Compositions, methods, and systems for promoting zinc solubilization and zinc uptake in plants

Abstract

The present disclosure provides methods and systems for production of biostimulants that promote zinc solubilization in plants. Embodiments described include methods of making a biostimulant composition in a bioreactor system that includes two or more containers arranged in series. The bioreactor system may include an established population of a zinc-solubilizing microbial strain. The method may include operating the bioreactor system by transferring into the system an aqueous feedstock that comprises a microbial consortium, transferring working fluid between containers of the system, and collecting a product. The method may further include maintaining a concentration of a zinc-solubilizing microbial strain in the system.

Description

COMPOSITIONS, METHODS, AND SYSTEMS FOR PROMOTING ZINC SOLUBILIZATION AND ZINC UPTAKE IN PLANTSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 613,548, filed December 21, 2023, which application is incorporated herein by reference.BACKGROUND

[0002] The disclosure is generally related to biostimulant compositions and methods of using such biostimulant compositions to promote plant growth and enhance zinc uptake in plants.

[0003] Promoting efficient production of food crops and other crops is an important goal for environmental and economic reasons. Plant growth promoting products sourced from organic materials can help to enhance crop growth, improve the efficacy of agricultural products, such as fertilizers, and reduce the environmental impacts of synthetic fertilizers and climate change. There exists a need for plant growth promoting biostimulant compositions that use abundant and available organic feedstocks.SUMMARY

[0004] In an aspect, the present disclosure provides a method of making a biostimulant composition, the method comprising: (a) providing a bioreactor system comprising two or more containers arranged in a series, each of the two or more containers comprising a volume of a working fluid, wherein a first container comprises a first working fluid that includes a first microbial consortium and an established population of a zinc solubilizing bacterial strain; (b) operating the bioreactor system for a duration of time by: (i) transferring into the first container an aqueous feedstock comprising a second microbial consortium; (ii) transferring a portion of the working fluid out of each of the two or more containers into either a subsequent container of the bioreactor system or a product outflow stream; (iii) maintaining a concentration of the zinc solubilizing bacterial strain throughout the duration of time in at least the first container at at least 80% of a concentration of the zinc solubilizing bacterial strain at the beginning of the duration of time; and (iv) collecting at least a portion of the product outflow stream as the biostimulant composition; wherein the duration of time is at least 5 days; and wherein the zinc solubilizing bacterial strain is not present in the aqueous feedstock or any other input into the bioreactor system during the duration of time at a concentration that is higher than 1% of the concentration of the zinc solubilizing microbe in the first container.

[0005] In some embodiments, the method further comprises maintaining in at least the first container a concentration of a zinc-containing molecule of at least 5 mg / L. In some embodiments, the first microbial consortium comprises a first portion of microbes and a second portion of microbes, and wherein the method further comprises maintaining a concentration of a zinc-containing molecule in at least the first container that suppresses growth of the first portion of microbes relative to the zinc solubilizing bacterial strain. In some embodiments, the first portion of microbes comprises microbes that are not zinc solubilizers and / or are not zinc tolerant. In some embodiments, the second portion of microbes comprises microbes that are zinc solubilizers and / or are zinc tolerant. In some embodiments, the zinc-containing molecule is zinc oxide, zinc sulfide, zinc carbonate, zinc phosphate, zinc chloride, or zinc sulphate. In some embodiments, the zinc-solubilizing bacterial strain is of the genus Bacillus. In some embodiments, the zinc-solubilizing bacterial strain is of the species Bacillus safensis o Bacillus megaterium. In some embodiments, the zinc-solubilizing bacterial strain is one of the following:(a) a Bacillus safensis strain having one or more of the following: (i) a 16S rRNA gene sequence at least 95% identical to SEQ ID NO: 1; (ii) a gyrB gene sequence at least 95% identical to SEQ ID NO: 4; and (iii) a rpoB gene sequence at least 95% identical to SEQ ID NO: 7; (b) a Bacillus megaterium strain having one or more of the following: (i) a 16S rRNA gene sequence at least 95% identical to SEQ ID NO: 2; (ii) a gyrB gene sequence at least 95% identical to SEQ ID NO: 5; and (iii) a rpoB gene sequence at least 95% identical to SEQ ID NO: 8; or (b) a Bacillus megaterium strain having one or more of the following: (i) a 16S rRNA gene sequence at least 95% identical to SEQ ID NO: 3; (ii) a gyrB gene sequence at least 95% identical to SEQ ID NO: 6; and (iii) a rpoB gene sequence at least 95% identical to SEQ ID NO: 9. In some embodiments, the zinc-solubilizing bacterial strain is the Bacillus safensis strain deposited under ATCC Accession No. PTA-127681, the Bacillus megaterium strain deposited under ATCC Accession No. PTA-127683, or the Bacillus megaterium strain deposited under ATCC Accession No. PTA-127682. In some embodiments, the zinc solubilizing bacterial strain is not present in the aqueous feedstock or any other input into the bioreactor system during the duration of time at a concentration of greater than 100 CFU / ml. In some embodiments, the zinc solubilizing bacterial strain is not present in the aqueous feedstock or any other input into the bioreactor system during the duration of time. In some embodiments, the maintaining of step(b)(iii) comprises maintaining the concentration of the zinc solubilizing bacterial strain at at least IxlO3CFU / ml. In some embodiments, before step (b), the first container further comprises an established population of other zinc solubilizing microbes that are not the zinc solubilizing bacterial strain, and wherein step (b)(iii) further comprises maintaining a concentration of theother zinc solubilizing microbes in at least the first container throughout the duration of time at at least IxlO4CFU / ml or at at least 80% of a concentration of the other zinc solubilizing microbes at the beginning of the duration of time, wherein the other zinc solubilizing microbes are not added to the bioreactor system during the duration of time at a concentration that is higher than 1% of the concentration of the other zinc solubilizing microbes in the first container. In some embodiments, the other zinc solubilizing microbes are not present in the aqueous feedstock or any other input into the bioreactor system at a concentration of greater than 104CFU / ml. In some embodiments, the population of the other zinc solubilizing microbes in the first container is at least IxlO3CFU / ml at the beginning of the duration of time. In some embodiments, the method further comprises before step (a), adding an inoculum of the zinc solubilizing bacterial strain to the bioreactor system, wherein the inoculum of the zinc solubilizing bacterial strain produces an initial population of the zinc solubilizing bacterial strain of at least 0.5xl04CFU / ml in at least one container. In some embodiments, before adding the inoculum of the zinc solubilizing bacterial strain, the concentration of the zinc solubilizing bacterial strain is less than IxlO2CFU / ml. In some embodiments, the aqueous feedstock further comprises an organic material at least partially digestible by microbes present in at least one of the containers. In some embodiments, before the transferring of step (b)(i), the organic material had been partially digested by microbes endogenous to the organic material. In some embodiments, the method further comprises digesting the organic material in two or more serially connected containers before the transferring of step (b)(i). In some embodiments, the organic material comprises manure and / or material produced by microbial digestion of manure. In some embodiments, the aqueous feedstock further comprises an inorganic material. In some embodiments, the inorganic material comprises rock phosphate particles. In some embodiments, prior to the transferring of step (b)(i), the rock phosphate particles had been partially digested by microbes present in the aqueous feedstock. In some embodiments, the method further comprises partially digesting the rock phosphate particles in two or more serially connected containers before the transferring of step (b)(i). In some embodiments, the second microbial consortium comprises at least IxlO5CFU / ml. In some embodiments, the second microbial consortium comprises microbes derived from manure and / or from rock phosphate particles. In some embodiments, the operating of step (b) further comprises producing microbial metabolites that directly or indirectly promote zinc solubilization in a plant growth medium. In some embodiments, the transferring of step (b)(i), the transferring of step (b)(ii), and the collecting of step (b)(iv) are performed continuously throughout the duration of time. In some embodiments, the transferring of step (b)(i), the transferring of step (b)(ii), and the collecting of step (b)(iv) areperformed periodically throughout the duration of time. In some embodiments, the method further comprises adding one or more carbon sources to at least one container of the bioreactor system. In some embodiments, the one or more carbon sources are included in the aqueous feedstock. In some embodiments, the method further comprises maintaining a gluconic acid concentration and / or glucose concentration in at least one container of the bioreactor system at a concentration of at least 0.2% w / v in relation to the volume of working fluid in the at least one container. In some embodiments, the method further comprises adding one or more nitrogen sources to at least one container of the bioreactor system. In some embodiments, the one or more nitrogen sources comprise one or more of ammonium sulfate, ammonium chloride, ammonium nitrate, sodium nitrate, yeast extract, yeast, or any combination thereof. In some embodiments, the bioreactor system comprises a clarifier container comprising a clarifier working fluid. In some embodiments, the method further comprises separating a supernatant portion of the clarifier working fluid from a floc portion of the clarifier working fluid within the clarifier container. In some embodiments, the separating comprises gravity separation. In some embodiments, the method further comprises folding the floc portion of the clarifier working fluid. In some embodiments, the folding further comprises releasing a population of the zinc solubilizing bacterial strain into the supernatant portion without introducing floc solids into the supernatant portion. In some embodiments, the folding is performed by folding wipers in a bottom portion of the clarifier container. In some embodiments, the operating further comprises transferring the floc portion from the clarifier container to an earlier container in the bioreactor system. In some embodiments, the product outflow stream comprises the supernatant portion of the clarifier working fluid. In some embodiments, the method further comprises producing at least IxlO4CFU / ml of the zinc solubilizing bacterial strain in the product outflow stream. In some embodiments, the bioreactor system comprises the first container comprising a volume of a first working fluid, a second container comprising a volume of a second working fluid, and a third container comprising a volume of a third working fluid. In some embodiments, the first container comprises an outlet port fluidly connected to an inlet port of the second container and the second container comprises an outlet port fluidly connected to an input port of the third container. In some embodiments, the third container comprises an outlet port fluidly connected to a clarifier container. In some embodiments, the method further comprises maintaining the volume of each of the first working fluid, the second working fluid, and the third working fluid constant throughout the duration of time. In some embodiments, step (b) comprises operating the bioreactor system in a hydraulically balanced manner. In some embodiments, the transferring of step (b)(i), the transferring of step (b)(ii), and the collecting of step (b)(iv) are driven by gravity.In some embodiments, the operating comprises maintaining a flow rate that results in a hydraulic retention time of the bioreactor system of at least 5 days. In some embodiments, the operating comprises maintaining the product outflow stream at a flow rate of at least 100 gallons per day. In some embodiments, the volume of working fluid in each of the two or more containers is at least 100 gallons. In some embodiments, at least one of the two or more containers is a fluidized bed reactor. In some embodiments, at least one of the two or more containers is a packed bed reactor. In some embodiments, the method further comprises maintaining at least one of the two or more containers under microaerobic conditions. In some embodiments, the bioreactor system is operated continuously for at least 90 days.

[0006] In some embodiments, one or more species of one or more of the following genera are among five most abundant species in the second microbial consortium: Haliscomenobacter , I. ew ine Ila. Caldilinea, Terri monas. and Acidobacterium . In some embodiments, one or more of the following species are among five most abundant species in the microbial consortium: Lewinella cohaerens. Thauera phenylacelica. Thauera mechernichensis. Solitalea canadensis, and Nitrospira moscoviensis. In some embodiments, the second microbial consortium comprises microbes endogenous to the organic material. In some embodiments, at least one of the first working fluid, the second working fluid, or the third working fluid are circulated within the respective containers. In some embodiments, the method further comprises maintaining the pH of at least one of the first working fluid, the second working fluid, or the third working fluid between 6 and 9 throughout the duration of time. In some embodiments, the aqueous feedstock does not include the zinc solubilizing bacterial strain at a concentration higher than 10 CFU / ml. In some embodiments, the zinc solubilizing bacterial strain is not added to the bioreactor system during the duration of time at a concentration that is higher than 10 CFU / ml. In some embodiments, the bioreactor system comprises at least one container placed in the series before the first container. In some embodiments, the method further comprises producing a population of sporulated bacteria in the product outflow stream. In some embodiments, the method further comprises producing a population of the zinc solubilizing bacterial strain in the product outflow stream that is sporulated. In some embodiments, the population of the zinc solubilizing bacterial strain that is sporulated comprises at least IxlO3CFU / ml. In some embodiments, the method further comprises adding an additional population of the zinc solubilizing bacterial strain to the biostimulant product. In some embodiments, the method further comprises making at least a portion of the aqueous feedstock by a method comprising: (c) transferring water, rock phosphate, and, optionally, products of digestion of manure by microbes derived from the manure into a fourth container comprising a volume of a fourth working fluid; (d) transferring aportion of the fourth working fluid into a fifth container comprising a fifth working fluid; (e) transferring into the fifth container: (i) a liquid comprising (A) a third microbial consortium comprising microbes derived from manure, and (B) digestion products produced by anaerobic digestion of the manure by the microbes; (ii) manure; and (iii) yeast. In some embodiments, the method further comprises transferring a portion of the fifth working fluid into a sixth container comprising a sixth working fluid, and transferring a portion of the sixth working fluid into a seventh container comprising a seventh working fluid. In some embodiments, the method further comprises separating a portion of the seventh working fluid into a feedstock floc portion and a feedstock supernatant portion. In some embodiments, the method further comprises transferring the feedstock floc portion to the fourth container. In some embodiments, the method further comprises maintaining the fourth container, the fifth container, the sixth container, and / or the seventh container under aerobic conditions. In some embodiments, the fourth container, the fifth container, the sixth container, and / or the seventh container are fluidized bed reactors, wherein the rock phosphate is continuously circulated within the fourth container, the fifth container, the sixth container, and / or the seventh container. In some embodiments, a total volume of material added to the fourth container over a given time period is equal to a total volume of the fourth working fluid transferred to the fifth container over a same time period.

[0007] In another aspect, the present disclosure provides a bioreactor system comprising: (a) a stream of an aqueous feedstock in fluid communication with a first container comprising a volume of a first working fluid, wherein the aqueous feedstock comprises a first microbial consortium, wherein the first working fluid comprises an established population of a zinc solubilizing bacterial strain and a second microbial consortium, wherein a concentration of the zinc solubilizing bacterial strain in the first working fluid is at least 100 times higher than a concentration of the zinc solubilizing bacterial strain in the aqueous feedstock stream and in any other input into the bioreactor system; (b) one or more additional containers arranged in a series that includes the first container, wherein each of the one or more additional containers comprises a volume of a working fluid and is in fluid communication with at least one other container in the series, and wherein at least one of the one or more additional containers comprises a product outflow stream port; and (c) a product outflow stream in fluid communication with the product outflow stream port.

[0008] In some embodiments, at least the first working fluid comprises a zinc-containing molecule at a concentration of at least 5 mg / L. In some embodiments, the second microbial consortium comprises a first portion of microbes and a second portion of microbes, wherein at least the first working fluid comprises a zinc-containing molecule at a concentration thatsuppresses growth of the first portion of microbes relative to the zinc solubilizing bacterial strain. In some embodiments, the first portion of microbes comprises microbes that are not zinc solubilizers and / or are not zinc tolerant. In some embodiments, the second portion of microbes comprises microbes that are zinc solubilizers and / or are zinc tolerant. In some embodiments, the zinc-containing molecule is zinc oxide, zinc sulfide, zinc carbonate, zinc phosphate, zinc chloride, or zinc sulphate. In some embodiments, the zinc-solubilizing bacterial strain is of the genus Bacillus. In some embodiments, the zinc-solubilizing bacterial strain is of the species Bacillus safensis o Bacillus megaterium. In some embodiments, the zinc-solubilizing bacterial strain is one of the following: (a) a Bacillus safensis strain having one or more of the following: (i) a 16S rRNA gene sequence at least 95% identical to SEQ ID NO: 1; (ii) a gyrB gene sequence at least 95% identical to SEQ ID NO: 4; and (iii) a rpoB gene sequence at least 95% identical to SEQ ID NO: 7; (b) a Bacillus megaterium strain having one or more of the following: (i) a 16S rRNA gene sequence at least 95% identical to SEQ ID NO: 2; (ii) a gyrB gene sequence at least 95% identical to SEQ ID NO: 5; and (iii) a rpoB gene sequence at least 95% identical to SEQ ID NO: 8; or (b) a Bacillus megaterium strain having one or more of the following: (i) a 16S rRNA gene sequence at least 95% identical to SEQ ID NO: 3; (ii) a gyrB gene sequence at least 95% identical to SEQ ID NO: 6; and (iii) a rpoB gene sequence at least 95% identical to SEQ ID NO: 9. In some embodiments, the zinc-solubilizing bacterial strain is the Bacillus safensis strain deposited under ATCC Accession No. PTA-127681, the Bacillus megaterium strain deposited under ATCC Accession No. PTA-127683, or the Bacillus megaterium strain deposited under ATCC Accession No. PTA-127682. In some embodiments, the bioreactor system is a continuous flow bioreactor system and the stream of the aqueous feedstock is a continuous stream. In some embodiments, each of the volume of the working fluids is constant. In some embodiments, each of the first container and the one or more additional containers comprises a concentration of the zinc-solubilizing bacterial strain that remains at least IxlO4CFU / ml during operation of the bioreactor system. In some embodiments, the aqueous feedstock and any other input into the bioreactor system does not comprise the zinc- solubilizing bacterial strain or does not comprise a concentration of the zinc-solubilizing bacterial strain at level higher than 100 CFU / ml. In some embodiments, the first microbial consortium comprises at least IxlO4CFU / ml of microbes. In some embodiments, the aqueous feedstock further comprises an organic material digestible by microbes present in the containers. In some embodiments, the organic material comprises manure or material derived from manure. In some embodiments, the aqueous feedstock further comprises rock phosphate particles. In some embodiments, the first microbial consortium comprises microbes derived from manureand / or rock phosphate particles. In some embodiments, the container comprising the product outflow stream port is a clarifier container configured to separate a portion of a working fluid in the clarifier container into a supernatant portion and a floc portion. In some embodiments, the clarifier container comprises one or more floc folding flights configured to agitate settled floc in the clarifier container without resuspending solids in the floc portion into the supernatant portion. In some embodiments, the bioreactor system further comprises a floc return stream that flows from the clarifier to an earlier container in the series. In some embodiments, the product outflow stream comprises the supernatant portion. In some embodiments, the product outflow stream comprises at least IxlO4CFU / ml of the zinc-solubilizing bacterial strain. In some embodiments, the product outflow stream comprises at least IxlO2CFU / ml of a sporulated form of the zinc-solubilizing bacterial strain. In some embodiments, the product outflow stream comprises a total dry weight of 0.2 to 2.5 mg / ml. In some embodiments, the product outflow stream has a chemical oxygen demand between 80 to 500 mg / L. In some embodiments, the product outflow stream has an electrical conductivity between 0.1 and 1.5 mS / cm. In some embodiments, the first working fluid comprises glucose and / or gluconic acid at a concentration of at least 0.2% w / v. In some embodiments, the first working fluid and / or the working fluid in at least one of the one or more additional containers comprises microaerobic conditions. In some embodiments, a biostimulant composition can be made by the method or the system described herein. In some embodiments, method of promoting plant growth may comprise contacting a plant, seed, or plant growth medium with the biostimulant composition. In some embodiments, a method of increasing an amount of solubilized zinc may be available to a plant, the method may comprise contacting a plant, seed, or plant growth medium with the biostimulant composition.

[0009] In another aspect, the present disclosure provides a composition comprising: (a) a Bacillus safensis strain having one or more of the following: (i) a 16S rRNA gene sequence at least 95% identical to SEQ ID NO: 1; (ii) a gyrB gene sequence at least 95% identical to SEQ ID NO: 4; and (iii) a rpoB gene sequence at least 95% identical to SEQ ID NO: 7; and (b) a carrier. In some embodiments, the Bacillus safensis strain is the strain deposited under ATCC Accession No. PTA-127681, or an isolated clone thereof. In some embodiments, the composition further comprises products of digestion of an organic substrate by the Bacillus safensis strain. In some embodiments, the carrier comprises a fertilizer. In some embodiments, the carrier is a solid coated by the Bacillus safensis strain. In some embodiments, the carrier is further coated by a micronutrient. In some embodiments, the micronutrient is zinc oxide, zinc sulfide, zinc carbonate, or zinc phosphate. In some embodiments, the carrier is a liquid. In some embodiments, the carrier further comprises an adjuvant selected from a wetting agent, spreadingagent, dispersing agent, sticking agent, dust control agent, or adhesive. In some embodiments, the concentration of the Bacillus safensis strain in the composition ranges from IxlO3to IxlO11cfu / ml. In some embodiments, the concentration of the Bacillus safensis strain in the composition ranges from IxlO4to IxlO6cfu / ml.

[0010] In another aspect, the present disclosure provides an isolated strain of the species Bacillus safensis having one or more of the following: (a) a 16S rRNA gene sequence at least 95% identical to SEQ ID NO: 1; (b) a gyrB gene sequence at least 95% identical to SEQ ID NO: 4; and (c) a rpoB gene sequence at least 95% identical to SEQ ID NO: 7. In some embodiments, the B. safensis strain is the strain deposited under ATCC Accession No. PTA- 127681, or an isolated clone thereof.

[0011] In another aspect, the present disclosure provides a composition comprising: (a) a Bacillus megaterium strain having one or more of the following: (i)a 16S rRNA gene sequence at least 95% identical to SEQ ID NO: 2; (ii) a gyrB gene sequence at least 95% identical to SEQ ID NO: 5; and (iii) a rpoB gene sequence at least 95% identical to SEQ ID NO: 8; and (b) a carrier.

[0012] In some embodiments, the Bacillus megaterium strain is the strain deposited under ATCC Accession No. PTA-127683, or an isolated clone thereof. In some embodiments, the isolated strain further comprises products of digestion of an organic substrate by the Bacillus megaterium strain. In some embodiments, the carrier comprises a fertilizer. In some embodiments, the carrier is a solid coated by the Bacillus megaterium strain. In some embodiments, the carrier is further coated by a micronutrient. In some embodiments, the micronutrient is zinc oxide, zinc sulfide, zinc carbonate, or zinc phosphate. In some embodiments, the carrier is a liquid. In some embodiments, the carrier further comprises an adjuvant selected from a wetting agent, spreading agent, dispersing agent, sticking agent, dust control agent, or adhesive. In some embodiments, the concentration of the Bacillus megaterium strain in the composition ranges from IxlO3to IxlO11cfu / ml. In some embodiments, the concentration of the Bacillus megaterium strain in the composition ranges from IxlO4to IxlO6.

[0013] In another aspect, the present disclosure provides an isolated strain of the species Bacillus megaterium having one or more of the following: (a) a 16S rRNA gene sequence at least 95% identical to SEQ ID NO: 2; (b) a gyrB gene sequence at least 95% identical to SEQ ID NO: 5; and (c) a rpoB gene sequence at least 95% identical to SEQ ID NO: 8.

[0014] In some embodiments, the 7>. megaterium strain is the strain deposited under ATCC Accession No. PTA-127683, or an isolated clone thereof.

[0015] In another aspect, the present disclosure provides a composition comprising: (a) a Bacillus megaterium strain having one or more of the following: (i)a 16S rRNA gene sequence at least 95% identical to SEQ ID NO: 3; (ii) a gyrB gene sequence at least 95% identical to SEQ ID NO: 6; and (iii) a rpoB gene sequence at least 95% identical to SEQ ID NO: 9; and (b) a carrier.

[0016] In some embodiments, the Bacillus megaterium strain is the strain deposited under ATCC Accession No. PTA-127682, or an isolated clone thereof. In some embodiments, composition further comprising products of digestion of an organic substrate by the Bacillus megaterium strain. In some embodiments, the carrier comprises a fertilizer. In some embodiments, the carrier is a solid coated by the Bacillus megaterium strain. In some embodiments, the carrier is further coated by a micronutrient. In some embodiments, the micronutrient is zinc oxide, zinc sulfide, zinc carbonate, or zinc phosphate. In some embodiments, the carrier is a liquid. In some embodiments, the carrier further comprises an adjuvant selected from a wetting agent, spreading agent, dispersing agent, sticking agent, dust control agent, or adhesive. In some embodiments, the concentration of the Bacillus megaterium strain in the composition ranges from IxlO3to IxlO11cfu / ml. In some embodiments, the concentration of the Bacillus megaterium strain in the composition ranges from IxlO4to IxlO6.

[0017] In another aspect, the present disclosure provides an isolated strain of the species Bacillus megaterium having one or more of the following: a 16S rRNA gene sequence at least 95% identical to SEQ ID NO: 3; a gyrB gene sequence at least 95% identical to SEQ ID NO: 6; and a rpoB gene sequence at least 95% identical to SEQ ID NO: 9.

[0018] In some embodiments, the 7>. megaterium strain is the strain deposited under ATCC Accession No. PTA-127682, or an isolated clone thereof. In some embodiments, a method for promoting growth of a plant growing in a medium, the method may comprise contacting the plant or the medium with the biostimulant, the composition, or a composition comprising the isolated strain as disclosed herein. In some embodiments, the contacting causes increases the amount of solubilized zinc available to the plant by at least 5%. In some embodiments, the composition increases plant growth by at least 5% as compared to a control. In some embodiments, the composition increases uptake of a nutrient by the plant by at least 5% as compared to a control. In some embodiments, the nutrient is zinc, and wherein the uptake of zinc by the plant is increased by at least 5% as compared to a control. In some embodiments, the nutrient is phosphate. In some embodiments, the nutrient is sulfur, potassium, magnesium, calcium, boron, manganese, iron, and / or copper. In some embodiments, the medium is soil or a hydroponic medium.

[0019] In some embodiments, a method of remedying a zinc deficiency in a plant growth medium, the method comprises: (a) measuring a concentration of soluble zinc in the plant growth medium that is less than 0.5 ppm; and (b) after step (a), contacting the plant growth medium with the biostimulant, the composition, or a composition comprising the isolated strain disclosed herein.

[0020] Provided herein is a method of promoting plant growth, comprising: (a) contacting a plant and / or medium in which the plant is growing with a composition comprising one or more compounds comprising l-hexadecyl-2,3-di-o-acethyl glycerol, diisodecyl phthalate, allothreonine, (S)-(-)-.alpha.-(l-Naphthyl)ethylamine, indoleacetic acid (indole-3 -acetic acid), salicylanilide, 25-Hydroxycholesterol, 4-(2-Aminophenyl)-2,4-dioxobutanoic acid, 13,14- dihydro-15-keto PGD2, 4-Ethyloctanoic acid, phthalic acid, alpha-Ketoisovaleric acid, datiscetin, Vai-Ala, N-methylundec-10-enamide, His-Ile-Lys-Arg, methylcarbamyl PAF, homogentisic acid, Ala-Gly-Leu-Val-Ser, Arg-Val-Met, chaps, 2-Phenylethanol, prostaglandin D3, ritalinic acid, 5-Hydroxyindoleacetaldehyde, azithromycin dihydrate, Pro-Ala-Phe, (4R,7S,7aR,12bS)-3-methyl-2,4,4a,7,7a,13-hexahydro-lH-4,12-methanobenzofuro[3,2- e]isoquinoline-7,9-diol, Glu-Pro-Thr, 2,6-Naphthalenediol, 3 -Aminoquinoline, acylated phloroglucinol, 1-Pyrrolysine, tanikolide, Val-Arg-Glu, 3 -Methyladipic acid, 10- Propoxydecanoic acid, 7-hydroxy-3-(2-methoxyphenyl)-4H-chromen-4-one, N-methylundec-10- enamide, prosta-5,13-dien-l-oic acid, 9, 11 -epidioxy- 15 -hydroperoxy-,(5Z,9alpha,l 1 alpha, 13E, 15 S)-, Ile-Phe-Val-Lys, gamma-Glutamyl-Se-methylselenocysteine;5- L-Glutamyl-Se-methylselenocysteine, Trp-Ala-Lys, pantoyllactone glucoside, vasicinone, LPC(O- 16:0 / 2:0), Sphingosine 1-phosphate, coenzyme Q6, dihydrocortisol, dibutylone hydrochloride, LPE(18:0 / 0:0), Nialamide, androstanedione, 12-(2,3-Dihydroxycyclopentyl)-2- dodecanone, Androst-5-ene-3, 17-dione, hydroxytyrosol, 4-Hydroxy cinnamic acid, 2,4,5- Trihydroxytoluene, isopropamide, (1R,2R,5R,8R,9S,10R,12S)-12-Hydroxy-1 l-methyl-6- methylidene-16-oxo-15-oxapentacyclo[9.3.2.15,8.01,10.02,8]heptadecane-9-carboxylic acid, Allopurinol riboside, l-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3- phosphoethanolamine, Uridine triacetate, JWH 018 7-hydroxyindole metabolite-d9, 1,6- Dimethylphenazine, 2-(p-Bromophenyl)-8-methyl-8H-thieno(2,3-b)indole, 3,4-Dihydroxy- phenyl glycol, dodecyl ci s-9,10-epoxy octadecanoate, heptacosane, lauric acid, lignoceric acid, tetratriacontane, tryptophol, one or more derivatives thereof, or a combination thereof. In some embodiments, the contacting comprises contacting the plant with the composition. In some embodiments, the contacting comprises contacting a plant seed with the composition. In some embodiments, the contacting comprises contacting a leaf of the plant with the composition. Insome embodiments, the medium comprises a soil, a hydroponic medium, turface, or isolite. In some embodiments, the contacting causes an increase in plant growth of the plant by at least 10 percent as compared to a plant growth of the plant when the plant or the medium is not contacted with the composition comprising one or more compounds. In some embodiments, the composition further comprises an adjuvant selected from the group consisting of a wetting agent, spreading agent, dispersing agent, sticking agent, dust control agent, and adhesive. In some embodiments, the composition is configured to enhance an availability of zinc in soil. In some embodiments, the composition is configured to enhance zinc uptake by plant tissues. In some embodiments, the composition is configured to promote zinc use efficiency.Provided herein is a composition for promoting plant growth, comprising: (a) at least one microbial strain comprising Bacillus safensis o Bacillus megalerium and (b) one or more compounds comprising l-hexadecyl-2,3-di-o-acethyl glycerol, diisodecyl phthalate, allothreonine, (S)-(-)-.alpha.-(l-Naphthyl)ethylamine, indoleacetic acid (indole-3 -acetic acid), salicylanilide, 25-Hydroxycholesterol, 4-(2-Aminophenyl)-2,4-dioxobutanoic acid, 13,14- dihydro-15-keto PGD2, 4-Ethyloctanoic acid, phthalic acid, alpha-Ketoisovaleric acid, datiscetin, Vai-Ala, N-methylundec-10-enamide, His-Ile-Lys-Arg, methylcarbamyl PAF, homogentisic acid, Ala-Gly-Leu-Val-Ser, Arg-Val-Met, chaps, 2-Phenylethanol, prostaglandin D3, ritalinic acid, 5-Hydroxyindoleacetaldehyde, azithromycin dihydrate, Pro-Ala-Phe, (4R,7S,7aR,12bS)-3-methyl-2,4,4a,7,7a,13-hexahydro-lH-4,12-methanobenzofuro[3,2- e]isoquinoline-7,9-diol, Glu-Pro-Thr, 2,6-Naphthalenediol, 3 -Aminoquinoline, acylated phloroglucinol, 1-Pyrrolysine, tanikolide, Val-Arg-Glu, 3 -Methyladipic acid, 10- Propoxydecanoic acid, 7-hydroxy-3-(2-methoxyphenyl)-4H-chromen-4-one, N-methylundec-10- enamide, prosta-5,13-dien-l-oic acid, 9, 11 -epidioxy- 15 -hydroperoxy-,(5Z,9alpha,l 1 alpha, 13E, 15 S)-, Ile-Phe-Val-Lys, gamma-Glutamyl-Se-methylselenocysteine;5- L-Glutamyl-Se-methylselenocysteine, Trp-Ala-Lys, pantoyllactone glucoside, vasicinone, LPC(O- 16:0 / 2:0), Sphingosine 1-phosphate, coenzyme Q6, dihydrocortisol, dibutylone hydrochloride, LPE(18:0 / 0:0), Nialamide, androstanedione, 12-(2,3-Dihydroxycyclopentyl)-2- dodecanone, Androst-5-ene-3, 17-dione, hydroxytyrosol, 4-Hydroxy cinnamic acid, 2,4,5- Trihydroxytoluene, isopropamide, (1R,2R,5R,8R,9S,10R,12S)-12-Hydroxy-1 l-methyl-6- methylidene-16-oxo-15-oxapentacyclo[9.3.2.15,8.01,10.02,8]heptadecane-9-carboxylic acid, Allopurinol riboside, l-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3- phosphoethanolamine, Uridine triacetate, JWH 018 7-hydroxyindole metabolite-d9, 1,6- Dimethylphenazine, 2-(p-Bromophenyl)-8-methyl-8H-thieno(2,3-b)indole, 3,4-Dihydroxy- phenyl glycol, dodecyl ci s-9,10-epoxy octadecanoate, heptacosane, lauric acid, lignoceric acid,tetratriacontane, tryptophol, one or more derivatives thereof, or a combination thereof. In some embodiments, the at least one microbial strain comprises Bacillus safensis strain comprises one or more of the following: (i) a 16S rRNA gene sequence at least 95% identical to SEQ ID NO: 1; (ii) a gyrB gene sequence at least 95% identical to SEQ ID NO: 4; or (iii) a rpoB gene sequence at least 95% identical to SEQ ID NO: 7. In some embodiments, the at least one microbial strain comprises Bacillus megaterium strain comprises one or more of the following: (i) a 16S rRNA gene sequence at least 95% identical to SEQ ID NO: 2; (ii) a gyrB gene sequence at least 95% identical to SEQ ID NO: 5; or (iii) a rpoB gene sequence at least 95% identical to SEQ ID NO: 8. In some embodiments, the at least one microbial strain comprises Bacillus megaterium strain comprises one or more of the following: (i) a 16S rRNA gene sequence at least 95% identical to SEQ ID NO: 3; (ii) a gyrB gene sequence at least 95% identical to SEQ ID NO: 6; or (iii) a rpoB gene sequence at least 95% identical to SEQ ID NO: 9. In some embodiments, the composition further comprises a carrier. In some embodiments, the carrier is formulated for application to a plant and / or medium in which the plant is growing. In some embodiments, the composition further comprises an adjuvant selected from the group consisting of a wetting agent, spreading agent, dispersing agent, sticking agent, dust control agent, and adhesive. In some embodiments, the composition is configured to enhance an availability of zinc in soil. In some embodiments, the composition is configured to enhance a zinc uptake by plant tissues. In some embodiments, the composition is configured to promote zinc use efficiency.

[0021] Provided herein is a composition for promoting plant growth, comprising: (a) two or more compounds comprising froml-hexadecyl-2,3-di-o-acethyl glycerol, diisodecyl phthalate, allothreonine, (S)-(-)-.alpha.-(l-Naphthyl)ethylamine, indoleacetic acid (indole-3 -acetic acid), salicylanilide, 25-Hydroxycholesterol, 4-(2-Aminophenyl)-2,4-dioxobutanoic acid, 13,14- dihydro-15-keto PGD2, 4-Ethyloctanoic acid, phthalic acid, alpha-Ketoisovaleric acid, datiscetin, Vai-Ala, N-methylundec-10-enamide, His-Ile-Lys-Arg, methylcarbamyl PAF, homogentisic acid, Ala-Gly-Leu-Val-Ser, Arg- Vai-Met, chaps, 2-Phenylethanol, prostaglandin D3, ritalinic acid, 5-Hydroxyindoleacetaldehyde, azithromycin dihydrate, Pro-Ala-Phe, (4R,7S,7aR,12bS)-3-methyl-2,4,4a,7,7a,13-hexahydro-lH-4,12-methanobenzofuro[3,2- e]isoquinoline-7,9-diol, Glu-Pro-Thr, 2,6-Naphthalenediol, 3 -Aminoquinoline, acylated phloroglucinol, 1-Pyrrolysine, tanikolide, Val-Arg-Glu, 3 -Methyladipic acid, 10- Propoxydecanoic acid, 7-hydroxy-3-(2-methoxyphenyl)-4H-chromen-4-one, N-methylundec-10- enamide, prosta-5,13-dien-l-oic acid, 9, 11 -epidioxy- 15 -hydroperoxy-,(5Z,9alpha, 11 alpha, 13E, 15 S)-, Ile-Phe-Val-Lys, gamma-Glutamyl-Se-methylselenocysteine;5-L-Glutamyl-Se-methylselenocysteine, Trp-Ala-Lys, pantoyllactone glucoside, vasicinone, LPC(O- 16:0 / 2:0), Sphingosine 1-phosphate, coenzyme Q6, dihydrocortisol, dibutylone hydrochloride, LPE(18:0 / 0:0), Nialamide, androstanedione, 12-(2,3-Dihydroxycyclopentyl)-2-dodecanone, Androst-5-ene-3, 17-dione, hydroxytyrosol, 4-Hydroxycinnamic acid, 2,4,5-Trihydroxytoluene, isopropamide, (lR,2R,5R,8R,9S,10R,12S)-12-Hydroxy-ll-methyl-6-methylidene-16-oxo-15- oxapentacyclo[9.3.2.15,8.01,10.02,8]heptadecane-9-carboxylic acid, Allopurinol riboside, 1- hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3 -phosphoethanolamine, Uridine triacetate, JWH 018 7-hydroxyindole metabolite-d9, 1,6-Dimethylphenazine, 2-(p- Bromophenyl)-8-methyl-8H-thieno(2,3-b)indole, 3,4-Dihydroxy-phenyl glycol, dodecyl cis- 9, 10-epoxy octadecanoate, heptacosane, lauric acid, lignoceric acid, tetratriacontane, tryptophol, one or more derivatives thereof, or a combination thereof; and (ii) a carrier. In some embodiments, the carrier is formulated for application to a plant or medium in which the plant is growing. In some embodiments, the carrier comprises a fertilizer. In some embodiments, the fertilizer is a solid. In some embodiments, carrier is a liquid. In some embodiments, the composition further comprises an adjuvant selected from the group consisting of wetting agent, spreading agent, dispersing agent, sticking agent, dust control agent, and adhesive. In some embodiments, the composition is configured to enhance an availability of zinc in soil. In some embodiments, the composition is configured to enhance zinc uptake by plant tissues. In some embodiments, the composition is configured to promote zinc use efficiency.

