Nutritional Compositions For Plants And Soils

Information

  • Patent Application
  • 20190194081
  • Publication Number
    20190194081
  • Date Filed
    December 19, 2016
    7 years ago
  • Date Published
    June 27, 2019
    5 years ago
Abstract
The current application relates to a liquid fertilizer composition for application to plants and soils, comprising an autothermal thermophilic aerobic bioreaction product from a liquid fraction of poultry manure and further to a method of improving health and productivity of a plant or crop using said composition and to a method of conditioning soil using said composition.
Description
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 nutrients analysis (source: Biol. & Agric. Eng. Dept. NC State University,


January 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
% dry basis
74
80
43
73
66
43-80


Solids


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
% dry basis
32
26

18

18-32


Protein


Total
col/100 gm
7.32E+11
1.06E+11

5.63E+11


Bacteria


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 1B







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%, 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 bio-reactor 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 dry 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
889 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 108 colony-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 1011 or 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 (nematicidal), 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 NO3 to 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, O-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-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, 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, 1st Ed., (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.










Process
Range of Operational




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









Process
Range of Operational



Parameter
Parameters
Notes





Decanting Centrifuge
3250 RPM Max



Influent volume
25-30 gallons per
Slurry from mixer



minute
being pumped into




centrifuge


Effluent volume
25% of input manure by
Liquid fraction



weight is extracted as
exiting the



finely suspended solids
centrifuge


Solids separation
75% of input manure by
Solids fraction



weight
discharge


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












Process
Range of Operational






Parameter
Parameters



Notes















Data collection Record
1 minute to



How frequent the PLC records



30 minutes



data


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 6PSI


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 4-20ma







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









Process
Range of Operational



Parameter
Parameters
Notes





Pasteurization
65 to 100° C.
Steam injection



5 to 60 minutes


Filtration Step 1
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









Process
Range of Operational



Parameter
Parameters
Notes





Axial Turbine
50-68 RPM
42 inch axial turbine shaft,


mixing

1.5 HP 1 to 1.6 turnovers




per minute




Interval programmed mixing




cycle


Filtration
50-74 micron
Final filtration prior to


Step 3

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 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.









Info

















3-Cake*
3-Cake**



Nutrients
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−1 resolution.



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−1 to 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 biomarker groups in samples from the process of Example 1.









Concentration (nmol g−1)


















Sample
FAME
Actin
G−
G+
Fungi
AM 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
245.44
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
412.97
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;


Actin, Actinobacteria;


G−, gram negative bacteria;


G+, gram positive bacteria;


AM Fungi, arbuscular mycorrhizal fungi;


TBB, total bacterial biomass;


TMB, total microbial 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 (%)














Sample
FAME
Actin
G−
G+
Fungi
AM Fungi
Protists

















1-Raw
44.1
0.60
6.8
35.1
13.0
0.02
0.32


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;


Actin, Actinobacteria;


G−, gram negative bacteria;


G+, gram positive bacteria;


AM Fungi, arbuscular mycorrhizal 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., St. 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−1 controlled 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/

Lipids/


Carboxylic






Sugar
Growth
fatty


acids/


Sample
Sugar
alcohol
factor
acids
amines
phenolics
organic acids
nucleosides
other



















1-Raw
66705
39217
11772
225618
1376373
63343
2825680
34567
300420


2-Slurry
60431
30507
16992
378225
1984846
26576
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
1991126
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-anhydro-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. I


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 the 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 or tremalose, is a natural alpha-linked disaccharide



formed by an α,α-1,1-glucoside bond between two α-glucose units.


