relies on the fact that certain molecules absorb specific frequencies determined by the shape and configuration of absorbing molecules. FTIR was used to examine how the raw product was transformed as it underwent the different steps of a production process described herein, e.g., in Example 1. In particular, IR spectra from the mid-IR region (i.e., 600 to 1800 cm⁻¹) were used to evaluate the structural or biochemical changes that may have occurred throughout the production process. To accomplish this, a 5% mixture of material from each of samples 1-6 was prepared by freeze-drying followed by grinding and thorough mixing with potassium bromide (KBr). IR spectra were recorded using a NICOLET 6700 FTIR (Thermo Fisher Scientific Inc., Waltham, Mass., USA) equipped with a SMART collector diffuse reflectance accessory and MCT/A detector. In these examples, each sample spectra was an average of 254 spectra collected with 4 cm⁻¹resolution.
FIG. 2 depicts a significant reduction in the characteristic fingerprint region of proteins (1750-1500 cm⁻¹) and the bands associated with phospholipids (1250-1220 cm⁻¹), glycopeptides, ribose, polysaccharides and phosphodiesters (1200-1000 cm⁻¹) during the production process. When compared to the Raw sample, spectra from the formulated samples 6 and 7 (i.e., Formulated Unpasteurized and Formulated Post-Pasteurized, respectively) had greater signal intensities from the phospholipid, glycopeptide, ribose and polysaccharides region and less from the proteins region. The reduction in proteins and increase in phospholipids are likely indicative of the microbial driven decomposition taking place during the production process. Decomposition of components in the ‘as-received’ material (e.g., raw bedding and feathers) during the production process resulted in an increase in components of microbial origin (i.e., phospholipids found in microbe cell walls) and those resulting from decomposition (e.g., phosphodiesters).

Example 3. Microbial Communities Present in Samples 1-7

Samples 1-7 (except for sample 5B) from the process described in Example 1 were analyzed for microbial community composition. To identify and characterize the microbial communities that are present in these samples, microbial biomarkers were analyzed using phospholipid fatty acid (PLFA) analysis. While concentration of biomarker groups may increase or decrease within a particular stage, they may do so disproportionally. As such, relative abundance (i.e., the concentration of each biomarker group relativized to the total microbial biomass (TMB) within each sample) was used to evaluate how the microbial community composition within each sample changes throughout the process. Interpreting the data prepared in these two ways (i.e, concentration and proportional abundance) was expected to prove useful. For example, the percent biomarker for one category may decrease even though its absolute concentration increases. As such, the composition of the microbial community within a sample may be more telling of its function, or functional potential, than the concentration of any one biomarker group alone.
Phospholipids were extracted from each of the samples using the high-throughput methodology described by Buyer and Sasser (2012. Applied Soil Ecology, 61:127-130), the content of which is incorporated by reference herein in its entirety. Briefly, phospholipids were extracted from freeze dried samples in Bligh-Dyer extractant containing an internal 19:0 (1,2-dinonadecanoyl-sn-glycero-3-phosphocholine) standard for 2 hours by rotating end-over-end followed by centrifugation for 10 minutes. Then, the liquid phase from each sample was transferred to 13×100 mm test tubes and 1.0 ml chloroform and deionized water were added. After vortexing for 10 seconds, the samples were centrifuged for 10 minutes and the top phase removed, while the lower phase containing the phospholipids was evaporated to dryness. Lipid separation was achieved by solid phase extraction (SPE) using a 96 well SPE plate (Phenomenex, Torrance, Calif., USA). The dried samples were dissolved in 1 ml hexane and loaded onto the SPE column followed by two 1 ml additions of chloroform and 1 ml of acetone. Phospholipids were then eluted from the column into new vials using a 0.5 ml of a 5:5:1 methanol:chloroform:H₂O mixture. A transesterification reagent (0.2 ml) was then added, and the samples were incubated at 37° C. for 15 minutes. After incubation, acetic acid (0.075M) and chloroform (0.4 ml) were added to each sample. Each sample was quickly vortexed and then allowed to separate, after which the bottom phase was removed and evaporated to dryness. The extract was then dissolved in 0.7 μl of hexane and the fatty acid methyl esters (FAME) detected on an AGILENT 7890 gas chromatograph (GC) equipped with automatic sampler, an Agilent 7693 Ultra 2 column, and a flame ionization detector (Agilent Technologies, Wilmington, Del., USA). The carrier gas was ultra-high-purity hydrogen gas with a column split ratio of 30:1. The oven temperature was increased from 190° C. to 285° C. and then to 310° C. at a rate of 10° C./min and 60° C./min, respectively. FAME identities and relative percentages were automatically calculated using MIDI methods (Sherlock Microbial Identification System version 6.2, MIDI Inc., Newark, Del., USA) described by Buyer and Sasser (2012).
As shown in FIG. 3 and Table 9, the greatest concentration of total microbial biomass (i.e., the total of the bacterial biomass and the fungi biomass) was detected in both the Raw and Centrate samples followed by the T24, Slurry and Formulated Unpasteurized samples. The Cake and Formulated Post-Pasteurized samples had the least concentration of total microbial biomass. Shown in Tables 9 and 10 are the PLFA results for actinobacteria, gram positive bacteria, gram negative bacteria, fungi, arbuscular mycorrhizal fungi, and protists. General fatty acid methyl ester (FAME) biomarkers are those found across multiple microbial biomarker groups and are not assigned to any one group. However, FAME biomarkers are included in calculations of total microbial biomass. Actinobacteria are a phylum of gram positive bacteria that are distinctive for the significant role they play in soil nutrient cycling. Several Actinobacteria species have been identified that produce growth promoting compounds (see, e.g., Strap (2012)).
The concentration of total fungal biomass (TFB) of the first four steps of the production process (i.e., samples 1-4 or Raw, Slurry, Cake, and Centrate, respectively) was in the range from about 110 nmol g⁻¹to about 150 nmol g⁻¹, but then decreased greatly in the T24 sample. The lowest concentration of TFB among the samples tested was found in the Formulated Post-Pasteurized sample. The Formulated Post-Pasteurized sample follows the pasteurization step in the production process, which likely explains the reduction in TFB and overall reduction in the concentration of all microbial biomarker groups in this sample.

TABLE 9

Concentration of microbial groups in samples from the process of Example 1.

Concentration (umol g⁻¹)

						AM
Sample	FAME	Actin	G−	G+	Fungi	Fungi	Protists	TBB	TFB	TMB	F:B

1-Raw	496.52	6.81	76.80	394.74	146.55	0.2809	3.56	974.86	146.83	1125.25	0.1305
2-Slurry	484.68	6.06	71.92	115.88	108.62	0.2500	4.88	678.55	108.87	792.30	0.1374
3-Cake	345.44	2.03	28.50	28.47	107.68	B.D.	2.76	304.44	107.68	414.88	0.2596
4-Centrate	831.61	11.19	141.33	270.39	134.45	0.5509	8.96	1254.52	135.01	1398.48	0.0965
5A-T24	412.87	4.32	68.84	387.97	24.43	B.D.	0.69	874.10	24.43	899.22	0.0272
6-Form	228.79	1.40	33.35	254.12	11.59	0.0917	0.28	517.67	11.68	529.63	0.0221
7-Post	121.03	1.34	14.42	218.31	6.03	B.D.	0.13	355.10	6.03	361.26	0.0167

Form, Formulated Unpasteurized sample; Post, Formulated Post-Pasteurized sample; FAME, fatty acid methyl esters; Activ, Actinobacteria; G−, gram negative bacteria; G+, gram positive bacteria; AM Fungi, arbuscular mycorrbizal fungi; TBB, total bacterial biomass; TMB, total microbal biomass; TFB, total fungal biomass; F:B, fungus to bacteria ratio

General fungal biomass made up a greater portion of the total microbial biomass in samples 1-3 compared to all other samples tested. As shown in Table 10, the Cake (3) sample had the greatest relative abundance of fungi making up over a quarter of the total microbial biomass present. The proportion of Gram positive bacteria decreased from 35% in the Raw (1) sample to its lowest, 6.9%, in the Cake (3) sample.

TABLE 10

Microbial community composition based on relative abundance of PLFA biomarker groups.

Relative Abundance (%)

						AM
Sample	FAME	Actin	G−	G+	Fungi	Fungi	Protists

1-Raw	44.1	0.60	6.8	35.1	13.0	0.02	0.032
2-Slurry	61.2	0.77	9.1	14.6	13.7	0.03	0.62
3-Cake	59.2	0.49	6.9	6.9	26.0	0.00	0.67
4-Centrate	59.5	0.80	10.1	19.3	9.6	0.04	0.64
5A-T24	45.9	0.48	7.7	43.1	2.7	0.00	0.08
6-Form	43.2	0.26	6.3	48.0	2.2	0.02	0.05
7-Post	33.5	0.37	4.0	60.4	1.7	0.00	0.04

Form, Formulated Unpasteurized sample; Post, Formulated Post-Pasteurized sample; FAME, fatty acid methyl esters; Activ, Actinobacteria; G−, gram negative bacteria; G+, gram positive bacteria; AM Fungi, arbuscular mycorrbizal fungi

Example 4. Identification of Metabolites

Samples 1-7 (except sample 5B) were collected as described above and analyzed via Gas chromatography mass spectrometry (GC/MS) to provide insight into how the chemical profiles differ between the different process steps and as a first-approximation of the chemical composition in the samples. GC time of flight MS (GC-TOF-MS) is a commonly used mass spectrometric method for determining the chemical composition within a complex matrix. The approach used herein was untargeted in that the analysis was not aimed at identifying any one particular class of compounds, but rather provided an approximation of all the chemicals present in the sample. The ability to identify the compounds from their mass spectra was dependent on the quality and size of the database of compounds with known mass spectra.
In preparation for GC analysis, samples were extracted in acetonitrile, dried down in a SPEEDVAC vacuum concentrator (ThermoFisher Scientific Inc., Waltham, Mass., USA), and then derivatized for GC TOF-MS according to Sana et al. (Metabolomics, 2010, 6:451-465), the content of which is incorporated by reference herein in its entirety. An AGILENT 6890 gas chromatograph coupled to a PEGASUS IV TOF mass spectrometer (Agilent, Böblingen, Germany) was used to analyze the composition. A GERSTEL CIS4 with dual MPS injector with a multipurpose sample (MPS2) dual rail was used to inject 0.5 μL of the sample into the GERSTEL CIS cold injection system (Gerstel, Muehlheim, Germany). The injector was operated in splitless mode with a flow rate of 10 μl/s and then by opening the split vent after 25 seconds. Next, the temperature was increased from 50° C. to 250° C. at a rate of 12° C./s. For separation, a 30 m long, 0.25 mm i.d. Rtx-5Sil MS column was used with an additional 10 m integrated guard column (0.25 μm of 5% diphenyl film and an additional 10 m integrated guard column; Restek, Bellefonte, Pa.). The carrier gas (99.9999% pure Helium) was used with a built-in purifier (Airgas, Radnor Pa.) set at constant flow rate of 1 ml/min. The oven temperature was held constant at 50° C. for 1 min and then ramped at 20° C./min to 330° C. and held constant for 5 minutes. Mass spectrometry was performed on a PEGASUS IV TOF mass spectrometer (LECO Corp., S. Joseph, Mich.) with the transfer line temperature between gas chromatograph and mass spectrometer maintained at 280° C. The electron impact ionization energy was −70 eV, and the ion source temperature was 250° C. MS data were acquired from m/z 85-500 at 17 spectra s⁻¹controlled by the LECOCHROMA TOF software vs. 2.32 (LECO Corp., St. Joseph, Mich.). Data were preprocessed immediately after acquisition and stored as .cdf files. Automated metabolite annotation was performed using the BinBase metabolic annotation database as described in Fiehn et al., (“Setup and Annotation of Metabolomic Experiments by Integrating Biological and Mass Spectrometric Metadata” in LECTURE NOTES IN COMPUTER SCIENCE, vol. 3615 pp. 224-239 (2005)), the content of which is incorporated herein by reference in its entirety. The relative abundance of the compounds was calculated via peak height normalized to the sum intensity of all identified peaks. As one skilled in the art will appreciate, peak height is a more precise for identifying low abundance metabolites. Hierarchical clustering was used to group the identified root exudate compounds into clusters using a Ward's minimum variance method as described in Ward (J. Am. Statistical Assoc., 1963, 58:236-244), the content of which is incorporated by reference herein in its entirety. The results are presented as dendrograms, and color maps were generated after clustering to show how the metabolite levels vary between the different stages of the production process. The identity and distribution of known compounds in each of the samples is discussed first and then followed by a discussion of the unknowns.
Known compounds. Of the 706 unique compounds identified in the untargeted GC analysis, 252 were positively identified (FIG. 4 ). The dendrogram to the right of the heat map in FIG. 4 indicates which samples are most similar to each other. The Centrate (4) and T24 (5A) samples had the greatest relative peak abundance for a large majority of the chemicals identified and were unique among the other steps in the process. The relative abundance of chemicals in the Formulated Post-Pasteurized (7) and Formulated Unpasteurized (6) samples were similar, and both were similar to the Slurry (2) and Cake (3) samples. Chemicals in fractions 2 and 3, and 6 and 7 were similar to those in the Raw (1) sample.
The 252 identified chemicals were classified into distinct chemical classes including sugars, polyol/sugar alcohols, growth factors, lipids/fatty acids, amines, phenolics, carboxylic and organic acids, and nucleosides. Those compounds not fitting any of these classes were categorized as “other”. The relative peak abundance of each of these compound classes within each of the samples is depicted in Table 11. The Raw sample had a large proportion of carboxylic and organic acids, the proportion of which were appreciably reduced in the Slurry, Cake and Centrate samples, which were dominated by amines and a greater proportion of lipids and fatty acids. In the T24 sample, the proportion of carboxylic and organic acids rebounded as did the proportion of unclassified chemicals (i.e., other). The chemical composition and amounts thereof in the Formulated Unpasteurized and Formulated Post-Pasteurized samples were very similar. Shown in Table 12 is a list of the known chemicals identified and grouped by functional class. Table 13 provides the relative peak abundance data of the identified known chemicals for each sample tested.