[0022] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.INCORPORATION BY REFERENCE

[0023] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0025] FIG. 1 depicts a schematic labeling of components of a Z ST system with a single reactor.

[0026] FIG. 2 depicts a serialized ZST system featuring labeling of a series of reactors and other components.

[0027] FIG. 3 depicts another serialized ZST system featuring a complete mixed reactor and a series of fluidized bed reactors.

[0028] FIG. 4 depicts another serialized ZST system featuring a complete mixed reactor and a series of packed bed reactors with scaffolding.

[0029] FIG. 5 is a graph depicting pH, glucose consumption (in g / L), and zinc solubilization (in mg / L), over time, wherein glucose is added to a ZST batch system A.

[0030] FIG. 6 is a graph depicting pH, glucose consumption (in g / L), and zinc solubilization (in mg / L), over time, wherein glucose is added to a ZST batch system B.

[0031] FIG. 7 is a graph depicting pH, glucose consumption (in g / L), and zinc solubilization (in mg / L), over time, wherein glucose is added to a ZST batch system C.

[0032] FIG. 8 is a graph depicting pH, glucose consumption (in g / L), and zinc solubilization (in mg / L), over time, wherein glucose is added to a ZST batch system D.

[0033] FIG. 9 is a graph showing the comparison of solubilized Zn2+ (in mg / L) in solution between the ZST 2.0 fluidized bed reactor (FBR) and the ZST 2.0 packed bed reactor (PBR) across different phases.

[0034] FIG. 10 is a graph showing the comparison of dissolved oxygen (in mg / L) between the ZST fluidized bed reactor (FBR) and the ZST packed bed reactor (PBR) across different phases.

[0035] FIG. 11 is a table listing the survivability of sixteen bacterial isolates, including MS4666, in a zinc fertilizer (Circa™ Zn).

[0036] FIG. 12 is a graph showing Zn solubilization (mg / L) capacity of target isolates MS4666, MS4687, and MS4689 at different inoculation concentrations using different carbon-sources for growth.

[0037] FIGs. 13A-13B are a series of graphs showing measures of Zn uptake and plant growth in navy beans treated with MAP fertilizer coated with one of four different treatments: water (UTC), Circa™ Zn, MS4666, and MS4666 with Circa™ Zn. FIG. 13A shows the amount of zinc uptake (pg / plant) for navy beans. FIG. 13B shows total biomass, shoot dry weights, and root dry weights across conditions.

[0038] FIGs. 14A-14B are a series of graphs showing measures of plant growth in com treated with MAP fertilizer coated with one of four different treatments: water (UTC), Circa™ Zn, MS4666, and MS4666 with Circa™ Zn. FIG. 14A shows results of measuring zinc uptake per plant for each condition. FIG. 14B shows total biomass, shoot dry weights, and root dry weights across conditions.

[0039] FIGs. 15A-15B are a series of graphs showing evaluation of Zn uptake and plant growth promotion in corn treated with MAP fertilizer coated with one of four different treatments: water (UTC), Circa™ Zn, MS4666, and MS4666 with Circa™ Zn. FIG. 15A shows results of measuring zinc uptake per plant for each condition. FIG. 15B shows total biomass, shoot dry weights, and root dry weights across conditions.

[0040] FIG. 16 is a graph showing the uptake of nitrogen, phosphorus, and potassium (pg / plant) in com treated with MAP fertilizer coated with one of four different treatments: water (UTC), Circa™ Zn (CZN), MS4666, and MS4666 with CZN. For each macronutrient condition, left to right bars are: UTC, CZN, MS4666, MS4666 + CZN.

[0041] FIG. 17 is a graph showing plant growth promotion (measured as dry weights (g)) in com fertilized with MAP fertilizer coated with one of four different treatments: water (UTC), Circa™ Zn, MS4666, and MS4666 with Circa™ Zn. Dry weights were measured for the total biomass, shoot, and root. For each treatment condition, left to right bars are: root, stem, and total biomass.

[0042] FIGs. 18A-18D show measures of growth from com treated with various microbial isolates which were applied in-furrow at planting. Measure were corn height (FIG. 18A), fresh weight (FIG. 18B), and dry weight (FIG. 18C) across control, MS4666, and other isolates (isolate concentration of IE x 104cfu / ml). FIG. 18D is a graph depicting the dry weight analysis results from the tables in FIG. 18A-18C. For each isolate condition, left to right bars are: root, stem, and total.

[0043] FIG. 19 shows in vitro zinc solubilization from solutions from three ZST systems at different concentrations of zinc oxide (ZnO).

[0044] FIG. 20 depicts zinc solubilization capacity of intact solutions from ZST systems and their filter-sterilized (metabolites only) counterparts at a fixed 0.1% ZnO concentration.

[0045] FIG. 21 shows the zinc made available (in mg / L) from MESZ fertilizer prills coated with ZST 2.0 FBR at different application rates.

[0046] FIG. 22 shows the percentage of zinc made soluble from soil fertilized with ZnO after treatment with different concentrations of ZST 1.5.

[0047] FIG. 23 shows the differences in Arabidopsis growth (in average leaf area(cm2)) across intact and filter-sterilized (F / S) solutions for all ZST systems. UTC and Accomplish LM are used as controls.

[0048] FIGs. 24A-24C depict effects of different treatments on the dry biomass of com.Percentages above the bars denote the percent change from UTC. An asterisk denotes significant difference from UTC. FIG. 24A shows the percent change of total dry biomass from UTC across multiple treatments. FIG. 24B depicts the average shoot dry biomass. FIG. 24C depicts the average root dry biomass.

[0049] FIGs. 25A-25C are a series of graphs showing the effects of the loading input components and supernatant (SPN) of the ZST system on plant growth production in corn. FIG. 25A shows total biomass treated with loading inputs and ZST 1.5 SPN. FIG. 25B shows total biomass with fertilizer, loading inputs plus Circa™ Zn. and ZST 1.5 SPN plus Circa™ Zn. FIG. 25C shows total biomass with of Circa™ Zn alone, ZST 1.5 SPN alone, and ZST 1.5 SPN plus Circa™ Zn.

[0050] FIGs. 26A-26B are a series of graphs depicting zinc uptake (in pg / plant) in corn after treatment with ZST SPN or isolate input (MS4666) compared to UTC and Circa™ Zn (CZN). FIG. 26A shows zinc uptake from the input into the ZST system (MS4666) and ZST SPN compared to UTC and CZN alone. FIG. 26B shows zinc uptake from the isolate input into the ZST system in combination with CZN (MS4666 CZN) and from ZST 1.5 SPN with CZN.

[0051] FIG. 27 shows a series of graphs depicting total corn grain yield (in g) across different ZST systems, untreated control (UTC), and Circa™ Zn (CZN) fertilizer alone.

[0052] FIG. 28 depicts the total zinc (ug) measured per ear of corn across different ZST systems, untreated control (UTC), and Circa™ Zn (CZN) fertilizer alone and in combination with ZST systems.

[0053] FIGs. 29A-29B are a series of graphs depicting the total macronutrients (nitrogen, phosphorus, and potassium) measured in mg per ear of corn across different ZST systems, untreated control (UTC), and Circa™ Zn (CZN) fertilizer alone. FIG. 29A shows results for ZST systems without CZn. FIG. 29B shows results for CZN alone and ZST systems with CZn. For each condition, left to right bars are: UTC, ZST 1.5, ZST 2.0 FBR, and ZST 2.0 PBR.

[0054] FIGs. 30A-30B are tables showing macronutrient and micronutrient content in the com grain across ZST treatment systems using an untreated control (UTC) (FIG. 30A) and using CZN fertilizer (FIG. 30B).

[0055] FIGs. 31A-31C are a series of graphs with harvest yield for three vegetables: broccoli (FIG. 31A), cabbage (FIG. 31B), and lettuce (FIG. 31C), measured as boxes / acre.

[0056] FIGs. 32A-32C show a series of graphs with plant growth promotion results of MS4666. Treatment with MS4666 shows superior com yield compared to that from untreated controls (FIGs. 32A-32B). Treatment with MS4666 at a rate of 2.0 qt / acre shows superior soybean yield compared to that from untreated controls (FIG. 32C).

[0057] FIG. 33 shows counts of isolate MS4666 over time following inoculation at a higher concentration and recirculation of floc.

[0058] FIG. 34 shows a distance-based neighbor joining phylogenetic tree based on 16s rRNA gene.

[0059] FIG. 35 shows results from a zinc solubilization capacity in vitro assay between the target isolate MS4666, PST Floc, and ZST-1.0 Batch solutions using PST Floc (Batch-A) or PST Floc with MS4666 (Batch-B).

[0060] FIG. 36 shows soil analysis results of Zn Mehlich-3 extractable Zn (Zn-bioavailability) bioavailability between target isolates, at 1.00E+4 cfu / mL and 1.00E+6 CFU / mL, and control treatments.

[0061] FIG. 37 shows a graph depicting concentrations of total bacteria and Zn-solubilizers across ZST- 1.5 process and base product.

[0062] FIG. 38 shows measurements of Zn2+ made soluble from ZST-1.5 Reactor 1 (Rl) and ZST-1.5 Base Product. * indicates p < 0.0001.

[0063] FIG. 39 shows concentration of Zn2+ found in ZST-2.0 FBR and ZST-2.5 reactors and base product.

[0064] FIG. 40A shows results measuring Zn2+ made soluble in ZET experiment from each treatment condition (ZST-2.5 BP, ZST-2.5 BP+MS4666, and ZST-2.0 FBR BP). FIG. 40B shows results measuring Zn2+ made soluble in ZST experiment from each treatment condition (UTC, ZST-2.5 BP, ZST-2.5 BP+MS4666, and ZST-2.0 FBR BP).

[0065] FIG. 41A shows Arabidopsis average leaf area (cm2) for plant growth promotion. FIG.41B shows Arabidopsis average leave area (cm2) for abiotic (salt) stress relief. FIG. 41C shows Arabidopsis average leaf area (cm2) for abiotic (cold) stress relief.

[0066] FIG. 42A and FIG. 42B show principal component analysis (PCA) based on gas chromatography-mass spectrometry (GC-MS) analysis of ZST-sIP and ZST-non sIP baseproducts of intact sample (FIG. 42A) and filter-sterilized sample (FIG. 42B). FIG. 42C shows a Venn Diagram of the number of up-regulated compounds in the intact and filter-sterilized samples of ZST-sIP over ZST-non sIP from GC-MS analysis. FIG. 42D and FIG. 42E show PCA based on LC-MS analysis of ZST-sIP and ZST-non sIP base products of intact sample (FIG. 42D) and filter-sterilized sample (FIG. 42E). FIG. 42F shows a Venn Diagram of the number of up-regulated compounds in the intact and filter-sterilized samples of ZST-sIP over ZST-non sIP from LC-MS analysisDETAILED DESCRIPTION

[0067] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0068] Described herein are systems and methods that employ microbial digestion of various feedstocks. A system of the present disclosure may comprise a continuous system capable of serialized isolate production (e.g., sIP system). The isolate production of the sIP system can occur within a mixed consortium of microbes. The target isolates of a sIP digestion system may become enriched in the microbial environment and may demonstrate improved efficacy and functionality. The main targeted functionality may be zinc solubilization of bound zinc from soil or fertilizer and improved nutrient uptake in plants. A target isolate may possess commercially valuable properties and can be introduced into a continuous (e.g., serialized) reactor system comprised of a complex microbial consortia that has been modified for functionality (e.g., for zinc solubilization). Without wishing to be bound by theory, a target isolate may provide a performance benefit to a microbial community of the digestions systems described herein, providing a chemical and / or functional synergistic relationship as it grows in the system. A digestion system described herein may comprise a zinc solubilization technology (ZST) system, with a target function to enrich a population or populations of zinc-solubilizing microbes, metabolites, or any combination thereof.

[0069] The products of digestion methods and systems described herein can include microbes and metabolites produced by microbial digestion of feedstock substrates. In some embodiments, the products of digestion methods and systems described herein can comprise biostimulant compositions that have plant growth promoting properties when applied to plants or to a medium in which plants are growing (e.g., soil). In some embodiments, methods and systemsdescribed herein are arranged to selectively promote growth of microbes that have a desired plant growth promoting property themselves or that produce metabolites that have the desired plant growth promoting property, such that the biostimulant product has the desired plant growth promoting property. Applications of the products of the digestion systems described herein may be on dry-fertilizers, applied in conjunction with the application of fertilizers, in formulations with additional components including liquid fertilizers or micronutrient coating formulations, and / or in foliar applications. Applications of the products of the digestion systems described herein may be to a part of a plant, such as a shoot, a stem, a leaf, a lateral bud, a terminal bud, a flower, a leaf axil, a root (e.g., a primary root, a lateral root, a root hair, a root cap), or any combination thereof. These and other features of embodiments disclosed herein are described in more detail below.A. Definitions

[0070] In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments. However, one skilled in the art will understand that the embodiments provided may be practiced without these details. Unless the context requires otherwise, throughout the specification and claims which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense, that is, as “including, but not limited to.” As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise. Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.

[0071] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” should be assumed to mean an acceptable error range for the particular value.

[0072] The term “culturing”, as used herein, can refer to the propagation of organisms on or in media of various kinds. Non-limiting examples of suitable media include tryptic soy agar (TSA), zinc agar, nutrient medium, lysogeny broth (LB medium), and / or plate count agar.

[0073] As used herein, the term “enriched culture” of an isolated microbial strain can refer to a microbial culture wherein the total microbial population of the culture contains a percentage of a target isolated strain. An enriched culture may comprise a percentage of a target isolated strain and a population of microbes enriched for a particular functionality (e.g., zinc solubilization). In some embodiments, an enriched culture may comprise a percentage of a target isolated strain, a population of microbes enriched for a particular functionality, and metabolites enriched for a particular functionality (e.g., zinc solubilization). The enriched culture may comprise a percentage of a total bacteria population in a container of digestion system described herein. The enriched culture may comprise a percentage of a total bacteria population in an output product (e.g., biostimulant) described herein. An enriched culture may comprise an increased amount of a target isolated strain and / or a target population of microbes compared to a total microbial population of a culture. An enriched culture may comprise a growing population of a target isolated strain and a population of microbes enriched for a particular functionality (e.g., zinc solubilization) over a time period. In some embodiments, an enriched culture can refer to a microbial culture wherein the total microbial population of the culture contains at least about 0.001%, at least about 0.01%, at least about 0.05%, at least about 0.1%, at least about 0.5%, at least about 1%, at least about 1.5%, at least about 2%, at least about 2.5%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, or at least about 75% of a target isolated strain, a population of microbes enriched for a particular functionality, metabolites enriched for a particular functionality, or any combination thereof. In some embodiments, an enriched culture can refer to a microbial culture wherein the total microbial population of the culture contains at most about 75%, at most about 50%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, at most about 2.5%, at most about 2%, at most about 1.5%, at most about 1%, or at most about 0.5% of a target isolated strain, a population of microbes enriched for a particular functionality, metabolites enriched for a particular functionality, or any combination thereof. In some embodiments, an enriched culture can refer to a microbial culture wherein the total microbial population of the culture contains from about 0.5% to about 75% of a target isolated strain, a population of microbes enriched for a particular functionality, metabolites enriched for a particular functionality, or any combination thereof. In some embodiments, an enriched culture can refer to a microbial culture wherein the total microbial population of the culture contains from about 0.5% to about 1%, about 0.5% to about 2%, about 0.5% to about 3%, about 0.5% to about 5%, about 0.5% to about 10%, about 0.5% to about 15%, about 0.5% to about 20%, about 0.5% to about 25%, about 0.5% to about 50%, about 0.5% to about 60%, about 0.5% to about75%, about 1% to about 2%, about 1% to about 3%, about 1% to about 5%, about 1% to about 10%, about 1% to about 15%, about 1% to about 20%, about 1% to about 25%, about 1% to about 50%, about 1% to about 60%, about 1% to about 75%, about 2% to about 3%, about 2% to about 5%, about 2% to about 10%, about 2% to about 15%, about 2% to about 20%, about 2% to about 25%, about 2% to about 50%, about 2% to about 60%, about 2% to about 75%, about 3% to about 5%, about 3% to about 10%, about 3% to about 15%, about 3% to about 20%, about 3% to about 25%, about 3% to about 50%, about 3% to about 60%, about 3% to about 75%, about 5% to about 10%, about 5% to about 15%, about 5% to about 20%, about 5% to about 25%, about 5% to about 50%, about 5% to about 60%, about 5% to about 75%, about 10% to about 15%, about 10% to about 20%, about 10% to about 25%, about 10% to about 50%, about 10% to about 60%, about 10% to about 75%, about 15% to about 20%, about 15% to about 25%, about 15% to about 50%, about 15% to about 60%, about 15% to about 75%, about 20% to about 25%, about 20% to about 50%, about 20% to about 60%, about 20% to about 75%, about 25% to about 50%, about 25% to about 60%, about 25% to about 75%, about 50% to about 60%, about 50% to about 75%, or about 60% to about 75% of a target isolated strain, a population of microbes enriched for a particular functionality, metabolites enriched for a particular functionality, or any combination thereof.

[0074] The term “composition” as used herein can refer to a combination of an active agent (e.g., a microbial strain described herein) and at least one other compound, carrier, or composition, which can be inert (for example, a detectable agent or liquid carrier) or active, such as, but not limited to, a fertilizer, nutrient, or pesticide. A microbial composition refers to a composition comprising at least one microbial species. A composition may comprise microbial metabolites generated in a microbial consortium of a digestion system described herein.

[0075] An “effective amount”, as used herein, can refer to an amount sufficient to effect beneficial and / or desired results. An effective amount can be administered in one or more administrations. The expression “effective microorganism” used herein in reference to a microorganism is intended to mean that the subject strain exhibits a degree of promotion of plant health, growth and / or yield, at a statistically significant level, that of an untreated control. In some instances, the expression “an effective amount” is used herein in reference to that quantity of microbial treatment which can be used to obtain a beneficial or desired result relative to that occurring in an untreated control under suitable conditions of treatment as described herein. For example, the expression “an agriculturally effective amount” is used herein in reference to that quantity of microbial treatment which can be used to obtain an agriculturally beneficial or desired result relative to that occurring in an untreated control under suitable conditions oftreatment as described herein. The effective amount of an agricultural formulation or composition that may be applied for the improvement of plant health, growth and / or yield, can be readily determined.

[0076] A “carrier” as used herein can refer to a substance or a composition that support the survival of the microbes. Such carriers may be either organic or non-organic.

[0077] “Percentage of sequence identity”, as used herein, can be determined by comparing two optimally locally aligned sequences over a comparison window defined by the length of the local alignment between the two sequences. The amino acid sequence in the comparison window may comprise additions or deletions (e. g., gaps or overhangs) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.

[0078] Local alignment between two sequences may include segments of each sequence that are deemed to be sufficiently similar according to a criterion that depends on the algorithm used to perform the alignment (e. g. BLAST). The percentage of sequence identity is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (Add. APL. Math. 2:482, 1981), by the global homology alignment algorithm of Needleman and Wunsch (J Mol. Biol. 48:443, 1970), by the search for similarity method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85:2444, 1988), by heuristic implementations of these algorithms (NCBI BLAST, WU-BLAST, BLAT, SIM, BLASTZ), or by inspection. Given that two sequences have been identified for comparison, GAP and BESTFIT may be employed to determine their optimal alignment. Typically, the default values of 5.00 for gap weight and 0.30 for gap weight length are used. The term “substantial sequence identity” between polynucleotide or polypeptide sequences refers to polynucleotide or polypeptide comprising a sequence that has at least about 50% sequence identity, at least about 60% sequence identity, at least about 70% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity compared to a reference sequence using the programs. In addition, pairwise sequence homology or sequence similarity, as used, refers to the percentage of residues that are similar between two sequences aligned. Families of amino acid residues having similarside chains have been well defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Query nucleic acid and amino acid sequences can be searched against subject nucleic acid or amino acid sequences residing in public or proprietary databases. Such searches can be done using the National Center for Biotechnology Information Basic Local Alignment Search Tool (NCBI BLAST v 2.18) program. The NCBI BLAST program is available on the internet from the National Center for Biotechnology Information (blast.ncbi.nlm.nih.gov / Blast.cgi). Typically the following parameters for NCBI BLAST can be used: Filter options set to “default”, the Comparison Matrix set to “BLOSUM62”, the Gap Costs set to “Existence: 11, Extension: 1”, the Word Size set to 3, the Expect (E threshold) set to le-3, and the minimum length of the local alignment set to 50% of the query sequence length. Sequence identity and similarity may also be determined using GenomeQuest™ software (Gene-IT, Worcester Mass. USA).

[0079] The term “plant growth promotion” (e.g., “PGP”) can refer to processes that can promote plant health, growth and / or yield. In some embodiments, PGP can encompass a wide range of improved plant properties, including but not limited to, improved nitrogen fixation, improved phosphate uptake, improved zinc uptake, improved root development, increased leaf area, increased plant yield, increased uptake of macronutrients, increased uptake of micronutrients, increased seed germination, enhancing seed germination, enhancing early plant development, improving root growth, improving shoot growth, improving plant height, increasing nutrient uptake, mitigating transplant shock, improving plant reproduction, improving soil microbial activity, increased photosynthesis, increased abundance of functional enzymes, increased dry biomass, or an increase in accumulated biomass of the plant. In some embodiments, the microbial strains, isolates, cultures, compositions or synthetic consortia as described herein improve nutrient uptake, plant health and vigor, improve root development, increase leaf area, increase plant yield, increased uptake of macronutrients, increased uptake of micronutrients, increase seed germination, increased abundance of functional enzymes, increased dry biomass, or an increase in accumulated biomass of the plant. In some embodiments, the microbial strains, isolates, cultures, or compositions as described herein increase the size or mass of a plant or parts thereof, as compared to a control plant, or a plant that has not been treated with a substance, or parts thereof or as compared to a predetermined standard. In some embodiments,the microbial strains, isolates, cultures, compositions or synthetic consortia as described herein improve the health, vigor and yield of a plant, as compared to a control plant or a plant that has not been treated with a substance, but also can survive and multiply in microhabitats associated with the root surface.

[0080] As used herein, the term “yield” can refer to the amount of harvestable plant material or plant-derived product, and is normally defined as the measurable produce of economic value of a crop.

[0081] For crop plants, “yield” can also mean the amount of harvested material per acre or unit of production. Yield may be defined in terms of quantity or quality. The harvested material may vary from crop to crop, for example, it may be seeds, above ground biomass, roots, fruits, cotton fibers, any other part of the plant, or any plant-derived product which is of economic value.

[0082] In some embodiments, microbial strains, isolates, cultures, and compositions, and biostimulant compositions described herein may be used to promote improved crop or product quality, including food quality. Improving crop quality may include improving characteristics that make a crop or product more marketable such as, for example, a desired color, size, or shape. Improving crop quality may also including making a food product with desired nutritional, nutraceutical, and / or pharmaceutical characteristics. Improving crop or product quality may include adding valuable attributes that may increase price. Improving crop or product quality may include increasing value per harvest unit, such as, for example, providing increased oil, sugar, starch, or protein yield per unit weight of harvested crop or product.

[0083] In some embodiments, the microbial strains, isolates, cultures and compositions according to the embodiments of this application can lead to plant growth promotion or plant growth improvement that is an at least 5% increase, at least 10% increase, at least 25% increase, at least 50% increase, at least 75% increase, or at least a 100% increase in the property being measured. In some embodiments, the microbial strains, isolates, cultures and compositions according to the embodiments of this application lead to plant growth promotion or plant growth improvement that is an at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% increase in the property being measured. In some embodiments, the microbial strains, isolates, cultures and compositions of this application may produce an above stated percentage increase in zinc uptake, an above stated increase in zinc solubilization capacity an above stated increase in total root weight, or in leaf area or in plant product yield (e.g., an above stated percentage increase in plant product weight).

[0084] A “control plant”, as used herein, can provide a reference point for measuring changes in phenotype of the subject plant, and may be any suitable plant cell, seed, plant component, plant tissue, plant organ or whole plant. A control plant may comprise, but is not limited to, (a) a plant which is genetically identical to the subject plant but which is not exposed to the same treatment (e.g., inoculant treatment) as the subject plant or (b) the subject plant itself, under conditions in which it has not been exposed to a particular treatment such as, for example, an inoculant or combination of inoculants and / or other chemicals. A treated plant may comprise a plant that has had an inoculum of a microbe or a biostimulant composition as described herein applied to any part of the plant (e.g., seed, stem, root, shoot, leaf, or combination thereof). A treated plant may comprise a plant that has had an inoculum of a microbe or a biostimulant composition as described herein applied using an in-furrow application. A treated plant may comprise a plant that has had an inoculum of a microbe or a biostimulant composition as described herein applied using a side-dress application a broadcast application, a y-drop application, a tape application, or a fertigation application. A treated plant may comprise a plant that has had an inoculum of a microbe or a biostimulant composition as described herein applied to the soil. An untreated plant may comprise a plant that that has not had an inoculum of a microbe or a biostimulant composition as described herein applied directly or indirectly.

[0085] “Inoculant” as used herein can refer to any culture or preparation that comprises at least one microorganism. In some embodiments, an inoculant (sometimes as microbial inoculant, or soil inoculant) is an agricultural addition that uses beneficial microbes (including, but not limited to endophytes) to promote plant health, growth and / or yield. Many of the microbes suitable for use in an inoculant form symbiotic relationships with the target crops where both parties benefit (mutualism). For example, an isolated microbial strain as described herein may benefit from carbon sources from the roots of a plant and the plant may benefit from metabolites generated by metabolism of the microbe. Without wishing to be bound by theory, a plant may be colonized by the isolate and the colonization of the roots may block plant pathogens from accessing the roots and / or it could provide plant growth benefits to the plant. An inoculant (e.g., inoculum of a microbe / microbial strain) can be added at one time point during an operating of a digestion system process. An inoculant (e.g., inoculum of a microbe / microbial strain) can be added at multiple time points during an operating of a digestion system process.

[0086] The term “serialized isolate production”, (e.g., sIP), can refer to specialized manipulated continuous serialized reactors that may enable the growth and enrichment of the microbes, isolates, target isolates, and / or microorganisms as described herein.

[0087] The term “floc” can refer to a mass formed by the aggregation of a number of fine suspended particles. For example, a floc can comprise organic materials recovered from a feedstock, waste, wastewater, and / or sludge material of a fluid used in a digestion system. A floc can comprise biosolids and / or particles from digestion products of organic materials. Floc can comprise an aggregated mass of microorganisms (e.g., bacteria).

[0088] The term “whole broth” (e.g., WB) can refer to a blend of supernatant and floc at a ratio for use in the technologies as described herein. A whole broth may comprise microbial populations (e.g., zinc-solubilizing microbes), enzymes, fungi, biosolids, or any combination thereof. For example, bacterial genera of a whole broth may comprise Haliscomenobacter, Lewinella, Caldilinea, Terrimonas, Acidobacterium, Lewinella cohaerens, Thauera phenylacetica, Thauera mechernichensis, Solitalea canadensis, Nitrospira moscoviensis, or any combination thereof. A whole broth may have plant growth promotion properties. For example, a whole broth may have nitrogen-fixation capacity.

[0089] The terms “microbial consortium” or “microbial population” can refer to a group of microorganisms in an environment. Consortiums may be endosymbiotic or ectosymbiotic. Microorganisms in a microbial consortium can include, but are not limited to, bacteria, fungi, yeasts, lichens, algae, protozoa, archaea, and / or molds.

[0090] The term “supernatant” (e.g., “base product”) can refer to the final product of the digestion system. The supernatant can be measured for amount of a microbial isolate, number of members within a microbial consortium, or types and amount of microbial metabolites with plant growth promotion capacity.

[0091] The term “load rate” can refer to a rate at which a source material is introduced into a digestion system. In some embodiments, load rate may refer to “organic load rate” or “hydraulic load rate”. Organic load rate comprises a rate at which feedstock (e.g., organic feedstock) is introduced into the system. Hydraulic load rate comprises a rate at which a hydraulic source is introduced into the system.

[0092] The term “internal recycle rate” can refer to a rate at which a working fluid is recycled within a phase space.

[0093] The term “hydraulic feed rate” can refer to a rate at which working fluid is transferred between phase spaces.

[0094] The term “hydraulic dwell time” can refer to an amount of time that a working fluid is present in a phase space.

[0095] The term “working fluid” can refer to a fluid substance supporting and transporting biology and nutrients through a system of containers. For example, a working fluid maycomprisen organic materials, microorganisms (e.g., microbes and / or metabolites), biosolids, macronutrients, micronutrients, organic nutrients, inorganic nutrients, or any combination thereof. A working fluid can comprise a solution that flows throughout a digestion system and may provide an enriched environment for microbes of the digestion system.

[0096] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” can apply to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0097] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” can apply to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.B. Microbial Digestion Methods and Systems

[0098] Certain embodiments disclosed herein include methods and systems in which microbes comprised in microbial consortia digest substances provided in a feedstock. The digestion systems may be comprised of a series of separate, fluidly connected containers, also referred to herein as “reactors.” In each reactor, a different microbial consortium may be established and maintained throughout continuous operation of the digestion system. The unique microbial consortia present in each reactor may provide for different physiological activities in the different reactors. Thus, different steps in digestion of a feedstock may be performed in different reactors, which may result in (1) a more complete digestion — i.e., more complete breakdown of macromolecules in the feedstock — than other types of digestion systems, and / or (2) production of a variety of microbial digestion products having plant growth promoting properties (e.g., ability to enhance or improve soil available zinc via zinc solubilization, zinc uptake by plant tissues, and / or otherwise promote zinc use efficiency). In some embodiments, conditions that may allow for an enrichment of a microbial community with a targeted functionality (e.g., zinc solubilization) may comprise a retention time, addition of a selective pressure, hydraulic flow rate, one or more carbon sources, one or more nitrogen sources, pH, nutrient mix, microaerobic conditions, recirculation of a working fluid, or any combination thereof.

[0099] In some embodiments, the bioreactor system (e.g., the digestion system) comprises an established population of one or more zinc-solubilizing microbial strains in one or morecontainers of the system. An “established population” of a particular microbial strain is a population that remains within an operating bioreactor system without replenishing the microbial strain from outside the bioreactor system. In some embodiments, an established population is one that has not been diminished by more than about 1, 3, 5, 10, 15, 20, or 25% during continuous operation of the bioreactor system for at least about 5, 10, 15, 20, 25, 30, 60, or 90 days without adding a population of the microbial strain to the bioreactor system at a concentration higher than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 25, 50, or 100 CFU / ml. In some embodiments, an established population of a microbial strain has been established by making one or more inoculations of the microbial strain into one or more containers of the bioreactor system. In some embodiments, a measurement (e.g., a concentration or bacterial count) of an established population may be performed for a digestion system. In some embodiments, an established population is a population that is derived from a microbial population that was inoculated into the system at least about 5, 10, 20, 30, 40, 50, 60, or 90 days prior to a measurement of the established population.

[0100] In some embodiments, a bioreactor system comprises at least one microbial strain. In some embodiments, a bioreactor system comprises at least one zinc-solubilizing microbial strain. In some embodiments, a bioreactor system comprises an established population of a first zinc-solubilizing microbial strain and an established population of a second zinc-solubilizing microbial strain. In some embodiments, the bioreactor system further comprises an established population of a third zinc-solubilizing microbial strain. The established populations of the respective microbial strains may be established in individual or combined inoculations into the bioreactor system. An individual inoculation may comprise one inoculum of a microbial strain (e.g., microbe). A combined inoculation may comprise an inoculum comprising at least two microbial strains. The combined inoculation may comprise the same isolated microbial strains. The combined inoculation may comprise an isolated microbial strain and non-isolated microbial strain. The combined inoculation may comprise two or more isolated microbial strains.

[0101] In some embodiments, a reactor or a series of reactors (e.g., serialized assembly of reactors) functions to contribute to the growth of one or more microbes having desired plant growth promoting properties and / or to the production of digestion products having plant growth promoting properties. The system can comprise 1, 2, 3, 4, 5, 6, or more reactors. In some embodiments, the operation of a digestion system may lead to growth of one or more microbes having a desired plant growth promoting effect. The one or more microbes may be one or more isolated microbes added separately as an inoculum to the digestion system. The one or more microbes may also be input into the system as part of a feed material that includes a mixture ofmicrobes. The one or more microbes may be endogenous to an organic material such as, for example, a manure, a plant, a lignocellulosic material, or an algae. The one or more microbes may also be endogenous to other types of feed materials, such as rock phosphate. In some embodiments, endogenous microbes are those microbes naturally present in feedstock material (e.g., a manure, a plant, a lignocellulosic material, or an algae). These microbes may naturally reside in a closed system and / or are present in the ecosystem of the feedstock material.

[0102] Inputs into digestion systems may include one or more of water, a microbial inoculum (e.g., inoculum of a microbe or inoculum of a microbial strain), nutrients (e.g., one or more sources of carbon, nitrogen, phosphorous, etc.), and a digestion substrate. Fluid within reactors of a digestion system may be referred to herein as a “working fluid.” In continuous operation, each reactor may have a constant volume of working fluid therein, with the rate of fluid flowing into each reactor matching the rate of fluid flowing out of each reactor. As each reactor may include a different microbial consortium and have different conditions from other reactors, the working fluid within each reactor may be considered to be distinct from working fluids within the other reactors. The total volume of working fluid within a digestion system may be referred to herein as the “total working volume” of the digestion system.

[0103] Digestion substrates included in an input stream into a digestion system may include, for example, organic materials that can be digested by microbes in the digestion system. Such organic materials may include, for example, manure, lignocellulosic material, wastewater biosolids, food waste, energy crops, yeast, guano, agricultural waste, algae, or any combination thereof. The manure may be chicken manure, cow manure, horse manure, sheep manure, alpaca manure, rabbit manure, and / or pig manure. In some embodiments, the manure is a mixture of one, two, three, or more manures. Digestion substrates input into digestion systems may have been subject to a partial digestion before being input into the system. Thus, the input into the system may include products of digestion of an original digestion substrate by microbes endogenous to the original digestion substrate, as well as digestible materials still present in the input. In some embodiments, digestion substrates included in an input stream may include an inorganic substrate. The inorganic substrate may include, for example, sand, vermiculite, perlite, and / or pumice. In some embodiments, the inorganic substrates comprises a mineral. In some embodiments, the inorganic substrate comprises rock phosphate. In some embodiments, an input stream may include a source of zinc such as, for example, an insoluble form of zinc.

[0104] In some embodiments, a microbial inoculum comprises a single isolated microbe. An isolated microbe may comprise a microbe generated outside of a natural environment (e.g., by plating methods or culture mediums). In some embodiments, the microbial inoculum maycomprise between 1 and 5 isolated microbes (e.g., microbial strains). In some embodiments, the inoculum may comprise 1, 2, 3, 4 or 5 isolated microbes. In some embodiments, the inoculum may comprise greater than 5 isolated microbes. In some embodiments, in addition to one or more isolated microbes, a microbial inoculum input into a digestion system may include a complex mixture of microbes, which may include at least 5, 10, 20, 25, 50, 100, 200, 225, 250, 275, 300, 350, 400, or more species of microbes.

[0105] An inoculum of a microbe as described herein may have at least one plant growth promotion property (e.g., a property of plant growth). A plant growth promotion property may comprise shoot biomass, root biomass, nutrient uptake, crop yield, leaf area, chlorophyll content, photosynthetic activity, zinc uptake, zinc solubilization, micronutrient uptake, or total biomass. A digestion system may be configured to enhance production of the inoculum of the microbe. A microbe may be a bacterial species, a fungal species, or an algal species.

[0106] In some embodiments, the inoculum of a microbe may comprise at least two isolated microbes. In some embodiments, the inoculum of a microbe may comprise at least one isolated microbe and at least one non-isolated microbe. In some embodiments, the inoculum of a microbe may comprise 1, 2, 3, 4, 5, or more microbes. An inoculum of a microbe may be transferred to a first container (e.g., reactor) of a digestion system one time, two times, three times, four times, five times, or more. An inoculum of a microbe may be provided to a second container, a third container, a fourth container, a fifth container, a sixth container, or any container of a system described herein.

[0107] An inoculum of a microbe may have a concentration of at least about, at most about, or about 1.0 x 102cfu / ml, 1.0 x 103cfu / ml, 1.0 x 104cfu / ml, 1.0 x 105cfu / ml, 1.0 x 106cfu / ml, 1.0 x 107cfu / ml, 1.0 x 108cfu / ml, 1.0 x 109cfu / ml, 1.0 x 1010cfu / ml, 1.0 x 1011cfu / ml, or 1.0 x 1012cfu / ml, or a range between any of these two values, prior to transferring to a first container of a digestion system. An inoculum of a microbe may have a concentration of at least about, at most about, or about 1.0 x 102cfu / ml, 1.0 x 103cfu / ml, 1.0 x 104cfu / ml, 1.0 x 105cfu / ml, 1.0 x 106cfu / ml, 1.0 x 107cfu / ml, 1.0 x 108cfu / ml, 1.0 x 109cfu / ml, 1.0 x 1010cfu / ml, 1.0 x 1011cfu / ml, or 1.0 x 1012cfu / ml, or a range between any of these two values, after incubation in a digestion system described herein.