UDP-glucuronic 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








1-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 Cupuaçu butter.


arachidonic acid
is a polyunsaturated omega-6 fatty acid 20:4. It is structurally related to the saturated



arachidic acid found in Cupuaçu 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


cyclohexylamine


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)4NH2 that 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 be 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-hydroxyglutaric 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-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-
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.


chenodeoxycholic 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, an acidifier, or a curing, pickling, or leavening agent.


glutaric acid


glycolic acid
Glycolic acid; chemical formula C2H4O3, is the smallest α-hydroxy acid.


hexuronic acid


hydrocinnamic acid
Phenylpropanoic acid or hydrocinnamic acid is a carboxylic acid with the formula



C9H10O2 belonging to the class of phenylpropanoids.


isocitric acid
Isocitric acid is an organic compound closely related to citric acid.


isohexonic 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


1,2-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 metabolism by Pseudomonas sp


3-(3-hydroxyphenyl)-3-


hydroxypropionic acid


3-ureidopropionate
an intermediate in the metabolism of uracil


4-methylcatechol
4-Methylcatechol 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 com, and 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



C6H12O6 or (—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 formula 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 E 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.






























xanthurenic


γ
tocopherol
tocopherol



propane-




Sample
zymosterol
acid
xanthine
urea
tocopherol
alpha-
acetate
squalene
pyrophosphate
pyrogallol
1,3-diol
piperidone
phosphattext missing or illegible when filed





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
























parabanic

octadecyl
nicotinic
myo-

linoleic



glycerol-3-




Sample
acid
ononitol
glycerol
acid
inositol
methanolphosphate
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
8612
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





























5-










conduritol-

butane-


hydroxymethyl-


3-(3-hydroxyphenyl)-
2-
2-
2-
2,8-



beta-
butyrolactam
2,3-diol
benzoic
adipic
2-furoic
4-
3-
3-hydroxypropionic
ketoisocaproic
deoxytetronic
deoxypentitol
dihydroxy-


Sample
epoxide
NIST
NIST
acid
acid
acid NIST
methylcatechol
ureidopropionate
acid
acid
acid
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
4042
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-















dihydroxybutanoic
1-
1,2-




trans-4-


Sample
acid
methylhydantoin
cyclohexanedione
valine
tyrosine
tyramine
tryptophan
hydroxyproline
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
1684
718
24261
188
432
299
397
524
158
325


6 - form
111
1923
460
2077
449
14786
247
1630
1037
484
427
89
112






























N-











phenyl-

O-

methylglutamic
N-
N-
N-
N-
N-
n-acetyl-d-


Sample
putrescine
phenylethylamine
alanine
oxoproline
acetylserine
norvaline
acid
methylalanine
carbamylglutamate
acetylputrescine
acetylornithine
acetylglutamate
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-














N-acetyl-
acetylaspartic
methionine


Sample
D-galactosamine
acid
sulfoxide
methionine
maleimide
lysine
leucine
isoleucine
hydroxylamine
homoserine
glycylproline
glycine
glutamine





1 - raw
457
3560
812
3956
1124
3453
105313
71751
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
358
206
20161
177


4 - centrate
129
3017
1466
16298
1687
3747
276693
173099
93890
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
1779
162821
129
169
20540
147





























beta-




alpha-





glutamic




glutamic
beta-
aspartic

arachidonic
aminoadipic
alanine-


Sample
acid
ethanolamine
cysteine
cyclohexylamine
citrulline
acid
alanine
acid
asparagine
acid
acid
alanine
alanine





1 - raw
54262
2429
377
1201
745
16013
6955
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
328258


4 - centrate
46128
2405
666
1267
1634
3328
102212
142073
269
2953
1121
1971
1213184


5A - T24
1239
5128
472
2033
1173
734
128251
18549
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-
3-amino-
2,4-







glycerol-




5-
aminovaleric
isobutyric
diaminobutyric
1,3-
myristic
lignoceric
lauric
isoheptadecanoic
hexadecylglycerol
heptadecanoic
alpha-
D-erythrtext missing or illegible when filed


Sample
methoxytryptamine
acid
acid
acid
diaminopropane
acid
acid
acid
acid NIST
NIST
acid
phosphate
sphingositext missing or illegible when filed