TABLE 11

Relative peak abundance of the identified chemicals.

		Polyol/					Carboxylic
		Sugar	Growth	Lipids/			acids/organic
Sample	Sugar	alcohol	factor	fatty acids	amines	phenlies	acids	nucleosides	other

1-Raw	66705	39217	11772	225618	1376373	63343	2825680	34567	300420
2-Slurry	60431	30507	16992	278225	1984846	26476	497403	5169	154921
3-Cake	50141	14407	10742	383221	975961	12574	273535	3112	94318
4-Centrate	193671	73318	48621	778905	4007446	60056	1261583	25568	468090
5A-T24	43963	76236	206776	342180	2904018	32799	1991 126	24324	853369
6-Form	5774	7242	14057	196666	305760	5938	582619	1326	88778
7-Post	7010	6441	17710	145750	448978	5821	713499	1716	148832

Form, Formulated Unpasteurized; Post, Formulated Post-Pasteurized

TABLE 12

List of identified known chemicals.

Name	Representative Function

Sugars and Sugar Acids

3,6-anhydrous-D-galactose
beta-gentiobiose
cellobiose	a disaccharide with the formula [HOCH₂CHO(CHOH)₃]₂O, Cellobiose, a reducing
	sugar, consists of two β-glucose molecules linked by a β bond.
glucose
glucose-1-phosphate
glyceric acid	a natural three-carbon sugar acid
fructose
fucose	a hexose deoxy sugar with the chemical formula C₆H₁₂O₅. It is found on N-linked
	glycans on the mammalian, insect and plant cell surface, and is the fundamental sub-
	unit of the fucoidan polysaccharide.
galactose
isomaltose
isoribose
isothreonic acid	A sugar acid derived from therose
lactobionic acid	a sugar acid. It is a disaccharide formed from gluconic acid and galactose.
lyxose	Lyxose is an aldopentose—a monosaccharide containing five carbon atoms, and
	including an aldehyde functional group. 1
maltose
maltotriose
ribose
sucrose
tagatose	Tagatose is a functional sweetener. It is a naturally occurring monosaccharide,
	specifically a hexose. It is often found in dairy products, and is very similar in texture
	to sucrose and is 92% as sweet, but with only 38% of fire calories.
threonic acid	Threonic acid is a sugar acid derived from threose. The L-isomer is a metabolite of
	ascorbic acid. One study suggested that because L-threonate inhibits DKK1 expression
	in vitro, it may have potential in treatment of androgenic alopecia.
trehalose	Trehalose, also known as mycose ortremalose, is a natural alpha-linked disaccharide
	formed by an α,α-1,1-glucoside bond between two α-glucose units.
UDP-glacuronic acid	Uridine diphosphate glucuronic acid is a sugar used in the creation of polysaccharides
	and is an intermediate in the biosynthesis of ascorbic acid.
xylonic acid	Xylonic acid is a sugar acid that can be obtained by the complete oxidation of xylose
xylonic acid isomer	Xylonic acid is a sugar acid that can be obtained by the complete oxidation of xylose
xylose	a monosaccharide of the aldopentose type, which means that it contains five carbon
	atoms and includes a formyl functional group.
xylulose	Xylulose is a ketopentose, a monosaccharide containing five carbon atoms, and
	including a ketone functional group. It has the chemical formula C₅H₁₀O₅.

Polyols/Sugar Alcohols

1-deoxyerythritol
1-hexadecanol
2-deoxyerythritol	A polyol
deoxypentitol
diglycerol
erythritol	sugar alcohol that has been approved for use as a food additive in
	the United States and throughout much of the world.
glycerol
hexitol
lyxitol
mannitol	an osmotic diuretic that is metabolically inert in humans and occurs naturally, as a
	sugar or sugar alcohol, in fruits and vegetables
pinitol	a cyclic polyol. It is a known anti-diabetic agent isolated from Sutherlandia frutescens
	leaves. Gall plant tannins can be differentiated by their content of pinitol.
threitol	a four-carbon sugar alcohol with the molecular formula C₄H₁₀O₄. It is primarily used
	as an intermediate in the chemical synthesis of other compounds.
xylitol	a sugar alcohol used as a sweetener.

Growth Factors

indole-3-acetate (IAA)	the most common. naturally-occurring, plant hormone of the auxin class.
5-hydroxy-3-indoleacetic	is the main metabolite of serotonin
acid
6-hydroxynicotinic acid	an intermediate in the oxidation of nicotonic acid by Pseudomonas fluorescens
citramalic acid	Citramalic acid and salicylic acid in sugar beet root exudates solubilize soil
	phosphorus
galactinol	Galactinol is a Signaling Component of the Induced Systemic Resistance Caused by
	Pseudomonas chlororaphis O6 Root Colonization
pantothenic acid
salicylic acid	a monohydroxybenzoic acid, a type of phenolic acid and a beta hydroxy acid; widely
	used in organic synthesis and functions as a signaling moledule.

Lipids/Fatty Acids

l-monoolein	one of the most important lipids in the fields of drug delivery, emulsion stabilization
	and protein crystallization
1-monopalmitin
1-monostearin
2-monoolein
arachidic acid	is a polyunsaturated omega-6 fatty acid 20:4. It is structurally related to the saturated
	arachidic acid found in Cupuac̨u butter.
arachidonic acid	is a polyunsaturated omega-6 fatty acid 20:4. It is structurally related to the saturated
	arachidic acid found in Cupuac̨u butter.
beta-hydroxymyristic acid	also called tetradecanoic acid, is a common saturated fatty acid
beta- sitosterol	one of several phytosterols with chemical structures similar to that of cholesterol.
	Sitosterols are white, waxy powders with a characteristic odor.
capric acid	a saturated fatty acid (no double bond so in shorthand 10:0) member of the sub-group
	called medium chain fatty acids (MCFA), from 6 to 12 carbon atoms.
caprylic acid	common name for the eight-carbon saturated fatty acid known by the systematic name
	octanoic acid
cerotinic acid	A longchain fatty acid found in natural waxes, wool fat, and certain lipids.
cholesterol	a sterol (or modified steroid),^[4]a lipid molecule and is biosynthesized by all
	animal cells because it is an essential structural component of all animal cell
	membranes that is required to maintain both membrane structural integrity and fluidity
cis-gondoic acid
dihydrocholesterol
D-erythro-sphingosine
glycerol-alpha-phosphate
heptadecanoic acid	or margaric acid, is a saturated fatty acid. Its molecular formula is CH₃(CH₂)₁₅COOH
hexadecylglycerol
isoheptadecanoic acid	Heptadecanoic acid, or margaric acid, is a saturated fatty acid, its molecular formula is
	CH₃(CH₂)₁₅COOH
lauric acid	Lauric acid or systematically, dodecanoic acid, is a saturated fatty acid with a 12-
	carbon atom chain,
lignoceric acid	or tetracosanoic acid, is the saturated fatty acid with formula C₂₃H₄₇COOH. It is found
	in wood tar, various cerebrosides, and in small amounts in most natural fats.
linoleic acid	Conjugated linoleic acids are a family of at least 28 isomers of linoleic acid found
	mostly in the meat and dairy products derived from ruminants.
myristic acid	also called tetradecanoic acid, is a common saturated fatty acid with the molecular
	formula CH₃(CH₂)₁₂COOH.
nonadecanoic acid	a 19-carbon long-chain saturated fatty acid
octadecanol
oleamide	amide of the fatty acid oleic acid
oleic acid	a fatty acid that occurs naturally in various animal and vegetable fats and oils. I
palmitic acid	or hexadecanoic acid in IUPAC nomenclature, is the most common fatty acid found in
	animals, plants and microorganisms.
palmitoleic acid	a common constituent of the glycerides of human adipose tissue
pelargonic acid
pentadecanoic acid	a saturated fatty acid. Its molecular formula is CH₃(CH₂)₁₃COOH. It is rare in nature,
	being found at the level of 1.2% in the milk fat from cows.
squalene	a natural 30-carbon organic compound originally obtained for commercial purposes
	primarily from shark liver oil, although plant sources are now used as well, including
	amaranth seed, rice bran, wheat germ, and olives
stearic acid	a saturated fatty acid with an 18-carbon chain
stigmasterol	Stigmasterol is an unsaturated phytosterol occurring in the plant fats or oils
	Pasteurization will inactivate stigmasterol.

Amines

valine	an α-amino acid that is used in the biosynthesis of proteins.
aminomalonate	an enzyme inhibitor
1,3-diaminopropane	trimethylenediamine, is a simple diamine with the formula (CH₂)₃(NH₂)₂.
2,4-diaminobutyric acid
3-aminoisobutyric acid	a product formed by the catabolism of thymine
5-aminovaleric acid
5-methoxytryptamine	also known as mexamine, is a tryptamine derivative closely related to the
	neurotransmitters serotonin and melatonin
alanine	an α-amino acid that is used in the biosynthesis of proteins.
alpha-aminoadipic acid	an intermediate in the α-Aminoadipic acid pathway for the metabolism
	oflysine and saccharopine
asparagine	an α-amino acid that is used in the biosynthesis of proteins.
aspartic acid	also known as aspartate, is an α-amino acid that is used in the biosynthesis of proteins
beta-alanine	a naturally occurring beta amino acid, which is an amino acid in which the amino
	group is at the β-position from the carboxylate group
beta-glutamic acid	an α-amino acid that is used in the biosynthesis of proteins.
citrulline	organic compound citrulline is an α-amino acid. Its name is derived from citrullus, the
	Latin word for watermelon, from which it was first isolated in 1914
cyclobexylamine
cysteine	a semi-essential proteinogenic amino acid with the formula HO₂CCHCH₂SH.
ethanolamine
glutamic acid	an α-amino acid that is used in the biosynthesis of proteins.
glutamine	an α-amino acid that is used in the biosynthesis of proteins.
glycine	an α-amino acid that is used in the biosynthesis of proteins.
glycyl proline
homoserine	Homoserine is an α-amino acid with the chemical formula HO₂CCHCH₂CH₂OH.
	L-Homoserine is not one of the common amino acids encoded by DNA it differs from
	the proteinogenic amino acid serine by insertion of an additional —CH₂—
	unit into the backbone.
hydroxylamine	Hydroxylamine is an inorganic compound with the formula NH₂OH.
isoleucine	an α-amino acid that is used in the biosynthesis of proteins
leucine	an α-amino acid used in the biosynthesis of proteins.
lysine	an α-amino acid that is used in the biosynthesis of proteins.
maleimide	Maleimide is a chemical compound with the formula H₂C₂(CO)₂NH. This unsaturated
	imide is an important building block in organic synthesis.
methionine	Methionine is an essential amino acid in humans. Like other essential amino acids this
	means that a restriction of dietary intake to zero will eventually lead to death.
methionine sulfoxide	Methionine sulfoxide is the organic compound with the formula CH₃SCH₂CH₂-
	CHCO₂H.. It occurs naturally although it is formed post-translationally.
N-acetyl-D-galactosamine	N-Acetylgalactosamine, is an amino sugar derivative of galactose
n-acetyl-d-hexosamine
N-acetylaspartic acid
N-acetylglutamate	In prokaryotes, lower eukaryotes and plants it is the first intermediate in the
	biosynthesis of arginine
N-acetylputrescine
N-carbamylglutamate	an affective precursor of arginine
N-methylalanine
N-methylglutamic acid	chemical derivative of glutamic acid in which a methyl group has been added to the
	amino group. It is an intermediate in methane metabolism.
norvaline	an amino acid with the formula CH₃(CH₂)₂CHCO₂H. The compound is an isomer of
	the more common amino acid valine.
O-acetylserine	s the α-amino acid with the chemical formula HO₂CCHCH₂OCCH₃. It is an
	intermediate in the biosynthesis of the common amino acid cysteine in bacteria and
	plants.
oxoproline
phenylalanine	an α-amino acid used in the biosynthesis of proteins
phenylethylamine
putrescine	Putrescine, or tetramethylenediamine, is a foul-smelling organic chemical compound
	NH₂(CH₂)₄NH₂that is related to cadaverine; both are produced by the breakdown of
	amino acids in living and dead organisms and both are toxic in large doses
serine	an α-amino acid that is used in the biosynthesis of proteins.
spermidine	Spermidine is a polyamine compound found in ribosomes and living tissues, and
	having various metabolic functions within organisms.
taurine	Taurine, or 2-aminoethanesulfonic acid, is an organic compound that is widely
	distributed in animal tissues. It is a major constituent of bile and can ire found in the
	large intestine, and accounts for up to 0.1% of total human body weight.
threonine	an α-amino acid that is used in the biosynthesis of proteins.
thymine	one of the four nucleobases in the nucleic acid of DNA that are represented by the
	letters G-C-A-T. The others are adenine, guanine, and cytosine. Thymine is also
	known as 5-methyluracil, a pyrimidine nucleobase.
trans-4-hydroxyproline	a common non-proteinogenic amino acid
tryptophan	an α-amino acid that is used in the biosynthesis of proteins.
tyramine	Tyramine, also known by several other names, is a naturally occurring monoamine and
	trace amine derived from the amino acid tyrosine. Tyramine acts as a catecholamine
	releasing agent.
tyrosine	Tyrosine or 4-hydroxyphenylalanine is one of the 22 amino acids that are used by cells
	to synthesize proteins. it is a non-essential amino acid with a polar side group.