[0108] In some embodiments, the inoculum comprises a single microbe. In some embodiments, the inoculum may comprise between 1 and 5 microbes. In some embodiments, the inoculum may comprise at least 1, at least 2, at least 3, at least 4, at least 5, or more microbes. In some embodiments, the inoculum may comprise at most 5, at most 4, at most 3, at most 2, or at most 1 microbe(s).

[0109] In some embodiments, the feedstock (e.g., aqueous organic feedstock) and microbial inoculum may be transferred to the first reactor separately. In some embodiments, the feedstock (e.g., aqueous organic feedstock) is transferred to the first reactor before the microbial inoculum. In some embodiments, the microbial inoculum is transferred to the first reactor before the feedstock (e.g., aqueous organic feedstock). In some embodiments, the feedstock (e.g., aqueous organic feedstock) and microbial inoculum may be transferred to the first reactor together.

[0110] In some embodiments, the aqueous feedstock may not contain the target isolate strain (e.g., an inoculum of the microbe). For example, the aqueous feedstock may not contain the target isolate strain prior to transfer to a first container. The concentration of the target isolate microbial strain may be 0 cfu / ml. In some embodiments, the aqueous feedstock may contain the target isolate strain prior to transfer to a first container. In some embodiments, the aqueous feedstock may contain at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1%, at most about 0.5%, at most about 0.1%, at most about 0.05%, at most about, 0.04%, at most about 0.03%, at most about 0.02%, at most about 0.01%, at most about 0.008%, at most about 0.005%, at most about 0.004%, at most about 0.003%, at most about 0.002%, at most about 0.001%, at most about 0.0001%, or less than about 0.0001% of the target isolate strain. In some embodiments, the aqueous feedstock may not include the microbial strain (e.g., the zinc-solubilizing microbial strain) at a concentration higher than about 1 CFU / ml, 2 CFU / ml, 3 CFU / ml, 4 CFU / ml, 5 CFU / ml, 6 CFU / ml, 7 CFU / ml, 8 CFU / ml, 9 CFU / ml, 10 CFU / ml, 11 CFU / ml, 12 CFU / ml, 13 CFU / ml, 14 CFU / ml, 15 CFU / ml, 20 CFU / ml, 25 CFU / ml, 30 CFU / ml, 40 CFU / ml, or 50 CFU / ml.[OHl] In some embodiments, the digestion system comprises a clarifier chamber or clarifier tank (CLF). The clarifier may comprise a single in-flow port and a single out-flow port. The clarifier may comprise a single in-flow port and multiple out-flow ports. In some embodiments, the clarifier comprises floc-folding wipes which rotate and release microbes that have been immobilized in the floc without introducing solids in the supernatant. The floc-folding wipers may move a working fluid in the clarifier to re-suspend microbes within the working fluid. In some embodiments, the microbes and / or an amount of target isolate strain may be re-suspended in the solution in the clarifier and transferred to the supernatant (e.g., base product). In some embodiments, the clarifier further comprises a flow line to return floc to the first reactor. The flow line may comprise a conduit from the clarifier to a container or combination of containers of the digestion system (e.g., a first container, a second container, a third container, a fourth container, a fifth container, a sixth container, or any combination thereof). The clarifier may return flow to any container (e.g., a first container, a second container, a third container, a fourthcontainer, a fifth container, a sixth container, or any combination thereof) of a digestion system to provide a recirculation of working fluid. The working fluid recirculated from the clarifier may comprise a different microbial community (e.g., different amounts of microbes) than a working fluid of another container in the digestion system (e.g., a first working fluid, a second working fluid, a third working fluid, a fourth working fluid, a fifth working fluid, and / or a sixth working fluid). Without wishing to be bound by theory, the recirculation of flow from the clarifier to a container of the digestion system may help enrich a microbial community of a microbial consortium of a digestion system by providing working fluid from the clarifier to a different point (e.g., container) of the system. The recirculated working fluid may comprise organic materials, microbes of a microbial consortium, a target isolate, metabolites, or any combination thereof.

[0112] In the clarifier, a floc portion of a working fluid (e.g., a clarifier working fluid) may separate from a supernatant portion of a working fluid. The floc-folding wipers of the clarifier may help in separating the working fluid of the clarifier. In some embodiments, the separating may comprise gravity separation. The floc may settle on the bottom of the clarifier and the supernatant may be collected.

[0113] Biostimulant compositions produced by a digestion process as described herein may be used as-is or may be further processed before being used. For example, the outflow from the digestion system, referred to herein as “base product,” may be concentrated, sterilized, filtered, pasteurized, dehydrated before being used, or any combination of these. In some embodiments, the base product may be concentrated 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, or more. In some embodiments, the base product may be filter sterilized to remove any bacteria or other microbes in the composition.

[0114] In an aspect, provided herein is a method comprising transferring a feedstock (e.g., aqueous organic feedstock) into a first container. An inoculum of a microbe may be transferred into a first container. A feedstock (e.g., a aqueous organic feedstock) may be transferred into a first container. A feedstock (e.g., a aqueous organic feedstock) and an inoculum of a microbe may be transferred into a first container. The first container may comprise a volume of a first working fluid. The feedstock (e.g., aqueous organic feedstock) may comprise a microbial consortium. The feedstock (e.g., aqueous organic feedstock) may comprise digestion products produced by digestion of an organic material. The feedstock (e.g., aqueous organic feedstock) may comprise a microbial consortium and digestion products produced by digestion of an organic material. The organic material may be digested by one or more microbes in the microbial consortium. The organic material may be digested by a population of microbes of theinoculum of a microbe, such as a zinc solubilizing target isolate. The digestion products described herein may comprise sugars (e.g., xylose, mannose, glucose, or any combination thereof), metabolites generated by microbes of the working fluid, fatty acids, dead microorganisms, fragments of dead microorganisms, microorganism fermentation products, enzymes, biological plant growth regulators, organic acids, chelators, or any combination thereof. The method may further comprise incubating the inoculum of a microbe under conditions that selectively promotes growth of the microbes and increases the population of the microbes. The method may further comprise incubating the inoculum of a microbe under conditions that selectively promotes growth of at least a portion of microbes in the microbial consortium. The terms “microbial digestion” and “digestion” can be used interchangeably.

[0115] In some embodiments, the digestion is anaerobic digestion. In some embodiments, the digestion is aerobic digestion. In some embodiments, the digestion is microaerobic digestion. In some embodiments, the digestion is aerobic digestion, microaerobic digestion, anaerobic digestion, or some combination thereof. Without wishing to be bound by theory, it is believed that during the digestion process, microbes digest the biomolecules and other nutrients present in the manure, yeast, kelp, and / or produce digestion products that include compounds that promote plant growth and soil health. In some embodiments of a digestion process, the feedstock (e.g., organic feedstock) may be mixed with water to make a feedstock (e.g., organic feedstock) for an anaerobic digestion system. The anaerobic digestion system may include a mixing tank in which the feedstock (e.g., organic feedstock) is mixed to make a fluid feed mixture or working fluid. In some embodiments, the fluid feed mixture may include manure, water, and Saccharomyces cerevisiae yeast. In some embodiments, anaerobic digestion comprises a process by which bacteria break down organic biomaterials in the absence of oxygen. The biostimulant may also contain microbes that contribute to the plant-beneficial properties of the biostimulant product. The microbes in the biostimulant product may be derived from the microbial population present in the feedstock (e.g., organic feedstock).

[0116] In an exemplary system of the present disclosure, a series of reactors functions to contribute to the growth of an inoculum of a microbe (e.g., isolate) having desired plant growth promoting properties. A series of reactors (e.g., serialized assembly of reactors) may also function to contribute to the production of microbial metabolites having desired plant growth promoting properties. The digestion system described herein can enrich an inoculum of a target microbe, a population of microbes within a microbial consortium with plant-growth promotion properties (e.g., zinc solubilization), metabolites produced by the enriched population of the microbial consortium with plant-growth promotion properties (e.g., zinc solubilization), or anycombination thereof. This system provides added benefits to other digestion systems in that it can target a functional community of microbes and / or metabolites with specific functionality and enrich and / or maintain the community in the digestion system. The system can comprise two, three, four, five, or more reactors chambers (e.g., containers or chambers). Without wishing to be bound by theory, the serialized reactors enable the growth and enrichment of proprietary specialist target microbes with optimal plant growth promoting properties. The system may direct a flow of working fluid comprising an inoculum, carbon source, and / or nutrient source from an input feedstock (e.g., organic feedstock) to produce a base product (BP). A hydraulic source can flow into a reactor via in-flow port to comprise a first working fluid in a reactor tank. A substance providing selective pressure can flow into a reactor via in-flow port to comprise a first working fluid in a reactor tank. A hydraulic source and a substance providing selective pressure can flow into a reactor via in-flow port to comprise a first working fluid in a reactor tank. A hydraulic source may input into a first reactor or any reactor of the system. In some embodiments, a hydraulic source may input (e.g., flow) into a tank or container prior to a first reactor. In some embodiments, the container may comprise a “complete mixed reactor” (CMR). Other inputs into a system described herein may flow into any reactor of the system, including but not limited to a first reactor, a second reactor, a third reactor, or any other reactor following a first reactor.

[0117] An inoculum of a microbe (e.g., a target microbial strain) may incubate in a reactor (e.g., container) of a digestion system described herein. In some embodiments, the inoculum of a microbe may be incubated under conditions that selectively enrich and / or maintain a concentration of the microbial strain in the digestions system. These conditions to selectively enrich and / or maintain a population of the microbial strain may comprise an addition of a selective pressure source. In some cases, the selective pressure can be a zinc source. In some case, the zinc source may be a form of insoluble zinc. Upon addition of a selective pressure (e.g., zinc source), the inoculation of a zinc-solubilizing microbial strain may solubilize the insoluble zinc and create a concentration of high zinc ion. This environment of high zinc ion concentration may further enrich a microbial population with a target functionality (e.g., zinc solubilization). In some cases, the population of the microbial strain may survive in the digestion system in a vegetative or sporulated state (e.g., a dormant state in the system). In some cases, the selective pressure source (e.g., selective pressure) may establish an environment to enhance the survival of the population of the microbial strain. Without wishing to be bound by theory, the selective pressure source may shift the complex microbial consortia of the digestion system to enrich at least a portion of microbes within a microbial consortium with plant growth promotionproperties (e.g., zinc solubilization). Incubation of the inoculum of the microbe and / or the portion of microbes within a microbial consortium with plant growth promotion properties may further generate metabolites with plant growth promotion properties (e.g., zinc solubilization).

[0118] The inoculum of the microbe may comprise a zinc-solubilizing microbe that can be maintained at a higher ion concentration in a working fluid caused by addition of the selective pressure. Survival of an inoculum of a microbe may comprise the inoculum of a microbe configured to maintain its initial amount in the environment caused by addition of the selective pressure source. Survival of an inoculum of a microbe may comprise an instance where an amount of the inoculum of the microbe is alive (e.g., maintained) at the end of a retention period of the digestion system (e.g., in a reactor or clarifier chamber). Without wishing to be bound by theory, the inoculum of a microbe may not be susceptible to the change in ionic concentration or may have solubilizing properties for the ion which may allow it to survive in the working fluid. Following addition of the selective pressure, at least a portion of microbes of the microbial consortium may enrich (e.g., grow or increase in number). These microbes of the portion of microbes in the microbial consortium may have zinc solubilization capacities. Following addition of the selective pressure source, other microbes of the working fluid may reduce in number as the microbes of the microbial consortium are unable to survive (e.g., are susceptible) in the ionic environment. A proportion of the zinc-solubilizing microbial strain relative to at least a portion of the microbes in a microbial consortium may be maintained in a first container of a digestion system. A proportion of the zinc-solubilizing microbial strain relative to at least a portion of the microbes in a microbial consortium may be maintained in a second, third, fourth, fifth, sixth, seventh, or eighth container of a digestion system. A maintained zinc-solubilizing microbial strain may change its amount in a working fluid of a digestion system less than about 0.001%, less than about 0.01%, less than about 0.1%, less than about 0.5%, less than about 1%, less than about 5%, or less than about 10% over a measured time period.

[0119] In some cases, the change in ionic concentration caused by the addition of the selective pressure may promote the growth of microbes or at least a portion of microbes in the microbial consortium. These microbes may be zinc-solubilizing microbes. An amount of microbes or at least a portion of microbes in the microbial consortium may grow by at least about, at most about, or about 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 30%, or a range between any of these values, following addition of the selective pressure. In some cases, at least a portion of zinc-solubilizing microbes in the microbial consortium may enrich and / or grow in the system without addition of the inoculum of the microbial strain.

[0120] A selective pressure source may be added to a digestion system on a first day of a digestion process. In some embodiments, a selective pressure source may be added to the digestion system daily. In some embodiments, a selective pressure source may be added to the digestion system every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, and / or weekly.

[0121] Without wishing to be bound by theory, a high zinc concentration may inhibit growth of a microbe and / or a population of microbes unless the zinc can be solubilized. Nutrients (e.g., macronutrients, micronutrients, inorganic nutrients, or any combination thereof) can be present in the digestion system to provide an environment for bacterial growth. The addition of a selective pressure (e.g., increased zinc ionic concentration) with inputs to facilitate microbial growth can allow for enrichment of zinc solubilizers in the working fluid of a digestion system described herein. The zinc solubilizers can comprise the inoculum of the microbe (e.g., a population of the inoculum of the microbe), zinc-solubilizing microbes of a microbial consortium, zinc-solubilizing metabolites produced by the inoculum of the microbe and / or the zinc-solubilizing microbes of the microbial consortium, or any combination thereof.

[0122] An inoculum of a microbe described herein may contact (e.g., be applied to) a plant. In some embodiments, the contacting of an inoculum of a microbe to a plant may enhance at least one plant growth promotion property of the plant. In some embodiments, one, two, three, four, or more inoculums of a microbe may be transferred to a digestion system. An inoculum of a microbe and another inoculum of a microbe may be the same. An inoculum of a microbe and another inoculum of a microbe may be different. In some embodiments, the inoculum of the microbe and the feedstock (e.g., aqueous organic feedstock) are transferred to a container of the digestion system at the same time. In some embodiments, the inoculum of the microbe and the feedstock (e.g., aqueous organic feedstock) are not transferred to a container of the digestion system at the same time. In some embodiments, the inoculum of the microbe is transferred to a container of the digestion system prior to the feedstock (e.g., aqueous organic feedstock). In some embodiments, the inoculum of the microbe is transferred to a container of the digestion system after the feedstock (e.g., aqueous organic feedstock).

[0123] In some embodiments, the selective pressure can enrich a population of zinc-solubilizing microbes (e.g., zinc-solubilizer) in a container and / or containers of a digestion system. A population of zinc-solubilizing microbes can comprise an inoculum of a target zinc-solubilizing microbe, zinc-solubilizing microbes of a microbial consortium, zinc-solubilizing metabolites, or any combination thereof.

[0124] As a working fluid flows through a digestion system, an absolute number of zinc- solubilizing microbes may increase. In some embodiments, an absolute number of zinc solubilizers can be higher in a second container compared an absolute number of zinc solubilizers in a first container. In some embodiments, an absolute number of zinc solubilizers can be higher in a third container compared an absolute number of zinc solubilizers in a first container. In some embodiments, an absolute number of zinc solubilizers can be higher in a fourth container compared an absolute number of zinc solubilizers in a first container. In some embodiments, an absolute number of zinc solubilizers can be higher in a fifth container compared an absolute number of zinc solubilizers in a first container. In some embodiments, an absolute number of zinc solubilizers can be higher in a sixth container compared an absolute number of zinc solubilizers in a first container. In some embodiments, an absolute number of zinc solubilizers can be higher in a seventh container compared an absolute number of zinc solubilizers in a first container. In some embodiments, an absolute number of zinc solubilizers can be higher in an eighth container compared an absolute number of zinc solubilizers in a first container. In some embodiments, an absolute number of zinc solubilizers can be higher in a ninth container compared an absolute number of zinc solubilizers in a first container. In some embodiments, an absolute number of zinc solubilizers can be higher in a tenth container compared an absolute number of zinc solubilizers in a first container.

[0125] As working fluid flows through a digestion system, a proportion of zinc-solubilizer relative to a total population of bacteria may increase. In some embodiments, a proportion of zinc-solubilizer relative to a total population of bacteria can be higher in a second container compared a proportion of zinc-solubilizer relative to a total population of bacteria in a first container. In some embodiments, a proportion of zinc-solubilizer relative to a total population of bacteria can be higher in a third container compared a proportion of zinc-solubilizer relative to a total population of bacteria in a first container. In some embodiments a proportion of zinc- solubilizer relative to a total population of bacteria can be higher in a fourth container compared a proportion of zinc-solubilizer relative to a total population of bacteria in a first container. In some embodiments, a proportion of zinc-solubilizer relative to a total population of bacteria can be higher in a fifth container compared a proportion of zinc-solubilizer relative to a total population of bacteria in a first container. In some embodiments, a proportion of zinc-solubilizer relative to a total population of bacteria can be higher in a sixth container compared a proportion of zinc-solubilizer relative to a total population of bacteria in a first container. In some embodiments, a proportion of zinc-solubilizer relative to a total population of bacteria can be higher in a seventh container compared a proportion of zinc-solubilizer relative to a totalpopulation of bacteria in a first container. In some embodiments, a proportion of zinc-solubilizer relative to a total population of bacteria can be higher in an eighth container compared a proportion of zinc-solubilizer relative to a total population of bacteria in a first container. In some embodiments, a proportion of zinc-solubilizer relative to a total population of bacteria can be higher in a ninth container compared a proportion of zinc-solubilizer relative to a total population of bacteria in a first container. In some embodiments, a proportion of zinc-solubilizer relative to a total population of bacteria can be higher in a tenth container compared a proportion of zinc-solubilizer relative to a total population of bacteria in a first container.

[0126] An inoculum of a microbe may generate metabolites in a digestion system as described herein. Microbes of the microbial consortium and / or the inoculum of the microbe may be metabolized by catalytic enzymes to produce metabolites. Metabolites may be generated by microbial metabolism. Metabolites may be generated by enzymes catalyzing biochemical reactions of the organic substrates of the feedstock (e.g., aqueous organic feedstock) in a working fluid of a digestion system as described herein. The metabolites generated by the inoculum of the microbe may have a plant growth promotion property. The metabolites generated by the inoculum of the microbe may have two or more plant growth promotion properties. The plant growth promotion properties may comprise shoot biomass, root biomass, nutrient uptake, crop yield, deaminase activity, acid production, leaf area, chlorophyll content, heat tolerance, cold tolerance, drought tolerance, or salt tolerance, or total biomass. Metabolites may be used in biostimulant compositions and / or may be applied to plants.

[0127] In some embodiments, the feedstock (e.g., aqueous organic feedstock) comprises metabolites. In some embodiments, the feedstock (e.g., aqueous organic feedstock) comprises metabolites produced by microbes endogenous to the feedstock (e.g., organic feedstock). Primary metabolites can include carbohydrates, proteins, fats, vitamins, and nucleic acid components. Metabolites can further comprise alkaloids, amino acids, biogenic amines, carboxylic acids, cresols, terpenoids, phenols (e.g., flavonoids, coumarins, tannins, lignans, stilbenes, or chromones), polyketides, eicosanoids, hormones or derivatives thereof, indoles or derivatives thereof, nucleobases, citric acid, ceramides, diglycerides, triglycerides, amides, alkanes, alcohols, stearates, sterols, organic acids or fatty acids. In some embodiments, metabolites comprise sugars and / or fatty acids. Sugars can comprise fructose, hexose, galactose, glucose, lactose, maltose, sucrose, and / or xylose. Fatty acids can comprise stearic acid, lauric acid, myristic acid, palmitic acid, octadecenoic acid, octadecadienoic acid, oleic acid, arachidic acid, behenic acid, erucic acid, adrenic acid, tricosanoic acid, lignoceric acid, nervonic acid, nonadecanoic acid, arachidic acid, myristolic acid or hydroxylated myristic acid.

[0128] Metabolites generated by the inoculum of the microbe or by at least a portion of microbes of the microbial consortium may be present in a supernatant (e.g., base product) of a digestion system. In some embodiments, the metabolites may be present by weight in a volume of solution (e.g., in mg in 100 ml). In some embodiments, a weight of metabolites per 100 ml of a base product solution can be at least about 10 mg, at least about 20 mg, at least about 30 mg, at least about 40 mg, at least about 50 mg, at least about 60 mg, at least about 70 mg, at least about 80 mg, at least about 90 mg, at least about 100 mg, at least about 120 mg, at least about 140 mg, at least about 160 mg, at least about 180 mg, at least about 200 mg, at least about 220 mg, at least about 240 mg, at least about 260 mg, at least about 280 mg, at least about 300 mg, at least about 400 mg, at least about 500 mg, or greater than about 500 mg. In some embodiments, a weight of metabolites per 100 ml of a base product solution can be at most about 500 mg, at most about 400 mg, at most about 300 mg, at most about 280 mg, at most about 260 mg, at most about 240 mg, at most about 220 mg, at most about 200 mg, at most about 180 mg, at most about 160 mg, at most about 140 mg, at most about 120 mg, at most about 100 mg, at most about 90 mg, at most about 80 mg, at most about 70 mg, at most about 60 mg, at most about 50 mg, at most about 40 mg, at most about 30 mg, at most about 20 mg, at most about 10 mg, or less than about 10 mg. In some embodiments, a weight of metabolites per 100 ml of a base product solution can be from about 10 mg to about 500 mg. In some embodiments, a weight of metabolites per 100 ml of a base product solution can be from about 10 mg to about 20 mg, about 10 mg to about 30 mg, about 10 mg to about 40 mg, about 10 mg to about 50 mg, about 10 mg to about 75 mg, about 10 mg to about 100 mg, about 10 mg to about 125 mg, about 10 mg to about 150 mg, about 10 mg to about 175 mg, about 10 mg to about 250 mg, about 10 mg to about 500 mg, about 20 mg to about 30 mg, about 20 mg to about 40 mg, about 20 mg to about 50 mg, about 20 mg to about 75 mg, about 20 mg to about 100 mg, about 20 mg to about 125 mg, about 20 mg to about 150 mg, about 20 mg to about 175 mg, about 20 mg to about 250 mg, about 20 mg to about 500 mg, about 30 mg to about 40 mg, about 30 mg to about 50 mg, about 30 mg to about 75 mg, about 30 mg to about 100 mg, about 30 mg to about 125 mg, about 30 mg to about 150 mg, about 30 mg to about 175 mg, about 30 mg to about 250 mg, about 30 mg to about 500 mg, about 40 mg to about 50 mg, about 40 mg to about 75 mg, about 40 mg to about 100 mg, about 40 mg to about 125 mg, about 40 mg to about 150 mg, about 40 mg to about 175 mg, about 40 mg to about 250 mg, about 40 mg to about 500 mg, about 50 mg to about 75 mg, about 50 mg to about 100 mg, about 50 mg to about 125 mg, about 50 mg to about 150 mg, about 50 mg to about 175 mg, about 50 mg to about 250 mg, about 50 mg to about 500 mg, about 75 mg to about 100 mg, about 75 mg to about 125 mg, about 75 mg to about 150 mg,about 75 mg to about 175 mg, about 75 mg to about 250 mg, about 75 mg to about 500 mg, about 100 mg to about 125 mg, about 100 mg to about 150 mg, about 100 mg to about 175 mg, about 100 mg to about 250 mg, about 100 mg to about 500 mg, about 125 mg to about 150 mg, about 125 mg to about 175 mg, about 125 mg to about 250 mg, about 125 mg to about 500 mg, about 150 mg to about 175 mg, about 150 mg to about 250 mg, about 150 mg to about 500 mg, about 175 mg to about 250 mg, about 175 mg to about 500 mg, or about 250 mg to about 500 mg.

[0129] The term “feedstock” (e.g., “organic feedstock”) described herein can refer to raw biomaterials such as carbon compounds, proteins, and / or carbohydrates. The feedstock may be organic feedstock. The feedstock may be inorganic feedstock. The organic feedstock may be an aqueous organic feedstock. In some cases, the organic feedstock may comprise one or more inorganic nutrients. In some embodiments, the feedstock (e.g., organic feedstock) may comprise organic substrates comprising cottonseed, algae, neem, orange seed, linseedjojoba, kusum, rubber seed, alfalfa, sugarcane, Opuntia, coffee, Deccan hemp, or any combination thereof. In some embodiments, the feedstock can comprise an inorganic feedstock. In some embodiments, the feedstock (e.g., organic feedstock) may include, but is not limited to, manure, kelp, lignocellulose, wastewater biosolids, food waste, energy crops, glucose solution, ammonium sulfate, oils, fats, grease, or any combination thereof. In some embodiments, the feedstock is added at the beginning of the system (e.g., into a first reactor and / or a CMR). In some embodiments, the feedstock is added in a middle reactor of the system (e.g., not in the first or last reactor of the system). In some embodiments, the feedstock is added once to the system. In some embodiments, the feedstock is added two, three, four, or more times to the system. In some embodiments, the feedstock is added continuously to the system. In some embodiments, the feedstock (e.g., organic feedstock) is a composition of one raw biomaterial. In some embodiments, the feedstock (e.g., organic feedstock) is a blend of two, three, four, five, six, seven, eight, nine, ten, or more biomaterials.

[0130] Feedstock (e.g., organic feedstock) comprising carbon and nitrogen sources can flow into a reactor tank. Feedstock (e.g., organic feedstock) comprising carbon and nitrogen sources can flow into a reactor tank via a conduit (e.g., a pipe). The feedstock (e.g., organic feedstock) may further comprise microbial strain having a desired plant growth promoting property (e.g., a zinc- solubilizing microbial strain). In some embodiments, the feedstock (e.g., aqueous feedstock or aqueous organic feedstock) can comprise a concentration of the microbial strain (e.g., the zinc- solubilizing microbial strain) of at most about 15 CFU / ml, at most about 14 CFU / ml, at most about 13 CFU / ml, at most about 12 CFU / ml, at most about 11 CFU / ml, at most about 10CFU / ml, at most about 9 CFU / ml, at most about 8 CFU / ml, at most about 7 CFU / ml, at most about 6 CFU / ml, at most about 5 CFU / ml, at most about 4 CFU / ml, at most about 3 CFU / ml, at most about 2 CFU / ml, at most about 1 CFU / ml, or less than about 1 CFU / ml.

[0131] In some embodiments, a reactor tank circulates working fluid within itself to recycle working fluid, wherein reactor tanks can comprise out-flow pipes to circulate and recycle working fluid within each tank. Ports and piping between tanks can assist in transferring working fluid to adjacent reactor tanks. A working fluid in a final clarifier of a system may transfer from the reactor tank to a clarifier may produce a supernatant (e.g., base product). Working fluid flows through the serialized reactor system which can aid in selective growth of the added isolate and other microbes present that have the same property as the added isolate. Base product from the clarifier can be accessed and further analyzed for microbial composition. In a digestion system described herein, a working fluid may flow from a mixing chamber through at least one reaction and to a clarifier chamber.

[0132] A feedstock (e.g., organic feedstock) can comprise digestion products from digestion of organic substrates present in the feedstock (e.g., organic feedstock). Organic substrates may improve stability of the fluid feed mixture. Organic substrates can comprise coconut coir, peat moss, hemp, and / or wood fiber. In some embodiments, organic substrates comprise raw biomaterials present in the feedstock (e.g., aqueous organic feedstock). A digestion system may comprise a plurality of microbes and / or microorganisms derived from digestion of organic substrates in a feedstock (e.g., aqueous organic feedstock).

[0133] A feedstock (e.g., organic feedstock) described herein may comprise various organic and / or biological materials. In some embodiments, the feedstock (e.g., organic feedstock) further comprises Saccharomyces cerevisiae yeast, Saccharomyces arboricola yeast, Saccharomyces mikatae yeast, Saccharomyces jurei yeast, Saccharomyces eubayanus yeast, Saccharomyces kudriavzevii yeast, Saccharomyces uvarum yeast, or any combination thereof. In some embodiments, the feedstock (e.g., organic feedstock) may be an aqueous mixture of at least one feedstock material and water. In some embodiments, the feedstock (e.g., organic feedstock) may be an aqueous mixture of cow manure, S. cerevisiae yeast, water, or any combination thereof. A feedstock can be an organic feedstock. An organic feedstock can be an aqueous organic feedstock (e.g., an organic feedstock comprising water).

[0134] Parameters of the digestion system, such as flow rate and the solids content of the feedstock (e.g., organic feedstock), may be varied to achieve desired properties in the outflow biostimulant base product. In some embodiments, the hydraulic source is water. In some embodiments, the hydraulic source is a base product of another system. In some embodiments,the hydraulic source is a combination of water and a base product of another system. Water from the hydraulic source can be added to the feedstock (e.g., organic feedstock) of the digestion system to make an aqueous organic feedstock.

[0135] In some embodiments, the feedstock (e.g., aqueous organic feedstock) may further comprise a inorganic substrate. In some embodiments, the feedstock (e.g., aqueous organic feedstock) may include more than one inorganic substrate. The inorganic substrate may improve stability of the fluid feed mixture. In some embodiments, the inorganic substrate comprises sand, vermiculite, perlite, diatomaceous earth, and / or pumice. In some embodiments, the inorganic substrates comprises a mineral. In some embodiments, the inorganic substrate comprises rock phosphate. In some embodiments, the inorganic substrate can be zinc oxide.

[0136] In some embodiments, loading inputs into a reactor can comprise a carbon source, a nitrogen source, a flour, an isolate, or any combination thereof. In some embodiments, the loading inputs comprise recycled floc from the system. In some embodiments, the loading inputs comprise a whole broth (WB). The inoculum of a microbe as described herein may metabolize the carbon source. Metabolism of carbon by the inoculum of the microbe may comprise transfer of carbon-based moieties of the carbon source to substrates in the working fluid.

[0137] The inoculum of a microbe as described herein may metabolize the nitrogen source. Metabolism of nitrogen by the inoculum of the microbe may comprise transfer of nitrogen-based moieties of the nitrogen source to substrates in the working fluid. In some embodiments, the carbon source may be transferred to a first container of the digestion system. In some embodiments, the carbon source may be transferred to a second, third, fourth, fifth, or sixth container of the digestion system. In some embodiments, the nitrogen source may be transferred to a first container of the digestion system. In some embodiments, the nitrogen source may be transferred to a second, third, fourth, fifth, or sixth container of the digestion system.

[0138] In some embodiments, the feedstock (e.g., organic feedstock) is mixed within a reactor. In some embodiments, the feedstock (e.g., organic feedstock) is mixed outside of a reactor. In some embodiments, the feedstock (e.g., organic feedstock) is mixed between one, two, three, or more reactors. In some embodiments, the feedstock (e.g., organic feedstock) is a homogenous mixture.

[0139] In some embodiments, the feedstock (e.g., organic feedstock) further comprises a microbial consortium. The terms “microbe”, “microbial strain” and “microorganism” may refer to microscopic organisms, comprising bacteria, fungi, lichens, algae, protozoa, archaea, molds, or any combination thereof. The terms “microbe” and microorganism” may be used interchangeably herein. The feedstock (e.g., organic feedstock) can comprise a microbialconsortium with 2, 3, 4, 5, 6, 7, 8, 9, 10, or more microorganisms. The feedstock (e.g., organic feedstock) can comprise a microbial consortium with 2, 3, 4, 5, 6, 7, 8, 9, 10, or more groups of microorganisms. The feedstock (e.g., organic feedstock) can comprise a microbial consortium with 1 group of microorganisms. The microbial consortium can comprise different microorganisms. The microbial consortium can comprise the same microorganism. The microbes in the consortia may be derived from the microbes originally present within the feedstock (e.g., organic feedstock). The microbes may digest the manure, yeast, other organic raw materials, or any combination thereof to produce digestion products.

[0140] In some embodiments, microbes can be added to the start of the system (e.g., into the first reactor). In some embodiments, microbes can be added to the middle of the system (e.g., into a reactor that is not the first reactor or the final reactor of the system) or microbes can be added to the end of the system (e.g., into the final reactor). Microbes can be added concurrently with the feedstock (e.g., organic feedstock). Microbes can be added separately from the feedstock (e.g., organic feedstock). In some embodiments, microbes may be added to the system with the feedstock (e.g., organic feedstock) in the same reactor. In some embodiments, microbes may be added to the system with the feedstock (e.g., organic feedstock) in different reactors. In some embodiments, microbes may be added to the system prior to the feedstock (e.g., organic feedstock). In some embodiments, microbes may be added to the system after the feedstock (e.g., organic feedstock). In some embodiments, a period of time between addition of microbes to the system and addition of feedstock (e.g., organic feedstock) to the system can be at least about 1 minute, at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 30 minutes, at least about 40 minutes, at least about 50 minutes, or at least about 1 hours. In some embodiments, a period of time between addition of microbes to the system and addition of feedstock (e.g., organic feedstock) to the system can be at most about 2 hours, at most about 1 hours, at most about 45 minutes, at most about 30 minutes, at most about 15 minutes, at most about 10 minutes, at most about 5 minutes, at most about 1 minute, or at most about 30 seconds.

[0141] A microbe may have nutrient solubilization properties and / or plant growth promotion properties. For example, a microbe may increase plant growth, increase shoot and / or root biomass, increase crop yield, increase soil enzymatic activity, increase photosynthesis efficiency, lower heavy metal uptake, and / or decrease soil pH. A microbe may enhance plant growth on land with high salinity, on land with heavy metal contamination, or on land with drought conditions. A microbe (e.g., isolate) described herein may have zinc solubilization properties.

[0142] In some embodiments, the digestion system may comprise a retention time. A retention time may comprise a time an inoculum of a microbe spends in a digestion system or a time an inoculum of a microbe spends following transfer into a first container and until collection from the digestion system. A longer retention time may be advantageous for growth or enrichment of an inoculum of a microbe of the digestion system. A shorter retention time may be advantageous for growth or enrichment of an inoculum of a microbe of the digestion system. A retention time of a digestion system may comprise at least about, at most about, or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 6 months, 9 months, 1 year, 2 years, 3 years, 4 years, 5 years, or 10 years, or a range between any of these values.

[0143] In the first reactor, the working fluid may be agitated at a rate that allows heavier or undigested solids to settle to the bottom. An outlet at the top of the first reactors may allow the fluid to flow into the second reactor. An outlet at the bottom of the first reactor may transfer the settled solids back into a reactor tank. Each of the reactors in the series of reactors may have submerged scaffolding that provide a surface for biofilm growth. The scaffolding can be referred to as “fixed media substrates”. The flow of fluid from reactor to reactor may comprise a plug flow model, in which particles of an input fluid have the same velocity and direction of motion. In some embodiments, the flow of fluid in the digestion system is driven by gravity. In some embodiments, the flow of fluid in the digestion system is driven by a pump. The outflow from the top of the last reactor, may be used to create a product. Products of the methods and systems as described herein can be biostimulants. Biostimulants can promote plant growth, improve plant quality, or improve soil quality.C. Reactors

[0144] A digestion process to produce the biostimulant may be performed in a digestion system that includes a tank, a container, a vessel (e.g., reactor), or a series of tanks, a series of containers, or a series of vessels (e.g., reactors) through which the feedstock continuously flows. A reactor can be a fluidly connected container, system, vessel, or tank in which microbial consortia including the microbes, isolates, and / or microorganisms as described herein can be grown. A reactor can be separate or continuous. A reactor may be a physical containment system arranged in a discrete order to favor growth of particular microbes. Types of reactors can include, but are not limited to, fluidized-bed reactors (FBRs) or packed-bed reactors (PBRs).

[0145] In some embodiments, reactors may be arranged so that fluid can flow from an outflow port of a reactor into an adjacent reactor or tank. Fluid from near the top of the working fluid ina reactor may flow into the next reactor continuously. Fluid from the middle of a reactor may flow into the next reactor continuously. Fluid from the bottom of a reactor may flow into the next reactor continuously. Fluid may also be reintroduced from any outflow source into the same reactor. In some embodiments, an outflow port is between 0.1 and 35 inches below the top of the working fluid within a reactor. In some embodiments, an outflow port is at least about 1 inch, at least about 2 inches, at least about 5 inches, at least about 10 inches, at least about 20 inches, at least about 50 inches, at least about 100 inches, at least about 250 inches, at least about 500 inches, at least about 750 inches, at least about 1,000 inches, at least about 2,500 inches, at least about 5,000 inches, at least about 7,500 inches, or at least about 10,000 inches below the top of the working fluid within a reactor. In some embodiments, an outflow port is at most about 10,000 inches, at most about 7,500 inches, at most about 5,000 inches, at most about 2,500 inches, at most about 1,000 inches, at most about 750 inches, at most about 500 inches, at most about 250 inches, at most about 100 inches, at most about 50 inches, at most about 20 inches, at most about 10 inches, at most about 5 inches, at most about 2 inches, or at most about 1 inch below the top of the working fluid within a reactor. In some embodiments, the rate of outflowing product from a digestion system may match the rate of inflowing feedstock, providing for a hydraulically balanced flow throughout the system. A reactor within the system may have a unique, stable microbial consortium with distinct physiological characteristics and digestion capabilities as compared to consortia in other tanks in the system. A reactor within the system may have the same microbial consortium with similar physiological characteristics and digestion capabilities as another reactor within the system. Each reactor within the system may have the same volume capacity. Each reactor within the system may have a different volume capacity. The digestion system may comprise at least two reactors. The digestion system may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more reactors. Reactors of a digestion system may be arranged as a serialized assembly of reactors. A serialized assembly of reactors may have conduits (e.g., ports or outputs) connecting each reactor to an adjacent reactor and / or container. A serialized assembly of reactors may have a continuous flow of working fluid through each reactor to the adjacent reactor.

