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

Abstract

The present disclosure provides methods and systems for producing a bio-stimulant that promotes zinc solubilization in plants. Described embodiments include methods of making a bio-stimulant composition in a bioreactor system comprising two or more vessels arranged in a series. The bioreactor system can include a population of established zinc solubilizing microbial strains. The methods can include operating the bioreactor system by transferring an aqueous feedstock comprising a microbial consortium into the system, transferring a working fluid between vessels of the system, and collecting a product. The methods can also include maintaining a concentration of zinc solubilizing microbial strains in the system.

Description

[0001] Cross-reference to related applications This application claims the benefit of U.S. Provisional Application No. 63 / 613,548, filed December 21, 2023, which is incorporated herein by reference. Background Technology

[0002] This disclosure generally relates 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 both environmental and economic reasons. Plant growth promoters derived from organic materials can help enhance crop growth, improve the efficacy of agricultural products such as fertilizers, and reduce the environmental impact of synthetic fertilizers and mitigate climate change. There is a need for plant growth-promoting biostimulant compositions that utilize abundant and readily available organic raw materials. Summary of the Invention

[0004] In one aspect, this disclosure provides a method for preparing a biostimulant composition, the method comprising: (a) providing a bioreactor system comprising two or more containers arranged in series, each of the two or more containers comprising a volume of working fluid, wherein a first container comprises a first working fluid containing a first microbial aggregate and an established population of zinc-solubilizing bacterial strains; (b) operating the bioreactor system for a duration by: (i) transferring an aqueous feedstock containing a second microbial aggregate into the first container; (ii) transferring a portion of the working fluid from the two containers. (iii) transferring each of one or more containers to a subsequent container of the bioreactor system or to a product effluent; (iv) maintaining the concentration of the zinc-solubilizing bacteria strain in at least the first container during the duration at at least 80% of the concentration of the zinc-solubilizing bacteria strain at the start of the duration; and (iv) collecting at least a portion of the product effluent as the biostimulant composition; wherein the duration is at least 5 days; and wherein during the duration, the concentration of the zinc-solubilizing bacteria strain present in the aqueous feedstock or any other input to the bioreactor system is not higher than 1% of the concentration of the zinc-solubilizing microorganisms in the first container.