Phenolics

3,4-dihydroxybenzoic acid
4-hydroxybenzoate
catechol
cis-caffeic acid	consists of both phenolic and acrylic functional groups. It is found in all plants
	because it is a key intermediate in the biosynthesis oflignin, one of the principal
	components of plant biomass and its residues
ferulic acid	hydroxycinnamic acid, a type of organic compound. It is an abundant phenolic
	phytochemical found in plant cell wall components such as arabinoxylans as covalent
	side chains.
hydroquinone	Hydroquinone has a variety of uses principally associated with its action as a reducing
	agent that is soluble in water.
phenol
tyrosol	Tyrosol is a phenylethanoid, a derivative of phenethyl alcohol. It is a natural phenolic
	antioxidant present in a variety of natural sources. The principal source in the human
	diet is olive oil.
vanillic acid	Vanillic acid is a dihydroxybenzoic acid derivative used as a flavoring agent. It is an
	oxidized form of vanillin. It is also an intermediate in the production of vanillin from
	ferulic acid.

Carboxylic Acids/Organic Acids

2-hydroxy-2-
methylbutanoic acid
2-hydroxyadipic acid
2-hydroxybutanoic acid	a hydroxybutyric acid with the hydroxyl group on the carbon adjacent to the carboxyl.
2-bydroxyglutaric acid
2-hydroxyhexanoic acid
2-hydroxyvaleric acid
2-isopropylmalic acid	an intermediate in the biosynthesis of leucine
2-ketoadipic acid
2-methylglyceric acid
2-picolinic acid	organic compound with the formula C₅H₄N. It is a derivative of pyridine with a
	carboxylic acid substituent at the 2-position. It is an isomer of nicotinic acid, which
	has the carboxyl side chain at the 3-position.
3-(3-hydroxyphenyl)
propionic acid
3-(4-hydroxypheny)
propionic acid
3-hydroxy-3-
methylglutaric acid
3-hydroxybenzoic acid
3-hydroxybutyric acid
3 -hydroxypalmitic acid
3-hydroxyphenylacetic acid
3-hydroxypropionic acid
3-phenyllactic acid
3,4-dihydroxycinnamic
acid

3,4-dihydroxy-
hydrocinnamic acid
3,4-dihydroxy-	a metabolite of the neurotransmitter dopamine
phenylacetic acid
4-aminobutyric acid
4-hydroxybutyric acid
4-hydroxymandelic acid
4-hydroxyphenylacetic	a chemical compound found in olive oil and beer. In industry the chemical is an
acid	intermediate used to synthesize atenolol and 3,4-dihydroxyphenylacetic acid
4-pyridoxic acid
aconitic acid
adipic acid	the organic compound with the formula (CH₂)₄(COOH)₂. From an industrial
	perspective, it is the most important dicarboxylic acid:
alpha-ketoglutarate	one of two ketone derivatives of glutaric acid. Its anion, α-ketoglutarate is an
	important biological compound. α-Ketoglutarate is one of the most important
	nitrogen transporters in metabolic pathways.
behenic acid	a carboxylic acid, the saturated fatty acid with formula C₂₁H₄₃COOH.
benzoic acid	a colorless crystalline solid and a simple aromatic carboxylic acid.
chenodeeoxycholic acid	a bile acid. It occurs as a white crystalline substance insoluble in water but soluble in
	alcohol and acetic acid
citric acid
digalacturonic acid
fumaric acid	or trans-butenedioic acid is the chemical compound with the formula
	HO₂CCH—CHCO₂H. This white crystalline compound is one of two isomeric
	unsaturated dicarboxylic acids, the other being maleic acid.
gluconic acid	an organic compound with molecular formula C6H12O7 and condensed structural
	formula HOCH₂(CHOH)₄COOH. Gluconic acid, gluconate salts, and gluconate esters
	occur widely in nature because such species arise from the oxidation of glucose
gluconic acid lactone	also known as gluconolactone, is a food additive with the E number E575 used as a
	sequestrant, and acidifier, or a curing, pickling, or leavening agent.
glutaric acid
glycolic acid	Glycolic acid; chemical formala C₂H₄O₃, is the smallest α-hydroxy acid.
hexuronic acid
hydrocinnamic acid	Phenylpropanoic acid or hydrocinnamic acid is a carboxylic acid with the formula
	C₁₃H₁₀O₂belonging to the class of phenylpropanoids.
isocitric acid	Isocitric acid is an organic compound closely related to citric acid.
isobexonic acid	Hexanoic acid is the carboxylic acid derived from hexane with the general formula
	C₅H₁₁COOH.
isopentadecanoic acid
kynurenic acid	a product of the normal metabolism of amino acid L-tryptophan. It has been shown
	that kynurenic acid possesses neuroactive activity.
lactic acid	an organic compound with the formula CH₃CHCO₂H.
malic acid	Malic acid is an organic compound with the molecular formula C₄H₆O₅. It is a
	dicarboxylic acid that is made by all living organisms, contributes to the pleasantly
	sour taste of fruits, and is used as a food additive.
malonic acid	Malonic acid is a dicarboxylic acid with structure CH₂(COOH)₂.
methylmaleic acid	Maleic acid or cis-butenedioic acid is an organic compound that is a dicarboxylic acid,
	a molecule with two carboxyl groups
oxalic acid
oxamic acid
phenylacetic acid	a white solid with a disagreeable odor. Endogeneously, it is a catabolite of
	phenylalanine.
pimelic acid	Pimelic acid is the organic compound with the formula HO₂C(CH₂)₅CO₂H.
	Derivatives of pimelic acid are involved in the biosynthesis of the amino acid called
	lysine
pipecolinic acid	a small organic molecule which accumulates in pipecolic acidemia. It is the carboxylic
	acid of piperidine.
pyrrole-2-carboxylic acid
pyruvic acid	Pyruvic acid is the simplest of the alpha-keto acids, with, a carboxylic acid and a
	ketone functional group.
quinolinic acid	Quinolinic acid, also known as pyridine-2,3-dicarboxylic acid, is a dicarboxylic acid
	with a pyridine backbone
ribonic acid	obtained by oxidation of ribose
succinic acid	Succinic acid is a dicarboxylic acid with chemical formula (CH₂)₂(CO₂H)₂.
sulfuric acid
tartaric acid	Tartaric acid is a white crystalline organic acid that occurs naturally in many plants,
	most notably in grapes. Its salt, potassium bitartrate, commonly known as cream of
	tartar, develops naturally in the process of winemaking.
uric acid	Uric acid is a heterocyclic compound of carbon, nitrogen, oxygen, and hydrogen with
	the formula C₅H₄N₄O₃. It forms ions and salts known as urates and acid urates, such
	as ammonium acid urate.
urocanic acid	Urocanic acid is an intermediate in the catabolism of L-histidine.

Nucleosides

thymidine	Thymidine is a pyrimidine deoxynucleoside. Deoxythymidine is the DNA nucleoside
	T, which pairs with deoxyadenosine in double-stranded DNA. In cell biology it is used
	to synchronize the cells in G1/early S phase.
5,6-dihydrouracil	an intermediate in the catabolism of uracil
7-methylguanine NIST	a modified purine nucleoside. It is a methylated version of guanosine and when found
	in human urine, it may be a biomarker of some types of cancer.
adenine	nucleobase. Its derivatives have a variety of roles in biochemistry including cellular
	respiration.
adenosine	purine nucleoside composed of a molecule of adenine attached to a ribosesugar
	molecule (ribofuranose) moiety via a β-N₉-glycosidic bond.
cytosin	Cytosine is one of the four main bases found in DNA and RNA, along with adenine,
	guanine, and thymine. It is a pyrimidine derivative, with a heterocyclic aromatic ring
	and two substituents attached. The nucleoside of cytosine is cytidine
guanine	one of the four main nucleobases found in the nucleic acids DNA and RNA, the others
	being adenine, cytosine, and thymine.
pseudo uridine	Pseudouridine is an isomer of the nucleoside uridine in which the uracil is attached via
	a carbon—carbon instead of a nitrogen—carbon glycosidic bond. It is the most
	prevalent of the over one hundred different modified nucleosides found in RNA.
uracil	one of the four nucleobases in the nucleic acid of RNA that are represented by the
	letters A, G, C and U. The others are adenine, cytosine, and guanine. In RNA, uracil
	binds to adenine via two hydrogen bonds.

Others

zymosterol	Zymosterol is a cholesterol intermediate in the cholesterol biosynthesis. Disregarding
	some intermediate compounds lanosterol can be considered a precursor of zymosterol
	in the cholesterol synthesis pathway
1-methylhydantoin
112-cyclohexanedione
2-deoxypentitol NIST
2-deoxytetronic acid
2-ketoisocaproic acid	an intermediate in the metabolism of leucine
2,3-dihydroxybutanoic
acid NIST

2,8-dihydroxyquinoline	Product of quinoline metabol ism by Pseudomonas sp
3-(3-hydroxyphenyl)-3-
hydroxypropionic acid
3-ureidopropionate	an intermediate in the metabolism of uracil
4-methylcatechol	4-Methyleatechol is a chemical compound. It is a component of castoreum, the exudate
	from the castor sacs of the mature beaver.
5-hydroxymethyl-2-furoic	A byproduct of the fungus Aspergillus and probably other species of fungi and yeast as
acid NIST	well
butane-2,3-diol NIST	2,3-Butanediol has three stereoisomers. all of which are colorless, viscous liquids.
	Butanediols have applications as precursors to various plastics and pesticides. is
	produced by a variety of microorganisms in a process known as butanediol
	fermentation. It is found naturally in cocoa butter, in the roots of Ruta graveolens,
	sweet corn, anti in rotten mussels.
butyrolactam NIST	a chemical compound from the group of lactams. Butyrolactam, the lactam of the γ-
	aminobutyric acid ( GABA ), an inhibitory neurotransmitter, and it can be obtained
	by hydrolysis are converted to GABA.
conduritol-beta-epoxide
creatinine
daidzein	structurally belongs to the group of isoflavones
glycerol-3-galactoside
hypoxanthine	a naturally occurring purine derivative. It is occasionally found as a constituent of
	nucleic acids, where it is present in the anticodon of tRNA in the form of its
	nucleoside inosine.
isothreitol
lanosterol	a tetracyclic triterpenoid and is the compound from which all animal and fungi steroids
	are derived.
methanolphosphate
myo-inositol	Inositol or cyclohexane-1,2.3,4,5,6-hexol is a chemical compound with formula
	C₆H₁₂O₆or (—CHOH—)₆, a six-fold alcohol of cyclohexane
nicotinic acid	Nicotinic acid and its amide nicotinamide are the common, forms of the B-
	vitamin niacin (vitamin B3).
octadecylglycerol
ononitol	Ononitol is a cyclitol. It is a 4-O-methyl -myo-inositol and is a constituent of
	Medicago sativa.
parabanic acid NIST
phosphate
piperidone	a derivative of piperidine with the molecular formula C₅H₉NO. 4-Piperidone is used as
	an intermediate in the manufacture of chemicals and pharmaceutical drugs.
propane-1,3-diol NIST	1,3-Propanediol is the organic compound with the formala CH₂(CH₂OH)₂. This three-
	carbon diol is a colorless viscous liquid that is miscible with water.
pyrogallol	Pyrogallol is an organic compound with the formula C₆H₃(OH)₃. It is a white solid
	although because of its sensitivity toward oxygen, samples are typically brownish. It is
	one of three isomeric benzenetriols
pyrophosphate	pyrophosphate is a phosphorus oxyanion
tocopherol acetate	Tocopheryl acetate, also known as vitamin B acetate, is a common vitamin supplement
	with the molecular formula C₃₁H₅₂O₃. It is the ester of acetic acid and tocopherol.
	It is often used in dermatological products such as skin creams.
tocopherol alpha-	α-Tocopherol is a type of tocopherol or vitamin E
tocopherol gamma-	γ-Tocopherol is one of the chemical compounds that is considered vitamin E
urea
xanthine	Xanthine, is a purine base found in most human body tissues and fluids and in other
	organisms. A number of stimulants are derived from xanthine, including caffeine and
	theobromine. Xanthine is a product on the pathway of purine degradation.
xanthurenic acid.	Xanthurenic acid, or xanthurenate, is a chemical shown to induce gametogenesis of
	Plasmodium falciparum, the parasite that causes malaria, It is found in the gut of the
	Anopheles mosquito.