[0146] In some embodiments, a reactor may have a single in-flow port and a single out-flow port. In some embodiments, a reactor may have multiple in-flow ports and out-flow ports. In some embodiments, a reactor may have a single in-flow port and multiple out-flow ports. In some embodiments, a reactor may have multiple in-flow ports and a single out-flow port. A reactor may have another in-flow port to provide a carbon source and / or consortium inoculum. An in-flow port may be present at any location of a reactor of the digestion system. An in-flowport may be present at the top of the reactor or at the bottom of the reactor. An out-flow port may be present at the top of the reactor or at the bottom of the reactor. In some embodiments, the out-flow ports or in-flow ports described herein comprise pipes, pumps, ventilations, or other systems known to those skilled in the art for transferring fluid from one vessel to another.

[0147] A reactor may have a single fluid connection. A reactor may have multiple fluid connections. The fluid connections may be present at the top of the working fluid in each reactor or near the top of the working fluid in each reactor. In some embodiments, the reactor may have flow from the bottom of the container back to the top to prevent build-up of sludge in the bottom of the reactor. In some embodiments, a reactor may have stirrers in the bottom of the container. In some embodiments, a reactor may have wipers in the bottom of the container. The wipers may stir the feedstock and prevent clogging within the reactor. The wipers may fold floc and ease floc return.

[0148] In some embodiments, the reactors may comprise packed bed reactors. In some embodiments, each of the packed bed reactors has an open cell design to allow free movement of working fluid. A fixed media (e.g., scaffolding) may be secured to the inside of each packed bed reactor. The fixed media comprises materials that increase the contact surface area for the communities of microbes with working fluid. The fixed media also provides a stable platform for anchoring biofilm. The packed bed reactors may be packed with scaffolding to increase surface area within the reactor. The scaffolding within the reactor may increase biofilm. The packed bed reactor may improve contact between the biofilm and substrates within the reactor. The fixed media can be of several types, including durable plastic, polyvinyl chloride (PVC), metal, metal alloy, glass, glass compounds, fiberglass, or any suitably robust inert material. The design and configuration of the fixed media can assume various geometric patterns that allow working fluid to freely move through each packed bed reactor and prevents fouling. Free flow supports controlled hydraulic shearing which in time promotes even distribution of working fluid. In this embodiment, the fixed media is dispersed throughout a cross sectional area of each packed bed reactor.

[0149] The scaffolding may comprise tubes, rings, or other packing materials. In some embodiments, the packed bed reactors provided herein may comprise a bundle of tubes or columns. In some embodiments, the scaffolding may comprise hexagonal, grid-like, perforated tubing, or any combination thereof. Without wishing to be bound by theory, hexagonal, gridlike, and / or perforated scaffolding can increase the surface area and the flow through the columns within the container. In some embodiments, the tubes or columns of the scaffolding can comprise a diameter between 0.25 and 50 inches. In some embodiments, the scaffolding cancomprise a diameter of at least about 0.5 inches, at least about 0.6 inches, at least about 0.7 inches, at least about 0.8 inches, at least about 0.9 inches, at least about 1 inch, at least about 2 inches, at least about 3 inches, at least about 4 inches, at least about 5 inches, at least about 10 inches, at least about 15 inches, at least about 20 inches, at least about 25 inches, at least about 30 inches, at least about 40 inches, at least about 50 inches, at least about 60 inches, or at least about 75 inches. In some embodiments, the scaffolding can comprise a diameter of at most about 75 inches, at least about 60 inches, at most about 50 inches, at most about 40 inches, at most about 30 inches, at most about 25 inches, at most about 20 inches, at most about 15 inches, at most about 10 inches, at most about 5 inches, at most about 4 inches, at most about 3 inches, at most about 2 inches, at most about 1 inches, at most about 0.9 inches, at most about 0.8 inches, at most about 0.7 inches, at most about 0.6 inches, or at most about 0.5 inches. Without wishing to be bound by theory, a system with packed bed reactors may improve production of bacterial isolates or other microbes. In some embodiments, reactors without scaffolding (i.e., reactors that are not packed bed reactors) improve production of bacterial isolates or other microbes or improve digestion of digestible substrates.

[0150] In some embodiments, the reactors may comprise fluidized bed reactors. In fluidized bed reactors, solid particles may be circulated within working fluid of the reactors, which may provide a surface for microbial colonization. Such particles may include, for example, particles of an inorganic substrate such as rock phosphate particles. In some embodiments, the fluidized bed reactors may be the same volume. In some embodiments, the fluidized bed reactors may be different volumes. In some embodiments, the fluidized bed reactors increase uniformity of particle mixing within the digestion system. The solid material of the fluidized bed reactor can have intrinsic fluid-like properties and allow for a more complete mixing. Reduction or elimination of radial and axial concentration gradients can provide for better fluid-solid contact and can achieve better uniformity of particle mixing. In some embodiments, the fluidized bed reactors increase the uniformity of temperature gradients within the digestion system. Without wishing to be bound by theory, the open container of the fluidized bed reactor can provide for a reduction in isolated hot or cold spots that may develop during mixing of a working fluid in the container. Hot spots can be areas of localized temperature changes during heating of a working fluid. Cold spots can be areas of localized temperature changes during cooling of a working fluid. The reduction in isolated temperature changes from the configuration of the fluidized bed reactor can provide for a more uniform temperature distribution of the fluid.

[0151] The flow rate of the digestion system may be chosen to allow for sufficient dwell time within each of the reactor for a stable and unique microbial consortium to form within each ofthe reactors. In some embodiments, working fluid in each reactor is continuously recycled at a rate ratio in a range of approximately 25: 1 to 35: 1, 25 to 35 gallons per minute of the recycle rate to one gallon per minute of the hydraulic feed rate. In some embodiments, working fluid in each reactor is continuously recycled at a rate ratio of at least about, at most about, or about 10: 1, at least about 12: 1, at least about 14: 1, at least about 16: 1, at least about 18: 1, at least about 20: 1, at least about 22: 1, at least about 24: 1, at least about 26: 1, at least about 28: 1, at least about 30: 1, at least about 32: 1, at least about 34: 1, at least about 36: 1, at least about 38: 1, at least about 40: 1, at least about 45: 1, or at least about 50: 1, or a range between any of these two values. Working fluid may be recycled by a pump to prevent solids settling and to provide sufficient velocity and hydraulic shear to prevent excessive buildup and sloughing of biofilm. A digestion system provided herein may comprise a first flow rate, second flow rate, third flow rate, fourth flow rate, fifth flow rate, sixth flow rate, or seventh flow rate.

[0152] Reactors can be maintained at specific temperatures which may aid in digestion and growth of microbial consortia within the system. In some embodiments, a temperature of a reactor is at least about 15°C, at least about 20°C, at least about 21°C, at least about 22°C, at least about 23°C, at least about 24°C, at least about 25°C, at least about 26°C, at least about 27°C, at least about 28°C, at least about 29°C, at least about 30°C, at least about 35°C, at least about 40°C, at least about 45°C, or at least about 50°C. In some embodiments, a temperature of a reactor is at most about 50°C, at most about 45°C, at most about 40°C, at most about 35°C, at most about 30°C, at most about 29°C, at most about 28°C, at most about 27°C, at most about 26°C, at most about 25 °C, at most about 24°C, at most about 23 °C, at most about 22°C, at most about 21°C, at most about 20°C, or at most about 15°C.

[0153] In some embodiments, a temperature of a reactor is about 15°C to about 45°C. In some embodiments, a temperature of a reactor is about 15°C to about 20°C, about 15°C to about 22°C, about 15°C to about 24°C, about 15°C to about 26°C, about 15°C to about 28°C, about 15°C to about 30°C, about 15°C to about 32°C, about 15°C to about 34°C, about 15°C to about 36°C, about 15°C to about 40°C, about 15°C to about 45°C, about 20°C to about 22°C, about 20°C to about 24°C, about 20°C to about 26°C, about 20°C to about 28°C, about 20°C to about 30°C, about 20°C to about 32°C, about 20°C to about 34°C, about 20°C to about 36°C, about 20°C to about 40°C, about 20°C to about 45°C, about 22°C to about 24°C, about 22°C to about 26°C, about 22°C to about 28°C, about 22°C to about 30°C, about 22°C to about 32°C, about 22°C to about 34°C, about 22°C to about 36°C, about 22°C to about 40°C, about 22°C to about 45°C, about 24°C to about 26°C, about 24°C to about 28°C, about 24°C to about 30°C, about 24°C to about 32°C, about 24°C to about 34°C, about 24°C to about 36°C, about 24°C to about 40°C,about 24°C to about 45°C, about 26°C to about 28°C, about 26°C to about 30°C, about 26°C to about 32°C, about 26°C to about 34°C, about 26°C to about 36°C, about 26°C to about 40°C, about 26°C to about 45°C, about 28°C to about 30°C, about 28°C to about 32°C, about 28°C to about 34°C, about 28°C to about 36°C, about 28°C to about 40°C, about 28°C to about 45°C, about 30°C to about 32°C, about 30°C to about 34°C, about 30°C to about 36°C, about 30°C to about 40°C, about 30°C to about 45°C, about 32°C to about 34°C, about 32°C to about 36°C, about 32°C to about 40°C, about 32°C to about 45°C, about 34°C to about 36°C, about 34°C to about 40°C, about 34°C to about 45°C, about 36°C to about 40°C, about 36°C to about 45°C, or about 40°C to about 45°C.

[0154] Reactors may be maintained under aerobic, microaerobic, or anaerobic conditions. The series of reactors in a digestion system may have different aerobic conditions. The series of reactors in a digestion system may have the same aerobic conditions. In some embodiments, a reactor may have the same aerobic condition as an adjacent reactor. In some embodiments, a reactor may have a different aerobic condition than an adjacent reactor. In some embodiments, a digestion system may have aerobic, microaerobic, anaerobic conditions, or any combination thereof.

[0155] In some embodiments, aerobic conditions comprise conditions with a dissolved oxygen measurement of greater than 2 mg / L. In some embodiments, aerobic conditions comprise conditions with a dissolved oxygen measurement of at least about 2 mg / L mg / L, at least about 3 mg / L mg / L, at least about 4 mg / L, at least about 5 mg / L, at least about 6 mg / L, at least about 7 mg / L, at least about 8 mg / L, at least about 9 mg / L, at least about 10 mg / L, at least about 12 mg / L, at least about 14 mg / L, at least about 15 mg / L, or greater than about 15 mg / L. In some embodiments, aerobic conditions comprise conditions with a dissolved oxygen measurement from about 2 mg / L to about 15 mg / L. In some embodiments, aerobic conditions comprise conditions with a dissolved oxygen measurement from about 2 mg / L to about 3 mg / L, about 2 mg / L to about 4 mg / L, about 2 mg / L to about 5 mg / L, about 2 mg / L to about 6 mg / L, about 2 mg / L to about 7 mg / L, about 2 mg / L to about 8 mg / L, about 2 mg / L to about 9 mg / L, about 2 mg / L to about 10 mg / L, about 2 mg / L to about 12 mg / L, about 2 mg / L to about 14 mg / L, about 2 mg / L to about 15 mg / L, about 3 mg / L to about 4 mg / L, about 3 mg / L to about 5 mg / L, about 3 mg / L to about 6 mg / L, about 3 mg / L to about 7 mg / L, about 3 mg / L to about 8 mg / L, about 3 mg / L to about 9 mg / L, about 3 mg / L to about 10 mg / L, about 3 mg / L to about 12 mg / L, about 3 mg / L to about 14 mg / L, about 3 mg / L to about 15 mg / L, about 4 mg / L to about 5 mg / L, about 4 mg / L to about 6 mg / L, about 4 mg / L to about 7 mg / L, about 4 mg / L to about 8 mg / L, about 4 mg / L to about 9 mg / L, about 4 mg / L to about 10 mg / L, about 4 mg / L to about 12 mg / L, about 4mg / L to about 14 mg / L, about 4 mg / L to about 15 mg / L, about 5 mg / L to about 6 mg / L, about 5 mg / L to about 7 mg / L, about 5 mg / L to about 8 mg / L, about 5 mg / L to about 9 mg / L, about 5 mg / L to about 10 mg / L, about 5 mg / L to about 12 mg / L, about 5 mg / L to about 14 mg / L, about 5 mg / L to about 15 mg / L, about 6 mg / L to about 7 mg / L, about 6 mg / L to about 8 mg / L, about 6 mg / L to about 9 mg / L, about 6 mg / L to about 10 mg / L, about 6 mg / L to about 12 mg / L, about 6 mg / L to about 14 mg / L, about 6 mg / L to about 15 mg / L, about 7 mg / L to about 8 mg / L, about 7 mg / L to about 9 mg / L, about 7 mg / L to about 10 mg / L, about 7 mg / L to about 12 mg / L, about 7 mg / L to about 14 mg / L, about 7 mg / L to about 15 mg / L, about 8 mg / L to about 9 mg / L, about 8 mg / L to about 10 mg / L, about 8 mg / L to about 12 mg / L, about 8 mg / L to about 14 mg / L, about 8 mg / L to about 15 mg / L, about 9 mg / L to about 10 mg / L, about 9 mg / L to about 12 mg / L, about 9 mg / L to about 14 mg / L, about 9 mg / L to about 15 mg / L, about 10 mg / L to about 12 mg / L, about 10 mg / L to about 14 mg / L, about 10 mg / L to about 15 mg / L, about 12 mg / L to about 14 mg / L, about 12 mg / L to about 15 mg / L, or about 14 mg / L to about 15 mg / L. In some embodiments, aerobic conditions comprise conditions with a dissolved oxygen measurement of between 2 mg / L and 10 mg / L. In some embodiments, microaerobic conditions comprise conditions with a dissolved oxygen measurement of less than 2 mg / L. In some embodiments, microaerobic conditions comprise conditions with a dissolved oxygen measurement of at most about 1.99 mg / L, at most about 1.8 mg / L, at most about 1.6 mg / L, at most about 1.5 mg / L, at most about 1.4 mg / L, at most about 1.3 mg / L, at most about 1.2 mg / L, at most about 1.1 mg / L, at most about 1 mg / L, at most about 0.9 mg / L, at most about 0.8 mg / L, at most about 0.7 mg / L, at most about 0.6 mg / L, at most about 0.5 mg / L, at most about 0.4 mg / L, at most about 0.3 mg / L, at most about 0.2 mg / L, at most about 0.1 mg / L, or less than about 0.1 mg / L but not 0 mg / L. In some embodiments, microaerobic conditions comprise conditions with a dissolved oxygen measurement from about 0.1 mg / L to about 1.99 mg / L. In some embodiments, microaerobic conditions comprise conditions with a dissolved oxygen measurement from about 0.1 mg / L to about 0.2 mg / L, about 0.1 mg / L to about 0.3 mg / L, about 0.1 mg / L to about 0.4 mg / L, about 0.1 mg / L to about 0.5 mg / L, about 0.1 mg / L to about 0.8 mg / L, about 0.1 mg / L to about 1 mg / L, about 0.1 mg / L to about 1.2 mg / L, about 0.1 mg / L to about 1.4 mg / L, about 0.1 mg / L to about 1.6 mg / L, about 0.1 mg / L to about 1.8 mg / L, about 0.1 mg / L to about 1.99 mg / L, about 0.2 mg / L to about 0.3 mg / L, about 0.2 mg / L to about 0.4 mg / L, about 0.2 mg / L to about 0.5 mg / L, about 0.2 mg / L to about 0.8 mg / L, about 0.2 mg / L to about 1 mg / L, about 0.2 mg / L to about 1.2 mg / L, about 0.2 mg / L to about 1.4 mg / L, about 0.2 mg / L to about 1.6 mg / L, about 0.2 mg / L to about 1.8 mg / L, about 0.2 mg / L to about 1.99 mg / L, about 0.3 mg / L to about 0.4 mg / L, about 0.3 mg / L to about 0.5 mg / L, about 0.3 mg / L to about 0.8 mg / L, about 0.3 mg / L to about 1mg / L, about 0.3 mg / L to about 1.2 mg / L, about 0.3 mg / L to about 1.4 mg / L, about 0.3 mg / L to about 1.6 mg / L, about 0.3 mg / L to about 1.8 mg / L, about 0.3 mg / L to about 1.99 mg / L, about 0.4 mg / L to about 0.5 mg / L, about 0.4 mg / L to about 0.8 mg / L, about 0.4 mg / L to about 1 mg / L, about 0.4 mg / L to about 1.2 mg / L, about 0.4 mg / L to about 1.4 mg / L, about 0.4 mg / L to about 1.6 mg / L, about 0.4 mg / L to about 1.8 mg / L, about 0.4 mg / L to about 1.99 mg / L, about 0.5 mg / L to about 0.8 mg / L, about 0.5 mg / L to about 1 mg / L, about 0.5 mg / L to about 1.2 mg / L, about 0.5 mg / L to about 1.4 mg / L, about 0.5 mg / L to about 1.6 mg / L, about 0.5 mg / L to about 1.8 mg / L, about 0.5 mg / L to about 1.99 mg / L, about 0.8 mg / L to about 1 mg / L, about 0.8 mg / L to about 1.2 mg / L, about 0.8 mg / L to about 1.4 mg / L, about 0.8 mg / L to about 1.6 mg / L, about 0.8 mg / L to about 1.8 mg / L, about 0.8 mg / L to about 1.99 mg / L, about 1 mg / L to about 1.2 mg / L, about 1 mg / L to about 1.4 mg / L, about 1 mg / L to about 1.6 mg / L, about 1 mg / L to about 1.8 mg / L, about 1 mg / L to about 1.99 mg / L, about 1.2 mg / L to about 1.4 mg / L, about 1.2 mg / L to about 1.6 mg / L, about 1.2 mg / L to about 1.8 mg / L, about 1.2 mg / L to about 1.99 mg / L, about 1.4 mg / L to about 1.6 mg / L, about 1.4 mg / L to about 1.8 mg / L, about 1.4 mg / L to about 1.99 mg / L, about 1.6 mg / L to about 1.8 mg / L, about 1.6 mg / L to about 1.99 mg / L, or about 1.8 mg / L to about 1.99 mg / L. In some embodiments, anaerobic conditions comprise conditions with a dissolved oxygen measurement of 0 mg / L.

[0156] Reactors may be maintained at different pH levels within a digestion system. Reactors may be maintained at the same pH levels within a digestion system. The pH of a reactor in a digestion system may be at least about 4.0, at least about 4.5, at least about 5.0, at least about5.5, at least about 6.0, at least about 6.5, at least about 7.0, at least about 7.5, at least about 8.0, at least about 8.5, at least about 9.0, at least about 9.5, or at least about 10.0. In some embodiments, the pH of a reactor in a digestion system may be at most about 10.0, at most about9.5, at most about 9.0, at most about 8.5, at most about 8.0, at most about 7.5, at most about 7.0, at most about 6.5, at most about 6.0, at most about 5.5, at most about 5.0, at most about 4.5, or at most about 4.0.

[0157] In some embodiments, the pH of a reactor in a digestion system may be about 3 to about 9. In some embodiments, the pH of a reactor in a digestion system may be about 3 to about 3.5, about 3 to about 4, about 3 to about 4.5, about 3 to about 5, about 3 to about 5.5, about 3 to about 6, about 3 to about 6.5, about 3 to about 7, about 3 to about 7.5, about 3 to about 8, about 3 to about 9, about 3.5 to about 4, about 3.5 to about 4.5, about 3.5 to about 5, about 3.5 to about5.5, about 3.5 to about 6, about 3.5 to about 6.5, about 3.5 to about 7, about 3.5 to about 7.5, about 3.5 to about 8, about 3.5 to about 9, about 4 to about 4.5, about 4 to about 5, about 4 to about 5.5, about 4 to about 6, about 4 to about 6.5, about 4 to about 7, about 4 to about 7.5,about 4 to about 8, about 4 to about 9, about 4.5 to about 5, about 4.5 to about 5.5, about 4.5 to about 6, about 4.5 to about 6.5, about 4.5 to about 7, about 4.5 to about 7.5, about 4.5 to about 8, about 4.5 to about 9, about 5 to about 5.5, about 5 to about 6, about 5 to about 6.5, about 5 to about 7, about 5 to about 7.5, about 5 to about 8, about 5 to about 9, about 5.5 to about 6, about 5.5 to about 6.5, about 5.5 to about 7, about 5.5 to about 7.5, about 5.5 to about 8, about 5.5 to about 9, about 6 to about 6.5, about 6 to about 7, about 6 to about 7.5, about 6 to about 8, about 6 to about 9, about 6.5 to about 7, about 6.5 to about 7.5, about 6.5 to about 8, about 6.5 to about 9, about 7 to about 7.5, about 7 to about 8, about 7 to about 9, about 7.5 to about 8, about 7.5 to about 9, or about 8 to about 9.

[0158] In some embodiments, the reactors may comprise distribution components (e.g., a distribution ring) that is subsurface of the discharge volume. The distribution component can reduce the amount of surface disruption, and / or keep the environment in the reactor anaerobic.D. Working Fluids and Microbial Consortia

[0159] A working fluid may comprise a fluidic substance that moves through a digestion system as described herein. A working fluid can comprise solid components, liquid components, gaseous components, or any combination thereof. A working fluid can comprise microbial consortia, isolated microbes or inoculum of a microbial strain (e.g., target isolates), additional organic and / or materials, or any combination thereof. The mixture of microbial consortia, isolated microbes (e.g., target isolates), additional organic and / or materials within a working fluid may allow for the expansion of microbes or act as a culture for an inoculum of a microbe to grow. A working fluid may comprise a pH, viscosity, temperature, surface tension, adhesion, and / or volume that enhances the growth and / or functioning of microbes or microorganisms. In some embodiments, a volume of working fluid within each reactor may be continuously replenished and drawn from. In some embodiments, a volume of working fluid within each reactor is replenished and drawn from in batches (e.g., discontinuously). In some embodiments, a working fluid in a first reactor may comprise a first working fluid. In some embodiments, a working fluid in a second reactor may comprise a second working fluid. In some embodiments, a working fluid in a third reactor may comprise a third working fluid. In some embodiments, a working fluid in a fourth reactor may comprise a fourth working fluid. In some embodiments, a working fluid in a fifth reactor may comprise a fifth working fluid. In some embodiments, at least a portion of the second working fluid may be transferred to the third reactor. In some embodiments, at least a portion of the third working fluid may be transferred to the fourth reactor. In some embodiments, at least a portion of the fourth working fluid may be transferredto the fifth reactor. In some embodiments, a working fluid may be mixed in a reactor (e.g., chamber or container) prior to a first reactor. In some embodiments, the working fluid in each reactor may be distinct from the working fluid in other reactors in the digestion system. Distinct working fluids may comprise different microbial populations. The different microbial populations may include different microbes, (e.g., bacteria, fungi, and / or algae). Distinct working fluids may comprise different concentrations of a target isolate. Distinct working fluids may comprise different concentrations of a carbon source and / or a nitrogen source. Distinct working fluids may comprise different microbial populations, different concentrations of a target isolate, different concentrations of a carbon source, different combinations of a nitrogen source, or any combination thereof. The working fluid in a reactor of a digestion system may be similar to a working fluid of a different reactor of the digestion system.

[0160] The working fluid within each reactor may comprise different enzymes, which may be produced by microbes within the working fluid. An enzyme within a working fluid may comprise a dehydrogenase, a hydrogenase, an oxidase, a catalase, a peroxidase, a phenol o- hydroxylase, a dextransucrase, an aminotransferase, a rhodanese, a carboxylesterase, a lipase, a phosphatase, a nuclease, a phytase, an aryl sulphatase, an amylase, a cellulase, an inulase, a xylanase, a dextranase, a levanase, a poly-galacturonase, a glucosidase, a galactosidase, an invertase, a peptidase, an asparaginase, a glutaminase, an amidase, a urease, an aspartate decarboxylase, a glutamate decarboxylase an aromatic amino acid decarboxylase, or any combination thereof. An enzyme within a working fluid may comprise nitrogenase, 1- aminocyclopropane-1 -carboxylate deaminase (e.g., ACC-deaminase), quinoprotein glucose dehydrogenase (e.g., PQQ or quinone), gluconate 2-dehydrogenase, cellulase, endo-l,3(4)-P- glucanase, and / or pectin lyase. The working fluid within each reactor may comprise different concentrations of enzymes. The working fluid within each reactor may comprise a different average abundance of an enzyme. An enzyme may be present at an average abundance between 0.001% to 1%. An enzyme may be present at an average abundance of less than 0.001%. An enzyme may be present at an average abundance of greater than 1%. In some embodiments, an enzyme may be present at an average abundance of at least about, at most about, or about 0.0001%, 0.001%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, or a range between any of these two values. The working fluid within each reactor may comprise enzymes with different enzymatic activity. Enzymatic activity may include, but is not limited to, nitrogen fixation, ammonia production, phosphate solubilization, zinc solubilization, and / or cell wall lysing.

[0161] A working fluid may comprise different digestion products from working fluid within other reactors of the system. In some embodiments, a working fluid may comprise digestion products from a feedstock (e.g., aqueous organic feedstock) and microbial consortium at least partially derived from a previous working fluid.

[0162] The pH of a working fluid within each reactor may be different from working fluid in other reactors. The pH of a working fluid within each reactor may be the same. The pH of a working fluid may be less than 6. The pH of a working fluid may be greater than 6. The pH of a working fluid may be in a range from 2 to 11. The pH of a working fluid may be at least about, at most about, or about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, or 11, or a range between any of these two values.

[0163] In some embodiments, a first microbial consortium may be established in a mixing chamber, in which various inputs may be mixed into a homogenous aqueous mixture to be input into a digestion reactor. In some embodiments, a digestions system described herein may comprise one or more mixing chambers in which a microbial consortium may be established. A mixing chamber may be fed an input or a series of inputs (e.g., target microbial strain, aqueous feedstock, carbon source, nitrogen source, nutrients, water, or any combination thereof) which may be mixed prior to being inputted into a first container (e.g., a first reactor). The first microbial consortium may be derived from microbes originally present in one or more digestion substrates and / or from other inputs into the mixing chamber. A first microbial consortium may be derived from inputs to a reactor in a digestion system described herein. The inputs may be from a whole broth (WB) from another digestion system (e.g., water-based phosphate solubilizing technology (PwST) or phosphate solubilizing technology (PST)). In some embodiments, a second microbial consortium is established in a first reactor. The second microbial consortium may be derived from microbes within the mixing chamber. In some embodiments, a third microbial consortium is established in a second reactor. The third microbial consortium may be derived from the first working fluid present in the first reactor and transferred to the second reactor. In some embodiments, a fourth microbial consortium is established in a third reactor. The fourth microbial consortium may be derived from the second working fluid present in the second reactor and transferred to the third reactor. In some embodiments, a fifth microbial consortium is established in a fourth reactor. The fifth microbial consortium may be derived from the fourth working fluid present in the fourth reactor and transferred to the fifth reactor. A microbial consortium can be present in any reactor of a digestion system described herein. A microbial consortium can be derived from the working fluid of a reactor of a digestion system described herein. A first microbial consortium may bederived from inputs to a first reactor and may be present in a base product of a digestion system. A first microbial consortium may be present in a first reactor, a second reactor, a third reactor, or a clarifier. Without wishing to be bound by theory, a first microbial consortium in a working fluid may shift its microbial population and form a second microbial consortium. A second microbial consortium may be present in a first reactor, a second reactor, a third reactor, or a clarifier. Without wishing to be bound by theory, a second microbial consortium in a working fluid may shift its microbial population and form a third microbial consortium. A third microbial consortium may be present in a first reactor, a second reactor, a third reactor, or a clarifier. Without wishing to be bound by theory, a third microbial consortium in a working fluid may shift its microbial population and form a fourth microbial consortium. A fourth microbial consortium may be present in a first reactor, a second reactor, a third reactor, or a clarifier. Without wishing to be bound by theory, a fourth microbial consortium in a working fluid may shift its microbial population and form a fifth microbial consortium. A fifth microbial consortium may be present in a first reactor, a second reactor, a third reactor, or a clarifier. Microbial consortia of the digestion system described herein may develop shifts in microbial communities based on conditions (e.g., nutrients, retention time, flow rate, pH, oxygen content, digestion products) of the reactors of the system and the working fluid. Without wishing to be bound by theory, as a working fluid incubates in a reactor of a bioreactor system described herein, microbes of a microbial consortium may become enriched, be maintained, or die which can shift a population of microbes in a microbial community in a working fluid and establish a new microbial consortium.

[0164] A portion of a first working fluid may be transferred to a second container of a serialized assembly of containers of a digestion system described herein. The working fluid of the second container may comprise a second working fluid. A portion of a second working fluid may be transferred to a third container of a serialized assembly of containers of a digestion system described herein. The working fluid of the third container may comprise a third working fluid. A portion of a third working fluid may be transferred to a fourth container of a serialized assembly of containers of a digestion system described herein. The working fluid of the fourth container may comprise a fourth working fluid. A portion of a fourth working fluid may be transferred to a fifth container of a serialized assembly of containers of a digestion system described herein. The working fluid of the fifth container may comprise a fifth working fluid. A portion of a fifth working fluid may be transferred to a sixth container of a serialized assembly of containers of a digestion system described herein. The working fluid of the sixth container may comprise a sixth working fluid.

[0165] The working fluid of a container in the digestion system may incubate in the container. A flow rate of the digestion system may increase or decrease a volume of working fluid. The working fluid of a first, second, third, fourth, fifth, or sixth container may increase in volume over a time period. The working fluid of a first, second, third, fourth, fifth, or sixth container may decrease in volume over a time period. The working fluid of a first, second, third, fourth, fifth, or sixth container may not increase or decrease in volume over a time period. The volume of working fluid in each of the containers of a digestion system may be the same. The volume of working fluid in each of the containers of a digestion system may be different. The volumes of the first working fluid, second working fluid, third working fluid, fourth working fluid, fifth working fluid, and / or sixth working fluid may be constant (e.g., unchanging over a time period). A constant volume may comprise a volume that does not increase or decrease over 1 minute, 5 minutes, 10 minutes, 30 minutes, 1 hours, 5 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, or 1 week.

[0166] As working fluid flows from each container of digestion system, the working fluid may comprise a flow rate. A first flow rate may comprise a flow rate of an aqueous feedstock inputted into a first container from a source outside the digestion system. A second flow rate may comprise a flow rate of a working fluid from a first container into a second container. A third flow rate may comprise a flow rate of a working fluid from a second container into a third container. A fourth flow rate may comprise a flow rate of a working fluid from a third container into a fourth container. A fifth flow rate may comprise a flow rate of a working fluid from a fourth container into a fifth container. A flow rate (e.g., first flow rate, second flow rate, third flow rate, fourth flow rate, fifth flow rate) may be at least about, at most about, or about 0.001, 0.003, 0.005, 0.01, 0.03, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0 gallons / min, or a range between any of these values.

[0167] A microbial consortium can comprise a population of microbes. The population of microbes can be generated from an input to the digestion system. An aqueous feedstock inputted into the digestion system may comprise a microbial consortium. Incubation of a working fluid in the digestion system may promote the growth of microbes within a microbial consortium (e.g., a first microbial consortium, a second microbial consortium, a third microbial consortium, a fourth microbial consortium, a fifth microbial consortium, a sixth microbial consortium). The microbes of a microbial consortium or at least a portion of microbes within the microbial consortium may have a desired plant growth promotion property. The plant growth promotion property may comprise shoot biomass, root biomass, nutrient uptake, crop yield, zinc solubilization, acid production, leaf area, chlorophyll content, photosynthetic activity, or total biomass.

[0168] Reactors of a digestion system may be fluidly connected. A portion of a working fluid in a first container may be transferred to a fluidly connected second container. A portion of a working fluid in a second container may be transferred to a fluidly connected third container. A portion of a working fluid in a third container may be transferred to a fluidly connected fourth container. A portion of a working fluid in a fourth container may be transferred to a fluidly connected fifth container. A portion of a working fluid in a fifth container may be transferred to a fluidly connected sixth container. In some embodiments, a transfer of working fluid between containers of a digestion system described herein may be continuous. A continuous flow of working fluid may comprise a flow of working fluid that does not stop or a flow of working fluid that stops for less than about 5 seconds, less than about 4 seconds, less than about 3 seconds, less than about 2 seconds, less than about 1 second, less than about 0.5 seconds, or less than about 0.1 seconds. A continuous flow of working fluid within a digestion system described herein (e.g., between containers of a digestion system) may have a first flow rate. A continuous flow of working fluid within a digestion system described herein (e.g., between containers of a digestion system) may have a second flow rate. In some embodiments, the first flow rate and the second flow rate are equal. A first flow rate may comprise a flow rate of fluid transferred from a source outside the digestion system into a first container. A second flow rate may comprise a rate of fluid flow from a first container to a second container. In some embodiments, an amount of working fluid and / or feedstock (e.g., aqueous organic feedstock) transferred into the first container over a time period is equal to an amount of working fluid and / or feedstock (e.g., aqueous organic feedstock) transferred into a second fluidly connected container over the same time period. In some embodiments, the first flow rate and the second flow rate are different. In some embodiments, a first flow rate may be faster than a second flow rate. In some embodiments, a first flow rate may be slower than a second flow rate. For example, flow rates may be different if an input stream into a first reactor is faster or slower than a stream of working fluid transferred from the first reactor to a second reactor. This difference in flow rates may result in different volumes of working fluid in containers of a digestion system.

[0169] In some embodiments, a first container of a digestion system comprises a constant volume. In some embodiments, a volume of a first container of a digestion system is different over time. In some embodiments, the flow rate between containers of the digestion system may maintain a constant volume in each container. A first container, a second container, a third container, a fourth container, a fifth container, and / or a sixth container may be maintained at a constant volume. A constant volume may be maintained by a continuous flow of fluid through a digestion system described herein. For example, similar flow rates of working fluid beingtransferred between containers of a digestion system may result in constant volumes of working fluid in the containers. This continuous flow of the bioreactor system may help enrich a microbial population with a targeted functionality (e.g., zinc solubilization) by not overflowing or depleting a working fluid in a container of the system.

[0170] In some embodiments, a digestion system may be inoculated with an inoculum of a microbe (e.g., an inoculum of a microbial strain). The inoculum of a microbial strain may be an isolated microbe. An isolated microbe can comprise a microbe grown or enriched outside of a natural environment (e.g., in a culture medium or a streak plate method). In some embodiments, the inoculum of a microbe may comprise a mixture of multiple isolated microbes. The inoculum of a microbe may comprise a mixture of at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, or more isolated microbes.

[0171] In some cases, a digestion system may not be reinoculated with an isolate (e.g., microbial strain) or combination of isolates following a first inoculation. Reinoculation of a digestion system may comprise providing a microbial strain following a previous inoculation.Reinoculation of a digestion system may comprise introducing a target isolate (e.g., microbial strain) in a container of the digestion system at a time point during operating of the digestion system. In some cases, a digestion system may be reinoculated with an isolate or combination of isolates at least every 20 days, at least every 50 days, at least every 100 days, at least every 200 days, at least every 300 days, at least every 400 days, at least every 500 days, or more. In some cases, a digestion system may be inoculated with an isolate or combination of isolates on day 1 of a digestion process and reinoculated 1, 2, 3, 4, 5, or more times after day 1 of the digestion process. In some cases, a digestion system may be reinoculated with a microbial strain described herein after operating the digestion system for a time period. For example, a digestion system may be reinoculated with a microbial strain described herein after operating the digestion system for a duration of time of at least about, at most about, or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 9 months, 12 months, 18 months, 24 months, 3 years, 4 years, 5 years, 10 years, or a range between any of these two values.

[0172] In some embodiments, the inoculation establishes an initial population of the target isolate having a concentration of at least, at most, or about IxlO2, 0.5xl03, IxlO3, 0.5xl04, IxlO4, 0.5xl05, IxlO5, 0.5xl06CFU / ml, or a range between any two of these values.

[0173] In some embodiments, the population of the microbial strain (e.g., concentration of the microbial strain) is maintained (e.g., retained) by at least about 0.00001%, at least about0.0001%, at least about 0.001%, at least about 0.01%, at least about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, or at least about 75% from the concentration of the microbial strain added into a container (e.g., a first container) of the digestion system. Biosolids (e.g., floc) may comprise small particles from a working fluid of a digestion system. The biosolids (e.g., floc) may accumulate in a clarifier chamber over time and separate from a supernatant (e.g., base product). In some embodiments, the population of the microbial strain may be retained in the floc (e.g., biosolids) of the digestion system. In some embodiments, a majority (95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%) of the inoculum of the microbe may be retained in the floc (e.g., biosolids) of the digestion system. Floc may be generated at any point during operating of a bioreactor system as described herein. For example, floc may be generated in a reactor of the bioreactor system (e.g., a first container, a second container, a third container, a fourth container, a fifth container, a sixth container, or any container of the system). For example, floc may be generated in a clarifier chamber of a bioreactor system. In some embodiments, floc may comprise at least a portion of zinc-solubilizing microbes generated in a digestion system described herein.