[0005] In some embodiments, the method further includes maintaining the concentration of zinc-containing molecules in at least the first container at at least 5 mg / L. In some embodiments, the first microbial aggregate comprises a first portion of microorganisms and a second portion of microorganisms, and the method further includes maintaining the concentration of zinc-containing molecules in at least the first container at a concentration that inhibits the growth of the first portion of microorganisms relative to the zinc-solubilizing bacterial strain. In some embodiments, the first portion of microorganisms comprises non-zinc solubilizers and / or non-zinc-tolerant microorganisms. In some embodiments, the second portion of microorganisms comprises zinc solubilizers and / or zinc-tolerant microorganisms. In some embodiments, the zinc-containing molecule is zinc oxide, zinc sulfide, zinc carbonate, zinc phosphate, zinc chloride, or zinc sulfate. In some embodiments, the zinc-solubilizing bacterial strain belongs to the genus *Bacillus*. In some embodiments, the zinc-solubilizing bacterial strain belongs to *Bacillus safranin* (…). Bacillus safensis ) or Bacillus megaterium ( Bacillus megaterium In some embodiments, the zinc-solubilizing bacterial strain is one of the following strains: (a) a *Bacillus saffron* strain having one or more of the following: (i) a 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 1; (ii) a 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 4. gyrB Gene sequence; and (iii) at least 95% identical to SEQ ID NO: 7. rpoB (a) Gene sequence; (b) Bacillus megaterium strains having one or more of the following: (i) a 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 2; (ii) a 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 5. gyrB Gene sequence; and (iii) at least 95% identical to SEQ ID NO: 8. rpoB Gene sequence; or (b) a Bacillus megaterium strain having one or more of the following: (i) a 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 3; (ii) a 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 6. gyrB Gene sequence; and (iii) at least 95% identical to SEQ ID NO: 9. rpoBGene sequence. In some embodiments, the zinc-solubilizing bacteria strain is a *Bacillus salsa* strain deposited with ATCC accession number PTA-127681, a *Bacillus megaterium* strain deposited with ATCC accession number PTA-127683, or a *Bacillus megaterium* strain deposited with ATCC accession number PTA-127682. In some embodiments, the concentration of the zinc-solubilizing bacteria strain present in the aqueous feedstock or any other input into the bioreactor system does not exceed 100 CFU / ml during the duration. In some embodiments, the zinc-solubilizing bacteria strain is not present in the aqueous feedstock or any other input into the bioreactor system during the duration. In some embodiments, maintaining step (b)(iii) includes maintaining the concentration of the zinc-solubilizing bacteria strain at at least 1 x 10⁻⁶ CFU / ml. 3 CFU / ml. In some embodiments, prior to step (b), the first container further includes an established community of other zinc-solubilizing microorganisms that are not zinc-solubilizing bacterial strains, and step (b)(iii) further includes maintaining the concentration of the other zinc-solubilizing microorganisms in at least the first container at at least 1 x 10⁻⁶ CFU / ml during the duration. 4 The concentration of other zinc-solubilizing microorganisms added to the bioreactor system during the duration is at least 80% of the CFU / ml or the initial concentration of other zinc-solubilizing microorganisms at the start of the duration, wherein the concentration of other zinc-solubilizing microorganisms added to the bioreactor system during the duration is no higher than 1% of the concentration of other zinc-solubilizing microorganisms in the first container. In some embodiments, the concentration of other zinc-solubilizing microorganisms present in the aqueous feedstock or any other input into the bioreactor system is no greater than 10. 4 CFU / ml. In some embodiments, at the start of the duration, the population of other zinc-solubilizing microorganisms in the first container is at least 1 x 10⁻⁶. 3 CFU / ml. In some embodiments, the method further includes, prior to step (a), adding an inoculum of a zinc-solubilizing bacterial strain to the bioreactor system, wherein the inoculum of the zinc-solubilizing bacterial strain produces at least 0.5 x 10⁻⁶ CFU / ml in at least one container. 4 An initial population of zinc-solubilizing bacteria strains at CFU / ml. In some embodiments, the concentration of zinc-solubilizing bacteria strains is less than 1 x 10⁻⁶ CFU / ml before adding the inoculum. 2CFU / ml. In some embodiments, the aqueous feedstock further comprises organic material that can be at least partially digested by microorganisms present in at least one of the containers. In some embodiments, the organic material has been partially digested by microorganisms endogenous to the organic material prior to the transfer in step (b)(i). In some embodiments, the method further comprises digesting the organic material in two or more containers connected in series prior to the transfer in 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 inorganic material. In some embodiments, the inorganic material comprises phosphate rock particles. In some embodiments, the phosphate rock particles have been partially digested by microorganisms present in the aqueous feedstock prior to the transfer in step (b)(i). In some embodiments, the method further comprises partially digesting the phosphate rock particles in two or more containers connected in series prior to the transfer in step (b)(i). In some embodiments, the second microbial aggregate comprises at least 1 x 102 5CFU / ml. In some embodiments, the second microbial aggregate comprises microorganisms derived from manure and / or phosphate rock particles. In some embodiments, the operation in step (b) further includes the production of microbial metabolites that directly or indirectly promote zinc solubilization in the plant growth medium. In some embodiments, the transfer in step (b)(i), the transfer in step (b)(ii), and the collection in step (b)(iv) are performed continuously during the duration. In some embodiments, the transfer in step (b)(i), the transfer in step (b)(ii), and the collection in step (b)(iv) are performed periodically during the duration. In some embodiments, the method further includes adding one or more carbon sources to at least one vessel of the bioreactor system. In some embodiments, the one or more carbon sources are contained in an aqueous feedstock. In some embodiments, the method further includes maintaining the gluconic acid and / or glucose concentration in at least one vessel of the bioreactor system at a concentration of at least 0.2% w / v relative to the volume of the working fluid in at least one vessel. In some embodiments, the method further includes adding one or more nitrogen sources to at least one vessel of the bioreactor system. In some embodiments, the one or more nitrogen sources include ammonium sulfate, ammonium chloride, ammonium nitrate, sodium nitrate, yeast extract, yeast, or any combination thereof. In some embodiments, the bioreactor system includes a clarifier vessel containing clarifier working fluid. In some embodiments, the method further includes separating a supernatant portion of the clarifier working fluid from a flocculent portion within the clarifier vessel. In some embodiments, the separation includes gravity separation. In some embodiments, the method further includes folding the flocculent portion of the clarifier working fluid. In some embodiments, the folding further includes releasing a colony of zinc-solubilizing bacteria strains into the supernatant portion without introducing flocculent solids into the supernatant portion. In some embodiments, the folding is performed using a folding wiper at the bottom portion of the clarifier vessel. In some embodiments, the operation further includes transferring the flocculent portion from the clarifier vessel to a forward vessel in the bioreactor system. In some embodiments, the product effluent includes the supernatant portion of the clarifier working fluid. In some embodiments, the method further includes generating at least 1x10 in the product effluent stream. 4A zinc-solubilizing bacterial strain at CFU / ml. In some embodiments, the bioreactor system includes: a first container containing a volume of a first working fluid; a second container containing a volume of a second working fluid; and a third container containing a volume of a third working fluid. In some embodiments, the first container includes an outlet port fluidly connected to an inlet port of the second container, and the second container includes an outlet port fluidly connected to an inlet port of the third container. In some embodiments, the third container includes an outlet port fluidly connected to a clarifier container. In some embodiments, the method further includes maintaining a constant volume of each of the first, second, and third working fluids during a duration. In some embodiments, step (b) includes operating the bioreactor system in a hydraulically balanced manner. In some embodiments, the transfer in step (b)(i), the transfer in step (b)(ii), and the collection in step (b)(iv) are gravity-driven. In some embodiments, the operation includes maintaining a flow rate resulting in a hydraulic residence time of at least 5 days for the bioreactor system. In some embodiments, the operation includes maintaining a product effluent flow rate of at least 100 gallons / day. In some embodiments, the volume of the 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 includes maintaining at least one of the two or more containers under microaerobic conditions. In some embodiments, the bioreactor system operates continuously for at least 90 days.