TABLE 13

Relative peak abundance of the identified chemicals

		xunthurenic			γ toco-	tocopherol	tocopherol				propane-
Sample	zymosterol	acid	xanthine	urea	pherol	alpha-	acetate	squalene	pyrophosphate	pyrogallol	1,3-diol	piperidone	phosphate

1-raw	124	2206	3857	44115	409	143	195	1938	291	153	1608	5557	147264
2-slurry	250	2867	1451	203	335	131	118	2047	85	249	3967	14247	6755
3-cake	392	1550	460	1698	368	103	183	1545	432	94	966	2516	7714
4-centrate	266	9381	15036	1880	707	644	140	5910	99	241	8121	55855	16207
5A-T24	494	15265	1179	78070	860	777	127	2731	66	3926	6877	235057	822
7-post	703	1812	76	16123	362	127	134	331	516	101	886	15782	11701
6-form	297	1936	101	11986	261	210	218	749	142	72	1737	9516	10211

	parahanic		octadecyl	nicotinc		methanol-	linoleic				glycerol-3-
Sample	acid	ononitol	glycerol	acid	myoinositol	phosphate	acid	lanosterol	isothreitol	hypoxanthine	galactoside	daidzein	creatinine

1-raw	5600	84	506	7516	4113	1232	7634	189	518	9292	367	1794	1012
2-slurry	2790	132	640	2293	6066	774	17598	206	1103	511	149	2411	368
3-cake	1391	74	526	799	1181	1522	27421	329	234	192	118	3214	628
4-centrate	14581	190	1668	5939	18353	1257	35434	398	374	8838	266	4713	2542
5A-T24	8223	179	197	2612	261	1368	7848	227	239	426	194	4706	2500
7-post	2147	104	129	308	155	1248	726	69	268	109	97	93	358
6-form	1236	123	120	287	269	943	560	124	141	97	144	535	218

								3-	3-(3-hydroxy-
			butane-			5-	4-	ureido-	phenyl)-3-	2-	2-	2-	2,8-
	conduritol-	butyrolactam	2,3-diol	benzoic	adipic	hydroxymethyl-	methyl-	propionic	hydroxy-	ketoisocaproic	deoxy-	deoxy-	dihydroxy-
Sample	beta-epoxide	NIST	NIST	acid	acid	2-furnic acid NIST	catechol	acid	propionic acid	acid	tetraonic acid	pentitol NIST	quinoline

1-raw	1171	3551	4743	22130	1475	2510	88	1356	158	1843	1471	482	5543
2-slurry	1160	4768	6389	49350	1685	1570	121	2642	182	1639	1981	547	9349
3-cake	541	1708	4072	19038	1244	578	132	824	111	773	818	329	5910
4-centrate	3234	19483	38143	147281	3353	4098	108	5987	148	1723	5021	1140	20640
5A-T24	2743	42076	11353	349321	5850	7981	831	8802	663	748	8090	1222	23729
7-post	69	2070	2293	82457	1829	317	169	74	136	669	919	144	393
6-form	133	2105	2122	34230	1322	680	95	117	177	327	670	150	1923

	2,3-	1-	1,2-					trans-4-
	dihydroxy-	methyl-	cyclo-					hydroxy-
Sample	butanoic acid	hydautoin	hexanedione	valine	tyrosine	tyramine	tryptophan	proline	thymine	threonine	taurine	spermidine	serine

1-raw	219	5487	476	148122	39321	12714	3966	2941	7456	1473	3460	642	5552
2-slurry	312	4360	1120	215093	56112	55231	14646	1605	2201	2643	4809	1132	1637
3-cake	187	1699	734	79230	13380	24219	2203	612	1177	921	2010	2077	673
4-centrate	427	8080	184	364196	167202	86503	42389	2225	6111	3816	12612	2728	939
5A-T24	239	6235	2255	35924	6980	184045	1103	14330	49364	530	1584	1467	692
7-post	83	1671	1074	16684	718	24261	188	432	299	297	524	159	325
6-form	111	1923	460	2077	449	14786	247	1630	1037	484	427	89	112

					O-		N-	N-	N-	N-	N-	N-
		phenyl-	phenyl-		acetyl-		methyl-	methyl-	carbamyl-	acetyl-	acetylor-	acetyl-	a-acetly-
Sample	putrescine	ethylamine	alamine	oxoproline	serine	norvaline	glutamic acid	alanine	glutamate	putrescine	nithine	glutamate	d-hexosamine

1-raw	6438	1212	17114	78015	563	8819	16053	18292	838	1293	862	542	299
2-slurry	24843	4233	42346	64355	459	27664	7310	79238	394	1922	443	451	264
3-cake	5855	3223	11700	16307	282	7145	2449	18176	250	620	271	380	251
4-centrate	100518	6502	118647	150331	584	46737	21025	170067	996	3244	746	1207	671
5A-T24	319316	15285	4335	5623	1301	7159	21666	30639	527	14262	1036	1228	177
7-post	1831	892	492	3158	242	255	5368	1723	215	1910	166	55	110
6-form	19225	908	313	2983	312	304	2863	3247	140	1000	117	96	225

	N-acetyl-	N-
	D-galactos-	acetylaspartic	methionine						hydroxyl-		glycyl
Sample	amine	acid	sulfoxide	methionine	maleimide	lysine	leucine	isoleucine	amine	homoserine	proline	glycine	glutamine

1-raw	457	3560	812	3956	1124	3453	105313	1751	94032	570	1030	24995	398
2-slurry	177	2465	502	6447	741	1474	136042	100706	114972	876	369	22034	316
3-cake	133	717	488	778	551	201	58600	35007	127228	258	206	20161	177
4-centrate	129	3017	1466	16298	1687	3747	276693	173099	39890	2047	858	44359	647
5A-T24	235	192	855	555	2567	4334	40139	37527	50164	1047	9872	7357	604
7-post	102	391	311	86	899	280	967	1566	169696	128	244	20423	172
6-form	109	201	233	88	649	119	840	179	162821	129	169	20540	147

	glutamic	ethanol-		cyclohexyl-		beta-glutamic-	beta-	aspartic		arachidonic	alpha-amino-	alanine-
Sample	acid	amine	cysteine	amine	citrulline	acid	alanine	acid	asparagine	acid	adipic acid	alanine	alanine

1-raw	54262	2429	377	1201	745	16013	69555	72811	177	912	1488	852	469853
2-slurry	26108	1555	465	757	612	1235	43191	30303	128	1164	427	743	635610
3-cake	3940	786	275	646	693	403	16981	13024	88	2001	281	450	32858
4-centrate	46128	2405	666	1267	1634	3328	102212	142073	269	2953	1121	1971	1213184
5A-T24	1239	5128	472	2033	1173	734	128251	18543	254	1418	1369	1276	19923
7-post	201	715	63	310	195	382	11765	2562	79	350	242	171	4237
6-form	145	561	122	401	318	221	15098	2653	54	381	134	309	1898

	5-meth-		3-amino-	2,4-diamin					isohepta-	hexadecyl-	hepta-	glycerol-
	oxytrypt-	5-amino-	isobuyric	obutyric	1,3-diamin-		lignoceric	lauric	decanoie	glycerol	decanoic	alpha-	D-erythro-
Sample	amine	valeric acid	acid	acid	opropane	mysteric acid	acid	acid	acid NIST	NIST	acid	phosphate	sphingosine

1-raw	173	56369	1722	1274	1122	1808	707	8513	2364	314	6573	451	455
2-slurry	199	234818	8851	1716	812	2584	863	10690	5445	419	11488	131	557
3-cake	166	164211	3989	373	1380	2422	758	9370	3432	390	9606	421	502
4-centrate	395	519628	30117	5038	5124	4693	1641	18833	9190	901	24725	258	1123
5A-T24	1004	1777927	55308	10797	3142	2993	396	11799	4490	139	9690	138	539
7-post	343	182439	2134	1993	129	732	308	6066	3791	157	2497	186	135
6-form	609	37594	2478	1474	415	1123	385	6089	1713	107	6627	230	92

	cis-						beta-				2-
	gondoic		cerotinic	caprylic	capric	beta-	hydroxy-	behenic	arachidic		deoxy-	1-mono-	1-mono-
Sample	acid	cholesterol	aci	acid	acid	sitosterol	myristic acid	acid	acid	2-monoolein	erythritol	stearin	palmitin

1-raw	205	10267	198	1941	849	19049	420	2625	4236	387	4044	265	461
2-slurry	205	18183	240	2456	1047	29404	455	4211	8206	744	8619	355	658
3-cake	192	15436	209	2169	812	33696	680	3222	6362	8519	2319	653	671
4-centrate	542	33560	411	2064	1183	72438	960	8288	15576	1133	23032	361	93
5A-T24	189	19440	258	4643	1740	35516	823	1497	2932	288	16646	225	220
7-post	101	5442	329	1466	718	8494	256	1673	2153	155	569	381	115
6-form	95	6676	113	2224	815	15261	235	1982	3010	243	1978	439	319

			penta-										xylonic
		stearic	decanoic	pelargonic	palmitoleic			oleamide		nonadecanoic	xylulose		acid
Sample	1-monoolein	acid	acid	acid	acid	palmitic acid	aleic acid	NIST	octadecanol	acid	NIST	xylose	isomer

1-raw	1227	86160	7441	26773	63	26272	8947	176	662	1761	654	35832	664
2-slurry	3637	142612	10110	28922	808	49136	22654	479	589	2318	265	487223	471
3-cake	19176	138957	10419	23977	967	53350	31479	683	650	1722	738	39206	133
4-centrate	4274	321790	25985	28458	1747	117161	52177	455	796	5057	1480	153622	1004
5A-T24	8606	102755	17150	28257	1078	39935	27484	417	650	1247	195	33098	138
7-post	9827	58018	1854	25173	56	10924	3050	126	465	533	87	3728	89
6-form	10125	80658	4993	22837	115	21366	5234	132	530	920	80	2503	129

	xylonic									lactobionic
Sample	acid	xylitol	trehalose	tagutose	sucrose	ribose	maltotriose	maltose	lyxose	acid	isoribose	isomaltose	galactose

1-raw	240	654	1559	497	51	2873	203	1278	7866	2219	876	99	5995
2-slurry	237	352	456	247	22	1102	77	527	3436	514	1033	107	773
3-cake	124	216	672	167	89	722	123	2482	2188	396	321	162	1308
4-centrate	406	1059	225	538	62	2467	92	808	7682	1823	4374	109	13319
5A-T24	100	490	171	912	55	3099	60	386	1482	151	2371	92	203
7-post	61	127	335	32	84	400	111	272	267	70	374	154	176
6-form	83	140	248	79	87	429	96	287	116	138	323	101	254

										UDP-
			beta-	3,6-auhydro-				arocanic		glucuronic	threonic
Sample	fucose	fructose	gentiobiose	D-galactase	glucose	cellobiose	vanillic acid	acid	uric acid	acid	acid	tartaric acid	sulfuric acid

1-raw	2159	259	1783	944	199	61	2212	630	113383	411	572	314	2371
2-slurry	932	135	841	181	224	148	4780	140	9746	269	311	87	1201
3-cake	347	85	533	129	368	170	2307	122	26661	149	118	103	236
4-centrate	2574	71	1497	459	3605	71	13154	340	2642	380	465	155	1260
5A-T24	205	117	288	350	54	116	704	190	12808	735	96	544	315
7-post	141	74	233	195	78	120	129	84	141	128	133	129	603
6-form	127	130	266	158	99	127	180	160	113	139	85	70	3137

					pyrrole-2-
	succinic	ribonic	quinolinic	pyruvic	carboxylic	pipercolinic		phenylacetic	oxamic		methylmaleic
Sample	acid	acid	acid	acid	acid	acid	pimelic acid	acid	acid	oxalic acid	acid	malonic acid	malic acid