[0174] In some embodiments, there may be at least about 1 log CFU / ml, at least about 2 logs CFU / ml, at least about 3 logs CFU / ml, at least about 4 logs CFU / ml, at least about 5 logs CFU / ml, or greater than about 5 logs CFU / ml increase in a concentration of the population of the microbial strain after inoculation in a container (e.g., first container) of the digestion system described herein. In some embodiments, there may be at most about 5 logs CFU / ml, at most about 4 logs CFU / ml, at most about 3 logs CFU / ml, at most about 2 logs CFU / ml, at most about I log CFU / ml, or less than about I log CFU / ml increase in a concentration of the population of the microbial strain after inoculation in a container (e.g., first container) of the digestion system described herein. In some embodiments, there may be from about I log CFU / ml to about 8 logs CFU / ml increase in a concentration of the population of the microbial strain after inoculation in a container (e.g., first container) of the digestion system described herein. In some embodiments, there may be from about I log CFU / ml to about 2 logs CFU / ml, about I log CFU / ml to about 3 logs CFU / ml, about I log CFU / ml to about 4 logs CFU / ml, about I log CFU / ml to about 5 logs CFU / ml, about I log CFU / ml to about 6 logs CFU / ml, about I log CFU / ml to about 7 logs CFU / ml, about I log CFU / ml to about 8 logs CFU / ml, about 2 logs CFU / ml to about 3 logs CFU / ml, about 2 logs CFU / ml to about 4 logs CFU / ml, about 2 logs CFU / ml to about 5 logs CFU / ml, about 2 logs CFU / ml to about 6 logs CFU / ml, about 2 logs CFU / ml to about 7 logsCFU / ml, about 2 logs CFU / ml to about 8 logs CFU / ml, about 3 logs CFU / ml to about 4 logsCFU / ml, about 3 logs CFU / ml to about 5 logs CFU / ml, about 3 logs CFU / ml to about 6 logsCFU / ml, about 3 logs CFU / ml to about 7 logs CFU / ml, about 3 logs CFU / ml to about 8 logsCFU / ml, about 4 logs CFU / ml to about 5 logs CFU / ml, about 4 logs CFU / ml to about 6 logsCFU / ml, about 4 logs CFU / ml to about 7 logs CFU / ml, about 4 logs CFU / ml to about 8 logsCFU / ml, about 5 logs CFU / ml to about 6 logs CFU / ml, about 5 logs CFU / ml to about 7 logsCFU / ml, about 5 logs CFU / ml to about 8 logs CFU / ml, about 6 logs CFU / ml to about 7 logsCFU / ml, about 6 logs CFU / ml to about 8 logs CFU / ml, or about 7 logs CFU / ml to about 8 logs CFU / ml increase in a concentration of the population of the microbial strain after inoculation in a container (e.g., first container) of the digestion system described herein.

[0175] In some embodiments, there may be at least about 1 log CFU / ml, at least about 2 logs CFU / ml, at least about 3 logs CFU / ml, at least about 4 logs CFU / ml, at least about 5 logs CFU / ml, or greater than about 5 logs CFU / ml decrease in a concentration of the population of the microbial strain after inoculation in a container (e.g., first container) of the digestion system described herein. In some embodiments, there may be at most about 5 logs CFU / ml, at most about 4 logs CFU / ml, at most about 3 logs CFU / ml, at most about 2 logs CFU / ml, at most about 1 log CFU / ml, or less than about 1 log CFU / ml decrease in a concentration of the population of the microbial strain after inoculation in a container (e.g., first container) of the digestion system described herein. In some embodiments, there may be from about 1 log CFU / ml to about 8 logs CFU / ml decrease in a concentration of the population of the microbial strain after inoculation in a container (e.g., first container) of the digestion system described herein. In some embodiments, there may be from about 1 log CFU / ml to about 2 logs CFU / ml, about 1 log CFU / ml to about 3 logs CFU / ml, about 1 log CFU / ml to about 4 logs CFU / ml, about 1 log CFU / ml to about 5 logs CFU / ml, about 1 log CFU / ml to about 6 logs CFU / ml, about 1 log CFU / ml to about 7 logs CFU / ml, about 1 log CFU / ml to about 8 logs CFU / ml, about 2 logs CFU / ml to about 3 logs CFU / ml, about 2 logs CFU / ml to about 4 logs CFU / ml, about 2 logs CFU / ml to about 5 logsCFU / ml, about 2 logs CFU / ml to about 6 logs CFU / ml, about 2 logs CFU / ml to about 7 logsCFU / ml, about 2 logs CFU / ml to about 8 logs CFU / ml, about 3 logs CFU / ml to about 4 logsCFU / ml, about 3 logs CFU / ml to about 5 logs CFU / ml, about 3 logs CFU / ml to about 6 logsCFU / ml, about 3 logs CFU / ml to about 7 logs CFU / ml, about 3 logs CFU / ml to about 8 logsCFU / ml, about 4 logs CFU / ml to about 5 logs CFU / ml, about 4 logs CFU / ml to about 6 logsCFU / ml, about 4 logs CFU / ml to about 7 logs CFU / ml, about 4 logs CFU / ml to about 8 logsCFU / ml, about 5 logs CFU / ml to about 6 logs CFU / ml, about 5 logs CFU / ml to about 7 logsCFU / ml, about 5 logs CFU / ml to about 8 logs CFU / ml, about 6 logs CFU / ml to about 7 logsCFU / ml, about 6 logs CFU / ml to about 8 logs CFU / ml, or about 7 logs CFU / ml to about 8 logs CFU / ml decrease in a concentration of the population of the microbial strain after inoculation in a container (e.g., first container) of the digestion system described herein.

[0176] Incubation of a microbial consortium in the digestion system under conditions of a selective pressure source (e.g., zinc source) may enrich a microbial community with plant growth promotion properties (e.g., zinc solubilizing microbes). Incubation of the inoculum of the microbe and / or the zinc-solubilizing microbes of the microbial consortium may generate metabolites with desired plant growth promotion properties (e.g., zinc-solubilizing metabolites. The inoculum of the microbe, zinc-solubilizing microbes of the microbial consortium, zinc- solubilizing metabolites, or any combination thereof may be zinc-solubilizers in the working fluid of the system and / or in the output product (e.g., base product) of the digestion system. A concentration of zinc solubilizers may increase throughout a retention time of a digestion system described herein. The concentration of zinc solubilizers may increase from a first container to a second container of the digestion system. The concentration of zinc solubilizers may increase from a first container to a third container of the digestion system. The concentration of zinc solubilizers may increase from a first container to a fourth container of the digestion system. The concentration of zinc solubilizers may increase from a first container to a fifth container of the digestion system. The concentration of zinc solubilizers may increase from a first container to a sixth container of the digestion system. In some embodiments, an output product (e.g., base product) may have a higher concentration of zinc solubilizers than a concentration of zinc solubilizers in a first container of a digestion system.

[0177] In some embodiments, a concentration of zinc-solubilizers may increase from a first container to a second container, a third container, a fourth container, a fifth container, and / or a sixth container of a digestion system described herein. In some embodiments, a concentration of zinc-solubilizers in a second container, a third container, a fourth container, a fifth container, and / or a sixth container may be at least about 50x, at least about lOOx, at least about 200x, at least about 250x, at least about 300x, at least about 400x, at least about 500x, at least about 600x, at least about 700x, at least about an 800x, at least about 900x, at least about lOOOx, at least about 1250x, at least about 1500x, at least about 2000x, or greater than about 2000x increased from a concentration of zinc-solubilizers in a first container of a digestion system described herein. In some embodiments, a concentration of zinc-solubilizers in a second container, a third container, a fourth container, a fifth container, and / or a sixth container may be at most about 2000x, at most about 1500x, at most about 1250x, at most about lOOOx, at most about 900x, at most about an 800x, at most about 700x, at most about 600x, at most about 500x,at most about 400x, at most about 300x, at most about 250x, at most about 200x, at most about lOOx, at most about 50x, or less than about 50x increased from a concentration of zinc- solubilizers in a first container of a digestion system described herein.

[0178] In some embodiments, a concentration of zinc-solubilizers in a second container, a third container, a fourth container, a fifth container, and / or a sixth container may be from about 25x to about 2,500x increased from a concentration of zinc-solubilizers in a first container of a digestion system described herein. In some embodiments, a concentration of zinc-solubilizers in a second container, a third container, a fourth container, a fifth container, and / or a sixth container may be from about 25x to about 50x, about 25x to about lOOx, about 25x to about 150x, about 25x to about 200x, about 25x to about 250x, about 25x to about 500x, about 25x to about 750x, about 25x to about l,000x, about 25x to about l,500x, about 25x to about 2,000x, about 25x to about 2,500x, about 50x to about lOOx, about 50x to about 150x, about 50x to about 200x, about 50x to about 250x, about 50x to about 500x, about 50x to about 750x, about 50x to about l,000x, about 50x to about l,500x, about 50x to about 2,000x, about 50x to about 2,500x, about lOOx to about 150x, about lOOx to about 200x, about lOOx to about 250x, about lOOx to about 500x, about lOOx to about 750x, about lOOx to about l,000x, about lOOx to about l,500x, about lOOx to about 2,000x, about lOOx to about 2,500x, about 150x to about 200x, about 150x to about 250x, about 150x to about 500x, about 150x to about 750x, about 150x to about l,000x, about 150x to about l,500x, about 150x to about 2,000x, about 150x to about 2,500x, about 200x to about 250x, about 200x to about 500x, about 200x to about 750x, about 200x to about l,000x, about 200x to about l,500x, about 200x to about 2,000x, about 200x to about 2,500x, about 250x to about 500x, about 250x to about 750x, about 250x to about l,000x, about 250x to about l,500x, about 250x to about 2,000x, about 250x to about 2,500x, about 500x to about 750x, about 500x to about l,000x, about 500x to about l,500x, about 500x to about 2,000x, about 500x to about 2,500x, about 750x to about l,000x, about 750x to about l,500x, about 750x to about 2,000x, about 750x to about 2,500x, about l,000x to about l,500x, about l,000x to about 2,000x, about l,000x to about 2,500x, about l,500x to about 2,000x, about l,500x to about 2,500x, or about 2,000x to about 2,500x increased from a concentration of zinc-solubilizers in a first container of a digestion system described herein.

[0179] In some embodiments, the absolute population of the microbial inoculum of the digestion system may not decrease by more than about 0.000001%, 0.00001%, 0.0001%, 0.001%, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25% after incubation. In some embodiments, the absolute population of the microbial inoculum of the digestion system may not decrease by more than about 0.000001%, 0.00001%, 0.0001%, 0.001%, 0.01%,0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25% after incubation for a retention period of a digestion system (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, or 5 years). In some embodiments, the absolute population of the microbial inoculum of the digestion system may not decrease by more than about 0.000001%, 0.00001%, 0.0001%, 0.001%, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25% after incubation without inoculation of additional microbe.

[0180] In some embodiments, a proportion of a concentration of zinc solubilizers relative to a total bacterial population count may increase across containers of a fluidly connected digestion system. In some embodiments, a proportion of a concentration zinc solubilizers relative to a total bacterial population count may increase from a first container to a second container of a digestion system described herein. In some embodiments, a proportion of a concentration zinc solubilizers relative to a total bacterial population count may increase from a first container to a third container of a digestion system described herein. In some embodiments, a proportion of a concentration zinc solubilizers relative to a total bacterial population count may increase from a first container to a fourth container of a digestion system described herein. In some embodiments, a proportion of a concentration zinc solubilizers relative to a total bacterial population count may increase from a first container to a fifth container of a digestion system described herein. In some embodiments, a proportion of a concentration zinc solubilizers relative to a total bacterial population count may increase from a first container to a sixth container of a digestion system described herein.

[0181] Zinc solubilization can result in the production of zinc ions (Zn2+) as an insoluble source of zinc is solubilized by microbes described herein. A measure of zinc solubilization and capacity of microbes to solubilize zinc can be an amount of zinc ions (Zn2+) made soluble. In some embodiments, the zinc solubilization capacity (e.g., as Zn2+mg / L made soluble) of a working solution may increase in a digestion system provided herein. In some embodiments, the zinc solubilization capacity (e.g., as Zn2+mg / L made soluble) of a working solution may be at least about 50x higher, at least about 75x higher, at least about lOOx higher, at least about 150x higher, at least about 200x higher, at least about 250x higher, at least about 300x higher, at least about 350x higher, at least about 400x higher, at least about 450x higher, at least about 500x higher, at least about 600x higher, at least about 700x higher, at least about 800x higher, at least about 900x higher, at least about lOOOx higher, or greater than about l,000x higher in a second container, third container, or fourth container, compared to that in a first container of the digestion system. In some embodiments, the zinc solubilization capacity (e.g., as Zn2+mg / Lmade soluble) of a working solution may be at most about l,000x higher, at most about 900x higher, at most about 800x higher, at most about 700x higher, at most about 600x higher, at most about 500x higher, at most about 450x higher, at most about 400x higher, at most about 350x higher, at most about 300x higher, at most about 250x higher, at most about 200x higher, at most about 150x higher, at most about lOOx higher, at most about 75x higher, at most about 50x higher, or less than about 50x higher in a second container, third container, or fourth container, compared to that in a first container of the digestion system.

[0182] In some embodiments, a working solution may be obtained from a reactor of a digestion system and tested for zinc solubilizing capacity (e.g., quantified as Zn2+mg / L made soluble). In some embodiments, a working solution of a digestion system described herein may have a measure of Zn2+made soluble of at least about 50 mg / L, at least about 100 mg / L, at least about 150 mg / L, at least about 200 mg / L, at least about 250 mg / L, at least about 300 mg / L, at least about 350 mg / L, at least about 400 mg / L, at least about 450 mg / L, at least about 500 mg / L, at least about 600 mg / L, at least about 700 mg / L, at least about 800 mg / L, or greater than about 800 mg / L. In some embodiments, a working solution of a digestion system described herein may have a measure of Zn2+made soluble of at most about 800 mg / L, at most about 700 mg / L, at most about 600 mg / L, at most about 500 mg / L, at most about 450 mg / L, at most about 400 mg / L, at most about 350 mg / L, at most about 300 mg / L, at most about 250 mg / L, at most about 200 mg / L, at most about 150 mg / L, at most about 100 mg / L, at most about 50 mg / L, or less than about 50 mg / L. In some embodiments, a working solution of a digestion system described herein may have a measure of Zn2+ made soluble from about 20 mg / L to about 750 mg / L. In some embodiments, a working solution of a digestion system described herein may have a measure of Zn2+ made soluble from about 20 mg / L to about 50 mg / L, about 20 mg / L to about 100 mg / L, about 20 mg / L to about 150 mg / L, about 20 mg / L to about 200 mg / L, about 20 mg / L to about 250 mg / L, about 20 mg / L to about 300 mg / L, about 20 mg / L to about 350 mg / L, about 20 mg / L to about 400 mg / L, about 20 mg / L to about 450 mg / L, about 20 mg / L to about 500 mg / L, about 20 mg / L to about 750 mg / L, about 50 mg / L to about 100 mg / L, about 50 mg / L to about 150 mg / L, about 50 mg / L to about 200 mg / L, about 50 mg / L to about 250 mg / L, about 50 mg / L to about 300 mg / L, about 50 mg / L to about 350 mg / L, about 50 mg / L to about 400 mg / L, about 50 mg / L to about 450 mg / L, about 50 mg / L to about 500 mg / L, about 50 mg / L to about 750 mg / L, about 100 mg / L to about 150 mg / L, about 100 mg / L to about 200 mg / L, about 100 mg / L to about 250 mg / L, about 100 mg / L to about 300 mg / L, about 100 mg / L to about 350 mg / L, about 100 mg / L to about 400 mg / L, about 100 mg / L to about 450 mg / L, about 100 mg / L to about 500 mg / L, about 100 mg / L to about 750 mg / L, about 150 mg / L to about 200 mg / L, about 150 mg / Lto about 250 mg / L, about 150 mg / L to about 300 mg / L, about 150 mg / L to about 350 mg / L, about 150 mg / L to about 400 mg / L, about 150 mg / L to about 450 mg / L, about 150 mg / L to about 500 mg / L, about 150 mg / L to about 750 mg / L, about 200 mg / L to about 250 mg / L, about 200 mg / L to about 300 mg / L, about 200 mg / L to about 350 mg / L, about 200 mg / L to about 400 mg / L, about 200 mg / L to about 450 mg / L, about 200 mg / L to about 500 mg / L, about 200 mg / L to about 750 mg / L, about 250 mg / L to about 300 mg / L, about 250 mg / L to about 350 mg / L, about 250 mg / L to about 400 mg / L, about 250 mg / L to about 450 mg / L, about 250 mg / L to about 500 mg / L, about 250 mg / L to about 750 mg / L, about 300 mg / L to about 350 mg / L, about 300 mg / L to about 400 mg / L, about 300 mg / L to about 450 mg / L, about 300 mg / L to about 500 mg / L, about 300 mg / L to about 750 mg / L, about 350 mg / L to about 400 mg / L, about 350 mg / L to about 450 mg / L, about 350 mg / L to about 500 mg / L, about 350 mg / L to about 750 mg / L, about 400 mg / L to about 450 mg / L, about 400 mg / L to about 500 mg / L, about 400 mg / L to about 750 mg / L, about 450 mg / L to about 500 mg / L, about 450 mg / L to about 750 mg / L, or about 500 mg / L to about 750 mg / L.

[0183] Population of the microbe may be measured using methods including but not limited to spectrophotometers, cell counting, measures of turbidity, hemocytometers, electronic enumeration, measures of cell mass, measures of cell activity, cfu morphology on general nutrient media or selective and / or differentiating media, PCR, semi-quantitative PCR, and qPCR.

[0184] Zinc-solubilizing microbes may be identified using methods using agar plates or liquid testing solutions. Microbes may be tested on agar plates with insoluble zinc and the measurement of zinc solubilization capacity (e.g., amount of zinc made soluble), usually by the diameter of the zone of clearing around the microbial colony on the agar plate after incubation, may classify a microbe as capable of zinc solubilization. The greater the diameter, the more zinc was solubilized by the microbial colony. Microbes may be tested in liquid solution containing insoluble zinc to measure zinc solubilization capacity, by measuring the zinc made soluble after incubation. Microbes are inoculated in liquid zinc-media containing an insoluble source of zinc. After incubation, the solubilized zinc is measured in the form of Zn2+ using instruments such as Inductively Couples Plasma (ICP) Atomic Emission Spectrometer.

[0185] The addition of the target isolate (e.g., the inoculum of the microbial strain / microbe) can have an added benefit in improving target functionality of working solution in a reactor of the digestion system and / or in the output base product of the digestion system. In some embodiments, addition of the inoculum of the microbial strain can improve the zinc solubilization capacity of working fluids and base product of a digestion system compared toworking fluids and base product of a digestion system without an inoculum of the microbial strain .

[0186] In some embodiments, a working solution and / or base product of a digestion system inoculated with a target isolate may have an at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, or greater than 80% increase in Zn2+concentration in comparison with that of a working solution and / or base product of a digestion system not inoculated with a target isolate. In some embodiments, a working solution and / or base product of a digestion system inoculated with a target isolate may have an at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15% or less than about 15% increase in Zn2+concentration in comparison with that of a working solution and / or base product of a digestion system not inoculated with a target isolate.

[0187] In some embodiments, a working solution and / or base product of a digestion system inoculated with a target isolate may have from about 10% to about 80% increase in Zn2+concentration in comparison with that of a working solution and / or base product of a digestion system not inoculated with a target isolate. In some embodiments, a working solution and / or base product of a digestion system inoculated with a target isolate may have from about 10% to about 15%, about 10% to about 20%, about 10% to about 25%, about 10% to about 30%, about 10% to about 35%, about 10% to about 40%, about 10% to about 45%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 15% to about 20%, about 15% to about 25%, about 15% to about 30%, about 15% to about 35%, about 15% to about 40%, about 15% to about 45%, about 15% to about 50%, about 15% to about 60%, about 15% to about 70%, about 15% to about 80%, about 20% to about 25%, about 20% to about 30%, about 20% to about 35%, about 20% to about 40%, about 20% to about 45%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 25% to about 30%, about 25% to about 35%, about 25% to about 40%, about 25% to about 45%, about 25% to about 50%, about 25% to about 60%, about 25% to about 70%, about 25% to about 80%, about 30% to about 35%, about 30% to about 40%, about 30% to about 45%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 35% to about 40%, about 35% to about 45%, about 35% to about 50%, about 35% to about 60%, about 35% to about 70%, about 35% to about 80%,about 40% to about 45%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 45% to about 50%, about 45% to about 60%, about 45% to about 70%, about 45% to about 80%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 60% to about 70%, about 60% to about 80%, or about 70% to about 80% increase in Zn2+concentration in comparison with that of a working solution and / or base product of a digestion system not inoculated with a target isolate.

[0188] A digestion system may be inoculated with a microbial strain at the start of a digestion system, which may allow for the microbial strain to flow through the system and working fluids of the reactors. Without wishing to be bound by theory, a digestion system inoculated with a microbial strain may increase a zinc solubilization capacity of a working fluid and / or a base product compared to a working fluid and / or base product of an otherwise identical digestion system with no inoculated microbial strain. Without wishing to be bound by theory, a digestion system inoculated with a microbial strain may increase a zinc solubilization capacity of a working fluid and / or a base product compared to a working fluid and / or base product of an otherwise identical digestion system with the microbial strain added (e.g., spiked) at the end of the system. Without wishing to be bound by theory, a digestion system inoculated with a microbial strain may increase a zinc solubilization capacity of a base product compared to a base product of an otherwise identical digestion system with the microbial strain added (e.g., spiked) at the end of the system. Incubation of the microbial strain in the digestion system may enrich the working fluid with zinc-solubilizing microbes and / or generate zinc-solubilizing metabolites.E. Microbial Isolates

[0189] Certain microorganisms of the present disclosure have all of the identifying characteristics of the deposited strains and, in particular, the identifying characteristics of being able to promote plant growth and / or yield as described herein. In particular, the certain microorganisms of the present disclosure can refer to the deposited microorganisms as described herein, and strains derived therefrom.

[0190] Zinc is an essential micronutrient for plant growth and development. Zinc acts as an antioxidant in plants and is important in carbohydrate metabolism, auxin metabolism, and other plant growth promoting properties. A small amount of zinc may be present in soil as soluble zinc, with the rest present as insoluble minerals and other complexes. Zinc solubilizing microorganisms can solubilize zinc through acidification, excretion of organic acids, vitamins, phytohormones, and amino acids, oxido-reductive systems, production of chelating agents, proton extrusion, or other mechanisms of bioaugmentation. In some embodiments, disclosedherein are zinc solubilizing microbial strains, including compositions that include such strains and methods of using such strains to promote plant growth.

[0191] In some embodiments, the microbial strain is from a Bacillus genus. In some embodiments, the microbial strain is from a Bacillus safensis species. In some embodiments, the microbial strain is from a bacterial species other th n Bacillus safensis. In some embodiments, the Bacillus safensis strain is deposited under ATCC Accession No. PTA-127681 or an isolated clone thereof. In some embodiments, the 16S rRNA gene of the microbial strain comprises the nucleotide sequence of SEQ ID NO.: 1. In some embodiments, the microbial strain comprises a 16S rRNA gene comprising a nucleotide sequence that exhibits at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the nucleotide sequences as set forth in SEQ ID NO.: 1. In some embodiments, the 16S rRNA gene of the microbial strain comprises a nucleotide sequence that exhibits at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the nucleotide sequences as set forth in SEQ ID NO.: 1. In some embodiments, the gyrB gene of the microbial strain comprises the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the microbial strain comprises a gyrB gene comprising a nucleotide sequence that exhibits at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to a nucleotide sequence as set forth in SEQ ID NO: 4. In some embodiments, the rpoB gene of the microbial strain comprises the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the microbial strain comprises a rpoB gene comprising a nucleotide sequence that exhibits at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to a nucleotide sequence as set forth in SEQ ID NO: 7. Some embodiments provide a genus of plant growth-promoting microorganisms comprising any of the DNA sequences described herein and which enhances the health, growth and / or yield of a plant, as described herein.

[0192] In some embodiments, the microbial strain is from a Bacillus megaterium species. In some embodiments, the microbial strain is from a bacterial species other xasx Bacillus megaterium. In some embodiments, the Bacillus megaterium strain is deposited under ATCCAccession No. PTA-127683 or an isolated clone thereof. In some embodiments, the 16S rRNA gene of the microbial strain comprises the nucleotide sequence of SEQ ID NO.: 2. In some embodiments, the microbial strain comprises a 16S rRNA gene comprising a nucleotide sequence that exhibits at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the nucleotide sequences as set forth in SEQ ID NO.: 2. In some embodiments, the 16S rRNA gene of the microbial strain comprises a nucleotide sequence that exhibits at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the nucleotide sequences as set forth in SEQ ID NO.: 2. In some embodiments, the gyrB gene of the microbial strain comprises the nucleotide sequence of SEQ ID NO: 5. In some embodiments, the microbial strain comprises a gyrB gene comprising a nucleotide sequence that exhibits at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to a nucleotide sequence as set forth in SEQ ID NO: 5. In some embodiments, the rpoB gene of the microbial strain comprises the nucleotide sequence of SEQ ID NO: 8. In some embodiments, the microbial strain comprises a rpoB gene comprising a nucleotide sequence that exhibits at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to a nucleotide sequence as set forth in SEQ ID NO: 8. Some embodiments provide a genus of plant growth-promoting microorganisms comprising any of the DNA sequences described herein and which enhances the health, growth and / or yield of a plant, as described herein.

[0193] In some embodiments, the microbial strain is from a Bacillus megaterium species. In some embodiments, the microbial strain is from a bacterial species other i &n Bacillus megaterium. In some embodiments, the Bacillus megaterium strain is deposited under ATCC Accession No. PTA-127682 or an isolated clone thereof. In some embodiments, the 16S rRNA gene of the microbial strain comprises the nucleotide sequence of SEQ ID NO.: 3. In some embodiments, the microbial strain comprises a 16S rRNA gene comprising a nucleotide sequence that exhibits at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, atleast 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the nucleotide sequences as set forth in SEQ ID NO.: 3. In some embodiments, the 16S rRNA gene of the microbial strain comprises a nucleotide sequence that exhibits at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the nucleotide sequences as set forth in SEQ ID NO.: 3. In some embodiments, the gyrB gene of the microbial strain comprises the nucleotide sequence of SEQ ID NO: 6. In some embodiments, the microbial strain comprises a gyrB gene comprising a nucleotide sequence that exhibits at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to a nucleotide sequence as set forth in SEQ ID NO: 6. In some embodiments, the rpoB gene of the microbial strain comprises the nucleotide sequence of SEQ ID NO: 9. In some embodiments, the microbial strain comprises a rpoB gene comprising a nucleotide sequence that exhibits at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to a nucleotide sequence as set forth in SEQ ID NO: 9. Some embodiments provide a genus of plant growth-promoting microorganisms comprising any of the DNA sequences described herein and which enhances the health, growth and / or yield of a plant, as described herein.F. Exemplary Digestion Systems for Production of Isolates

[0194] The present disclosure provides systems with conditions to produce biostimulant products with plant-growth promoting capabilities (e.g., improved zinc solubilization capacity). Zinc Solubilizing Technology (ZST) System

[0195] Embodiments of systems and methods described herein produce biostimulant products that may have a multi-modal way of promoting zinc uptake in plants. Biostimulant products produced by embodiments described herein may be used to promote plant growth by applying the products to plants and / or plant growth media (e.g., soil, fertilizer). One mode of action of products produced in some embodiments may be enhanced zinc uptake included in the products. Another mode of action may be increases in soil zinc and uptake of zinc stimulated by microbes and / or microbial metabolites present in the products produced in embodiments described herein.

[0196] In an aspect, the present disclosure provides a method, comprising: transferring a feedstock (e.g., aqueous organic feedstock) and an inoculum of a microbe that is capable of promoting zinc solubilization into a first container comprising a volume of a first working fluid. The aqueous organic feedstock may comprise: (i) a first microbial consortium; and / or (ii) digestion products produced by digestion of an organic material by microbes in the first microbial consortium. In some cases, the inoculum may be incubated at a zinc ion concentration that selectively promotes growth of the microbe. The population of the microbe may be increased. The population of the microbe may be decreased.

[0197] In an aspect, the present disclosure provides a method, comprising (a) transferring a feedstock (e.g., aqueous organic feedstock) and an inoculum of a microbe that is capable of promoting zinc solubilization into a first container comprising a volume of a first working fluid, wherein the feedstock (e.g., aqueous organic feedstock) comprises: (i) a first microbial consortium; and (ii) digestion products produced by digestion of an organic material by microbes in the first microbial consortium; and (b) incubating the inoculum at a zinc ion concentration that selectively promotes growth of the microbe, thereby increasing the population of the microbe. Incubation of the inoculum of the microbial strain may increase or maintain a population of the microbial strain relative to one or more microbes of the microbial consortium in the working fluid of the reactor of the digestion system.

[0198] In some embodiments, incubating the zinc-solubilizing microbial strain at a zinc ion concentration may selectively enhance the survival of zinc solubilizing microbes. In some embodiments, incubating the microbial consortium at a zinc ion concentration may increase the proportion of microbes that can tolerate zinc (e.g., zinc solubilizers or zinc-solubilizing microbes). Without wishing to be bound by theory, addition of a selective pressure having a zinc ion concentration may shift a microbial population towards microbes with zinc solubilizing properties.

[0199] In some embodiments, the zinc-solubilizing microbial strain (e.g., microbe) is capable of enhancing zinc uptake in plants or promoting soil zinc content and uptake of zinc from soil. In some embodiments, the conditions of the digestion system (e.g., pH, nutrients, flow rate, retention time, selective pressure, carbon source, nitrogen source, or any combination thereof) may promote growth of one or more microbes in the first microbial consortium that may be capable of promoting plant growth or zinc uptake in plants, or of generating metabolites capable of promoting plant growth or zinc uptake in plants. In some embodiments, during the incubating the microbe or one or more microbes in the first microbial consortium, metabolites may be produced capable of promoting plant growth and zinc solubilization. The metabolites may havezinc solubilizing capacity and increase an amount of zinc available to plants. The metabolites may enhance the presence of bioavailable zinc ions. An increase in zinc ions following incubation with zinc-solubilizing metabolites may be from zinc solubilization or stimulation of bacteria on a zinc source (e.g., ZnO). The available zinc for plants may promote growth, vigor, or yield of a plant. In some embodiments, the incubating increases a population of one or more microbes in the microbial consortium capable of promoting plant growth. In some embodiments, the feedstock (e.g., aqueous organic feedstock) further comprises an inorganic substrate. In some embodiments, the first microbial consortium further comprises microbes derived from the inorganic substrate. In some embodiments, the inorganic substrate comprises rock phosphate. In some embodiments, the microbe or microbes may be of the species Bacillus safensis, Bacillus amyloliquefaciens. Bacillus megaterium, Pseudomonas spp., Agrobacterium spp., Bacillus circulans. Burkholderia lata, Lysinibacillus spp., Burkholderia lalens. Bacillus cereus, B. aryabhaUai, Pseudomonas laiw enensis, Acinetobacter spp., Paenibacillus sonchi, Chitinophaga spp., Gluconobacter spp., Diazotrophicus spp., Klebsiella spp., Serratia spp., Thiobacillus spp. or any combination thereof. In some embodiments, the microbe is the Bacillus safensis strain deposited under ATCC Accession No. PTA-127681 (MS4666), the Bacillus megaterium strain deposited under ATCC Accession No. PTA-127683 (MS4689), or the Bacillus megaterium strain deposited under ATCC Accession No. PTA-127682 (MS4687). In some embodiments, the first working fluid comprises (a) a second microbial consortium derived from the feedstock (e.g., aqueous organic feedstock), and / or (b) digestion products produced by digestion of substances present in the organic feedstock by the first microbial consortium and the microbe(s). In some embodiments, the method further comprises transferring at least a portion of the first working fluid into a second container comprising a second working fluid and incubating the second working fluid in the second container. In some embodiments, the second working fluid comprises (a) a third microbial consortium derived from the first working fluid, and (b) digestion products produced by digestion of substances present in the first working fluid by the third microbial consortium and the microbe. In some embodiments, the amount of the feedstock (e.g., aqueous organic feedstock) transferred into the first container over a time period is equal to the amount of the first working fluid transferred into the second container over the same time period. In some embodiments, the amount of the feedstock (e.g., aqueous organic feedstock) transferred into the first container over a time period is equal to the amount of the first working fluid transferred into the second container over different time periods. In some embodiments, the amount of the feedstock (e.g., aqueous organic feedstock) transferred into the first container over a time period is not equal to the amount of the first working fluid transferred into thesecond container over the same time period. In some embodiments, the amount of the feedstock (e.g., aqueous organic feedstock) transferred into the first container over a time period is not equal to the amount of the first working fluid transferred into the second container over different time periods. In some embodiments, the volume of the first working fluid in the first container is maintained constant. In some embodiments, transferring the feedstock (e.g., aqueous organic feedstock) into the first container comprises continuously flowing the feedstock (e.g., aqueous organic feedstock) into the first container at a first flow rate, transferring the portion of the first working fluid into the second container comprises continuously flowing the portion of the first working fluid into the second container at a second flow rate, and the first flow rate and the second flow rate may be equal. In some embodiments, the method further comprises transferring at least a portion of the second working fluid to a third container comprising a third working fluid and incubating the third working fluid in the third container. In some embodiments, the method further comprises transferring at least a portion of the third working fluid into a fourth container comprising a fourth working fluid and incubating the fourth working fluid in the fourth container. In some embodiments, the first working fluid, the second working fluid, the third working fluid, and the fourth working fluid may be maintained at constant volumes. In some embodiments, the first working fluid, the second working fluid, the third working fluid, and the fourth working fluid may not be maintained at constant volumes. In some embodiments, a plant growth promoting product made by the method. In some embodiments, a method of promoting zinc solubilization which increases plant bioavailable zinc, comprises contacting the plant and / or a medium in which the plant is growing with the product.

[0200] The digestion process of the zinc solubilizing technology (ZST) may be an anerobic process. The digestion process of a ZST system may incorporate an aerobic phase, a microaerobic phase, or an anaerobic phase in any container of the digestion system. The digestion system described herein may comprise an aerobic process, a microaerobic process, an anaerobic process, or any combination thereof.

[0201] In some embodiments, a serialized set of reaction chambers may be used in a method of producing a biostimulant product, as described herein. In some embodiments, conditions within reactor chambers may be established to selectively promote the production of one or more microbes that have a specific desired plant growth promoting effect.ZST 1.0 System

[0202] FIG. 1 schematically illustrates an exemplary batch digestion system 100 with conditions (e.g., microbes) that produce biostimulant products that may have a multi-modal way of promoting zinc solubilization. The system 100 may comprise at least one reactor 110 and thereactor 110 may be a fluidized bed reactor or a packed bed reactor. Water may act as a hydraulic source. The reactor may comprise an input channel or a series of input channels that flows inputs 130, 140, 150, 160, 170, 180 into the digestion system. In some cases, the inputs can be added individually to the reactor 110. In some cases, the inputs may be added in combination with one another into the reactor 110. The inputs, as described herein, may comprise water, a carbon source 130, a nitrogen source 140, micronutrients 150, a microbial product or intermediate product of another digestion system (e.g., PST WB 160), a microbial isolate 170, a zinc source 180, or any combination thereof. The inputs may comprise other organic materials. An outflow port may extend from the reactor from which a biostimulant product or digestion product (e.g., base product) may be collected.

[0203] The system 100 may comprise a pH sensor, pH controller, or similar mechanic to monitor and / or control the pH of the reactor and / or working fluid within the reactor so that pH is maintained at a threshold. The pH sensor or controller may be automatic. In some embodiments, a pH in the reactor 110 may be between 4.0-9.0. In some embodiments, the pH in the reactor may be at least about 3.0, at least about 3.5, at least about 4.0, at least about 4.5, at least about 5.0, at least about 5.5, at least about 6.0, at least about 6.5, at least about 7.0, at least about 7.5, at least about 8.0, at least about 8.5, at least about 9.0, at least about 9.5, or at least about 10.0. In some embodiments, the pH in the reactor may be at most about 10.0, at most about 9.5, at most about 9.0, at most about 8.5, at most about 8.0, at most about 7.5, at most about 7.0, at most about 6.5, at most about 6.0, at most about 5.5, at most about 5.0, at most about 4.5, at most about 4.0, at most about 3.5, or at most about 3.0. In some embodiments, the pH in the reactor may be from about 2 to about 10. In some embodiments, the pH in the reactor may be from about 2 to about 3, about 2 to about 4, about 2 to about 4.5, about 2 to about 5, about 2 to about 5.5, about 2 to about 6, about 2 to about 6.5, about 2 to about 7, about 2 to about 8, about 2 to about 9, about 2 to about 10, about 3 to about 4, about 3 to about 4.5, about 3 to about 5, about 3 to about 5.5, about 3 to about 6, about 3 to about 6.5, about 3 to about 7, about 3 to about 8, about 3 to about 9, about 3 to about 10, about 4 to about 4.5, about 4 to about 5, about 4 to about 5.5, about 4 to about 6, about 4 to about 6.5, about 4 to about 7, about 4 to about 8, about 4 to about 9, about 4 to about 10, about 4.5 to about 5, about 4.5 to about 5.5, about 4.5 to about 6, about 4.5 to about 6.5, about 4.5 to about 7, about 4.5 to about 8, about 4.5 to about 9, about 4.5 to about 10, about 5 to about 5.5, about 5 to about 6, about 5 to about 6.5, about 5 to about 7, about 5 to about 8, about 5 to about 9, about 5 to about 10, about 5.5 to about 6, about 5.5 to about 6.5, about 5.5 to about 7, about 5.5 to about 8, about 5.5 to about 9, about 5.5 to about 10, about 6 to about 6.5, about 6 to about 7, about 6 to about 8, about 6 to about 9, about 6 to about10, about 6.5 to about 7, about 6.5 to about 8, about 6.5 to about 9, about 6.5 to about 10, about 7 to about 8, about 7 to about 9, about 7 to about 10, about 8 to about 9, about 8 to about 10, or about 9 to about 10.

[0204] An outflow port may be positioned on the top, middle, and / or bottom of the reactor 110. Additionally, the reactor may comprise another outflow port 180 for reintroducing working fluid back into the same reactor, and may be pumped back to just below the surface of the same reactor to maintain homogeneous conditions within the working solutions. In this manner, working fluid may be recycled within the digestion system 100. For example, working fluid from the reactor 110 may be reintroduced back into the reactor 110. Biosolids (e.g., floc) may be generated through the process. Biosolids may comprise organic materials recovered from the feedstock, waste, wastewater, and / or sludge material of a working fluid of a digestion system described herein. In some embodiments, floc can comprise biosolids. In some embodiments, floc may comprise biosolids and / or other solid particles from digestion products of organic materials. In some embodiments, floc can be a flocculated mass of microorganisms, extraceHular polymeric substance (EPS) and adsorbed organic and inorganic material. A flocculated mass can comprise an aggregated mass of microorganis s, extracellular polymeric substance (EPS) and adsorbed organic and inorganic material. Suspended particles from the organic substrates of the working fluid may aggregate to form floc. Floc may be produced throughout the fermentation process in the digestion system.