[0006] In some implementations, one or more species from one or more of the following genera are the five most abundant species in the second microbial aggregate: *Bacillus* ( Haliscomenobacter ), Lymanella ( Lewinella ), Thermophyton genus ( Caldilinea ), Terremonium ( Terrimonas ) and Acidobacteria ( Acidobacterium In some embodiments, one or more of the following species are the five most abundant species in the said microbial aggregate: *Lymania granatum* (… Lewinella cohaerens ), Phenylacetylcholinesterol ( Thauera phenylacetica ), Herxolium mesenyi Thauera mechernichensis Solitella caninica ( ) Solitalea canadensis ) and Moscow nitrifying bacteria ( Nitrospira moscoviensisIn some embodiments, the second microbial aggregate comprises microorganisms endogenously derived from the organic material. In some embodiments, at least one of the first, second, or third working fluids is circulated within its respective container. In some embodiments, the method further includes maintaining the pH of at least one of the first, second, or third working fluids between 6 and 9 during the duration. In some embodiments, the aqueous feedstock does not contain zinc-solubilizing bacterial strains at concentrations higher than 10 CFU / ml. In some embodiments, zinc-solubilizing bacterial strains are not added to the bioreactor system at concentrations higher than 10 CFU / ml during the duration. In some embodiments, the bioreactor system comprises at least one container arranged in series preceding the first container. In some embodiments, the method further includes generating a population of sporulated bacteria in the product effluent. In some embodiments, the method further includes generating a population of sporulated zinc-solubilizing bacterial strains in the product effluent. In some embodiments, the population of sporulated zinc-solubilizing bacterial strains comprises at least 1 x 102 3 CFU / ml. In some embodiments, the method further includes adding an additional population of zinc-solubilizing bacterial strains to the biostimulant product. In some embodiments, the method further includes preparing at least a portion of an aqueous feedstock by means of the following steps: (c) transferring water, phosphate rock, and optionally products of manure digestion by manure-derived microorganisms to a fourth container containing a volume of a fourth working fluid; (d) transferring a portion of the fourth working fluid to a fifth container containing a fifth working fluid; (e) transferring to the fifth container: (i) a liquid containing (A) a third microbial aggregate containing manure-derived microorganisms and (B) digestion products produced by anaerobic digestion of manure by microorganisms; (ii) manure; and (iii) yeast. In some embodiments, the method further includes transferring a portion of the fifth working fluid to a sixth container containing a sixth working fluid, and a portion of the sixth working fluid to a seventh container containing a seventh working fluid. In some embodiments, the method further includes separating a portion of the seventh working fluid into a feedstock flocculent portion and a feedstock supernatant portion. In some embodiments, the method further includes transferring a portion of the feed flocculent to a fourth container. In some embodiments, the method further includes maintaining the fourth, fifth, sixth, and / or seventh containers under aerobic conditions. In some embodiments, the fourth, fifth, sixth, and / or seventh containers are fluidized bed reactors, wherein the phosphate rock is continuously circulated within the fourth, fifth, sixth, and / or seventh containers. In some embodiments, the total volume of material added to the fourth container within a given time period is equal to the total volume of the fourth working fluid transferred to the fifth container within the same time period.