1-raw	10204	4045	611	2604	1413	6248	592	6274	847	419	84	234	1009
2-slurry	35486	1163	807	1091	1040	20909	795	23012	313	302	131	197	183
3-cake	8833	611	439	234	486	28830	274	8236	217	289	90	164	135
4-centrate	89353	2242	2348	1004	2494	47562	1904	70989	1323	312	127	262	529
5A-T24	3301	568	1077	414	4813	89907	3242	281239	237	159	243	84	64
7-post	813	178	222	240	555	13730	72	30947	271	339	163	132	211
6-form	270	161	116	128	483	2558	489	24223	236	434	120	95	100

			isopenta-							gluconic
		kynurenic	decanoic	isohexonic	isocitric	hydrocinnamic	hexuronic	glycollic	glutaric	acid	gluconic	fumaric	digalacturonic
Sample	lactic acid	acid	acid	acid	acid	acid	acid	acid	acid	lactone	acid	acid	acid

1-raw	13155	117	6259	1175	40930	10531	361	4507	1619	227	312	1341	859
2-slurry	38347	160	10309	101	1699	55305	355	7819	2580	86	113	555	238
3-cake	15905	169	11435	107	1075	18563	211	861	1421	109	92	445	275
4-centrate	38112	261	21808	269	6076	205865	1299	10953	7308	332	336	1732	421
5A-T24	2894	127	15099	86	105	485189	226	9142	17973	107	79	1966	244
7-post	1494	109	1590	92	6069	22784	135	1408	636	104	179	600	281
6-form	3246	222	1355	145	4328	46799	142	1663	365	50	129	250	301

				chenode-					4-hydroxy-	4-hydroxy-
		citramalic	cis-caffeic	oxycholic	amino-	alpha-	aconitic	4-pyridoxic	phenylacetic	mandelic	4-hydroxy-	4-amino-	3-phenyl-
Sample	citric acid	acid	acid	acid	malonate	ketoglutarate	acid	acid	acid	acid	butyric acid	butyric acid	lactic acid

1-raw	2502309	974	630	1632	673	497	7489	329	8593	2845	1064	7408	5715
2-slurry	114857	1381	325	658	386	57	1525	176	23575	4527	1907	14352	10077
3-cake	65268	637	253	1796	227	119	463	152	10453	2181	1381	3779	4955
4-centrate	367798	3380	1165	1133	557	182	8640	570	62632	13174	4060	31270	14186
5A-T24	568	180	3636	1098	203	109	228	1359	263795	2279	6425	46169	731
7-post	518160	139	169	761	114	163	682	97	22687	430	871	854	189
6-form	383142	99	320	907	101	154	402	176	18674	233	641	1330	136

	3-hydroxy-	3-hydroxy-	3-hydroxy-	3-hydroxy-	3-hydroxy-	3-hydroxy-3-	3,4-dihydroxy-	3,4-dihydroxy-	3,4-dihydroxy-	3-(4-hydroxy-	3-(3-hydroxy-		2-methyl-
	propionic	phenylacetic	palmitic	butyric	benzoic	methylglutaric	phenylacetic	hydrocinnamic	hydrocinnamic	phenyl)prop-	phenyl)prop-	2-picolinic	glyceric
Sample	acid	acid	acid	acid	acid	acid	acid	acid NIST	acid	ionic acid	ionic acid	acid	acid NIST

1-raw	12527	374	392	2466	632	124	521	834	70	7472	3275	733	252
2-slurry	6384	751	700	5892	1136	145	583	4078	182	28408	11886	787	880
3-cake	2765	319	970	2140	466	92	292	395	49	11938	4535	348	328
4-centrate	7419	1266	955	15381	2988	188	272	1766	269	72737	30451	2740	2468
5A-T24	19701	7324	1122	4566	8383	86	17383	251759	4833	110418	228118	962	1911
7-post	483	1748	365	235	2913	88	130	149	91	1805	11992	655	484
6-form	470	888	484	778	924	115	1386	539	581	1286	23338	830	373

				2-hydroxy-	2-hydroxy-			2-hydroxy-2-
	2-keto-	2-isopropyl-	2-hydroxy-	hexanoic	glutaric	2-hydroxy-	2-hydroxy-	methylbutanoic			pseudo
Sample	adipic acid	malic acid	valeric acid	acid	acid	butanoic acid	adipic acid	acid	uracil	thymidine	uridine	guanine	cytosin

1-raw	454	652	1986	8602	2057	5450	30	649	22560	210	505	1057	189
2-slurry	1327	386	4744	23456	1585	9471	386	506	2825	141	170	86	159
3-cake	980	222	3294	14480	440	8064	129	422	1861	168	85	151	80
4-centrate	3169	1212	13982	30227	5027	22646	445	1161	21818	137	212	192	134
5A-T24	14394	202	37160	10007	619	6464	48	4909	13195	356	1247	87	452
7-post	3908	71	10449	657	168	45652	108	631	213	161	116	103	116
6-form	1827	49	4177	458	55	44656	90	408	319	144	114	79	104

			7-methyl-
			guanine	5,6-di-			salicylic					isothreonic
Sample	adenosine	adenine	NIST	hydrouracil	threitol	stigmusterol	acid	pinitol	phenoll	munnitol	lyxitol	acid	hexitol

1-raw	228	9002	501	315	765	256	360	7214	1820	227	3721	1627	138
2-slurry	118	241	544	885	775	757	597	3963	3031	153	1792	581	319
3-cake	85	183	286	213	406	388	304	1594	2325	66	586	197	109
4-centrate	139	1185	1337	414	1778	1187	1387	10531	3460	314	6387	923	727
5A-T24	294	6404	1403	886	133	606	5080	10791	2172	205	2175	410	229
7-post	248	392	103	264	268	168	1638	179	1274	114	227	145	99
6-form	100	268	60	138	176	270	560	144	834	152	121	101	56

												6-hydroxy-
	glucose-1-		dihydro-		deoxy-		1-hexa-	1-dexoy-	panto-	indole-3-		nicotinic	5-hydroxy-
Sample	phosphat	erythritol	cholesterol	diglycerol	pentitol	catechol	decanol	erythritol	thenic acid	acetate	galactinol	acid	3-iIAA

1-raw	2172	6010	1074	4881	3096	202	322	5072	549	9271	736	924	292
2-slurry	774	3753	134	3520	1338	471	278	7899	301	14482	512	774	923
3-cake	183	1336	843	1173	427	142	410	3380	127	9265	682	273	395
4-centrate	1997	10139	197	3798	2800	600	445	22972	715	41797	2034	2420	1655
5A-T24	1298	277	1763	1309	2384	21393	890	24961	254	202467	186	1659	2210
7-post	292	126	298	489	82	81	194	569	97	17118	241	90	164
6-form	631	106	601	318	89	1086	374	1397	98	13338	240	96	285

Sample	tyrosol	hydroquinone	glycerol	glyceric acid	ferulic acid	4-hydroxybenzoate	3,4-dihydroxybenzoate

1-raw	1065	268	24265	4588	858	4757	27542
2-slurry	2293	401	9119	1022	460	10465	2816
3-cake	1043	235	3821	473	405	4984	1613
4-centrate	5949	607	12417	1888	1799	29693	7703
5A-T24	9284	3619	4009	1201	717	6719	7250
7-post	429	129	3483	399	103	792	486
6-form	712	215	2813	376	128	1131	563

Out of the 254 chemicals identified in the untargeted analysis, there were several compounds of potential interest for their known plant growth promoting properties (FIG. 5A and FIG. 5B). For example, there are numerous studies on the plant growth promoting properties of the phytohormone Indole-3-acetic acid and its derivatives (5-hydroxyl-3-indoleacetic acid, indole-3-acetate) and its production by certain rhizosphere bacteria (see, e.g., Patten and Glick, J. Microbiol., 1996, 42:207-220; Spaepen, et al., FEMS Microbiol. Rev., 2007, 31:425-448), the contents of each of which are incorporated by reference herein in their entireties. Citramalic acid and salicylic acid from sugar beet root exudates have been shown to solubilize soil phosphorus (Kharassani, et al., BMC Plant Biology, 2011, 11:121), the content of which is incorporated by reference herein in its entirety. In addition, Salicylic acid is known for its role in plant stress responses (An and Mou, J. Integrative Plant Biol., 2011, 53:412-428; Raskin, Annu. Rev. Plant Physiol. Plant Mol. Biol., 1992, 43:439-463), has been shown to activate the systemic acquired resistance pathway in plants (Meyer et al., 1999), and as a plant metabolite is known for its role in moderating the colonization of the rhizosphere microbiome (Lebeis et al., Science, 2015, 349:860-864), the contents of each of the references is incorporated by reference herein in their entireties. Galactinol is a signaling component of the induced systemic resistance caused by Pseudomonas chlororaphis 06 colonization.
The maximal relative peak abundance of many of these compounds were found in different steps of the production process described herein and not always in the final product. A first approach to identifying the “active” ingredients of the product could be to focus on determining the concentration of the compounds in the final product and evaluating via plant growth assays the how effective this dose is at promoting growth or altering root system architecture. Another approach could be to use the plant growth assays to screen samples from the different stages for plant growth promoting potential. In this way, if a differential growth response is seen in products from different steps of the production process, these could be potentially be commercialized as different products with different modes of action (e.g., improving P acquisition, favoring induced system resistance).
Unknown chemicals. The remaining unique chemicals detected in the liquid compositions did not exist in the Binbase database and were not identifiable. Comparison of the composition of unknown compounds in the samples from the different process steps resulted in a dendgrogram (FIG. 6 ) similar to what was found for the known compounds (FIG. 4 ). The Centrate (4) and T24 (5A) samples had the greatest relative peak abundance for a large majority of the chemicals identified and were unique among the other steps in the process. The relative abundance of chemicals in the Formulated Post-Pasteurized (7) and Formulated Unpasteurized (6) samples were similar. The Slurry (2) and Cake (3) samples were similar and, together, were similar to the Raw (1) sample.

Example 5. Analysis of a Liquid Product Sample Taken after 72 Hours in the Bioreactor, but Prior to the Primary Formulation Step

A T72 sample (5B) was prepared using the process of Example 1. Sample 4B is the liquid product obtained after 72 hours in the aerobic bioreactor 50, but taken prior to primary formulation 52 (see FIG. 1 ). To determine the nutrient content of this sample, the sample was sent to Midwest Laboratories, Inc. (Omaha, NF, USA) for nutrient analysis. The results are shown in Table 14 and Table 15.

TABLE 14

Nutrient analysis

			Total content
	Analysis	Analysis	lbs per ton
Nutrients	(as rec’d)	(dry weight)	(as rec’d)

Nitrogen
Total Nitrogen	%	0.57	18.39	11.4
Organic Nitrogen	%	0.26	8.42	5.2
Ammonium
Nitrogen	%	0.309	9.968	6.2
Nitrate Nitrogen	%	<0.01	—	—
Major and Secondary
Nutrients
Phosphorus	%	0.08	2.58	1.6
Potassium as K₂O	%	0.38	12.26	7.6
Sulfur	%	<0.05	—	—
Calcium	%	0.22	7.10	4.4
Magnesium	%	0.06	1.94	1.2
Sodium	%	0.070	2.258	1.4
Micronutrients
Zinc	ppm	27.5	887	—
Iron	ppm	79.1	2552	0.2
Manganese	ppm	<20	—	—
Copper	ppm	<20	—	—
Boron	ppm	<100	—	—
Other Properties
Moisture	%	96.90
Total Solids	%	3.10		62.0
Organic Matter	%	1.99	64.19	39.8
Ash	%	1.10	35.48	22.0
C:N Ratio		4:1
Total Carbon	%	2.50	80.65
Chloride	%	0.14	4.52
pH		6.9

ppm, parts per million

TABLE 15

Nutrient analysis-amino acids

	Level Found	Level Found		Reporting
Nutrient	(as rec’d)	(dry weight)	Units	Limit	Method