[0205] In some embodiments, the input composition, as described herein, may comprise a selective pressure or a zinc source. Without wishing to be bound by theory, the selective pressure source (e.g., zinc source) may shift the complex microbial consortia of the digestion system 100 to increased zinc solubilizing microbes. The selective pressure source may change the ionic concentration of the working fluid. A shift in the complex microbial consortia may occur to favor microbes of the microbial consortium with zinc-solubilizing properties. In some embodiments, the selective pressure may create a high ionic concentration which selectively inhibits the growth of some microbes within the working fluid, while promoting the growth or survival of other microbes (e.g., an inoculum of a microbe and / or zinc-solubilizing microbes of the microbial consortium) in the working fluid. The zinc solubilizing microbes may comprise microbes that can survive a higher zinc ion concentration in a working fluid caused by addition of the selective pressure. In some embodiments, the inoculum of a microbe may comprise a zinc solubilizing microbe. Addition of the selective pressure to the digestion system 100 may promote survival of the inoculum of the microbe. The terms “selective pressure source” and “selective pressure” may be used interchangeably. Addition of a selective pressure source (e.g.,zinc source) may increase a concentration of zinc within the working fluid of the digestion system 100.

[0206] An inoculum of a microbial strain in the digestion system 100 may be configured to survive as a population of the microbial strain in the increased concentration of zinc. Survival of a microbial strain can comprise a microbial strain that does not die in the presence of the high zinc concentration. Survival (e.g., maintenance) of a microbial strain can comprise a microbial strain that grows (e.g., increases in number) or does not fluctuate in concentration in the presence of the high zinc concentration. Survival (e.g., maintenance) of a population of the microbial strain can comprise a population of the microbial strain that dies at a slower rate relative to a rate of death of other microbes in the presence of the high zinc concentration. Maintenance of a microbial strain added to a digestion system herein can comprise percentage of a concentration of the microbial strain present in the base product (e.g., supernatant) of the system relative to an initial concentration added to the digestion system. Survival (e.g., maintenance) of a population of the microbial strain following addition of a selective pressure can comprise at least about 0.01%, at least about 0.05%, at least about 1%, at least about 1.5%, at least about 2%, at least about 2.5%, at least about 3%, at least about 3.5%, at least about 4%, at least about 4.5%, at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, or at least about 75% of an initial concentration of a population of the microbial strain present in the base product of the digestion system. An initial concentration may comprise a concentration of a population of the microbial strain added into a first container of the digestion system. Survival (e.g., maintenance) of a population of the microbial strain following addition of a selective pressure can comprise a concentration of the microbial strain that is at most about 75%, at most about 70%, at most about 60%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, or at most about 10% of a concentration of the microbial strain at the beginning of operating of the digestion system. Survival (e.g., maintenance) of a population of the microbial strain following addition of a selective pressure can comprise a concentration of the microbial strain that is from about 5% to about 80% of a concentration of the microbial strain at the beginning of operating of the digestion system. . Survival (e.g., maintenance) of a population of the microbial strain following addition of a selective pressure can comprise a concentration of the microbial strain that is from about 5% to about 10%, about 5% to about 15%, about 5% to about 20%, about 5% to about 25%, about 5% to about 30%, about 5% to about 35%, about 5% to about 40%, about 5% to about 50%, about 5% to about60%, about 5% to about 70%, about 5% to about 80%, about 10% to about 15%, about 10% to about 20%, about 10% to about 25%, about 10% to about 30%, about 10% to about 35%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 15% to about 20%, about 15% to about 25%, about 15% to about 30%, about 15% to about 35%, about 15% to about 40%, about 15% to about 50%, about 15% to about 60%, about 15% to about 70%, about 15% to about 80%, about 20% to about 25%, about 20% to about 30%, about 20% to about 35%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 25% to about 30%, about 25% to about 35%, about 25% to about 40%, about 25% to about 50%, about 25% to about 60%, about 25% to about 70%, about 25% to about 80%, about 30% to about 35%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 35% to about 40%, about 35% to about 50%, about 35% to about 60%, about 35% to about 70%, about 35% to about 80%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 60% to about 70%, about 60% to about 80%, or about 70% to about 80% of a concentration of the microbial strain at the beginning of operating of the digestion system .

[0207] One or more microbes of a microbial consortium in the digestion system 100 may not be able to survive the increased concentration of zinc. Incubating the working fluid comprising a microbial consortium with a selective pressure source (e.g., zinc source) may raise a zinc ion concentration of a working fluid. The population of the microbial strain may be tolerant to the increased zinc ion concentration. The increased zinc ion concentration may be toxic to a portion of microbes in a microbial consortium. A portion of microbes of the microbial consortium may be tolerant to the increased zinc ion concentration. Incubating the working fluid comprising a microbial consortium with a selective pressure source (e.g., zinc source) may increase or maintain an amount of a population of the microbial strain (e.g., a target isolate or isolated microbe) and decrease other microbes in the digestion system 100. Incubating the working fluid comprising a microbial consortium with a selective pressure source (e.g., zinc source) may increase or maintain an amount of zinc-solubilizing microbes in a microbial consortium of the working fluid. A maintained amount of a population of the microbial strain may comprise no change in an amount of a microbe across a duration of time. A maintained inoculum of a microbe may change its amount in a working fluid of a digestion system 100 less than 0.1%, less than 0.5%, less than 1%, less than 5%, or less than 10% over a duration of time. The duration of time may be at least about, at most about, or about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 12hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 1 month 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 12 months, 2 years, 3 years, 4 years, or 5 years or a range between any of these values. Without wishing to be bound by theory, the addition of a selective pressure source (e.g., zinc source) may increase a proportion of a population of the microbial strain relative to the microbes or at least a portion of the microbes in a microbial consortium in the digestion system 100. Without wishing to be bound by theory, incubating the microbial strain with the selective pressure source (e.g., zinc source) may decrease an amount of a population of the microbial strain less than a decrease of at least a portion of the microbes in a microbial consortium in the digestion system 100.

[0208] The zinc source may comprise zinc oxide (ZnO) and may be added to the digestion system 100 on a first day of a digestion process. In some cases, the zinc source (e.g., ZnO) may be added to the digestion system 100 daily. In some cases, the zinc source (e.g., ZnO) may be added to the digestion system 100 every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, and / or weekly. In some cases, the zinc source may comprise zinc carbonate, zinc sulfate, calamine ore, or any combination thereof. The zinc source (e.g., zinc oxide) may be added to the reactor at a concentration between 0.1-1.0% w / v. The concentration of the zinc source may be based on the concentration of all components of a working fluid of the system as described herein.

[0209] In some embodiments, the zinc source (e.g., zinc oxide) may be added to the reactor at a concentration of at least about 0.1% w / v, at least about 0.05% w / v, at least about 0.1% w / v, at least about 0.2% w / v, at least about 0.3% w / v, at least about 0.4% w / v, at least about 0.5% w / v, at least about 0.6% w / v, at least about 0.7% w / v, at least about 0.8% w / v, at least about 0.9% w / v, at least about 1.0% w / v, at least about 1.1 % w / v, at least about 1.2% w / v, at least about 1.3% w / v, at least about 1.4% w / v, or at least about 1.5% w / v. In some embodiments, the zinc source (e.g., zinc oxide) may be added to the reactor at a concentration of at most about 1.5% w / v, at most about 1.4% w / v, at most about 1.3% w / v, at most about 1.2% w / v, at most about 1.1% w / v, at most about 1.0% w / v, at most about 0.9% w / v, at most about 0.8% w / v, at most about 0.7% w / v, at most about 0.6% w / v, at most about 0.5% w / v, at most about 0.4% w / v, at most about 0.3% w / v, at most about 0.2% w / v, at most about 0.1% w / v, or at most about 0.05% w / v.

[0210] In some embodiments, the zinc source (e.g., zinc oxide) may be added to the reactor at a concentration of about 0.05% w / v to about 1% w / v. In some embodiments, the zinc source (e.g., zinc oxide) may be added to the reactor at a concentration of about 0.05% w / v to about 0.1%w / v, about 0.05% w / v to about 0.2% w / v, about 0.05% w / v to about 0.3% w / v, about 0.05% w / v to about 0.4% w / v, about 0.05% w / v to about 0.45% w / v, about 0.05% w / v to about 0.5% w / v, about 0.05% w / v to about 0.6% w / v, about 0.05% w / v to about 0.7% w / v, about 0.05% w / v to about 0.8% w / v, about 0.05% w / v to about 0.9% w / v, about 0.05% w / v to about 1% w / v, about 0.1% w / v to about 0.2% w / v, about 0.1% w / v to about 0.3% w / v, about 0.1% w / v to about 0.4% w / v, about 0.1% w / v to about 0.45% w / v, about 0.1% w / v to about 0.5% w / v, about 0.1% w / v to about 0.6% w / v, about 0.1% w / v to about 0.7% w / v, about 0.1% w / v to about 0.8% w / v, about 0.1% w / v to about 0.9% w / v, about 0.1% w / v to about 1% w / v, about 0.2% w / v to about 0.3% w / v, about 0.2% w / v to about 0.4% w / v, about 0.2% w / v to about 0.45% w / v, about 0.2% w / v to about 0.5% w / v, about 0.2% w / v to about 0.6% w / v, about 0.2% w / v to about 0.7% w / v, about 0.2% w / v to about 0.8% w / v, about 0.2% w / v to about 0.9% w / v, about 0.2% w / v to about 1% w / v, about 0.3% w / v to about 0.4% w / v, about 0.3% w / v to about 0.45% w / v, about 0.3% w / v to about 0.5% w / v, about 0.3% w / v to about 0.6% w / v, about 0.3% w / v to about 0.7% w / v, about 0.3% w / v to about 0.8% w / v, about 0.3% w / v to about 0.9% w / v, about 0.3% w / v to about 1% w / v, about 0.4% w / v to about 0.45% w / v, about 0.4% w / v to about 0.5% w / v, about 0.4% w / v to about 0.6% w / v, about 0.4% w / v to about 0.7% w / v, about 0.4% w / v to about 0.8% w / v, about 0.4% w / v to about 0.9% w / v, about 0.4% w / v to about 1% w / v, about 0.45% w / v to about 0.5% w / v, about 0.45% w / v to about 0.6% w / v, about 0.45% w / v to about 0.7% w / v, about 0.45% w / v to about 0.8% w / v, about 0.45% w / v to about 0.9% w / v, about 0.45% w / v to about 1% w / v, about 0.5% w / v to about 0.6% w / v, about 0.5% w / v to about 0.7% w / v, about 0.5% w / v to about 0.8% w / v, about 0.5% w / v to about 0.9% w / v, about 0.5% w / v to about 1% w / v, about 0.6% w / v to about 0.7% w / v, about 0.6% w / v to about 0.8% w / v, about 0.6% w / v to about 0.9% w / v, about 0.6% w / v to about 1% w / v, about 0.7% w / v to about 0.8% w / v, about 0.7% w / v to about 0.9% w / v, about 0.7% w / v to about 1% w / v, about 0.8% w / v to about 0.9% w / v, about 0.8% w / v to about 1% w / v, or about 0.9% w / v to about 1% w / v.

[0211] In some embodiments, the input composition to the digestion system 100 may comprise a carbon source 130. The carbon source may be glucose, malate, gluconic acid, lactose, sucrose, pyruvate, other simple sugars, or any combination thereof. In some cases, the carbon source may be added to the digestion system on a first day of a digestion process. In some cases, the carbon source may be added to the digestion system on a second day, third day, fourth day, or any day following a first day of a digestion process. The carbon source (e.g., glucose) may be added to the reactor to maintain a concentration range of 0.5%-3.0% w / v of carbon based on the total volume of the working fluid of the system. In some embodiments, the concentration of carbonthat may be maintained can be based on the total concentration of all components of the working fluid in the at a point during the digestion process of the digestion system 100.

[0212] In some embodiments, the carbon source (e.g., glucose) may be added to a reactor to maintain a concentration of carbon source in the working fluid of at least about 0.1% w / v, at least about 0.25% w / v, at least about 0.5% w / v, at least about 0.75% w / v, at least about 1.0% w / v, at least about 1.25% w / v, at least about 1.5% w / v, at least about 1.75% w / v, at least about 2.0% w / v, at least about 2.5% w / v, at least about 3.0% w / v, at least about 5.0% w / v, at least about 7.5% w / v, or at least about 10.0% w / v. In some embodiments, the carbon source (e.g., glucose) may be added to a reactor to maintain a concentration of carbon source in the working fluid of at most about 10.0% w / v, at most about 7.5% w / v, at most about 5.0% w / v, at most about 3.0% w / v, at most about 2.5% w / v, at most about 2.0% w / v, at most about 1.75% w / v, at most about 1.5% w / v, at most about 1.25% w / v, at most about 1.0% w / v, at most about 0.75% w / v, at most about 0.5% w / v, at most about 0.25% w / v, or at most about 10.0% w / v.

[0213] In some embodiments, the carbon source (e.g., glucose) may be added to a reactor to maintain a concentration of carbon source in the working fluid of about 0.1% w / v to about 5% w / v. In some embodiments, the carbon source (e.g., glucose) may be added to a reactor to maintain a concentration of carbon source in the working fluid of about 0.1% w / v to about 0.25% w / v, about 0.1% w / v to about 0.5% w / v, about 0.1% w / v to about 0.75% w / v, about 0.1% w / v to about 1% w / v, about 0.1% w / v to about 1.25% w / v, about 0.1% w / v to about 1.5% w / v, about 0.1% w / v to about 1.75% w / v, about 0.1% w / v to about 2% w / v, about 0.1% w / v to about 2.5% w / v, about 0.1% w / v to about 3% w / v, about 0.1% w / v to about 5% w / v, about 0.25% w / v to about 0.5% w / v, about 0.25% w / v to about 0.75% w / v, about 0.25% w / v to about 1% w / v, about 0.25% w / v to about 1.25% w / v, about 0.25% w / v to about 1.5% w / v, about 0.25% w / v to about 1.75% w / v, about 0.25% w / v to about 2% w / v, about 0.25% w / v to about 2.5% w / v, about 0.25% w / v to about 3% w / v, about 0.25% w / v to about 5% w / v, about 0.5% w / v to about 0.75% w / v, about 0.5% w / v to about 1% w / v, about 0.5% w / v to about 1.25% w / v, about 0.5% w / v to about 1.5% w / v, about 0.5% w / v to about 1.75% w / v, about 0.5% w / v to about 2% w / v, about 0.5% w / v to about 2.5% w / v, about 0.5% w / v to about 3% w / v, about 0.5% w / v to about 5% w / v, about 0.75% w / v to about 1% w / v, about 0.75% w / v to about 1.25% w / v, about 0.75% w / v to about 1.5% w / v, about 0.75% w / v to about 1.75% w / v, about 0.75% w / v to about 2% w / v, about 0.75% w / v to about 2.5% w / v, about 0.75% w / v to about 3% w / v, about 0.75% w / v to about 5% w / v, about 1% w / v to about 1.25% w / v, about 1% w / v to about 1.5% w / v, about 1% w / v to about 1.75% w / v, about 1% w / v to about 2% w / v, about 1% w / v to about 2.5% w / v, about 1% w / v to about 3% w / v, about 1% w / v to about 5% w / v, about 1.25% w / v to about 1.5%w / v, about 1.25% w / v to about 1.75% w / v, about 1.25% w / v to about 2% w / v, about 1.25% w / v to about 2.5% w / v, about 1.25% w / v to about 3% w / v, about 1.25% w / v to about 5% w / v, about 1.5% w / v to about 1.75% w / v, about 1.5% w / v to about 2% w / v, about 1.5% w / v to about 2.5% w / v, about 1.5% w / v to about 3% w / v, about 1.5% w / v to about 5% w / v, about 1.75% w / v to about 2% w / v, about 1.75% w / v to about 2.5% w / v, about 1.75% w / v to about 3% w / v, about 1.75% w / v to about 5% w / v, about 2% w / v to about 2.5% w / v, about 2% w / v to about 3% w / v, about 2% w / v to about 5% w / v, about 2.5% w / v to about 3% w / v, about 2.5% w / v to about 5% w / v, or about 3% w / v to about 5% w / v.

[0214] In some embodiments, the input composition to the digestion system 100 may comprise a nitrogen source 140. The nitrogen source may be ammonium sulfate, ammonium chloride, ammonium nitrate, sodium nitrate, yeast extract, yeast, or any combination thereof. In some cases, the nitrogen source may be added to the digestion system on a first day of a digestion process. In some cases, the nitrogen source may be added to the digestion system on a second day, third day, fourth day, or any day following a first day of a digestion process. The nitrogen source (e.g., ammonium sulfate) may be added to the reactor to maintain a concentration range of 0.05-0.2% w / v of nitrogen based on the total volume of the working fluid of the system. In some embodiments, the concentration of nitrogen that may be maintained can be based on the total concentration of all components of the working fluid in the at a point during the digestion process of the digestion system 100.

[0215] In some embodiments, the nitrogen source e.g., ammonium sulfate) may be added to the reactor to maintain a concentration of nitrogen source in the working fluid of at least about 0.005% w / v, at least about 0.01% w / v, at least about 0.02% w / v, at least about 0.03% w / v, at least about 0.04% w / v, at least about 0.05% w / v, at least about 0.055% w / v, at least about 0.06% w / v, at least about 0.065% w / v, at least about 0.07% w / v, at least about 0.075% w / v, at least about 0.1% w / v, at least about 0.125% w / v, at least about 0.15% w / v, at least about 0.175% w / v, at least about 0.2% w / v, at least about 0.225% w / v, at least about 0.25% w / v, at least about 0.275% w / v, at least about 0.3% w / v, at least about 0.4% w / v, at least about 0.5% w / v, at least about 0.75% w / v, or at least about 1.0% w / v. In some embodiments, the nitrogen source (e.g., ammonium sulfate) may be added to the reactor to maintain a concentration of nitrogen source in the working fluid of at most about 1.0% w / v, at most about 0.75% w / v, at most about 0.5% w / v, at most about 0.4% w / v, at most about 0.3% w / v, at most about 0.275% w / v, at most about 0.25% w / v, at most about 0.225% w / v, at most about 0.2% w / v, at most about 0.175% w / v, at most about 0.15% w / v, at most about 0.125% w / v, at most about 0.1% w / v, at most about 0.075% w / v, at most about 0.07% w / v, at most about 0.065% w / v, at most about 0.06% w / v, atmost about 0.055% w / v, at most about 0.05% w / v, at most about 0.04% w / v, at most about 0.03% w / v, at most about 0.02% w / v, at most about 0.01% w / v, or at most about 0.005% w / v.

[0216] In some embodiments, the nitrogen source (e.g., ammonium sulfate) may be added to the reactor to maintain a concentration of nitrogen source in the working fluid of about 0.03% w / v to about 1% w / v. In some embodiments, the nitrogen source (e.g., ammonium sulfate) may be added to the first reactor to maintain a concentration of nitrogen source in the working fluid of about 0.25% w / v to about 0.03% w / v, about 0.25% w / v to about 0.04% w / v, about 0.25% w / v to about 0.05% w / v, about 0.25% w / v to about 0.1% w / v, about 0.25% w / v to about 0.125% w / v, about 0.25% w / v to about 0.15% w / v, about 0.25% w / v to about 0.175% w / v, about 0.25% w / v to about 0.2% w / v, about 0.25% w / v to about 0.5% w / v, about 0.25% w / v to about 0.75% w / v, about 0.25% w / v to about 1% w / v, about 0.03% w / v to about 0.04% w / v, about 0.03% w / v to about 0.05% w / v, about 0.03% w / v to about 0.1% w / v, about 0.03% w / v to about 0.125% w / v, about 0.03% w / v to about 0.15% w / v, about 0.03% w / v to about 0.175% w / v, about 0.03% w / v to about 0.2% w / v, about 0.03% w / v to about 0.5% w / v, about 0.03% w / v to about 0.75% w / v, about 0.03% w / v to about 1% w / v, about 0.04% w / v to about 0.05% w / v, about 0.04% w / v to about 0.1% w / v, about 0.04% w / v to about 0.125% w / v, about 0.04% w / v to about 0.15% w / v, about 0.04% w / v to about 0.175% w / v, about 0.04% w / v to about 0.2% w / v, about 0.04% w / v to about 0.5% w / v, about 0.04% w / v to about 0.75% w / v, about 0.04% w / v to about 1% w / v, about 0.05% w / v to about 0.1% w / v, about 0.05% w / v to about 0.125% w / v, about 0.05% w / v to about 0.15% w / v, about 0.05% w / v to about 0.175% w / v, about 0.05% w / v to about 0.2% w / v, about 0.05% w / v to about 0.5% w / v, about 0.05% w / v to about 0.75% w / v, about 0.05% w / v to about 1% w / v, about 0.1% w / v to about 0.125% w / v, about 0.1% w / v to about 0.15% w / v, about 0.1% w / v to about 0.175% w / v, about 0.1% w / v to about 0.2% w / v, about 0.1% w / v to about 0.5% w / v, about 0.1% w / v to about 0.75% w / v, about 0.1% w / v to about 1% w / v, about 0.125% w / v to about 0.15% w / v, about 0.125% w / v to about 0.175% w / v, about 0.125% w / v to about 0.2% w / v, about 0.125% w / v to about 0.5% w / v, about 0.125% w / v to about 0.75% w / v, about 0.125% w / v to about 1% w / v, about 0.15% w / v to about 0.175% w / v, about 0.15% w / v to about 0.2% w / v, about 0.15% w / v to about 0.5% w / v, about 0.15% w / v to about 0.75% w / v, about 0.15% w / v to about 1% w / v, about 0.175% w / v to about 0.2% w / v, about 0.175% w / v to about 0.5% w / v, about 0.175% w / v to about 0.75% w / v, about 0.175% w / v to about 1% w / v, about 0.2% w / v to about 0.5% w / v, about 0.2% w / v to about 0.75% w / v, about 0.2% w / v to about 1% w / v, about 0.5% w / v to about 0.75% w / v, about 0.5% w / v to about 1% w / v, or about 0.75% w / v to about 1% w / v.

[0217] In some embodiments, the input composition to the digestion system 100 may comprise micronutrients 150. Micronutrients may comprise important elements for microorganisms to support physiological functions. The micronutrients may comprise less than about 1%, less than about 2%, less than about 3%, less than about 4%, or less than about 5% of a dry weight of a plant. Elemental micronutrients may comprise boron, zinc, manganese, copper, chlorine, molybdenum, or any combination thereof. The addition of micronutrients or compounds that provide micronutrients may enrich a population of the microbial strain and / or microbes of a microbial consortium of a working fluid of a digestion system as described herein. Addition of inorganic nutrients may enrich a population of the microbial strain and / or microbes of a microbial consortium of a working fluid of a digestion system as described herein. The inorganic nutrients may comprise potassium chloride (KC1), dipotassium phosphate (K2HPO4), magnesium sulfate (MgSC ), or any combination thereof. Potassium chloride may be added into the reactor at a concentration range of at least about, at most about, or about 0.005% w / v, 0.01% w / v, 0.015% w / v, 0.02% w / v, 0.025% w / v, 0.03% w / v, 0.035% w / v, 0.04% w / v, 0.045% w / v, 0.05% w / v, 0.055% w / v, 0.06% w / v, 0.07% w / v, 0.08% w / v, 0.09% w / v, or 0.1% w / v, or a range between any two of these values. Dipotassium phosphate may be added into the reactor at a concentration range of at least about, at most about, or about 0.005% w / v, 0.01% w / v, 0.015% w / v, 0.02% w / v, 0.025% w / v, 0.03% w / v, 0.035% w / v, 0.04% w / v, 0.045% w / v, 0.05% w / v, 0.055% w / v, 0.06% w / v, 0.07% w / v, 0.08% w / v, 0.09% w / v, or 0.1% w / v, or a range between any two of these values. Magnesium sulfate may be added into the reactor at a concentration range of at least about, at most about, or about 0.02% w / v, 0.03% w / v, 0.04% w / v, 0.05% w / v, 0.06% w / v, 0.07% w / v, 0.08% w / v, 0.09% w / v, 0.1% w / v, 0.125% w / v, 0.15% w / v, 0.175% w / v, 0.2% w / v, 0.25% w / v, 0.3% w / v, 0.35% w / v, 0.4% w / v, 0.45% w / v, 0.5% w / v, 0.75% w / v, or a range between any two of these values. In some embodiments, micronutrients may be added on a first day, a second day, a third day, a fourth day, and / or a later day of a digestion process using digestion system 100 based on the total volume of the working fluid of the system .

[0218] In some embodiments, a concentration of range of 0.1-5.0% v / v of floc of a Phosphate Solubilizing Technology (PST / PwST), PST WB, may also be added to the reactor on the span of the hydraulic retention time of the digestion system. A hydraulic retention time may comprise an amount of time a working fluid is maintained in a container (e.g., is not transferred out) of a digestion system as described herein. In some embodiments, the concentration range of PST WB input to the system may be at least about, at most about, or about 0.05% v / v, 0.1% v / v, 0.2% v / v, 0.3% v / v, 0.4%v, 0.5% v / v, 0.6% v / v, 0.7% v / v, 0.8% v / v, 0.9% v / v, 1.0% v / v, 1.5% v / v, 2.0% v / v, 2.5% v / v, 3.0% v / v, 3.5% v / v, 4.0% v / v, 4.5% v / v, 5.0% v / v, 5.5% v / v, 6.0% v / v, 6.5% v / v,7.0% v / v, 7.5% v / v, 10.0% v / v, 12.5% v / v, 15.0% v / v, or a range between any two of these values. In some cases, the PST WB provides a phosphorous source of rock phosphate to the working fluid of the digestion system 100. The PST WB can provide a first consortium to the digestion system 100 in order to populate a working fluid of a first reactor.

[0219] In some embodiments, the digestion system 100 may be inoculated with isolates MS4666, MS4689, MS4687, or any combination thereof. A digestion system 100 may be inoculated with zinc solubilizing spore former. A digestion system 100 may be inoculated with zinc solubilizing rhizobacteria. In some cases, a digestion system 100 may be inoculated with an isolate or combination of isolates once. In some cases, a digestion system 100 may not be reinoculated with an isolate or combination of isolates following a first inoculation. In some cases, a digestion system 100 may be reinoculated with an isolate or combination of isolates at least every 20 days, at least every 50 days, at least every 100 days, at least every 200 days, at least every 300 days, at least every 400 days, at least every 500 days, or more. In some cases, a digestion system 100 may be reinoculated with an isolate or combination of isolates at most every 500 days, at most every 400 days, at most every 300 days, at most every 200 days, at most every 100 days, at most every 50 days, at most every 20 days, or less. In some cases, a digestion system 100 may be inoculated with an isolate or combination of isolates on day 1 of a digestion process and reinoculated 1, 2, 3, 4, or 5 more times after day 1 of the digestion process. In some embodiments, the population of the microbe does not decrease by more than 60%, by more than 50%, by more than 45%, by more than 40%, by more than 35%, by more than 30%, by more than 25%, by more than 20%, by more than 15%, by more than 10%, or by more than 5% between a first and a second transferring of the isolate (e.g., microbe). In some embodiments, the population of the microbe is maintained by at least about 5%, at least 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, or at least about 75%.

[0220] The inoculum of a microbe may be an isolated microbe. An isolated microbe can comprise a microbe grown or enriched outside of a natural environment (e.g., in a culture medium or a streak plate method). In some embodiments, the inoculum of a microbe may comprise a mixture of multiple isolated microbes. The inoculum of a microbe may comprise a mixture of at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, or more isolated microbes.

[0221] Referring again to FIG. 1, a working fluid may be maintained within a reactor 110. A maintained working fluid may be a working fluid as it rests in a reactor 110 of a digestionsystem. Without wishing to be bound by theory, maintenance of a working fluid may support enrichment and / or growth of a population of the microbial strain or another component of a working fluid as described herein. In some embodiments, the volume of working fluid within the reactor 110 may be maintained at 5 gallons. In some embodiments, the volume of working fluid within the reactor may be maintained at least about 1 gallon, at least about 2 gallons, at least about 3 gallons, at least about 4 gallons, at least about 5 gallons, at least about 6 gallons, at least about 7 gallons, at least about 10 gallons, at least about 15 gallons, at least about 20 gallons, at least about 25 gallons, at least about 30 gallons, at least about 50 gallons, at least about 75 gallons, at least about 100 gallons, at least about 250 gallons, at least about 500 gallons, at least about 1,000 gallons, at least about 2,500 gallons, at least about 5,000 gallons, at least about 10,000 gallons, or more than about 10,000 gallons. In some embodiments, the volume of working fluid within the reactor may be maintained at most about 10,000 gallons, at most about 5,000 gallons, at most about 2,500 gallons, at most about 1,000 gallons, at most about 500 gallons, at most about 250 gallons, at most about 100 gallons, at most about 75 gallons, at most about 50 gallons, at most about 30 gallons, at most about 25 gallons, at most about 20 gallons, at most about 15 gallons, at most about 10 gallons, at most about 7 gallons, at most about 6 gallons, at most about 5 gallons, at most about 4 gallons, at most about 3 gallons, at most about 2 gallons, at most about 1 gallon, or less than about 1 gallon.

[0222] In some embodiments, the volume of working fluid within the reactor may be maintained from about 5 gallons to about 10,000 gallons. In some embodiments, the volume of working fluid within the reactor may be maintained from about 5 gallons to about 10 gallons, about 5 gallons to about 25 gallons, about 5 gallons to about 50 gallons, about 5 gallons to about 75 gallons, about 5 gallons to about 100 gallons, about 5 gallons to about 150 gallons, about 5 gallons to about 500 gallons, about 5 gallons to about 1,000 gallons, about 5 gallons to about 2,500 gallons, about 5 gallons to about 5,000 gallons, about 5 gallons to about 10,000 gallons, about 10 gallons to about 25 gallons, about 10 gallons to about 50 gallons, about 10 gallons to about 75 gallons, about 10 gallons to about 100 gallons, about 10 gallons to about 150 gallons, about 10 gallons to about 500 gallons, about 10 gallons to about 1,000 gallons, about 10 gallons to about 2,500 gallons, about 10 gallons to about 5,000 gallons, about 10 gallons to about 10,000 gallons, about 25 gallons to about 50 gallons, about 25 gallons to about 75 gallons, about 25 gallons to about 100 gallons, about 25 gallons to about 150 gallons, about 25 gallons to about 500 gallons, about 25 gallons to about 1,000 gallons, about 25 gallons to about 2,500 gallons, about 25 gallons to about 5,000 gallons, about 25 gallons to about 10,000 gallons, about 50 gallons to about 75 gallons, about 50 gallons to about 100 gallons, about 50 gallons to about 150gallons, about 50 gallons to about 500 gallons, about 50 gallons to about 1,000 gallons, about 50 gallons to about 2,500 gallons, about 50 gallons to about 5,000 gallons, about 50 gallons to about 10,000 gallons, about 75 gallons to about 100 gallons, about 75 gallons to about 150 gallons, about 75 gallons to about 500 gallons, about 75 gallons to about 1,000 gallons, about 75 gallons to about 2,500 gallons, about 75 gallons to about 5,000 gallons, about 75 gallons to about 10,000 gallons, about 100 gallons to about 150 gallons, about 100 gallons to about 500 gallons, about 100 gallons to about 1,000 gallons, about 100 gallons to about 2,500 gallons, about 100 gallons to about 5,000 gallons, about 100 gallons to about 10,000 gallons, about 150 gallons to about 500 gallons, about 150 gallons to about 1,000 gallons, about 150 gallons to about 2,500 gallons, about 150 gallons to about 5,000 gallons, about 150 gallons to about 10,000 gallons, about 500 gallons to about 1,000 gallons, about 500 gallons to about 2,500 gallons, about 500 gallons to about 5,000 gallons, about 500 gallons to about 10,000 gallons, about 1,000 gallons to about 2,500 gallons, about 1,000 gallons to about 5,000 gallons, about 1,000 gallons to about 10,000 gallons, about 2,500 gallons to about 5,000 gallons, about 2,500 gallons to about 10,000 gallons, or about 5,000 gallons to about 10,000 gallons.

[0223] In some embodiments, the working fluid in the reactor 110 may be recirculated (e.g., recycled) within the reactor of the digestion system 100. Working fluid may be recirculated via a conduit or output of a reactor and reintroduced into the same reactor. Without wishing to be bound by theory, recirculation of a working fluid within a reactor may support enrichment and / or growth of a population of the microbial strain or another component of a working fluid as described herein. In some embodiments, the rate of recirculation within the reactor may be between 4-9 gallons / min. In some embodiments, the rate of recirculation within the reactor 110 may be at least about 0.5 gallon / min, at least about 1 gallon / min, at least about 2 gallons / min, at least about 3 gallons / min, at least about 3.5 gallons / min, at least about 4 gallons / min, at least about 4.5 gallons / min, at least about 5 gallons / min, at least about 5.5 gallons / min, at least about 6 gallons / min, at least about 6.5 gallons / min, at least about 7 gallons / min, at least about 7.5 gallons / min, at least about 8 gallons / min, at least about 8.5 gallons / min, at least about 9 gallons / min, at least about 10 gallons / min, at least about 11 gallons / min, at least about 13 gallons / min, at least about 15 gallons / min, at least about 20 gallons / min, at least about 25 gallons / min, at least about 30 gallons / min, at least about 40 gallons / min, or at least about 50 gallons / min.

[0224] In some embodiments, the incubation time, i.e., the amount of time that the working fluid is retained in the digestion system 100 before the product is removed, of the digestion system 100 may be 7 days. In some embodiments, the incubation time of the digestion system may be atleast about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 25 days, at least about 30 days, or at least about 50 days. In some embodiments, the incubation time of the digestion system 100 may be at most about 50 days, at most about 30 days, at most about 25 days, at most about 20 days, at most about 19 days, at most about 18 days, at most about 17 days, at most about 16 days, at most about 15 days, at most about 14 days, at most about 13 days, at most about 12 days, at most about 11 days, at most about 10 days, at most about 9 days, at most about8 days, at most about 7 days, at most about 6 days, at most about 5 days, at most about 4 days, at most about 3 days, at most about 2 days, or at most about 1 day.

[0225] In some embodiments, the incubation time of the digestion system may be about 1 day to about 30 days. In some embodiments, the incubation time of the digestion system may be about 1 day to about 3 days, about 1 day to about 5 days, about 1 day to about 8 days, about 1 day to about 10 days, about 1 day to about 12 days, about 1 day to about 14 days, about 1 day to about 16 days, about 1 day to about 18 days, about 1 day to about 20 days, about 1 day to about 25 days, about 1 day to about 30 days, about 3 days to about 5 days, about 3 days to about 8 days, about 3 days to about 10 days, about 3 days to about 12 days, about 3 days to about 14 days, about 3 days to about 16 days, about 3 days to about 18 days, about 3 days to about 20 days, about 3 days to about 25 days, about 3 days to about 30 days, about 5 days to about 8 days, about 5 days to about 10 days, about 5 days to about 12 days, about 5 days to about 14 days, about 5 days to about 16 days, about 5 days to about 18 days, about 5 days to about 20 days, about 5 days to about 25 days, about 5 days to about 30 days, about 8 days to about 10 days, about 8 days to about 12 days, about 8 days to about 14 days, about 8 days to about 16 days, about 8 days to about 18 days, about 8 days to about 20 days, about 8 days to about 25 days, about 8 days to about 30 days, about 10 days to about 12 days, about 10 days to about 14 days, about 10 days to about 16 days, about 10 days to about 18 days, about 10 days to about 20 days, about 10 days to about 25 days, about 10 days to about 30 days, about 12 days to about 14 days, about 12 days to about 16 days, about 12 days to about 18 days, about 12 days to about 20 days, about 12 days to about 25 days, about 12 days to about 30 days, about 14 days to about 16 days, about 14 days to about 18 days, about 14 days to about 20 days, about 14 days to about 25 days, about 14 days to about 30 days, about 16 days to about 18 days, about 16 days to about 20 days, about 16 days to about 25 days, about 16 days to about 30 days, about 18 days to about 20 days,about 18 days to about 25 days, about 18 days to about 30 days, about 20 days to about 25 days, about 20 days to about 30 days, or about 25 days to about 30 days.

[0226] In some embodiments, a digestion system may comprise multiple digestion systems 100 with each reactor producing a base product. Each reactor may operate individually and produce a base product.ZST 1.5 / 1.5+ System

[0227] FIG. 2 schematically illustrates an example of a digestion system 300 with conditions (e.g., microbes) that produce biostimulant products that may have a multi-modal way of promoting plants to access bound sources of zinc and / or enhance the uptake zinc and other nutrients. The system 300 can comprise a first reactor 310, a second reactor 315, a third reactor 320, a fourth reactor 325, and / or a clarifier chamber 330 connected sequentially in which feedstock can continuously flow and microbial consortia as described herein can be grown. The first reactor, second reactor, third reactor, and / or fourth reactor may be fluidized bed reactors without the scaffolding or packed bed reactors with a scaffolding within the reactors. Water may act as a hydraulic source 340 and may be coupled to a first reactor 310 and provide continuous flow of water to the reactor. The system 300 may also comprise an input channel or a series of input channels that flows inputs 350, 360, 370, 380 into the digestion system. In some cases, the inputs can be added individually to the digestion system 300. In some cases, the inputs may be added in combination with one another into the digestion system. The inputs, as described herein, may comprise one or more of water, a microbial inoculum 350, a microbial product or intermediate product of another digestion system 360, a carbon source 370, a nitrogen source 380, micronutrients, a zinc source. The inputs may comprise other organic materials. In some cases, the inputs into the digestion system may be flown into the first reactor through a water tank. In other cases, the input composition can be added to the first reactor. In other cases, the input composition can be added to the second reactor.