1 - raw
173
56569
1722
1274
1122
1808
707
8517
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-









gondoic

cerotinic
caprylic
capric
beta-
hydroxymyristic
behenic
arachidic

2-
1-
1-


Sample
acid
cholesterol
aci
acid
acid
sitosterol
acid
acid
acid
2-monoolein
deoxyerythritol
monostearin
monopalmitin





1 - raw
205
10267
198
1941
849
19049
420
2625
4236
387
4044
265
461


2 - slurry
205
18183
240
2456
1047
39404
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




































xylonic



1-
stearic
pentadecanoic
pelargonic
palmitoleic
palmitic
oleic
oleamide

nonadecanoic
xylulose

acid


Sample
monoolein
acid
acid
acid
acid
acid
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
48723
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
tagatose
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






























3,6-





UDP-









beta-
anhydro-D-


vanillic
urocanic
uric
glucuronic
threonic
tartaric
sulfuric



Sample
fucose
fructose
gentiobiose
galactose
glucose
cellobiose
acid
acid
acid
acid
acid
acid
acitext missing or illegible when filed







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
pipecolinic
pimelic
phenylacetic
oxamic
oxalic
methylmaleic
malonic
malic



Sample
acid
acid
acid
acid
acid
acid
acid
acid
acid
acid
acid
acid
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
772
30947
271
339
163
132
211



6 - form
270
161
116
128
483
2558
489
24223
236
434
120
95
100

























lactic
kynurenic
isopentadecanoic
isohexonic
isocitric
hydrocinnamic
hexuronic
glycolic
glutaric
gluconic
gluconic
fumaric
digalacturonic


Sample
acid
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
7816
2580
86
113
555
238


3 - cake
15905
169
11435
107
1075
18563
211
861
1421
109
93
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
































4-
4-
4-
4-
3-



citric
citramalic
cis-caffeic
chenodeoxycholic

alpha-
aconitic
4-pyridoxic
hydroxyphenylacetic
hydroxymandelic
hydroxybutyric
aminobutyric
phenyllacttext missing or illegible when filed


Sample
acid
acid
acid
acid
aminomalonate
ketoglutarate
acid
acid
acid
acid
acid
acid
acid





1 - raw
2502309
974
630
1632
673
497
7489
329
8593
2845
1064
7508
5715


2 - slurry
114857
1381
625
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-
3-
3-
3-
3-
3-hydroxy-
3,4-
3,4-
3,4-
3-(4-
3-(3-





hydroxy-
hydroxy-
hydroxy-
hydroxy-
hydroxy-
3-methyl-
dihydroxy-
dihydroxy-
dihydroxy-
hydroxy-
hydroxy-

2-methylglyceric



propionic
phenylacetic
palmitic
butyric
benzoic
glutaric
phenylacetic
hydrocinnamic
cinnamic
phenyl)propionic
phenyl)propionic
2-picolinic
acid


Sample
acid
acid
acid
acid
acid
acid
acid
acid NIST
acid
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
2722
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-
2-
2-
2-
2-
2-
2-
2-hydroxy-2-








ketoadipic
isopropylmalic
hydroxyvaleric
hydroxyhexanoic
hydroxyglutaric
hydroxybutanoic
hydroxyadipic
methylbutanoic


Sample
acid
acid
acid
acid
acid
acid
acid
acid
uracil
thymidine
pseudouridine
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
758
55
44656
90
408
319
144
114
79
104




























7-
















methylguanine
5,6-


salicylic




isothreonic



Sample
adenosine
adenine
NIST
dihydrouracil
threitol
stigmasterol
acid
pinitol
phenol
mannitol
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
10781
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-




glucose-





1-
1-

indole-3-

hydroxynicotinic
5-hydroxy


Sample
1-phosphat
erythritol
dihydrocholesterol
diglycerol
deoxypentitol
catechol
hexadecanol
deoxyerythritol
pantothenic 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