Aspartic add	0.05	1.61	%	0.01	AOAC 994.12 (Alt. III)
Threonine	0.02	0.64	%	0.01	AOAC 994.12 (Alt. III)
Serine	0.02	0.64	%	0.01	AOAC 994.12 (Alt. III)
Glutamic acid	0.05	1.61	%	0.01	AOAC 994.12 (Alt. III)
Proline	0.01	0.32	%	0.01	AOAC 994.12 (Alt. III)
Glycine	0.02	0.64	%	0.01	AOAC 994.12 (Alt. III)
Alanine	0.03	0.97	%	0.01	AOAC 994.12 (Alt. III)
Cysteine		n.d.	%	0.01	AOAC 994.12 (Alt. I)
Valine	0.02	0.64	%	0.01	AOAC 994.12 (Alt. III)
Methionine		n.d.	%	0.01	AOAC 994.12 (Alt. I)
Isoleucine	0.03	0.97	%	0.01	AOAC 994.12 (Alt. III)
Leucine	0.04	1.29	%	0.01	AOAC 994.12 (Alt. III)
Tyrosine	0.05	1.61	%	0.01	AOAC 994.12 (Alt. III)
Phenylalanine	0.03	0.97	%	0.01	AOAC 994.12 (Alt. III)
Lysine (total)	0.04	1.29	%	0.01	AOAC 994.12 (Alt. III)
Histidine	0.03	0.97	%	0.01	AOAC 994.12 (Alt. III)
Arginine	0.06	1.94	%	0.01	AOAC 994.12 (Alt. III)
Tryptophan		n.d.	%	0.01	AOAC 988.15 (mod)
Erythromycin residue			ppm	0.05	FDA LIB 4438
Penicillin residue	n.d.		ppm	0.05	FDA LIB 4438
Chlorotetracycline (CTC) residue	n.d.		ppm	0.05	FDA LIB 4438
Virginiamycin residue	n.d.		ppm	0.05	FDA LIB 4438
Doxycycline (residue)	n.d.		ppm	0.050	FDA LIB 4438
Tetracycline (residue)	n.d.		ppm	0.050	FDA LIB 4438
Oxytetracycline (OTC) residue	n.d.		ppm	0.05	FDA LIB 4438
Protein	3.6	116	%	0.1	MWL FO 014
Ortho-phosphate (P2O5)	n.d.		%	0.10	AFPC 11-6
Poly-phosphate (P2O5)	n.d.		%	0.10	Calculation

n.d., not detected; ppm, parts per million

Example 6. Efficiency of the Filtration System

In some aspects, it is desirable to conduct a modified sieve analysis and measure retain material that is greater than a known sieve size of a quantity of product, e.g., to examine the efficacy of the pressure filter vessel used in the production method described in Example 1. In other aspects, such a modified sieve analysis can be used for quality control.
To determine the efficacy of the filtration step (e.g., the pressure filtration steps 60, 63 of Example 1 and FIG. 1 ) of the methods described herein, a modified sieve analysis was performed on the formulated liquid product produced as described in Example 1. Briefly, 9,000 mL of formulated liquid composition was collected in a pristine five gallon pail and promptly transferred to the laboratory to undergo the modified sieve analysis. Next, four 8 inch sieve, all stainless, half height, American Section of the International Association for Testing Materials (ASTM) standard stackable sieves were thoroughly cleansed and oven dried at 60° C. for 1 hour and then stacked on a vibratory bucket sieve unit. The vibratory motor was engaged, and 9,000 mL of product was poured such that it flowed freely through the stacked apparatus at a rate of 1,500 mL per minute. Upon complete passage of all material, the apparatus was permitted to run for an additional 60 seconds. FIG. 7A is a photograph of an exemplary filtering apparatus. The stackable sieves were collected and a gentle rinse of room temperature water of approximately 70° F. (approximately 21° C.) was sprayed over the top surface of the sieves to dislodge and cleanse any material less than the sieve rating. Retain material on each sieve was rinsed free with a direct stream of water into a vacuum filtration unit equipped with a 0.20 micron filter with a known weight. FIG. 7B is a photograph of an exemplary retain fraction collection procedure. Finally, sieve material and the 0.20 micron filter were removed from filter apparatus, dried at 101° C. for 24 hours, and weighed. The modified sieve test was performed using mesh sizes 230, 200, 170, and 140.
The data for the modified sieve mesh test sizes, micron rating, new filter weight in grams, processed filter weight in grams, retain and retain as a percentage of tested material are shown in Table 16. FIG. 7C is a photograph showing the retained material on an exemplary filtration disc used in pressure filtration of the process described herein. Depicted in FIG. 9 is a graphical representation of the data from the modified sieve test. These data show that pressure filtration used in the present method yields a finished liquid product that is within the specified 99.9% absolute filter rating of the 100 mesh filter cartridge.

TABLE 16

Sieve test results.

Sieve	Micron	New Filter	Processed Filter	Retain	% Retain (Retain
Mesh	Rating	(grams)	Dry (grams)	(grams)	grams/9,000 mL)

230	63	0.0966	0.1504	0.0538	0.000005978
200	74	0.0945	0.1473	0.0528	0.000005867
170	88	0.0933	0.1131	0.0198	0.000002200
140	105	0.0940	0.1348	0.0408	0.000004533

Example 7. Pathogen Challenge Study and GMO Analysis

To assess the viability of certain pathogenic and non-pathogenic bacteria in the liquid product produced by the methods described herein, a challenge study was conducted on samples following formulation, pasteurization, and filtration. The objective was to assess the viability of inoculated Salmonella ssp., Listeria ssp., E. coli O157:H7 and Generic E. coli ssp. in the liquid product. The specific organisms chosen for use in this study were Salmonella typhimurium, Listeria monocytogenes, E. coli O157:117, and generic E. coli mixed cultures. Growth media was inoculated with individual cultures of Salmonella typhimurium, Listeria monocytogenes, E. coli O157:H7, and generic E. coli from strains grown and cultured at Alliant Food Safety Labs, LLC (Farmington, Conn., USA). Cell suspensions were mixed to prepare inoculums containing approximately equal numbers of cells of each strain. The number of viable cells were verified by approved plate count methods well known in the art. Nine containers of each sample type preparation were inoculated with a composite culture at approximately 1,000,000 colony forming units (CFU) per gram of product with one separate container used for a negative control. After inoculation, all products were stored at cool warehouse temperatures (10° C.).
Formulated liquid composition was maintained at ambient temperatures (21° C.) with a pH 5.33 and a water activity of 0.910. To a sterile tube, 5 mL of the liquid was added and inoculated with about 1.0×10⁶colony forming units (CFU) per gram of liquid. The inoculum was prepared and concentrated into a 100 μl portion of solution. Then, 100 μl of inoculum was dispensed into each 5 mL tube with a sterile pipette. The negative control contained 100 μl sterile water. Inoculated samples were tested in triplicate at 1 minute, 24 hours, and 48 hours after inoculation using plating methods well known in the art.
Table 17 provides the baseline information for a sub-sample that was collected aseptically and tested on day 0 for Total Plate Count, Enterobacteriaceae plate count, Listeria spp., and Salmonella ssp. Tables 18-23 provide the data for the challenge test. The results show that the methods described herein produce a liquid product while reducing certain pathogenic bacteria present in raw manure (see FIGS. 9 A-D).

TABLE 17

Baseline information

Sample	Day	Results

Total Plate Count	0	<10 CFU/g
Enterobacteriaeeae
0	<10 CFU/g
Listeria spp.	0	Negative/25 g
Salmonella ssp.	0	Negative/25 g

CFU, colony forming units.

TABLE 18

Bacterial Strain

Bacterial Culture	Strain	Approximate Inoculums

Salmonella typhimurium	ATCC 133T1	2.0 × 10⁶cells per gram
		of product
Listeria monocytogenes	ATCC 19115	7.0 × 10⁵cells per gram
		of product
Escherichia coli (Migula)	ATCC 51813	1.0 × 10⁷cells per gram
Castellani and Chalmers		of product
Escherichia coli	ATCC 35150	1.3 × 10⁷cells per gram
O157:H7	ATCC 43888	of product

TABLE 19

S. fyphimurium challenge

Culture	CFU/gram		LOG Value	Time

Inoculum	1900000	1900000	1900000	1.9 × 10⁶	1.9 × 10⁶	1.9 × 10⁶	0 hr
S.	660000	360000	340000	6.6 × 10⁵	3.6 × 10⁵	3.4 × 10⁵	1 min
typhimurium	36000	32000	37000	3.6 × 10⁴	3.2 × 10⁴	3.7 × 10⁴	24 hrs
	7800	4600	8300	7.8 × 10³	4.6 × 10³	8.3 × 10³	48 hrs

TABLE 20

L. monocytogenes challenge

Culture	CFU/gram	LOG Value	Time

Inoculum	700000	700000	700000	7.0 × 10⁵	7.0 × 10⁵	7.0 × 10⁵	0 hr
L.	510000	480000	660000	5.1 × 10⁵	4.8 × 10⁵	6.6 × 10⁵	1 min
monocytogenes	<10	<10	<10	<1.0 × 10	<1.0 × 10	<1.0 × 10	24 hrs
	<10	<10	<10	1.0 × 10	<1.0 × 10	<1.0 × 10	48 hrs

indicates data missing or illegible when filed

TABLE 21

E. coli O157:H7 challenge

Culture	CFU/gram	LOG Value	Time

Inoculum	700000	700000	700000	7.0 × 10⁵	7.0 × 10⁵	7.0 × 10⁵	0 hr
L.	510000	480000	660000	5.1 × 10⁵	4.8 × 10⁵	6.6 × 10⁵	1 min
monocytogenes	<10	<10	<10	<1.0 × 10	<1.0 × 10	<1.0 × 10	24 hrs
	<10	<10	<10	<1.0 × 10	<1.0 × 10	<1.0 × 10	48 hrs

indicates data missing or illegible when filed

TABLE 22

non-pathogenic E. coli challenge

Culture	CFU/gram	LOG Value	Time

Inoculum	13000000	13000000	13000000	1.3 × 10⁷	1.3 × 10⁷	1.3 × 10⁷	0 hr
Generic E. coli	340000	530000	410000	3.4 × 10⁵	5.3 × 10⁵	4.1 × 10⁵	1 min
	220000	250000	240000	2.2 × 10⁵	2.5 × 10⁵	2.4 × 10⁵	24 hrs
	100000	110000	130000	1.0 × 10⁵	1.1 × 10⁵	1.3 × 10⁵	48 hrs

TABLE 23

Negative controls

Culture	CFU/gram	LOG Value	Time

Control	<10	Na	Na	3 × 10	Na	Na		0 hr
	<10	Na	Na	1 × 10	Na	Na		1 min
	<10	Na	Na	1 × 10	Na	Na	24 hrs
	<10	Na	Na	1 × 10	Na	Na		48 hrs

indicates data missing or illegible when filed

The formulated liquid product produced by the production method described herein was also tested for the presence of plant material from genetically modified organisms (GMO) using qualitative PCR. Qualitative PCR methods are well within the purview of the skilled artisan and will not be discussed further. The results of this analysis is shown in Table 24.

TABLE 24

35S Promoter/NOS Terminator/FMV Promoter QPCR Analysis

Test Component	Result

Corn/Maize DNA Reference	Not detected
Soy DNA Reference (additional)	Not detected
CaMV 35S Promoter	Not detected
NOS Terminator	Not detected
FMV Promoter	Not detected

Example &. Bacterial Enumeration and Morphology in T72 Samples

A T72 sample (see FIG. 1 , sample 5B) of liquid product produced after 72 hours in the bioreactor 50 and taken prior to formulation was analyzed for bacterial enumeration and colony observation. Serial dilutions were executed by protocol, with exception of final plating technique. A pour plate technique with 9 mL of molten 50° C. Trypticase soy agar (TSA) was used. T72 samples were collected from the bioreactor, and a series of 8 plates were inoculated for each sample. The plates from the 10⁻⁶dilution were the only plates observed for microbial growth since there were between 30 and 300 colonies per plate and therefore suitable for observation. Plates were incubated at 29° C. As shown in Table 2.5, the average CFU per ml was calculated as 1×10⁸or about 100,000,000.

TABLE 25

CFU calculations
CFU analysis base

	10{circumflex over ( )}⁻⁶plates
Sample #	(total colonies)

1	63 and 85
2	116 and 103
3	165 and 168
4	91
5	26 and 28
6	109 and 99
7	48 and 74

Calculations

Sample #	Average	CFU/mL

1	74	8.22E+07
2	109.5	1.22E+08
3	166.5	1.85E+08
4	91	1.01E+08
5	30	3.33E+07
6	101	1.12E+08
7	61	6.78E+07

Using microscopy techniques well within the purview of the skilled artisan, isolated colonies were observed on the TSA plates of the T72 samples. The form, elevation, margin, surface, opacity, and cosmogenesis of the bacterial colonies present on the TSA plates were recorded (see Table 26). Finally, isolated colony Gram staining was performed and analyzed microscopically. Staining for both Gram positive and Gram negative bacteria was performed. In addition, the shapes associated with Gram positive and Gram negative bacteria was recorded in Table 27.