[0228] The system 300 may comprise a pH sensor, pH controller, or similar mechanic to monitor and / or control the pH of the reactor and / or working fluid within the reactor so that pH is maintained at a threshold. The pH sensor or controller may be automatic. A buffer addition system may also be used to control the pH of the digestion system 300. For example, if the pH of the working fluid in the first reactor is below the threshold value, an automatic base (e.g., 3M of NaOH) may be added to the first reactor until the pH reaches the threshold value (e.g., at least about 7). In some embodiments, a pH in a reactor of digestion system 300 may be between 4.0- 9.0. In some embodiments, the pH in the reactor may be at least about 3.0, at least about 3.5, at least about 4.0, at least about 4.5, at least about 5.0, at least about 5.5, at least about 6.0, at leastabout 6.5, at least about 7.0, at least about 7.5, at least about 8.0, at least about 8.5, at least about 9.0, at least about 9.5, or at least about 10.0.

[0229] In some cases, fluid (e.g., working fluid) can flow in a hydraulically balanced manner. The clarifier chamber 330 produces biostimulant products or digestion products (e.g., base products) 390.

[0230] Fluid (e.g., working fluid) from an outflow port of the first reactor 310 can flow into the second reactor 315 continuously. Fluid from an outflow port of the second reactor 315 can flow into the third reactor 320 continuously. Fluid from an outflow port of the third reactor 320 can flow into the fourth reactor 325 continuously. Fluid from an outflow port of the fourth reactor 325 can into the clarifier chamber 330 continuously.

[0231] The outflow port may be positioned on the top, middle, and / or bottom of a reactor. Additionally, each reactor or clarifier chamber may comprise another outflow port for reintroducing fluid back into the same reactor or clarifier chamber, and may be pumped back to just below the surface of the same reactor to maintain homogeneous conditions within the working solutions. Reactor 1 may comprise another outflow port 395 for reintroducing fluid back into the Reactor 1. Reactor 2 may comprise another outflow port 396 for reintroducing fluid back into the Reactor 2. Reactor 3 may comprise another outflow port 397 for reintroducing fluid back into the Reactor 3. Reactor 4 may comprise another outflow port 398 for reintroducing fluid back into the Reactor 4. In some cases, the working fluid from each reactor is recirculated within each reactor from the bottom of the reactor back to just below the surface of the working solution to maintain a homogeneous environment for fermentation. For example, fluid from the first reactor 310 may be reintroduced back into the first reactor 310. Biosolids (e.g., floc) may be generated through the process. Biosolids may comprise organic materials recovered from the feedstock, waste, wastewater, and / or sludge material of a working fluid of a digestion system described herein. In some embodiments, floc can comprise biosolids. Floc may be produced throughout the fermentation process in the digestion system. Floc can be collected in a clarifier chamber and reintroduced to a container (e.g., reactor) of the system to serve as a seed of microbes. In some embodiments, floc can be a flocculated mass of microorganisms, extracellular polymeric substance (EPS) and adsorbed organic and inorganic material. A flocculated mass can comprise an aggregated mass of microorganisms, extracellular polymeric substance (EPS) and absorbed organic and inorganic material.

[0232] In some cases, the clarifier chamber 330 may comprise an outflow port for reintroducing fluid back to the first reactor 310. In some embodiments, the supernatant (or base product) from the clarifier chamber may be continuously collected, and at least a portion of the floc at thebottom of the clarifier can be returned to the first reactor. In some cases, the floc may be returned manually back to a first reactor 310. In some cases, the floc may be returned to any container of the digestion system 300.

[0233] In some embodiments, water may act as a hydraulic source 340 and, when coupled to the first reactor 310, provides continuous flow of water to the first reactor 310 at an electrical conductivity of at least about 100 microsiemens / centimeter (pS / cm), at least about 200 pS / cm, at least about 300 pS / cm, at least about 400 pS / cm, at least about 450 pS / cm, at least about 500 pS / cm, at least about 550 pS / cm, at least about 600 pS / cm, at least about 650 pS / cm, at least about 700 pS / cm, at least about 800 pS / cm, at least about 900 pS / cm, at least about 1000 pS / cm, at least about 1250 pS / cm, at least about 1500 pS / cm. In some embodiments, water may act as a hydraulic source 340 and, when coupled to the first reactor 310, provides continuous flow of water to the first reactor 310 at an electrical conductivity of at most about 1500 pS / cm, at most about 1250 pS / cm, at most about 1000 pS / cm, at most about 900 pS / cm, at most about 800 pS / cm, at most about 700 pS / cm, at most about 650 pS / cm, at most about 600 pS / cm, at most about 550 pS / cm, at most about 500 pS / cm, at most about 450 pS / cm, at most about 400 pS / cm, at most about 300 pS / cm, at most about 200 pS / cm, or at most about 100 pS / cm.

[0234] In some embodiments, water may act as a hydraulic source 340 and, when coupled to the first reactor 310, provides continuous flow of water to the first reactor 310 at an electrical conductivity of about 200 pS / cm to about 1,200 pS / cm. In some embodiments, water may act as a hydraulic source 340 and, when coupled to the first reactor 310, provides continuous flow of water to the first reactor 310 at an electrical conductivity of about 200 pS / cm to about 300 pS / cm, about 200 pS / cm to about 400 pS / cm, about 200 pS / cm to about 450 pS / cm, about 200 pS / cm to about 500 pS / cm, about 200 pS / cm to about 550 pS / cm, about 200 pS / cm to about 600 pS / cm, about 200 pS / cm to about 700 pS / cm, about 200 pS / cm to about 800 pS / cm, about 200 pS / cm to about 900 pS / cm, about 200 pS / cm to about 1,000 pS / cm, about 200 pS / cm to about 1,200 pS / cm, about 300 pS / cm to about 400 pS / cm, about 300 pS / cm to about 450 pS / cm, about 300 pS / cm to about 500 pS / cm, about 300 pS / cm to about 550 pS / cm, about 300 pS / cm to about 600 pS / cm, about 300 pS / cm to about 700 pS / cm, about 300 pS / cm to about 800 pS / cm, about 300 pS / cm to about 900 pS / cm, about 300 pS / cm to about 1,000 pS / cm, about 300 pS / cm to about 1,200 pS / cm, about 400 pS / cm to about 450 pS / cm, about 400 pS / cm to about 500 pS / cm, about 400 pS / cm to about 550 pS / cm, about 400 pS / cm to about 600 pS / cm, about 400 pS / cm to about 700 pS / cm, about 400 pS / cm to about 800 pS / cm, about 400 pS / cm to about 900 pS / cm, about 400 pS / cm to about 1,000 pS / cm, about 400 pS / cm to about 1,200 pS / cm, about 450 pS / cm to about 500 pS / cm, about 450 pS / cm to about 550 pS / cm,about 450 pS / cm to about 600 pS / cm, about 450 pS / cm to about 700 pS / cm, about 450 pS / cm to about 800 pS / cm, about 450 pS / cm to about 900 pS / cm, about 450 pS / cm to about 1,000 pS / cm, about 450 pS / cm to about 1,200 pS / cm, about 500 pS / cm to about 550 pS / cm, about 500 pS / cm to about 600 pS / cm, about 500 pS / cm to about 700 pS / cm, about 500 pS / cm to about 800 pS / cm, about 500 pS / cm to about 900 pS / cm, about 500 pS / cm to about 1,000 pS / cm, about 500 pS / cm to about 1,200 pS / cm, about 550 pS / cm to about 600 pS / cm, about 550 pS / cm to about 700 pS / cm, about 550 pS / cm to about 800 pS / cm, about 550 pS / cm to about 900 pS / cm, about 550 pS / cm to about 1,000 pS / cm, about 550 pS / cm to about 1,200 pS / cm, about 600 pS / cm to about 700 pS / cm, about 600 pS / cm to about 800 pS / cm, about 600 pS / cm to about 900 pS / cm, about 600 pS / cm to about 1,000 pS / cm, about 600 pS / cm to about 1,200 pS / cm, about 700 pS / cm to about 800 pS / cm, about 700 pS / cm to about 900 pS / cm, about 700 pS / cm to about 1,000 pS / cm, about 700 pS / cm to about 1,200 pS / cm, about 800 pS / cm to about 900 pS / cm, about 800 pS / cm to about 1,000 pS / cm, about 800 pS / cm to about 1,200 pS / cm, about 900 pS / cm to about 1,000 pS / cm, about 900 pS / cm to about 1,200 pS / cm, or about 1,000 pS / cm to about 1,200 pS / cm.

[0235] An amount of biosolids may be maintained in a digestion system. Maintenance of biosolids may support enrichment and / or growth of a population of the microbial strain, microbes of a microbial consortium, nutrients, or additional components of a digestion system as described herein. In some embodiments, as biosolids accumulate over time in the clarifier chamber 330, a range of at least about 20-25% biosolids v / v may be maintained in the digestion system. In some embodiments, additional floc may be harvested from the digestion system and removed. In some embodiments, at least about 5% biosolids v / v, at least about 10% biosolids v / v, at least about 15% biosolids v / v, at least about 16% biosolids v / v, at least about 17% biosolids v / v, at least about 18% biosolids v / v, at least about 19% biosolids v / v, at least about 20% biosolids v / v, at least about 21% biosolids v / v, at least about 22% biosolids v / v, at least about 23% biosolids v / v, at least about 24% biosolids v / v, at least about 25% biosolids v / v, at least about 26% biosolids v / v, at least about 27% biosolids v / v, at least about 28% biosolids v / v, at least about 29% biosolids v / v, at least about 30% biosolids v / v, at least about 35% biosolids v / v, at least about 40% biosolids v / v, at least about 45% biosolids v / v, or at least about 50% biosolids v / v may be maintained in the digestion system.

[0236] In some embodiments, at most about 50% biosolids v / v, at most about 45% biosolids v / v, at most about 40% biosolids v / v, at most about 35% biosolids v / v, at most about 30% biosolids v / v, at most about 29% biosolids v / v, at most about 28% biosolids v / v, at most about 27% biosolids v / v, at most about 26% biosolids v / v, at most about 25% biosolids v / v, at most about24% biosolids v / v, at most about 23% biosolids v / v, at most about 22% biosolids v / v, at most about 21% biosolids v / v, at most about 20% biosolids v / v, at most about 19% biosolids v / v, at most about 18% biosolids v / v, at most about 17% biosolids v / v, at most about 16% biosolids v / v, at most about 15% biosolids v / v, at most about 14% biosolids v / v, at most about 13% biosolids v / v, at most about 12% biosolids v / v, at most about 11% biosolids v / v, at most about 10% biosolids v / v, or at most about 5% biosolids v / v may be maintained in the digestion system.

[0237] In some embodiments, about 0.1% biosolids v / v to about 60% biosolids v / v may be maintained in the digestion system. In some embodiments, about 0.1% biosolids v / v to about 1% biosolids v / v, about 0.1% biosolids v / v to about 5% biosolids v / v, about 0.1% biosolids v / v to about 10% biosolids v / v, about 0.1% biosolids v / v to about 15% biosolids v / v, about 0.1% biosolids v / v to about 20% biosolids v / v, about 0.1% biosolids v / v to about 25% biosolids v / v, about 0.1% biosolids v / v to about 30% biosolids v / v, about 0.1% biosolids v / v to about 35% biosolids v / v, about 0.1% biosolids v / v to about 40% biosolids v / v, about 0.1% biosolids v / v to about 50% biosolids v / v, about 0.1% biosolids v / v to about 60% biosolids v / v, about 1% biosolids v / v to about 5% biosolids v / v, about 1% biosolids v / v to about 10% biosolids v / v, about 1% biosolids v / v to about 15% biosolids v / v, about 1% biosolids v / v to about 20% biosolids v / v, about 1% biosolids v / v to about 25% biosolids v / v, about 1% biosolids v / v to about 30% biosolids v / v, about 1% biosolids v / v to about 35% biosolids v / v, about 1% biosolids v / v to about 40% biosolids v / v, about 1% biosolids v / v to about 50% biosolids v / v, about 1% biosolids v / v to about 60% biosolids v / v, about 5% biosolids v / v to about 10% biosolids v / v, about 5% biosolids v / v to about 15% biosolids v / v, about 5% biosolids v / v to about 20% biosolids v / v, about 5% biosolids v / v to about 25% biosolids v / v, about 5% biosolids v / v to about 30% biosolids v / v, about 5% biosolids v / v to about 35% biosolids v / v, about 5% biosolids v / v to about 40% biosolids v / v, about 5% biosolids v / v to about 50% biosolids v / v, about 5% biosolids v / v to about 60% biosolids v / v, about 10% biosolids v / v to about 15% biosolids v / v, about 10% biosolids v / v to about 20% biosolids v / v, about 10% biosolids v / v to about 25% biosolids v / v, about 10% biosolids v / v to about 30% biosolids v / v, about 10% biosolids v / v to about 35% biosolids v / v, about 10% biosolids v / v to about 40% biosolids v / v, about 10% biosolids v / v to about 50% biosolids v / v, about 10% biosolids v / v to about 60% biosolids v / v, about 15% biosolids v / v to about 20% biosolids v / v, about 15% biosolids v / v to about 25% biosolids v / v, about 15% biosolids v / v to about 30% biosolids v / v, about 15% biosolids v / v to about 35% biosolids v / v, about 15% biosolids v / v to about 40% biosolids v / v, about 15% biosolids v / v to about 50% biosolids v / v, about 15% biosolids v / v to about 60% biosolids v / v, about 20% biosolids v / v to about 25% biosolids v / v, about 20% biosolids v / v to about 30%biosolids v / v, about 20% biosolids v / v to about 35% biosolids v / v, about 20% biosolids v / v to about 40% biosolids v / v, about 20% biosolids v / v to about 50% biosolids v / v, about 20% biosolids v / v to about 60% biosolids v / v, about 25% biosolids v / v to about 30% biosolids v / v, about 25% biosolids v / v to about 35% biosolids v / v, about 25% biosolids v / v to about 40% biosolids v / v, about 25% biosolids v / v to about 50% biosolids v / v, about 25% biosolids v / v to about 60% biosolids v / v, about 30% biosolids v / v to about 35% biosolids v / v, about 30% biosolids v / v to about 40% biosolids v / v, about 30% biosolids v / v to about 50% biosolids v / v, about 30% biosolids v / v to about 60% biosolids v / v, about 35% biosolids v / v to about 40% biosolids v / v, about 35% biosolids v / v to about 50% biosolids v / v, about 35% biosolids v / v to about 60% biosolids v / v, about 40% biosolids v / v to about 50% biosolids v / v, about 40% biosolids v / v to about 60% biosolids v / v, or about 50% biosolids v / v to about 60% biosolids v / v may be maintained in the digestion system.

[0238] In some embodiments, the input composition, as described herein, may comprise a selective pressure or a zinc source. Without wishing to be bound by theory, the selective pressure source (e.g., zinc source) may shift the complex microbial consortia of the digestion system 300 to increased zinc solubilizing microbes. The zinc solubilizing microbes may comprise microbes that can survive a higher zinc ion concentration in a working fluid caused by addition of the selective pressure. In some embodiments, the inoculum of the microbial strain may comprise a zinc solubilizing microbe. Addition of the selective pressure to the digestion system 300 may promote survival (e.g., maintenance) of the inoculum of the microbe. The terms “selective pressure source” and “selective pressure” may be used interchangeably. Addition of a selective pressure source (e.g., zinc source) may increase a concentration of zinc within the working fluid of the digestion system 300. A population of the microbial strain in the digestion system 300 may be configured to survive the increased concentration of zinc. At least a portion of microbes of a microbial consortium in the digestion system 300 may not be able to survive the increased concentration of zinc. A shift in the complex microbial consortia may occur to enrich microbes of the microbial consortium with zinc-solubilizing properties. In some embodiments, the selective pressure may create a high ionic concentration which selectively inhibits the growth of some microbes within the working fluid, while promoting the growth or survival of other microbes (e.g., a population of the microbial strain and / or zinc-solubilizing microbes of the microbial consortium) in the working fluid. Addition of a selective pressure source (e.g., zinc source) may increase or maintain an amount of a population of the microbial strain (e.g., a target isolate or isolated microbe) and decrease other microbes in the digestion system 300. A maintained amount of a population of the microbial strain may comprise nochange in an amount of a microbe across two time periods. A maintained amount of a population of the microbial strain may change its amount in a working fluid of a digestion system 300 less than 0.1%, less than 0.5%, less than 1%, less than 5%, or less than 10% over two time periods. Without wishing to be bound by theory, the addition of a selective pressure source (e.g., zinc source) may increase a proportion of a population of the microbial strain relative to the microbes or at least a portion of the microbes in a microbial consortium in the digestion system 300. Without wishing to be bound by theory, the addition of a selective pressure source (e.g., zinc source) may decrease an amount of a population of the microbial strain less than a decrease of microbes or at least a portion of the microbes in a microbial consortium in the digestion system 300. The zinc source may comprise zinc oxide (ZnO) and may be added to the digestion system 300 on a first day of a digestion process. In some cases, the zinc source (e.g., ZnO) may be added to the digestion system 300 daily. In some cases, the zinc source (e.g., ZnO) may be added to the digestion system 300 every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, and / or weekly. In some cases, the zinc source may comprise one or more of zinc carbonate, zinc sulfate, and / or calamine ore. The zinc source (e.g., zinc oxide) may be added to the first reactor 310, the second reactor 320, or another reactor of the digestion system 300. The zinc source (e.g., zinc oxide) may be added to the reactor at a concentration between 0.05-0.5% w / v. A concentration of the zinc source may be based on a total concentration of components of a working fluid of a digestion system 300. In some cases, the concentration range of the zinc source (e.g., zinc oxide) in the digestion system 300 may be based on the hydraulic retention time of the system. In some embodiments, the zinc source (e.g., zinc oxide) may be added to the reactor at a concentration of at least about 0.01% w / v, at least about 0.02% w / v, at least about 0.03% w / v, at least about 0.04% w / v, at least about 0.05% w / v, at least about 0.1% w / v, at least about 0.2% w / v, at least about 0.3% w / v, at least about 0.4% w / v, at least about 0.5% w / v, at least about 0.6% w / v, at least about 0.7% w / v, at least about 0.8% w / v, at least about 0.9% w / v, at least about 1.0% w / v, at least about 1.1 % w / v, at least about 1.2% w / v, at least about 1.3% w / v, at least about 1.4% w / v, or at least about 1.5% w / v. In some embodiments, the zinc source (e.g., zinc oxide) may be added to the reactor at a concentration of at most about 1.5% w / v, at most about 1.4% w / v, at most about 1.3% w / v, at most about 1.2% w / v, at most about 1.1% w / v, at most about 1.0% w / v, at most about 0.9% w / v, at most about 0.8% w / v, at most about 0.7% w / v, at most about 0.6% w / v, at most about 0.5% w / v, at most about 0.4% w / v, at most about 0.3% w / v, at most about 0.2% w / v, at most about 0.1% w / v, at most about 0.05% w / v, at most about 0.04% w / v, at most about 0.03% w / v, at most about 0.02% w / v, or at most about 0.01% w / v.

[0239] In some embodiments, the zinc source (e.g., zinc oxide) may be added to the reactor at a concentration of about 0.01% w / v to about 1% w / v. In some embodiments, the zinc source (e.g., zinc oxide) may be added to the reactor at a concentration of about 0.01% w / v to about 0.025% w / v, about 0.01% w / v to about 0.05% w / v, about 0.01% w / v to about 0.075% w / v, about 0.01% w / v to about 0.1% w / v, about 0.01% w / v to about 0.15% w / v, about 0.01% w / v to about 0.2% w / v, about 0.01% w / v to about 0.3% w / v, about 0.01% w / v to about 0.4% w / v, about 0.01% w / v to about 0.5% w / v, about 0.01% w / v to about 0.75% w / v, about 0.01% w / v to about 1% w / v, about 0.025% w / v to about 0.05% w / v, about 0.025% w / v to about 0.075% w / v, about 0.025% w / v to about 0.1% w / v, about 0.025% w / v to about 0.15% w / v, about 0.025% w / v to about 0.2% w / v, about 0.025% w / v to about 0.3% w / v, about 0.025% w / v to about 0.4% w / v, about 0.025% w / v to about 0.5% w / v, about 0.025% w / v to about 0.75% w / v, about 0.025% w / v to about 1% w / v, about 0.05% w / v to about 0.075% w / v, about 0.05% w / v to about 0.1% w / v, about 0.05% w / v to about 0.15% w / v, about 0.05% w / v to about 0.2% w / v, about 0.05% w / v to about 0.3% w / v, about 0.05% w / v to about 0.4% w / v, about 0.05% w / v to about 0.5% w / v, about 0.05% w / v to about 0.75% w / v, about 0.05% w / v to about 1% w / v, about 0.075% w / v to about 0.1% w / v, about 0.075% w / v to about 0.15% w / v, about 0.075% w / v to about 0.2% w / v, about 0.075% w / v to about 0.3% w / v, about 0.075% w / v to about 0.4% w / v, about 0.075% w / v to about 0.5% w / v, about 0.075% w / v to about 0.75% w / v, about 0.075% w / v to about 1% w / v, about 0.1% w / v to about 0.15% w / v, about 0.1% w / v to about 0.2% w / v, about 0.1% w / v to about 0.3% w / v, about 0.1% w / v to about 0.4% w / v, about 0.1% w / v to about 0.5% w / v, about 0.1% w / v to about 0.75% w / v, about 0.1% w / v to about 1% w / v, about 0.15% w / v to about 0.2% w / v, about 0.15% w / v to about 0.3% w / v, about 0.15% w / v to about 0.4% w / v, about 0.15% w / v to about 0.5% w / v, about 0.15% w / v to about 0.75% w / v, about 0.15% w / v to about 1% w / v, about 0.2% w / v to about 0.3% w / v, about 0.2% w / v to about 0.4% w / v, about 0.2% w / v to about 0.5% w / v, about 0.2% w / v to about 0.75% w / v, about 0.2% w / v to about 1% w / v, about 0.3% w / v to about 0.4% w / v, about 0.3% w / v to about 0.5% w / v, about 0.3% w / v to about 0.75% w / v, about 0.3% w / v to about 1% w / v, about 0.4% w / v to about 0.5% w / v, about 0.4% w / v to about 0.75% w / v, about 0.4% w / v to about 1% w / v, about 0.5% w / v to about 0.75% w / v, about 0.5% w / v to about 1% w / v, or about 0.75% w / v to about 1% w / v.

[0240] In some embodiments, the input composition to the digestion system 300 may comprise a carbon source 370. The carbon source may be glucose, malate, gluconic acid, lactose, sucrose, pyruvate, other simple sugars, or any combination thereof. In some cases, the carbon source may be added to the digestion system on a first day of a digestion process. In some cases, the carbon source may be added to the digestion system on a second day, third day, fourth day, and / or anyday following a first day of a digestion process. In some cases, the carbon source may be added to the digestion system daily. In some embodiments, the carbon source may be added to the second reactor 315 of the digestion system. In some embodiments, the carbon source is added to a first reactor 310, a third reactor 320, or a fourth reactor 325. The carbon source (e.g., glucose) may be added to the reactor to maintain a concentration range of 0.5%-3.0% w / v based on the span of the hydraulic retention time of the digestion system. In some embodiments, the concentration of carbon that may be maintained can be based on the total concentration of all components of the working fluid in the at a point during the digestion process of the digestion system 300.

[0241] In some embodiments, the carbon source (e.g., glucose) may be added to a reactor to maintain a concentration of carbon source in the working fluid of at least about 0.1% w / v, at least about 0.25% w / v, at least about 0.5% w / v, at least about 0.75% w / v, at least about 1.0% w / v, at least about 1.25% w / v, at least about 1.5% w / v, at least about 1.75% w / v, at least about 2.0% w / v, at least about 2.5% w / v, at least about 3.0% w / v, at least about 5.0% w / v, at least about 7.5% w / v, or at least about 10.0% w / v. In some embodiments, the carbon source (e.g., glucose) may be added to a reactor to maintain a concentration of carbon source in the working fluid of at most about 10.0% w / v, at most about 7.5% w / v, at most about 5.0% w / v, at most about 3.0% w / v, at most about 2.5% w / v, at most about 2.0% w / v, at most about 1.75% w / v, at most about 1.5% w / v, at most about 1.25% w / v, at most about 1.0% w / v, at most about 0.75% w / v, at most about 0.5% w / v, at most about 0.25% w / v, or at most about 10.0% w / v.

[0242] In some embodiments, the carbon source (e.g., glucose) may be added to a reactor to maintain a concentration of carbon source in the working fluid of about 0.1% w / v to about 5% w / v. In some embodiments, the carbon source (e.g., glucose) may be added to a reactor to maintain a concentration of carbon source in the working fluid of about 0.1% w / v to about 0.25% w / v, about 0.1% w / v to about 0.5% w / v, about 0.1% w / v to about 0.75% w / v, about 0.1% w / v to about 1% w / v, about 0.1% w / v to about 1.25% w / v, about 0.1% w / v to about 1.5% w / v, about 0.1% w / v to about 1.75% w / v, about 0.1% w / v to about 2% w / v, about 0.1% w / v to about 2.5% w / v, about 0.1% w / v to about 3% w / v, about 0.1% w / v to about 5% w / v, about 0.25% w / v to about 0.5% w / v, about 0.25% w / v to about 0.75% w / v, about 0.25% w / v to about 1% w / v, about 0.25% w / v to about 1.25% w / v, about 0.25% w / v to about 1.5% w / v, about 0.25% w / v to about 1.75% w / v, about 0.25% w / v to about 2% w / v, about 0.25% w / v to about 2.5% w / v, about 0.25% w / v to about 3% w / v, about 0.25% w / v to about 5% w / v, about 0.5% w / v to about 0.75% w / v, about 0.5% w / v to about 1% w / v, about 0.5% w / v to about 1.25% w / v, about 0.5% w / v to about 1.5% w / v, about 0.5% w / v to about 1.75% w / v, about 0.5% w / v to about 2% w / v, about0.5% w / v to about 2.5% w / v, about 0.5% w / v to about 3% w / v, about 0.5% w / v to about 5% w / v, about 0.75% w / v to about 1% w / v, about 0.75% w / v to about 1.25% w / v, about 0.75% w / v to about 1.5% w / v, about 0.75% w / v to about 1.75% w / v, about 0.75% w / v to about 2% w / v, about 0.75% w / v to about 2.5% w / v, about 0.75% w / v to about 3% w / v, about 0.75% w / v to about 5% w / v, about 1% w / v to about 1.25% w / v, about 1% w / v to about 1.5% w / v, about 1% w / v to about 1.75% w / v, about 1% w / v to about 2% w / v, about 1% w / v to about 2.5% w / v, about 1% w / v to about 3% w / v, about 1% w / v to about 5% w / v, about 1.25% w / v to about 1.5% w / v, about 1.25% w / v to about 1.75% w / v, about 1.25% w / v to about 2% w / v, about 1.25% w / v to about 2.5% w / v, about 1.25% w / v to about 3% w / v, about 1.25% w / v to about 5% w / v, about 1.5% w / v to about 1.75% w / v, about 1.5% w / v to about 2% w / v, about 1.5% w / v to about 2.5% w / v, about 1.5% w / v to about 3% w / v, about 1.5% w / v to about 5% w / v, about 1.75% w / v to about 2% w / v, about 1.75% w / v to about 2.5% w / v, about 1.75% w / v to about 3% w / v, about 1.75% w / v to about 5% w / v, about 2% w / v to about 2.5% w / v, about 2% w / v to about 3% w / v, about 2% w / v to about 5% w / v, about 2.5% w / v to about 3% w / v, about 2.5% w / v to about 5% w / v, or about 3% w / v to about 5% w / v.

[0243] In some embodiments, the input composition to the digestion system 300 may comprise a nitrogen source 380. The nitrogen source may be ammonium sulfate, ammonium chloride, ammonium nitrate, sodium nitrate, yeast extract, yeast, or any combination thereof. In some cases, the nitrogen source may be added to the digestion system on a first day of a digestion process. In some cases, the nitrogen source may be added to the digestion system on a second day, third day, fourth day, and / or any day following a first day of a digestion process. In some cases, the nitrogen source may be added to the digestion system daily. In some embodiments, the nitrogen source may be added to the second reactor 315 of the digestion system. In some embodiments, the nitrogen source is added to a first reactor 310, a third reactor 320, or a fourth reactor 325. The nitrogen source (e.g., ammonium sulfate) may be added to the reactor to maintain a concentration range of 0.05-0.2% w / v based on the span of the hydraulic retention time of the digestion system. In some embodiments, the concentration of nitrogen that may be maintained can be based on the total concentration of all components of the working fluid at a point during the digestion process of the digestion system 300.

[0244] In some embodiments, the nitrogen source (e.g., ammonium sulfate) may be added to the reactor to maintain a concentration range of nitrogen source in the working fluid of at least about 0.005% w / v, at least about 0.01% w / v, at least about 0.02% w / v, at least about 0.03% w / v, at least about 0.04% w / v, at least about 0.05% w / v, at least about 0.055% w / v, at least about 0.06% w / v, at least about 0.065% w / v, at least about 0.07% w / v, at least about 0.075% w / v, at leastabout 0.1% w / v, at least about 0.125% w / v, at least about 0.15% w / v, at least about 0.175% w / v, at least about 0.2% w / v, at least about 0.225% w / v, at least about 0.25% w / v, at least about 0.275% w / v, at least about 0.3% w / v, at least about 0.4% w / v, at least about 0.5% w / v, at least about 0.75% w / v, or at least about 1.0% w / v. In some embodiments, the nitrogen source (e.g., ammonium sulfate) may be added to the reactor to maintain a concentration of nitrogen source in the working fluid of at most about 1.0% w / v, at most about 0.75% w / v, at most about 0.5% w / v, at most about 0.4% w / v, at most about 0.3% w / v, at most about 0.275% w / v, at most about 0.25% w / v, at most about 0.225% w / v, at most about 0.2% w / v, at most about 0.175% w / v, at most about 0.15% w / v, at most about 0.125% w / v, at most about 0.1% w / v, at most about 0.075% w / v, at most about 0.07% w / v, at most about 0.065% w / v, at most about 0.06% w / v, at most about 0.055% w / v, at most about 0.05% w / v, at most about 0.04% w / v, at most about 0.03% w / v, at most about 0.02% w / v, at most about 0.01% w / v, or at most about 0.005% w / v.

[0245] In some embodiments, the nitrogen source (e.g., ammonium sulfate) may be added to the reactor to maintain a concentration of nitrogen source in the working fluid of about 0.03% w / v to about 1% w / v. In some embodiments, the nitrogen source (e.g., ammonium sulfate) may be added to the first reactor to maintain a concentration of nitrogen source in the working fluid of about 0.25% w / v to about 0.03% w / v, about 0.25% w / v to about 0.04% w / v, about 0.25% w / v to about 0.05% w / v, about 0.25% w / v to about 0.1% w / v, about 0.25% w / v to about 0.125% w / v, about 0.25% w / v to about 0.15% w / v, about 0.25% w / v to about 0.175% w / v, about 0.25% w / v to about 0.2% w / v, about 0.25% w / v to about 0.5% w / v, about 0.25% w / v to about 0.75% w / v, about 0.25% w / v to about 1% w / v, about 0.03% w / v to about 0.04% w / v, about 0.03% w / v to about 0.05% w / v, about 0.03% w / v to about 0.1% w / v, about 0.03% w / v to about 0.125% w / v, about 0.03% w / v to about 0.15% w / v, about 0.03% w / v to about 0.175% w / v, about 0.03% w / v to about 0.2% w / v, about 0.03% w / v to about 0.5% w / v, about 0.03% w / v to about 0.75% w / v, about 0.03% w / v to about 1% w / v, about 0.04% w / v to about 0.05% w / v, about 0.04% w / v to about 0.1% w / v, about 0.04% w / v to about 0.125% w / v, about 0.04% w / v to about 0.15% w / v, about 0.04% w / v to about 0.175% w / v, about 0.04% w / v to about 0.2% w / v, about 0.04% w / v to about 0.5% w / v, about 0.04% w / v to about 0.75% w / v, about 0.04% w / v to about 1% w / v, about 0.05% w / v to about 0.1% w / v, about 0.05% w / v to about 0.125% w / v, about 0.05% w / v to about 0.15% w / v, about 0.05% w / v to about 0.175% w / v, about 0.05% w / v to about 0.2% w / v, about 0.05% w / v to about 0.5% w / v, about 0.05% w / v to about 0.75% w / v, about 0.05% w / v to about 1% w / v, about 0.1% w / v to about 0.125% w / v, about 0.1% w / v to about 0.15% w / v, about 0.1% w / v to about 0.175% w / v, about 0.1% w / v to about 0.2% w / v, about 0.1% w / v to about 0.5% w / v, about 0.1% w / v to about 0.75% w / v, about 0.1% w / v to about 1% w / v, about 0.125% w / vto about 0.15% w / v, about 0.125% w / v to about 0.175% w / v, about 0.125% w / v to about 0.2% w / v, about 0.125% w / v to about 0.5% w / v, about 0.125% w / v to about 0.75% w / v, about 0.125% w / v to about 1% w / v, about 0.15% w / v to about 0.175% w / v, about 0.15% w / v to about 0.2% w / v, about 0.15% w / v to about 0.5% w / v, about 0.15% w / v to about 0.75% w / v, about 0.15% w / v to about 1% w / v, about 0.175% w / v to about 0.2% w / v, about 0.175% w / v to about 0.5% w / v, about 0.175% w / v to about 0.75% w / v, about 0.175% w / v to about 1% w / v, about 0.2% w / v to about 0.5% w / v, about 0.2% w / v to about 0.75% w / v, about 0.2% w / v to about 1% w / v, about 0.5% w / v to about 0.75% w / v, about 0.5% w / v to about 1% w / v, or about 0.75% w / v to about 1% w / v.

[0246] In some cases, the quantities added of the carbon and / or nitrogen sources to the system may be relative to the retention time of the system and can be adjusted accordingly if the flow rate changes. For example, a digestion system with a longer retention time may have a greater amount of a carbon source and / or nitrogen source added compared to an amount of a carbon source and / or nitrogen source added to a system with a short retention time.

[0247] In some embodiments, the input composition to the digestion system 300 may comprise micronutrients. Micronutrients may comprise important elements for microorganisms to support physiological functions. The micronutrients may comprise less than about 1%, less than about 2%, less than about 3%, less than about 4%, or les...

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method of making a biostimulant composition, the method comprising:(a) providing a bioreactor system comprising two or more containers arranged in a series, each of the two or more containers comprising a volume of a working fluid, wherein a first container comprises a first working fluid that includes a first microbial consortium and an established population of a zinc solubilizing bacterial strain;(b) operating the bioreactor system for a duration of time by:(i) transferring into the first container an aqueous feedstock comprising a second microbial consortium;(ii) transferring a portion of the working fluid out of each of the two or more containers into either a subsequent container of the bioreactor system or a product outflow stream;(iii) maintaining a concentration of the zinc solubilizing bacterial strain throughout the duration of time in at least the first container at at least 80% of a concentration of the zinc solubilizing bacterial strain at the beginning of the duration of time; and(iv) collecting at least a portion of the product outflow stream as the biostimulant composition; wherein the duration of time is at least 5 days; and wherein the zinc solubilizing bacterial strain is not present in the aqueous feedstock or any other input into the bioreactor system during the duration of time at a concentration that is higher than 1% of the concentration of the zinc solubilizing microbe in the first container.

2. The method of claim 1, further comprising maintaining in at least the first container a concentration of a zinc-containing molecule of at least 5 mg / L.

3. The method of claim 1, wherein the first microbial consortium comprises a first portion of microbes and a second portion of microbes, and wherein the method further comprises maintaining a concentration of a zinc-containing molecule in at least the first container that suppresses growth of the first portion of microbes relative to the zinc solubilizing bacterial strain.

4. The method of claim 3, wherein the first portion of microbes comprises microbes that are not zinc solubilizers and / or are not zinc tolerant.

5. The method of claim 3 or 4, wherein the second portion of microbes comprises microbes that are zinc solubilizers and / or are zinc tolerant.

6. The method of any one of claims 2-5, wherein the zinc-containing molecule is zinc oxide, zinc sulfide, zinc carbonate, zinc phosphate, zinc chloride, or zinc sulphate.

7. The method of any one of claims 1-6, wherein the zinc-solubilizing bacterial strain is of the genus Bacillus.

8. The method of any one of claims 1-7, wherein the zinc-solubilizing bacterial strain is of the species Bacillus safensis or Bacillus megaterium.

9. The method of any one of claims 1-8, wherein the zinc-solubilizing bacterial strain is one of the following:(a) a Bacillus safensis strain having one or more of the following:(i) a 16S rRNA gene sequence at least 95% identical to SEQ ID NO: 1;(ii) a gyrB gene sequence at least 95% identical to SEQ ID NO: 4; and(iii) a rpoB gene sequence at least 95% identical to SEQ ID NO: 7;(b) a Bacillus megaterium strain having one or more of the following:(i) a 16S rRNA gene sequence at least 95% identical to SEQ ID NO: 2;(ii) a gyrB gene sequence at least 95% identical to SEQ ID NO: 5; and(iii) a rpoB gene sequence at least 95% identical to SEQ ID NO: 8; or(b) a Bacillus megaterium strain having one or more of the following:(i) a 16S rRNA gene sequence at least 95% identical to SEQ ID NO: 3;(ii) a gyrB gene sequence at least 95% identical to SEQ ID NO: 6; and(iii) a rpoB gene sequence at least 95% identical to SEQ ID NO: 9.