4-
3,4-






glyceric
ferulic
hydroxy-
dihydroxy-


Sample
tyrosol
hydroquinone
glycerol
acid
acid
benzoate
benzoate





1 - raw
1065
268
24265
4588
858
4757
27542


2 - slur
2293
401
9119
1022
460
10465
2816


3 - cake
1043
235
3821
473
405
4984
1613


4 - centr
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






text missing or illegible when filed indicates data missing or illegible when filed







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 O6 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 5B 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, Nebr., USA) for nutrient analysis. The results are shown in Table 14 and Table 15.









TABLE 14







Nutrient analysis











Analysis (as
Analysis (dry
Total content lbs


Nutrients
rec'd)
weight)
per ton (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 acid
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.














New
Processed

% Retain


Sieve
Micron
Filter
Filter
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:H7, 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 1.0×106 colony 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. 9A-D).









TABLE 17







Baseline information











Sample
Day
Results
















Total Plate Count
0
<10
CFU/g



Enterobacteriaceae
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 13311
2.0 × l06 cells per gram of




product



Listeria monocytogenes

ATCC 19115
7.0 × l05 cells per gram of




product



Escherichia coli (Migula)

ATCC 51813
1.0 × 107 cells per gram of


Castellani and Chalmers

product



Escherichia coli O157:H7

ATCC 35150
1.3 × 107 cells per gram of



ATCC 43888
product
















TABLE 19








S. typhimurium challenge











Culture
CFU/gram
LOG Value
Time


















Inoculum
1900000
1900000
1900000
1.9 × 106
1.9 × 106
1.9 × 106
0
hr



S. typhimurium

660000
360000
340000
6.6 × 105
3.6 × 105
3.4 × 105
1
min



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. monocytogenes

510000
480000
660000
 5.1 × 105
 4.8 × 105
 6.6 × 105
1
min



<10
<10
<10
<1.0 × 101
<1.0 × 101
<1.0 × 101
24
hrs



<10
<10
<10
<1.0 × 101
<1.0 × 101
<1.0 × 101
48
hrs
















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. monocytogenes

510000
480000
660000
 5.1 × 105
 4.8 × 105
 6.6 × 105
1
min



<10
<10
<10
<1.0 × 101
<1.0 × 101
<1.0 × 101
24
hrs



<10
<10
<10
<1.0 × 101
<1.0 × 101
<1.0 × 101
48
hrs
















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
1 × 101
Na
Na
0
hr



<10
Na
Na
1 × 101
Na
Na
1
min



<10
Na
Na
1 × 101
Na
Na
24
hrs



<10
Na
Na
1 × 101
Na
Na
48
hrs









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 8. 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 10−6 dilution 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 25, the average CFU per ml was calculated as 1×108 or about 100,000,000.









TABLE 25





CFU calculations







CFU analysis base








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




















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













Cocci
Bacilli







Coccus (−)
Bacillus (+)



Diplococcus(+, −)
Diplobacillus (−)



Streptococcui (+, −)
Streptobacilli (+, −)



Staphylococci (+, −)










Example 9. Microbial Community Composition of T72 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 5B). 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 ml 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 N2 at 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 N2 at 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 N2 at 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 N2 at 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−1 material. 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 w; 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 T72 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 Total
Biomass
% of Total
Biomass
% of Total
Biomass
% of Total



PFLA ng/g
Biomass
PFLA ng/g
Biomass
PFLA ng/g
Biomass
PFLA 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


Rhizobia
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 (see 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.