TABLE 26

Bacterial colony observations

Form	Elevation	Margin	Surface	Opacity	Cosmogenesis

Circular	Raised	Undulate	Rough	Clear	White
Irregular	Convex	Curled	Dull	Opaque	Red
	Umbonate	Lobate	Wrinkled		Pink
		Entire	Glistening		Yellow
					Buff
					Purple

TABLE 27

Bacterial morphology observations

Cocci	Bacilli

Coccus (−)	Bacillus (+)
Diplococcus(+, −)	Diplobacillus (−)
Streptococcui (+, −)	Streptobacilli (+,−)
Staphylococci (+, −)

Example 9. Microbial Community Composition of T172 Samples

To assess the microbial community composition of the liquid product produced after 72 hours in the bioreactor 50 and taken prior to formulation, T72 samples were obtained as described in Example 1 (see FIG. 1 , sample 51B). Samples were homogenized with sterile ground glass and shipped overnight to Ward Laboratories, Inc. (Kearney, Nebr., USA) where PFLA testing was performed in triplicate.
PLFAs were analyzed according to the method of Clapperton et al. (Res. Newsletter, 2005, 1-2). Total lipids were extracted in test tubes by shaking approximately 2 g (dry weight equivalent) of frozen material in 9.5 ml dichloromethane (DCM):methanol (MeOH):citrate buffer (1:2:0.8 v/v) for 1 hour at 240 revolutions per minute (RPM). Then, 2.5 nil of DCM and 10 ml of a saturated KCl solution were added to each tube and shaken for 5 minutes. Tubes were then centrifuged at 3000 RPM for 10 min. The organic fraction was pipetted into clean vials and then dried under a flow of N₂at 37° C. in the fume hood. Samples were dissolved in 2 ml of DCM and stored at −20° C. for less than two weeks.
Lipid-class separation was conducted in silica gel columns. Samples were loaded onto columns and the vials washed twice with a small amount of DCM using a pipette. Care was taken to keep solvent level above the silica gel at all times. The neutral, glyco- and phospholipids fractions were eluted by sequential leaching with approximately 2 ml of DCM, 2 ml of acetone and 2 ml of methanol, respectively. The glycolipid fraction and neutral fraction were discarded and the phospholipids fraction was collected in a 4 ml vial. This fraction was dried under a flow of N₂at 37° C. in the fume hood, dissolved in a few ml of MeOH and then stored at −20° C.
Fatty acid methyl esters were created through mild acid methanolysis. Phospholipids fractions were dried under a flow of N₂at 37° C. in the fume hood. Half a Pasteur pipette full of MeOH/H₂SO₄(25:1 v/v) was added to the vials, which were placed in an 80° C. oven for 10 minutes, cooled to room temperature before the addition of approximately 2 ml of hexane with a Pasteur pipette. Vials were vortexed during 30 seconds and left to settle for 5 min before the lower fraction was discarded. Vials were vortexed for 30 seconds, left still for 5 min before the aqueous fraction was discarded entirely. Samples were dried under a flow of N₂at 37° C. in the fume hood. Vials were washed with 50 μl of hexane using a glass syringe, the samples transferred into 100 μl tapered glass inserts, placed inside a gas chromato-graph (GC) vial.
Samples were analyzed using a Agilent 7890A GC equipped with a 7693 autosampler and a flame ionization detector (FID). Hydrogen was the carrier gas (30 ml min⁻¹) and the column was a 50-m Varian Capillary Select FAME # cp7420. Sample (2 μl) injection was in 5:1 split mode. The injector was held at 250° C. and the FID at 300° C. The initial oven temperature, 190° C., was held for 5 minutes, raised to 210° C. at a rate of 2° C. min⁻¹, then raised from 210° C. to 250° C. at a rate of 5° C. min⁻¹, and held for 12 minutes.
Identification of peaks was based on comparison of retention times to known standards (Supelco Bacterial Acid Methyl Esters #47080-U, plus MJS Biolynx #MT1208 for 16:1ω5). The abundance of individual PLFAs was expressed as pg PLFA g⁻¹material. Amounts were derived from the relative area under specific peaks, as compared to the 19:0 peak value, which was calibrated according to a standard curve made from a range of concentrations of the 19:0 FAME standard dissolved in hexane. Fatty acids were named according to the w-designation described as follows: total number of carbons followed by a colon; the number of double bonds; the symbol c; the position of the first double bond from the methyl end of the molecule. Cis and trans isomers are indicated with c or t, respectively. Methyl (meth) and hydroxy (OH) groups are labelled at the beginning of the name where appropriate. Iso and anteiso forms are indicated by i- and a-, respectively. Table 28 shows the microbial community distribution of the 172 samples produced by the process described herein.

TABLE 28

Microbial community distribution of the T72 samples.

Control

Sample

1

Sample 2

Sample 3

Biomass	% of	Biomass	% of	Biomass	% of	Biomass	%of
PFLA	Total	PFLA	Total	PFLA	Total	PFLA	Total
ng/g	Biomass	ng/g	Biomass	ng/g	Biomass	ng/g	Biomass

Total Bacteria	21.72	19.01	1970.48	54.62	3944.72	55.4	4796.76	59.19
Gram (+)	0	0	1670.22	46.3	3415.11	47.65	4030.7	49.73
Actinomycetes	0	0	23.76	0.06	31.84	0.44	32.47	0.4
Gram (−)	21.72	19.01	300.26	8.32	529.62	7.39	766.06	9.45
Rbizobia	0	0	4.14	0.11	3.28	0.05	3.14	0.04
Total Fungi	0.01	0	507.06	14.06	769.68	10.74	776.31	9.58
Arbuscular	0	0.01	46.25	1.28	5.86	0.08	0	0
Saprophytes	0	0	460.81	12.77	763.83	10.66	776.31	9.58
Protozoa	0	0	2.18	0.06	3,27	0.05	0	0
Undifferentiated	92.54	80.98	1127.87	31.26	2449	34.17	2531.41	31.23

Example 10. The Effect of Heat on Microbial Community Composition

To determine the effect of heat on the change in microbial community composition of the liquid product produced by the process described herein, a T72 sample as described in Example 1 (se e FIG. 1 , sample 5B) was heated/pasteurized for 30 minutes at 95° C. The heated; pasteurized T72 sample was assessed for microbial content as compared to an unheated T72 sample. To assess microbial content, unheated T72 and heated/pasteurized T72 samples were homogenized with sterile ground glass and shipped overnight to Ward Laboratories, Inc. (Kearney, Nebr., USA) where PFLA testing was performed as described in Example 9. The microbial community composition of the unheated T72 and heated/pasteurized T72 samples were compared to a sterile substrate control sample. As shown in Table 29, the microbial community of the T72 sample both prior to and following heat treatment/pasteurization at 95° C. was predominantly comprised of Gram positive bacteria suggesting a link between thermophilic bacteria and Gram positive bacteria in the liquid product produced by the instant process.

TABLE 29

Microbial analysis of T72 samples.

		Pasteurized T72	Control
	T72 Sample
5B	Sample	Sample

	% of		% of		% of
Biomass	Total	Biomass	Total	Biomass	Total
PFLA ng/g	Biomass	PFLA ng/g	Biomass	PFLA ng/g	Biomass

Total Bacteria	2928.64	68.23	3377.97	53.46	6.25	16.85
Gram (+)	2540.42	59.18	2930.16	46.37	0	0
Actinomycetes	4.49	0.1	0	0	0	0
Gram (−)	388.21	9.04	447.81	7.09	6.25	16.85
Rhizobia	0	0	0	0	0	0
Total Fungi	77.68	1.81	178.41	2.82	1.66	4.46
Arbuscular	0	0	0	0	0	0
Saprophytes	77.68	1.81	178.41	2.82	1.66	4.46
Protozoa	0 1	0	0	0	0	0
Undifferentiated	1286.05	29.96	2762.56	43.72	29.22	78.69

Example 11. Product Use

The base product produced from the bioreactor was formulated to grade, filtered and finished as described in Example 1, to produce the two products described below.

- 1. Product 3-0-3-1S, with a guaranteed analysis by weight: 1.74% water soluble nitrogen, 1.26% water insoluble nitrogen, 3% soluble potash (KIO) and 1% sulfur, 9.6 lbs/gal.
- 2. Product 1.5-0-1, with a guaranteed analysis by weight: 0.6% water soluble nitrogen, 0.9% water insoluble nitrogen and 1% soluble potash (K₂O), 8.6 lbs/gal.
  The products were manufactured under conditions enabling OMRI listing for use in organic programs. The products were applied to selected crops, and results were observed.

High tunnel produce—conventional: A grower of commercial market vegetables applied Product 34-3-1S to selected crops grown in soil under high tunnel. The product was applied in drip fertigation in a solution of approximately 50 ppm of N with an injection rate of 1:15. The grower observed greater than 50% increase in growth/yield on high tunnel conventional tomatoes and cucumbers, as well as on field strawberries, as compared with the crops receiving approximately 150-200 ppm of N from synthetic fertilizer (10-10-10).
Winter Wheat—conventional: A conventional farmer in Oklahoma applied Product 3-0-3-1S as top dress on hard red winter wheat. The product was applied at 4-5 gallons per acre top dress and compared with another section of crop to which 46-18-18 (3 gal/acre) fertilizer was applied. The farmer averaged 35-45 bushels of high quality (higher protein and other grading factors) wheat, as compared with 70-80 bushels of lesser quality wheat from the alternative fertilizer, for essentially equal return. He also saw a healthier root system with more fine hairs, and improved soil organic matter. Product 3-0-3-1S was also deemed easy to use by the farmer.
Hay—transitioning to organic: A Wisconsin farmer transitioning to organic production applied Product 3-0-3-1S to first year transitional hay. The product was applied at 5 gallons per acre between cuttings, following early season application of a 1-0-3 liquid carbon-based fertilizer derived from sugar cane molasses, and kelp. He visually observed positive color and height differences between Product 3-0-3-1S treated and untreated crop within 2 weeks of application.
Hydroponics: A hydroponic grower in Michigan used Product 3-0-3-1S to grow organic produce. The product was used in a 1:200 product:water solution daily in initial growth stages, then Product 1.5-0-1 was applied at the same rate in final growth stages. The combination of nutrients, microbes and amino acids in both Product 3-0-3-1S and Product 1.5-0-1 enabled the grower to simplify his process and use one product instead of three, reducing labor and input costs. He also observed improved growth results as compared with crops previously grown. Finally, the grower noted no clogging of the hydroponic injection system and no odor problems, as compared with past usage of a fish emulsion fertilizer.
Spelt—organic: An organic farmer in Michigan used Product 3-0-3-1S to grow organic spelt. The product was applied at 7 gallons per acre at the booting stage. He observed positive visible color differences between Product 3-0-3-1S treated crop, as compared with crop treated with liquid fish fertilizer. He also noted ease of use and lack of clogging of foliar application equipment, as compared with past usage of fish fertilizers.
Corn—organic: An organic farmer in Pennsylvania used Product 3-0-3-1S to grow organic corn. The product was applied at 8 gallons per acre as an in-furrow starter. The farmer saw superior emergence with robust color—an indicator of nutrient sufficiency and a “strong start” to the organic crop. Untreated corn did not emerge as well and showed the same nutrient deficiency symptoms the farmer had observed in previous years. The farmer also noted excellent handling with “minimal issues of flowability.”
The present invention is not limited to the embodiments described and exemplified herein. It is capable of variation and modification within the scope of the appended claims.

Claims

1-36. (canceled)

37. A method of conditioning a selected soil, comprising:

a) selecting a soil for which conditioning is sought;

b) treating the soil with a liquid composition comprising an autothermal thermophilic aerobic bioreaction product from a liquid fraction of poultry manure slurry, the poultry manure slurry comprising at least about 80% by weight moisture content and subjected to an autothermal thermophilic aerobic bioreaction for at least about 1 day, wherein the autothermal thermophilic aerobic bioreaction product endogenously comprises at least one living species of plant growth promoting bacteria or fungi and at least one non-living substance that promotes plant growth;

c) measuring at least one parameter of conditioning in the treated soil, and

d) comparing the at least one measured parameter of conditioning in the treated soil with an equivalent measurement in an equivalent soil not treated with the liquid composition, or before treatment with the liquid composition;

wherein an improvement in the at least one measured parameter in the treated, as compared with the untreated soil, or with the soil prior to treatment, is indicative of conditioning the selected soil.

38. The method of claim 37, wherein the at least one living species of plant growth promoting bacteria comprises Rhizobium spp, Geobaccillus spp, Brevibacillus spp, Paenibacillus spp, Bacillus spp, Ureibacillus spp, or any combination thereof.

39. The method of claim 38, wherein the Brevibacillus spp is selected from the group consisting of Brevibacillus agri, Brevibacillus borsteinensis, Brevibacillus levickii, Brevibacillus thermoruber, and any combination thereof.

40. The method of claim 38, wherein the Paenibacillus spp is selected from the group consisting of Paenibacillus granivorans, Paenibacillus cookie, Paenibacillus borealis, Paenibacillus popilliae, Paenibacillus chinjuensis, and any combination thereof.

41. The method of claim 38, wherein the Geobaccillus spp is selected from the group consisting of Geobaccillus stearothermophilus, Geobaccillus thermoleovorans, Geobaccillus thermocaternuiatus, Geobaccillus thermodenitrificans, and any combination thereof.

42. The method of claim 37, wherein the at least one living species of plant growth promoting bacteria comprises Bacillus subtilis, Bacillus anthracis, Bacillus firmus, Bacillus arnyloliquelaciens, Bacillus licheniformis, Bacillus pumilus, Bacillus azotoformans, Ureibacillus thermosphaericus or any combination thereof.