10. The method of any one of claims 1-9, wherein the zinc-solubilizing bacterial strain is the Bacillus safensis strain deposited under ATCC Accession No. PTA-127681, the Bacillus megaterium strain deposited under ATCC Accession No. PTA-127683, or the Bacillus megaterium strain deposited under ATCC Accession No. PTA-127682.

11. The method of any one of claims 1-10, wherein the zinc solubilizing bacterial strain is not present in the aqueous feedstock or any other input into the bioreactor system during the duration of time at a concentration of greater than 100 CFU / ml.

12. The method of any one of claims 1-11, wherein the zinc solubilizing bacterial strain is not present in the aqueous feedstock or any other input into the bioreactor system during the duration of time.

13. The method of any one of claims 1-12, wherein the maintaining of step (b)(iii) comprises maintaining the concentration of the zinc solubilizing bacterial strain at at least IxlO3CFU / ml.

14. The method of any one of claims 1-13, wherein, before step (b), the first container further comprises an established population of other zinc solubilizing microbes that are not the zinc solubilizing bacterial strain, and wherein step (b)(iii) further comprises maintaining a concentration of the other zinc solubilizing microbes in at least the first container throughout the duration of time at at least IxlO4CFU / ml or at at least 80% of a concentration of the other zinc solubilizing microbes at the beginning of the duration of time, wherein the other zinc solubilizing microbes are not added to the bioreactor system during the duration of time at a concentration that is higher than 1% of the concentration of the other zinc solubilizing microbes in the first container.

15. The method of claim 14, wherein the other zinc solubilizing microbes are not present in the aqueous feedstock or any other input into the bioreactor system at a concentration of greater than 104CFU / ml.

16. The method of claim 14 or 15, wherein the population of the other zinc solubilizing microbes in the first container is at least IxlO3CFU / ml at the beginning of the duration of time.

17. The method of any one of claims 1-16, further comprising, before step (a), adding an inoculum of the zinc solubilizing bacterial strain to the bioreactor system, wherein the inoculum of the zinc solubilizing bacterial strain produces an initial population of the zinc solubilizing bacterial strain of at least 0.5xl04CFU / ml in at least one container.

18. The method of claim 17, wherein, before adding the inoculum of the zinc solubilizing bacterial strain, the concentration of the zinc solubilizing bacterial strain is less than IxlO2CFU / ml.

19. The method of any one of claims 1-18, wherein the aqueous feedstock further comprises an organic material at least partially digestible by microbes present in at least one of the containers.

20. The method of claim 19, wherein, before the transferring of step (b)(i), the organic material had been partially digested by microbes endogenous to the organic material.

21. The method of claim 19, further comprising digesting the organic material in two or more serially connected containers before the transferring of step (b)(i).

22. The method of any one of claims 19-21, wherein the organic material comprises manure and / or material produced by microbial digestion of manure.

23. The method of any one of claims 1-22, wherein the aqueous feedstock further comprises an inorganic material.

24. The method of claim 23, wherein the inorganic material comprises rock phosphate particles.

25. The method of claim 24, wherein, prior to the transferring of step (b)(i), the rock phosphate particles had been partially digested by microbes present in the aqueous feedstock.

26. The method of claim 24, further comprising partially digesting the rock phosphate particles in two or more serially connected containers before the transferring of step (b)(i).

27. The method of any one of claims 1-26, wherein the second microbial consortium comprises at least IxlO5CFU / ml.

28. The method of claim 27, wherein the second microbial consortium comprises microbes derived from manure and / or from rock phosphate particles.

29. The method of any one of claims 1-28, wherein the operating of step (b) further comprises producing microbial metabolites that directly or indirectly promote zinc solubilization in a plant growth medium.

30. The method of any one of claims 1-29, wherein the transferring of step (b)(i), the transferring of step (b)(ii), and the collecting of step (b)(iv) are performed continuously throughout the duration of time.

31. The method of any one of claims 1-30, wherein the transferring of step (b)(i), the transferring of step (b)(ii), and the collecting of step (b)(iv) are performed periodically throughout the duration of time.

32. The method of any one of claims 1-31, further comprising adding one or more carbon sources to at least one container of the bioreactor system.

33. The method of claim 32, wherein the one or more carbon sources are included in the aqueous feedstock.

34. The method of claim 32 or 33, further comprising maintaining a gluconic acid concentration and / or glucose concentration in at least one container of the bioreactor system at a concentration of at least 0.2% w / v in relation to the volume of working fluid in the at least one container.

35. The method of any one of claims 1-34, further comprising adding one or more nitrogen sources to at least one container of the bioreactor system.

36. The method of claim 35, wherein the one or more nitrogen sources comprise one or more of ammonium sulfate, ammonium chloride, ammonium nitrate, sodium nitrate, yeast extract, yeast, or any combination thereof.

37. The method of any one of claims 1-36, wherein the bioreactor system comprises a clarifier container comprising a clarifier working fluid.

38. The method of claim 37, further comprising separating a supernatant portion of the clarifier working fluid from a floc portion of the clarifier working fluid within the clarifier container.

39. The method of claim 38, wherein the separating comprises gravity separation.

40. The method of claim 38 or 39, further comprising folding the floc portion of the clarifier working fluid.

41. The method of claim 40, wherein the folding further comprises releasing a population of the zinc solubilizing bacterial strain into the supernatant portion without introducing floc solids into the supernatant portion.

42. The method of claim 40 or 41, wherein the folding is performed by folding wipers in a bottom portion of the clarifier container.

43. The method of any one of claims 38-42, wherein the operating further comprises transferring the floc portion from the clarifier container to an earlier container in the bioreactor system.

44. The method of any one of claims 38-43, wherein the product outflow stream comprises the supernatant portion of the clarifier working fluid.

45. The method of any one of claims 1-44, wherein the method further comprises producing at least IxlO4CFU / ml of the zinc solubilizing bacterial strain in the product outflow stream.

46. The method of any one of claims 1-45, wherein the bioreactor system comprises the first container comprising a volume of a first working fluid, a second container comprising a volume of a second working fluid, and a third container comprising a volume of a third working fluid.

47. The method of claim 46, wherein the first container comprises an outlet port fluidly connected to an inlet port of the second container and the second container comprises an outlet port fluidly connected to an input port of the third container.

48. The method of claim 47, wherein the third container comprises an outlet port fluidly connected to a clarifier container.

49. The method of any one of claims 46-48, further comprising maintaining the volume of each of the first working fluid, the second working fluid, and the third working fluid constant throughout the duration of time.

50. The method of any one of claims 1-49, wherein step (b) comprises operating the bioreactor system in a hydraulically balanced manner.

51. The method of any one of claims 1-50, wherein the transferring of step (b)(i), the transferring of step (b)(ii), and the collecting of step (b)(iv) are driven by gravity.

52. The method of any one of claims 1-51, wherein the operating comprises maintaining a flow rate that results in a hydraulic retention time of the bioreactor system of at least 5 days.

53. The method of any one of claims 1-52, wherein the operating comprises maintaining the product outflow stream at a flow rate of at least 100 gallons per day.

54. The method of any one of claims 1-53, wherein the volume of working fluid in each of the two or more containers is at least 100 gallons.

55. The method of any one of claims 1-54, wherein at least one of the two or more containers is a fluidized bed reactor.

56. The method of any one of claims 1-55, wherein at least one of the two or more containers is a packed bed reactor.

57. The method of any one of claims 1-56, further comprising maintaining at least one of the two or more containers under microaerobic conditions.

58. The method of any one of claims 1-57, wherein the bioreactor system is operated continuously for at least 90 days.

59. The method of any one of claims 1-58, wherein one or more species of one or more of the following genera are among five most abundant species in the second microbial consortium: Haliscomenobacler. I. ew ine Ila. Caldilinea, Terri monas. and Acidobacterium.

60. The method of any one of claims 1-59, wherein one or more of the following species are among five most abundant species in the microbial consortium: Lewinella cohaerens. Thauera phenylacelica. Thauera mechernichensis. Solitalea canadensis, and Nitrospira moscoviensis .

61. The method of any one of claims 19-60, wherein the second microbial consortium comprises microbes endogenous to the organic material.

62. The method of any one of claims 46-61, wherein at least one of the first working fluid, the second working fluid, or the third working fluid are circulated within the respective containers.

63. The method of any one of claims 46-62, further comprising maintaining the pH of at least one of the first working fluid, the second working fluid, or the third working fluid between 6 and 9 throughout the duration of time.

64. The method of any one of claims 1-63, wherein the aqueous feedstock does not include the zinc solubilizing bacterial strain at a concentration higher than 10 CFU / ml.

65. The method of any one of claims 1-64, wherein the zinc solubilizing bacterial strain is not added to the bioreactor system during the duration of time at a concentration that is higher than 10 CFU / ml.

66. The method of any one of claims 1-65, wherein the bioreactor system comprises at least one container placed in the series before the first container.

67. The method of any one of claims 1-66, further comprising producing a population of sporulated bacteria in the product outflow stream.

68. The method of any one of claims 1-67, further comprising producing a population of the zinc solubilizing bacterial strain in the product outflow stream that is sporulated.

69. The method of claim 68, wherein the population of the zinc solubilizing bacterial strain that is sporulated comprises at least IxlO3CFU / ml.

70. The method of any one of claims 1-69, further comprising adding an additional population of the zinc solubilizing bacterial strain to the biostimulant product.

71. The method of any one of claims 1-70, wherein the method further comprises making at least a portion of the aqueous feedstock by a method comprising:(c) transferring water, rock phosphate, and, optionally, products of digestion of manure by microbes derived from the manure into a fourth container comprising a volume of a fourth working fluid;(d) transferring a portion of the fourth working fluid into a fifth container comprising a fifth working fluid;(e) transferring into the fifth container:(i) a liquid comprising (A) a third microbial consortium comprising microbes derived from manure, and (B) digestion products produced by anaerobic digestion of the manure by the microbes;(ii) manure; and(iii) yeast.

72. The method of claim 71, further comprising transferring a portion of the fifth working fluid into a sixth container comprising a sixth working fluid, and transferring a portion of the sixth working fluid into a seventh container comprising a seventh working fluid.

73. The method of claim 72, further comprising separating a portion of the seventh working fluid into a feedstock floc portion and a feedstock supernatant portion.

74. The method of claim 73, further comprising transferring the feedstock floc portion to the fourth container.

75. The method of any one of claims 72-74, further comprising maintaining the fourth container, the fifth container, the sixth container, and / or the seventh container under aerobic conditions.

76. The method of any one of claims 72-75, wherein the fourth container, the fifth container, the sixth container, and / or the seventh container are fluidized bed reactors, wherein the rock phosphate is continuously circulated within the fourth container, the fifth container, the sixth container, and / or the seventh container.

77. The method of any one of claims 72-76, wherein a total volume of material added to the fourth container over a given time period is equal to a total volume of the fourth working fluid transferred to the fifth container over a same time period.

78. A bioreactor system comprising:(a) a stream of an aqueous feedstock in fluid communication with a first container comprising a volume of a first working fluid, wherein the aqueous feedstock comprises a first microbial consortium, wherein the first working fluid comprises an established population of a zinc solubilizing bacterial strain and a second microbial consortium, wherein a concentration of the zinc solubilizing bacterial strain in the first working fluid is at least 100 times higher than a concentration of the zinc solubilizing bacterial strain in the aqueous feedstock stream and in any other input into the bioreactor system;(b) one or more additional containers arranged in a series that includes the first container, wherein each of the one or more additional containers comprises a volume of a working fluid and is in fluid communication with at least one other container in the series, and wherein at least one of the one or more additional containers comprises a product outflow stream port; and(c) a product outflow stream in fluid communication with the product outflow stream port.

79. The system of claim 78, wherein at least the first working fluid comprises a zinc-containing molecule at a concentration of at least 5 mg / L.

80. The system of claim 78, wherein the second microbial consortium comprises a first portion of microbes and a second portion of microbes, wherein at least the first working fluid comprises a zinc-containing molecule at a concentration that suppresses growth of the first portion of microbes relative to the zinc solubilizing bacterial strain.

81. The system of claim 80, wherein the first portion of microbes comprises microbes that are not zinc solubilizers and / or are not zinc tolerant.

82. The system of claim 80 or 81, wherein the second portion of microbes comprises microbes that are zinc solubilizers and / or are zinc tolerant.

83. The system of any one of claims 79-82, wherein the zinc-containing molecule is zinc oxide, zinc sulfide, zinc carbonate, zinc phosphate, zinc chloride, or zinc sulphate.

84. The system of any one of claims 78-83, wherein the zinc-solubilizing bacterial strain is of the genus Bacillus.

85. The system of any one of claims 78-84, wherein the zinc-solubilizing bacterial strain is of the species Bacillus safensis o Bacillus megaterium.

86. The system of any one of claims 78-85, wherein the zinc-solubilizing bacterial strain is one of the following:(a) a Bacillus safensis strain having one or more of the following:(i) a 16S rRNA gene sequence at least 95% identical to SEQ ID NO: 1;(ii) a gyrB gene sequence at least 95% identical to SEQ ID NO: 4; and(iii) a rpoB gene sequence at least 95% identical to SEQ ID NO: 7;(b) a Bacillus megaterium strain having one or more of the following:(i) a 16S rRNA gene sequence at least 95% identical to SEQ ID NO: 2;(ii) a gyrB gene sequence at least 95% identical to SEQ ID NO: 5; and(iii) a rpoB gene sequence at least 95% identical to SEQ ID NO: 8; or(b) a Bacillus megaterium strain having one or more of the following:(i) a 16S rRNA gene sequence at least 95% identical to SEQ ID NO: 3;(ii) a gyrB gene sequence at least 95% identical to SEQ ID NO: 6; and(iii) a rpoB gene sequence at least 95% identical to SEQ ID NO: 9;87. The system of any one of claims 78-86, wherein the zinc-solubilizing bacterial strain is the Bacillus safensis strain deposited under ATCC Accession No. PTA-127681, the Bacillus megaterium strain deposited under ATCC Accession No. PTA-127683, or the Bacillus megaterium strain deposited under ATCC Accession No. PTA-127682.

88. The system of any one of claims 78-87, wherein the bioreactor system is a continuous flow bioreactor system and the stream of the aqueous feedstock is a continuous stream.

89. The system of any one of claims 78-88, wherein each of the volume of the working fluids is constant.

90. The system of any one of claims 78-89, wherein each of the first container and the one or more additional containers comprises a concentration of the zinc-solubilizing bacterial strain that remains at least IxlO4CFU / ml during operation of the bioreactor system.

91. The system of any one of claims 78-90, wherein the aqueous feedstock and any other input into the bioreactor system does not comprise the zinc-solubilizing bacterial strain or does not comprise a concentration of the zinc-solubilizing bacterial strain at level higher than 100 CFU / ml.

92. The system of any one of claims 78-91, wherein the first microbial consortium comprises at least IxlO4CFU / ml of microbes.

93. The system of any one of claims 78-92, wherein the aqueous feedstock further comprises an organic material digestible by microbes present in the containers.

94. The system of claim 93, wherein the organic material comprises manure or material derived from manure.

95. The system of any one of claims 78-94, wherein the aqueous feedstock further comprises rock phosphate particles.

96. The system of claim 95, wherein the first microbial consortium comprises microbes derived from manure and / or rock phosphate particles.

97. The system of any one of claims 78-96, wherein the container comprising the product outflow stream port is a clarifier container configured to separate a portion of a working fluid in the clarifier container into a supernatant portion and a floc portion.

98. The system of claim 97, wherein the clarifier container comprises one or more floc folding flights configured to agitate settled floc in the clarifier container without resuspending solids in the floc portion into the supernatant portion.

99. The system of claim 97 or 98, further comprising a floc return stream that flows from the clarifier to an earlier container in the series.

100. The system of any one of claims 97-99, wherein the product outflow stream comprises the supernatant portion.

101. The system of claim 100, wherein the product outflow stream comprises at least IxlO4CFU / ml of the zinc-solubilizing bacterial strain.

102. The system of claim 100 or 101, wherein the product outflow stream comprises at least IxlO2CFU / ml of a sporulated form of the zinc-solubilizing bacterial strain.

103. The system of any one of claims 100-102, wherein the product outflow stream comprises a total dry weight of 0.2 to 2.5 mg / ml.

104. The system of any one of claims 100-103, wherein the product outflow stream has a chemical oxygen demand between 80 to 500 mg / L.

105. The system of any one of claims 100-104, wherein the product outflow stream has an electrical conductivity between 0.1 and 1.5 mS / cm.

106. The system of any one of claims 78-105, wherein the first working fluid comprises glucose and / or gluconic acid at a concentration of at least 0.2% w / v.

107. The system of any one of claims 78-106, wherein the first working fluid and / or the working fluid in at least one of the one or more additional containers comprises microaerobic conditions.

108. A biostimulant composition made by the method of any one of claims 1-77 or the system of any one of claims 78 to 107.

109. A method of promoting plant growth comprising contacting a plant, seed, or plant growth medium with the biostimulant composition of claim 108.

110. A method of increasing an amount of solubilized zinc available to a plant, the method comprising contacting a plant, seed, or plant growth medium with the biostimulant composition of claim 108.

111. A composition comprising:(a) a Bacillus safensis strain having one or more of the following:(i) a 16S rRNA gene sequence at least 95% identical to SEQ ID NO: 1;(ii) a gyrB gene sequence at least 95% identical to SEQ ID NO: 4; and(iii) a rpoB gene sequence at least 95% identical to SEQ ID NO: 7; and(b) a carrier.

112. The composition of claim 111, wherein the Bacillus safensis strain is the strain deposited under ATCC Accession No. PTA-127681, or an isolated clone thereof.

113. The composition of claim 111 or 112, further comprising products of digestion of an organic substrate by the Bacillus safensis strain.

114. The composition of any one of claims 111-113, wherein the carrier comprises a fertilizer.

115. The composition of any one of claims 111-114, wherein the carrier is a solid coated by the Bacillus safensis strain.

116. The composition of claim 115, wherein the carrier is further coated by a micronutrient.

117. The composition of claim 116, wherein the micronutrient is zinc oxide, zinc sulfide, zinc carbonate, or zinc phosphate.

118. The composition of any one of claims 111-114, wherein the carrier is a liquid.

119. The composition of any one of claims 111-118, wherein the carrier further comprises an adjuvant selected from a wetting agent, spreading agent, dispersing agent, sticking agent, dust control agent, or adhesive.

120. The composition of any one of claims 111-119, wherein the concentration of the Bacillus safensis strain in the composition ranges from IxlO3to IxlO11cfu / ml.

121. The composition of any one of claims 111-120, wherein the concentration of the Bacillus safensis strain in the composition ranges from IxlO4to IxlO6cfu / ml.

122. An isolated strain of the species Bacillus safensis having one or more of the following:(a) a 16S rRNA gene sequence at least 95% identical to SEQ ID NO: 1;(b) a gyrB gene sequence at least 95% identical to SEQ ID NO: 4; and(c) a rpoB gene sequence at least 95% identical to SEQ ID NO: 7.

123. The isolated strain of claim 122, wherein the B. safensis strain is the strain deposited under ATCC Accession No. PTA-127681, or an isolated clone thereof.

124. A composition comprising:(a) a Bacillus megaterium strain having one or more of the following:(i) a 16S rRNA gene sequence at least 95% identical to SEQ ID NO: 2;(ii) a gyrB gene sequence at least 95% identical to SEQ ID NO: 5; and(iii) a rpoB gene sequence at least 95% identical to SEQ ID NO: 8; and(b) a carrier.

125. The composition of claim 124, wherein the Bacillus megaterium strain is the strain deposited under ATCC Accession No. PTA-127683, or an isolated clone thereof.

126. The composition of claim 124 or 125, further comprising products of digestion of an organic substrate by the Bacillus megaterium strain.

127. The composition of any one of claims 124-126, wherein the carrier comprises a fertilizer.

128. The composition of any one of claims 124-127, wherein the carrier is a solid coated by the Bacillus megaterium strain.

129. The composition of claim 128, wherein the carrier is further coated by a micronutrient.

130. The composition of claim 129, wherein the micronutrient is zinc oxide, zinc sulfide, zinc carbonate, or zinc phosphate.

131. The composition of any one of claims 124-130, wherein the carrier is a liquid.

132. The composition of any one of claims 124-131, wherein the carrier further comprises an adjuvant selected from a wetting agent, spreading agent, dispersing agent, sticking agent, dust control agent, or adhesive.

133. The composition of any one of claims 124-132, wherein the concentration of the Bacillus megaterium strain in the composition ranges from IxlO3to IxlO11cfu / ml.

134. The composition of any one of claims 124-133, wherein the concentration of the Bacillus megaterium strain in the composition ranges from IxlO4to IxlO6.

135. An isolated strain of the species Bacillus megaterium having one or more of the following:(a) a 16S rRNA gene sequence at least 95% identical to SEQ ID NO: 2;(b) a gyrB gene sequence at least 95% identical to SEQ ID NO: 5; and(c) a rpoB gene sequence at least 95% identical to SEQ ID NO: 8.

136. The isolated strain of claim 135, wherein the B. megaterium strain is the strain deposited under ATCC Accession No. PTA-127683, or an isolated clone thereof.

137. A composition comprising:(a) a Bacillus megaterium strain having one or more of the following:(i) a 16S rRNA gene sequence at least 95% identical to SEQ ID NO: 3;(ii) a gyrB gene sequence at least 95% identical to SEQ ID NO: 6; and(iii) a rpoB gene sequence at least 95% identical to SEQ ID NO: 9; and(b) a carrier.

138. The composition of claim 137, wherein the Bacillus megaterium strain is the strain deposited under ATCC Accession No. PTA-127682, or an isolated clone thereof.

139. The composition of claim 137 or 138, further comprising products of digestion of an organic substrate by the Bacillus megaterium strain.

140. The composition of any one of claims 137-139, wherein the carrier comprises a fertilizer.

141. The composition of any one of claims 137-140, wherein the carrier is a solid coated by the Bacillus megaterium strain.

142. The composition of claim 141, wherein the carrier is further coated by a micronutrient.

143. The composition of claim 142, wherein the micronutrient is zinc oxide, zinc sulfide, zinc carbonate, or zinc phosphate.

144. The composition of any one of claims 137-143, wherein the carrier is a liquid.

145. The composition of any one of claims 137-144, wherein the carrier further comprises an adjuvant selected from a wetting agent, spreading agent, dispersing agent, sticking agent, dust control agent, or adhesive.

146. The composition of any one of claims 137-145, wherein the concentration of the Bacillus megaterium strain in the composition ranges from IxlO3to IxlO11cfu / ml.

147. The composition of any one of claims 137-146, wherein the concentration of the Bacillus megaterium strain in the composition ranges from IxlO4to IxlO6.

148. An isolated strain of the species Bacillus megaterium having one or more of the following: a 16S rRNA gene sequence at least 95% identical to SEQ ID NO: 3; a gyrB gene sequence at least 95% identical to SEQ ID NO: 6; and a rpoB gene sequence at least 95% identical to SEQ ID NO: 9.

149. The isolated strain of claim 148, wherein the B. megaterium strain is the strain deposited under ATCC Accession No. PTA-127682, or an isolated clone thereof.

150. A method for promoting growth of a plant growing in a medium, the method comprising contacting the plant or the medium with the biostimulant composition of claim 108, the composition of any one of claims 111-121, 124-134, or 137-147, or a composition comprising the isolated strain of any one of claims 122, 123, 135, 136, 148, and 149.

151. The method of claim 150, wherein the contacting causes increases the amount of solubilized zinc available to the plant by at least 5%.

152. The method of claim 150 or 151, wherein the composition increases plant growth by at least 5% as compared to a control.

153. The method of any one of claims 150-152, wherein the composition increases uptake of a nutrient by the plant by at least 5% as compared to a control.

154. The method of claim 153, wherein the nutrient is zinc, and wherein the uptake of zinc by the plant is increased by at least 5% as compared to a control.

155. The method of claim 153, wherein the nutrient is phosphate.

156. The method of claim 153, wherein the nutrient is sulfur, potassium, magnesium, calcium, boron, manganese, iron, and / or copper.

157. The method of any one of claims 150-156, wherein the medium is soil or a hydroponic medium.

158. A method of remedying a zinc deficiency in a plant growth medium, the method comprising:(a) measuring a concentration of soluble zinc in the plant growth medium that is less than 0.5 ppm; and(b) after step (a), contacting the plant growth medium with the biostimulant composition of claim 108, the composition of any one of claims 111-121, 124-134, or 137-147, or a composition comprising the isolated strain of any one of claims 122, 123, 135, 136, 148, and 149.

159. A method of promoting plant growth, comprising:(a) contacting a plant or medium in which the plant is growing with a composition comprising one or more compounds comprising 1 -hexadecyl-2,3 -di-o-acethyl glycerol, diisodecyl phthalate, allothreonine, (S)-(-)-.alpha.-(l-Naphthyl)ethylamine, indoleacetic acid (indole-3 -acetic acid), salicylanilide, 25-Hydroxycholesterol, 4-(2-Aminophenyl)-2,4- dioxobutanoic acid, 13,14-dihydro-15-keto PGD2, 4-Ethyloctanoic acid, phthalic acid, alpha- Ketoisovaleric acid, datiscetin, Vai-Ala, N-methylundec-10-enamide, His-Ile-Lys-Arg, methylcarbamyl PAF, homogentisic acid, Ala-Gly-Leu-Val-Ser, Arg-Val-Met, chaps, 2- Phenyl ethanol, prostaglandin D3, ritalinic acid, 5-Hydroxyindoleacetaldehyde, azithromycin dihydrate, Pro-Ala-Phe, (4R,7S,7aR,12bS)-3-methyl-2,4,4a,7,7a,13-hexahydro-lH-4,12- methanobenzofuro[3,2-e]isoquinoline-7,9-diol, Glu-Pro-Thr, 2,6-Naphthalenediol, 3- Aminoquinoline, acylated phloroglucinol, 1-Pyrrolysine, tanikolide, Val-Arg-Glu, 3- Methyladipic acid, 10-Propoxy decanoic acid, 7-hydroxy-3-(2-methoxyphenyl)-4H-chromen-4- one, N-methylundec-10-enamide, prosta-5,13-dien-l-oic acid, 9,1 l-epidioxy-15-hydroperoxy-, (5Z,9alpha,l 1 alpha, 13E, 15 S)-, Ile-Phe-Val-Lys, gamma-Glutamyl-Se-methylselenocysteine;5- L-Glutamyl-Se-methylselenocysteine, Trp-Ala-Lys, pantoyllactone glucoside, vasicinone, LPC(O- 16:0 / 2:0), Sphingosine 1-phosphate, coenzyme Q6, dihydrocortisol, dibutylone hydrochloride, LPE(18:0 / 0:0), Nialamide, androstanedione, 12-(2,3-Dihydroxycyclopentyl)-2- dodecanone, Androst-5-ene-3, 17-dione, hydroxytyrosol, 4-Hydroxy cinnamic acid, 2,4,5- Trihydroxytoluene, isopropamide, (1R,2R,5R,8R,9S,10R,12S)-12-Hydroxy-1 l-methyl-6- methylidene-16-oxo-15-oxapentacyclo[9.3.2.15,8.01,10.02,8]heptadecane-9-carboxylic acid, Allopurinol riboside, l-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3- phosphoethanolamine, Uridine triacetate, JWH 018 7-hydroxyindole metabolite-d9, 1,6- Dimethylphenazine, 2-(p-Bromophenyl)-8-methyl-8H-thieno(2,3-b)indole, 3,4-Dihydroxy-phenyl glycol, dodecyl ci s-9,10-epoxy octadecanoate, heptacosane, lauric acid, lignoceric acid, tetratriacontane, tryptophol, one or more derivatives thereof, or combination thereof.

160. The method of claim 159, wherein the contacting comprises contacting the plant with the composition.

161. The method of claim 159 or 160, wherein the contacting comprises contacting a plant seed with the composition.

162. The method of any one of claims 159-161, wherein the contacting comprises contacting a leaf of the plant with the composition.

163. The method of any one of claims 159-162, wherein the medium comprises a soil, a hydroponic medium, turface, or isolite.

164. The method of any one of claims 159-163, wherein the contacting causes an increase in plant growth of the plant by at least 10 percent as compared to a plant growth of the plant when the plant or the medium is not contacted with the composition comprising one or more compounds.

165. The method of any one of claims 159-164, wherein the composition further comprises an adjuvant selected from the group consisting of a wetting agent, spreading agent, dispersing agent, sticking agent, dust control agent, and adhesive.

166. The method of any one of claims 159-165, wherein the composition is configured to enhance an availability of zinc in soil.

167. The method of any one of claims 159-166, wherein the composition is configured to enhance zinc uptake by plant tissues.

168. The method of any one of claims 159-167, wherein the composition is configured to promote zinc use efficiency.

169. A composition for promoting plant growth, comprising:(i) at least one microbial strain comprises Bacillus safensis o Bacillus megalerium and(ii) one or more compounds comprising l-hexadecyl-2,3-di-o-acethyl glycerol, diisodecyl phthalate, allothreonine, (S)-(-)-.alpha.-(l-Naphthyl)ethylamine, indoleacetic acid (indole-3 -acetic acid), salicylanilide, 25-Hydroxycholesterol, 4-(2-Aminophenyl)-2,4- dioxobutanoic acid, 13,14-dihydro-15-keto PGD2, 4-Ethyloctanoic acid, phthalic acid, alpha- Ketoisovaleric acid, datiscetin, Vai-Ala, N-methylundec-10-enamide, His-Ile-Lys-Arg, methylcarbamyl PAF, homogentisic acid, Ala-Gly-Leu-Val-Ser, Arg-Val-Met, chaps, 2- Phenyl ethanol, prostaglandin D3, ritalinic acid, 5-Hydroxyindoleacetaldehyde, azithromycin dihydrate, Pro-Ala-Phe, (4R,7S,7aR,12bS)-3-methyl-2,4,4a,7,7a,13-hexahydro-lH-4,12- methanobenzofuro[3,2-e]isoquinoline-7,9-diol, Glu-Pro-Thr, 2,6-Naphthalenediol, 3-Aminoquinoline, acylated phloroglucinol, 1-Pyrrolysine, tanikolide, Val-Arg-Glu, 3- Methyladipic acid, 10-Propoxy decanoic acid, 7-hydroxy-3-(2-methoxyphenyl)-4H-chromen-4- one, N-methylundec-10-enamide, prosta-5,13-dien-l-oic acid, 9,1 l-epidioxy-15-hydroperoxy-, (5Z,9alpha,l 1 alpha, 13E, 15 S)-, Ile-Phe-Val-Lys, gamma-Glutamyl-Se-methylselenocysteine;5- L-Glutamyl-Se-methylselenocysteine, Trp-Ala-Lys, pantoyllactone glucoside, vasicinone, LPC(O- 16:0 / 2:0), Sphingosine 1-phosphate, coenzyme Q6, dihydrocortisol, dibutylone hydrochloride, LPE(18:0 / 0:0), Nialamide, androstanedione, 12-(2,3-Dihydroxycyclopentyl)-2- dodecanone, Androst-5-ene-3, 17-dione, hydroxytyrosol, 4-Hydroxy cinnamic acid, 2,4,5- Trihydroxytoluene, isopropamide, (1R,2R,5R,8R,9S,10R,12S)-12-Hydroxy-1 l-methyl-6- methylidene-16-oxo-15-oxapentacyclo[9.3.2.15,8.01,10.02,8]heptadecane-9-carboxylic acid, Allopurinol riboside, l-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3- phosphoethanolamine, Uridine triacetate, JWH 018 7-hydroxyindole metabolite-d9, 1,6- Dimethylphenazine, 2-(p-Bromophenyl)-8-methyl-8H-thieno(2,3-b)indole, 3,4-Dihydroxy- phenyl glycol, dodecyl ci s-9,10-epoxy octadecanoate, heptacosane, lauric acid, lignoceric acid, tetratriacontane, tryptophol, one or more derivatives thereof, or combination thereof.

170. The composition of claim 169, wherein the at least one microbial strain comprises the Bacillus safensis, and comprises one or more of the following:(i) a 16S rRNA gene sequence at least 95% identical to SEQ ID NO: 1;(ii) a gyrB gene sequence at least 95% identical to SEQ ID NO: 4; or(iii) a rpoB gene sequence at least 95% identical to SEQ ID NO: 7.

171. The composition of claim 169, wherein the at least one microbial strain comprises the Bacillus megaterium, and comprises one or more of the following:(i) a 16S rRNA gene sequence at least 95% identical to SEQ ID NO: 2;(ii) a gyrB gene sequence at least 95% identical to SEQ ID NO: 5; or(iii) a rpoB gene sequence at least 95% identical to SEQ ID NO: 8.

172. The composition of claim 169, wherein the at least one microbial strain comprises the Bacillus megaterium, and comprises one or more of the following:(i) a 16S rRNA gene sequence at least 95% identical to SEQ ID NO: 3;(ii) a gyrB gene sequence at least 95% identical to SEQ ID NO: 6; or(iii) a rpoB gene sequence at least 95% identical to SEQ ID NO: 9.

173. The composition of any one of claims 169-172, further comprising a carrier.

174. The composition of claim 173, wherein the carrier is formulated for application to a plant or medium in which the plant is growing.

175. The composition of any one of claims 169-174, wherein the composition further comprises an adjuvant selected from the group consisting of a wetting agent, spreading agent, dispersing agent, sticking agent, dust control agent, and adhesive.

176. The composition of any one of claims 169-175, wherein the composition is configured to enhance an availability of zinc in soil.

177. The composition of any one of claims 169-176, wherein the composition is configured to enhance zinc uptake by plant tissues.

178. The composition of any one of claims 169-177, wherein the composition is configured to promote zinc use efficiency.

179. A composition for promoting plant growth, comprising:(i) two or more compounds comprising l-hexadecyl-2,3-di-o-acethyl glycerol, diisodecyl phthalate, allothreonine, (S)-(-)-.alpha.-(l-Naphthyl)ethylamine, indoleacetic acid (indole-3- acetic acid), salicylanilide, 25-Hydroxycholesterol, 4-(2-Aminophenyl)-2,4-dioxobutanoic acid, 13,14-dihydro-15-keto PGD2, 4-Ethyloctanoic acid, phthalic acid, alpha-Ketoisovaleric acid, datiscetin, Vai-Ala, N-methylundec-10-enamide, His-Ile-Lys-Arg, methylcarbamyl PAF, homogentisic acid, Ala-Gly-Leu-Val-Ser, Arg-Val-Met, chaps, 2-Phenylethanol, prostaglandin D3, ritalinic acid, 5-Hydroxyindoleacetaldehyde, azithromycin dihydrate, Pro-Ala-Phe, (4R,7S,7aR,12bS)-3-methyl-2,4,4a,7,7a,13-hexahydro-lH-4,12-methanobenzofuro[3,2- e]isoquinoline-7,9-diol, Glu-Pro-Thr, 2,6-Naphthalenediol, 3 -Aminoquinoline, acylated phloroglucinol, 1-Pyrrolysine, tanikolide, Val-Arg-Glu, 3 -Methyladipic acid, 10- Propoxydecanoic acid, 7-hydroxy-3-(2-methoxyphenyl)-4H-chromen-4-one, N-methylundec-10- enamide, prosta-5,13-dien-l-oic acid, 9, 11 -epidioxy- 15 -hydroperoxy-, (5Z,9alpha,l 1 alpha, 13E, 15 S)-, Ile-Phe-Val-Lys, gamma-Glutamyl-Se-methylselenocysteine;5- L-Glutamyl-Se-methylselenocysteine, Trp-Ala-Lys, pantoyllactone glucoside, vasicinone, LPC(O- 16:0 / 2:0), Sphingosine 1-phosphate, coenzyme Q6, dihydrocortisol, dibutylone hydrochloride, LPE(18:0 / 0:0), Nialamide, androstanedione, 12-(2,3-Dihydroxycyclopentyl)-2- dodecanone, Androst-5-ene-3, 17-dione, hydroxytyrosol, 4-Hydroxy cinnamic acid, 2,4,5- Trihydroxytoluene, isopropamide, (1R,2R,5R,8R,9S,10R,12S)-12-Hydroxy-1 l-methyl-6- methylidene-16-oxo-15-oxapentacyclo[9.3.2.15,8.01,10.02,8]heptadecane-9-carboxylic acid, Allopurinol riboside, l-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3- phosphoethanolamine, Uridine triacetate, JWH 018 7-hydroxyindole metabolite-d9, 1,6- Dimethylphenazine, 2-(p-Bromophenyl)-8-methyl-8H-thieno(2,3-b)indole, 3,4-Dihydroxy- phenyl glycol, dodecyl ci s-9,10-epoxy octadecanoate, heptacosane, lauric acid, lignoceric acid, tetratriacontane, tryptophol, one or more derivatives thereof, or combination thereof; and(ii) a carrier.

180. The composition of claim 179, wherein the carrier is formulated for application to a plant or medium in which the plant is growing.

181. The composition of claim 179 or 180, wherein the carrier comprises a fertilizer.

182. The composition of claim 181, wherein the fertilizer is a solid.

183. The composition of any one of claims 179-182, wherein the carrier is a liquid.

184. The composition of any one of claims 179-183, wherein the composition further comprises an adjuvant selected from a wetting agent, spreading agent, dispersing agent, sticking agent, dust control agent, and adhesive.

185. The composition of any one of claims 179-184, wherein the composition is configured to enhance an availability of zinc in soil.

186. The composition of any one of claims 179-185, wherein the composition is configured to enhance a zinc uptake by plant tissues.

187. The composition of any one of claims 179-186, wherein the composition is configured to promote zinc use efficiency.

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