T72 Sample 5B
Pasteurized T72 Sample
Control Sample














Biomass
% of Total
Biomass
% of Total
Biomass
% of 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
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 (K2O) 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 3-0-3-1S 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. A liquid composition for application to plants and soils, comprising an autothermal thermophilic aerobic bioreaction product from a liquid fraction of poultry manure, wherein the liquid composition endogenously comprises at least one biostimulant.
  • 2. (canceled)
  • 3. The composition of claim 1, wherein the biostimulant is selected from the group consisting of amino acids, bacteria, fungi and combinations thereof.
  • 4. The composition of claim 2, endogenously comprising at least one living species of plant growth promoting bacteria or fungi.
  • 5. The composition of claim 1, endogenously comprising at least one non-living substance that promotes plant growth selected from the group consisting of citramalic acid, salicylic acid, pantothenic acid, indole-3-acetic acid, 5-hydroxy-indole-3-acetic acid, galactinol, and any combination thereof.
  • 6. (canceled)
  • 7. The composition of claim 1, endogenously comprising 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.
  • 8. The composition of claim 7, wherein the non-living substance is selected from the group consisting of salicylic acid, phenolic compounds, and any combination thereof.
  • 9. The composition of claim 1, wherein the poultry manure is chicken manure.
  • 10. The composition of claim 1, wherein: (a) 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;(b) the poultry manure slurry is heated to between about 60° C. and about 75° C. for between about 1 hour and about 4 hours; and(c) 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.
  • 11-12. (canceled)
  • 13. The composition of claim 10, wherein the pre-determined time is between about 1 day and about 18 days.
  • 14. The composition of claim 10, endogenously comprising all macronutrients and micronutrients required for plant growth.
  • 15. The composition of claim 10, endogenously comprising less than 0.5 wt % phosphorus.
  • 16. The composition of claim 1, comprising at least one additive.
  • 17. The composition of claim 16, wherein the additive is selected from a macronutrient, a micronutrient, a biostimulant, a biocontrol agent, and any combination thereof.
  • 18. The composition of claim 1, wherein the composition is: (a) formulated for application to soil or a medium in which a plant is growing or will be grown;(b) formulated for application to a seed or plant part;(c) suitable for use in an organic program; or(d) any combination of (a)-(c).
  • 19-20. (canceled)
  • 21. A method of improving health or productivity of a selected plant or crop, comprising: 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, andd) 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;
  • 22. The method of claim 21, wherein the plant or crop is grown or cultivated in a medium selected from, soil, soil-less solid, hydroponic or aeroponic.
  • 23. The method of claim 21, wherein the treating comprises applying the composition: (a) to seeds of the plant,(b) to a medium in which the plant or crop is growing or will be planted;(c) to portions of the plant or crop(d) to a medium in which the plant or crop is growing or will be planted, wherein the application to the medium is pre-planting, pre-inoculation, or pre-emergence;(e) as a side dressing to a medium in which the plant is growing or will be planted;(f) in the course of irrigation;(g) as a direct application to the plant or crop; or(h) any combination of (a)-(g).
  • 24-25. (canceled)
  • 26. The method of claim 21, wherein: 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; andthe 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; orb) grown in the substantially same location during a different growing season; orc) grown in a different location during the same growing season; ord) grown in a different location during a different growing season.
  • 27-30. (canceled)
  • 31. A method of conditioning a selected soil, comprising: a) selecting a soil for which conditioning is sought and in which plants or crops are or will be planted;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, andd) 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;
  • 32. The method of claim 31, wherein: (a) the selected soil comprises at least one feature selected from compaction, nutrient deficiency, microbial deficiency, organic matter deficiency, and any combination thereof;(b) 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; or(c) the at least one measured parameter in the treated soil is compared with an equivalent parameter prior to treatment of the same soil and/or with an equivalent parameter in an equivalent untreated soil in substantially the same location or in a different location.
  • 33-36. (canceled)
CROSS REFERENCE TO RELATED APPLICATIONS

This 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.

PCT Information
Filing Document Filing Date Country Kind
PCT/US2016/067614 12/19/2016 WO 00
Provisional Applications (1)
Number Date Country
62270009 Dec 2015 US