43. The method of claim 37, wherein the at least one non-living substance that promotes plant growth is selected from the group consisting of 3,6-anhydro-D-galactose, beta-gentiobiose, cellobiose, glucose, glucose-1-phosphate, glyceric acid, fructose, fucose, galactose, isomaltose, isoribose, isothreonic acid, lactobionic acid, lyxose, maltose, maltotriose, ribose, sucrose, tagatose, threonic acid, trehalose, UDP-glucuronic acid, xylonic acid, xylonic acid isomer, xylose, xylulose, 1-deoxyerythritol, 1-hexadecanol, 2-deoxyerythritol, deoxypentitol, diglycerol, erythritol, glycerol, hexitol, lyxitol, mannitol, pinitol, threitol, xylitol, indole-3-acetate (IAA), 6-hydroxynicotinic acid, 1-monoolein, 1-monopalmitin, 1-monostearin, 2-monoolein, arachidic acid, arachidonic acid, beta-hydroxymyristic acid, beta-sitosterol, capric acid, caprylic acid, cerotinic acid, cholesterol, cis-gondoic acid, Dehydrocholesterol, D-erythro-sphingosine, glycerol-alpha-phosphate, heptadecanoic acid, hexadecylglycerol, isoheptadecanoic acid, lauric acid, lignoceric acid, linoleic acid, myristic acid, nonadecanoic acid, octadecanol, oleamide, oleic acid, palmitic acid, palmitoleic acid, pelargonic acid, pentadecanoic acid, squalene, stearic acid, stigmasterol, valine, aminomalonate, 1,3-diaminopropane, 2,4-diaminobutyric acid, 3-aminoisobutyric acid, 5-aminovaleric acid, 5-methoxytryptamine, alanine, alpha-aminoadipic acid, asparagine, aspartic acid, beta-alanine, beta-glutamic acid, citrulline, cyclohexylamine, cysteine, ethanolamine, glutamic acid, glutamine, glycine, glycyl proline, homoserine, hydroxylamine, isoleucine, leucine, lysine, maleimide, methionine, methionine sulfoxide, N-acetyl-D-galactosamine, n-acetyl-d-hexosamine, N-acetylaspartic acid, N-acetylglutamate, N-acetylputrescine, N-carbamylglutamate, N-methylalanine, N-methylglutamic acid, norvaline, O-acetylserine, oxoproline, phenylalanine, phenylethylamine, putrescine, serine, spermidine, taurine, threonine, thymine, trans-4-hydroxyproline, tryptophan, tyramine, tyrosine, 3,4-dihydroxybenzoic acid, 4-hydroxybenzoate, catechol, cis-caffeic acid, ferulic acid, hydroquinone, phenol, tyrosol, vanillic acid, 2-hydroxy-2-methylbutanoic acid, 2-hydroxyadipic acid, 2-hydroxybutanoic acid, 2-hydroxyglutaric acid, 2-hydroxyhexanoic acid, 2-hydroxyvaleric acid, 2-isopropylmalic acid, 2-ketoadipic acid, 2-methylglyceric acid, 2-picolinic acid, 3-(3-hydroxyphenyl) propionic acid, 3-(4-hydroxyphenyl) propionic acid, 3-hydroxy-3-methylglutaric acid, 3-hydroxybenzoic acid, 3-hydroxybutyric acid, 3-hydroxypalmitic acid, 3-hydroxyphenylacetic acid, 3-hydroxypropionic acid, 3-phenyllactic acid, 3,4-dihydroxycinnamic acid, 3,4-dihydroxy-hydrocinnamic acid, 3,4-dihydroxy-phenylacetic acid, 4-aminobutyric acid, 4-hydroxybutyric acid, 4-hydroxymandelic acid, 4-hydroxyphenylacetic acid, 4-pyridoxic acid, aconitic acid, adipic acid, alpha-ketoglutarate, behenic acid, benzoic acid, chenodeoxycholic acid, citric acid, digalacturonic acid, fumaric acid, gluconic acid, gluconic acid lactone, glutaric acid, glycolic acid, hexuronic acid, hydrocinnamic acid, isocitric acid, isohexonic acid, isopentadecanoic acid, kynurenic acid, lactic acid, malic acid, malonic acid, methylmaleic acid, oxalic acid, oxamic acid, phenylacetic acid, pimelic acid, pipecolinic acid, pyrrole-2-carboxylic acid, pyruvic acid, quinolinic acid, ribonic acid, succinic acid, sulfuric acid, tartaric acid, uric acid, urocanic acid, thymidine, 5,6-dihydrouracil, 7-methylguanine NIST, adenine, adenosine, cytosin, guanine, pseudo uridine, uracil, zymosterol, 1-methylhydantoin, 1,2-cyclohexanedione, 2-deoxypentitol NIST, 2-deoxytetronic acid, 2-ketoisocaproic acid, 2,3-dihydroxybutanoic acid NIST, 2,8-dihydroxyquinoline, 3-(3-hydroxyphenyl)-3-hydroxypropionic acid, 3-ureidopropionate, 4-methylcatechol, 5-hydroxymethyl-2-furoic acid NIST, butane-2,3-diol NIST, butyrolactam NIST, conduritol-beta-epoxide, creatinine, daidzein, glycerol-3-galactoside, hypoxanthine, isothreitol, lanosterol, methanolphosphate, myo-inositol, nicotinic acid, octadecylglycerol, ononitol, parabanic acid NIST, phosphate, piperidone, propane-1,3-diol NIST, pyrogallol, pyrophosphate, tocopherol acetate, tocopherol alpha, tocopherol gamma, urea, xanthine, xanthurenic acid, and any combination thereof.

44. The method of claim 37, wherein the at least one parameter of conditioning the soil is selected from the group consisting of soil organic matter, microbial diversity, nutrient profile, bulk density, porosity, water permeation, and any combination thereof.

45. The method of claim 37, wherein the autothermal thermophilic aerobic bioreaction product endogenously comprises at least one biostimulant.

46. The method of claim 45, wherein the biostimulant is selected from the group consisting of amino acids, bacteria, fungi and combinations thereof.

47. The method of claim 37, wherein the treating of the selected soil is done as a remediation to damaged or polluted soil.

48. The method of claim 37, wherein the treating of the selected soil is done to improve chemical properties of the treated soil.

49. The method of claim 37, wherein the treating of the selected soil is done to improve biological properties of the treated soil.

50. The method of claim 37, further comprising the step of e) planting plants or crops in the treated soil following treatment.

51. A method of preventing or reducing nematode infestation on a plant or plant roots, comprising:

a) providing a plant disposed in soil, wherein the plant comprises plant roots;

b) treating the soil in which the roots are disposed with a liquid composition comprising an autothermal thermophilic aerobic bioreaction product from a liquid fraction of poultry manure slurry, the poultry manure slurry comprising at least about 80% by weight moisture content and subjected to an autothermal thermophilic aerobic bioreaction for at least about 1 day, wherein the autothermal thermophilic aerobic bioreaction product endogenously comprises at least one living species of plant growth promoting bacteria or fungi and at least one non-living substance that promotes plant growth;

c) measuring at least one parameter of nematode infestation in the treated soil or on the plant roots, and

d) comparing the at least one measured parameter of nematode infestation in the treated soil or on the plant roots with an equivalent measurement in an equivalent soil or plant roots not treated with the liquid composition;

wherein an improvement in the at least one measured parameter in the treated, as compared with the untreated soil or plant roots is indicative of preventing or reducing nematode infestation the plant or plant roots.

52. The method of claim 51, wherein the at least one measured parameter comprises quantity of nematodes in the treated soil or on the plant roots.

53. The method of claim 51, wherein the at least one living species of plant growth promoting bacteria comprises at least one Bacillus spp.

54. The method of claim 53, wherein the at least one Bacillus spp comprises Bacillus firmus.

55. The method of claim 51, wherein the at least one non-living substance that promotes plant growth is selected from the group consisting of 3,6-anhydro-D-galactose, beta-gentiobiose, cellobiose, glucose, glucose-1-phosphate, glyceric acid, fructose, fucose, galactose, isomaltose, isoribose, isothreonic acid, lactobionic acid, lyxose, maltose, maltotriose, ribose, sucrose, tagatose, threonic acid, trehalose, UDP-glucuronic acid, xylonic acid, xylonic acid isomer, xylose, xylulose, 1-deoxyerythritol, 1-hexadecanol, 2-deoxyerythritol, deoxypentitol, diglycerol, erythritol, glycerol, hexitol, lyxitol, mannitol, pinitol, threitol, xylitol, indole-3-acetate (IAA), 6-hydroxynicotinic acid, 1-monoolein, 1-monopalmitin, 1-monostearin, 2-monoolein, arachidic acid, arachidonic acid, beta-hydroxymyristic acid, beta-sitosterol, capric acid, caprylic acid, cerotinic acid, cholesterol, cis-gondoic acid, Dehydrocholesterol, D-erythro-sphingosine, glycerol-alpha-phosphate, heptadecanoic acid, hexadecylglycerol, isoheptadecanoic acid, lauric acid, lignoceric acid, linoleic acid, myristic acid, nonadecanoic acid, octadecanol, oleamide, oleic acid, palmitic acid, palmitoleic acid, pelargonic acid, pentadecanoic acid, squalene, stearic acid, stigmasterol, valine, aminomalonate, 1,3-diaminopropane, 2,4-diaminobutyric acid, 3-aminoisobutyric acid, 5-aminovaleric acid, 5-methoxytryptamine, alanine, alpha-aminoadipic acid, asparagine, aspartic acid, beta-alanine, beta-glutamic acid, citrulline, cyclohexylamine, cysteine, ethanolamine, glutamic acid, glutamine, glycine, glycyl proline, homoserine, hydroxylamine, isoleucine, leucine, lysine, maleimide, methionine, methionine sulfoxide, N-acetyl-D-galactosamine, n-acetyl-d-hexosamine, N-acetylaspartic acid, N-acetylglutamate, N-acetylputrescine, N-carbamylglutamate, N-methylalanine, N-methylglutamic acid, norvaline, O-acetylserine, oxoproline, phenylalanine, phenylethylamine, putrescine, serine, spermidine, taurine, threonine, thymine, trans-4-hydroxyproline, tryptophan, tyramine, tyrosine, 3,4-dihydroxybenzoic acid, 4-hydroxybenzoate, catechol, cis-caffeic acid, ferulic acid, hydroquinone, phenol, tyrosol, vanillic acid, 2-hydroxy-2-methylbutanoic acid, 2-hydroxyadipic acid, 2-hydroxybutanoic acid, 2-hydroxyglutaric acid, 2-hydroxyhexanoic acid, 2-hydroxyvaleric acid, 2-isopropylmalic acid, 2-ketoadipic acid, 2-methylglyceric acid, 2-picolinic acid, 3-(3-hydroxyphenyl) propionic acid, 3-(4-hydroxyphenyl) propionic acid, 3-hydroxy-3-methylglutaric acid, 3-hydroxybenzoic acid, 3-hydroxybutyric acid, 3-hydroxypalmitic acid, 3-hydroxyphenylacetic acid, 3-hydroxypropionic acid, 3-phenyllactic acid, 3,4-dihydroxycinnamic acid, 3,4-dihydroxy-hydrocinnamic acid, 3,4-dihydroxy-phenylacetic acid, 4-aminobutyric acid, 4-hydroxybutyric acid, 4-hydroxymandelic acid, 4-hydroxyphenylacetic acid, 4-pyridoxic acid, aconitic acid, adipic acid, alpha-ketoglutarate, behenic acid, benzoic acid, chenodeoxycholic acid, citric acid, digalacturonic acid, fumaric acid, gluconic acid, gluconic acid lactone, glutaric acid, glycolic acid, hexuronic acid, hydrocinnamic acid, isocitric acid, isohexonic acid, isopentadecanoic acid, kynurenic acid, lactic acid, malic acid, malonic acid, methylmaleic acid, oxalic acid, oxamic acid, phenylacetic acid, pimelic acid, pipecolinic acid, pyrrole-2-carboxylic acid, pyruvic acid, quinolinic acid, ribonic acid, succinic acid, sulfuric acid, tartaric acid, uric acid, urocanic acid, thymidine, 5,6-dihydrouracil, 7-methylguanine NIST, adenine, adenosine, cytosin, guanine, pseudo uridine, uracil, zymosterol, 1-methylhydantoin, 1,2-cyclohexanedione, 2-deoxypentitol NIST, 2-deoxytetronic acid, 2-ketoisocaproic acid, 2,3-dihydroxybutanoic acid NIST, 2,8-dihydroxyquinoline, 3-(3-hydroxyphenyl)-3-hydroxypropionic acid, 3-ureidopropionate, 4-methylcatechol, 5-hydroxymethyl-2-furoic acid NIST, butane-2,3-diol NIST, butyrolactam NIST, conduritol-beta-epoxide, creatinine, daidzein, glycerol-3-galactoside, hypoxanthine, isothreitol, lanosterol, methanolphosphate, myo-inositol, nicotinic acid, octadecylglycerol, ononitol, parabanic acid NIST, phosphate, piperidone, propane-1,3-diol NIST, pyrogallol, pyrophosphate, tocopherol acetate, tocopherol alpha, tocopherol gamma, urea, xanthine, xanthurenic acid, and any combination thereof.

56. The method of claim 51, wherein the autothermal thermophilic aerobic bioreaction product endogenously comprises at least one biostimulant.