[0007] In another aspect, this disclosure provides a bioreactor system comprising: (a) an aqueous feed stream in fluid communication with a first container containing a first working fluid of a certain volume, wherein the aqueous feed stream comprises a first microbial aggregate, wherein the first working fluid comprises a population of established zinc-solubilizing bacteria strains and a second microbial aggregate, wherein the concentration of zinc-solubilizing bacteria strains in the first working fluid is at least 100 times the concentration of zinc-solubilizing bacteria strains in the aqueous feed stream and any other input to the bioreactor system; (b) one or more additional containers arranged in series, each of the one or more additional containers comprising a first container and in fluid communication with at least one other container in the series, wherein at least one of the one or more additional containers includes a product effluent port; and (c) a product effluent stream in fluid communication with the product effluent port.

[0008] In some embodiments, at least the first working fluid contains zinc-containing molecules at a concentration of at least 5 mg / L. In some embodiments, the second microbial aggregate comprises a first portion of microorganisms and a second portion of microorganisms, wherein at least the first working fluid contains zinc-containing molecules, the concentration of which inhibits the growth of the first portion of microorganisms relative to zinc-solubilizing bacterial strains. In some embodiments, the first portion of microorganisms comprises non-zinc solubilizers and / or non-zinc-tolerant microorganisms. In some embodiments, the second portion of microorganisms comprises zinc solubilizers and / or zinc-tolerant microorganisms. In some embodiments, the zinc-containing molecules are zinc oxide, zinc sulfide, zinc carbonate, zinc phosphate, zinc chloride, or zinc sulfate. In some embodiments, the zinc-solubilizing bacterial strains belong to the genus Bacillus. In some embodiments, the zinc-solubilizing bacterial strains belong to the genus Bacillus (Sargassum). Bacillus safensis ) or Bacillus megaterium ( Bacillus megaterium In some embodiments, the zinc-solubilizing bacterial strain is one of the following strains: (a) a *Bacillus saffron* strain having one or more of the following: (i) a 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 1; (ii) a 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 4. gyrB Gene sequence; and (iii) at least 95% identical to SEQ ID NO: 7. rpoB (a) Gene sequence; (b) Bacillus megaterium strains having one or more of the following: (i) a 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 2; (ii) a 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 5. gyrB Gene sequence; and (iii) at least 95% identical to SEQ ID NO: 8. rpoBGene sequence; or (b) a Bacillus megaterium strain having one or more of the following: (i) a 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO:3; (ii) a 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO:6. gyrB Gene sequence; and (iii) at least 95% identical to SEQ ID NO: 9. rpoB Gene sequence. In some embodiments, the zinc-solubilizing bacteria strain is *Bacillus saffron* strain deposited with ATCC accession number PTA-127681, *Bacillus megaterium* strain deposited with ATCC accession number PTA-127683, or *Bacillus megaterium* strain deposited with ATCC accession number PTA-127682. In some embodiments, the bioreactor system is a continuous flow bioreactor system, and the aqueous feed stream is continuous. In some embodiments, the volume of each of the working fluids is constant. In some embodiments, each of the first container and one or more additional containers contains at least 1 x 10⁻⁶ cells / mL during operation of the bioreactor system. 4 The zinc-solubilizing bacterial strains are present at a concentration of CFU / ml. In some embodiments, the aqueous feedstock and any other inputs into the bioreactor system do not contain zinc-solubilizing bacterial strains, or do not contain zinc-solubilizing bacterial strains at concentrations higher than 100 CFU / ml. In some embodiments, the first microbial aggregate contains at least 1 x 10⁻⁶ CFU / ml. 4 Microorganisms at CFU / ml. In some embodiments, the aqueous feedstock also comprises organic material that can be digested by microorganisms present in the container. In some embodiments, the organic material includes manure or manure-derived material. In some embodiments, the aqueous feedstock also comprises phosphate rock particles. In some embodiments, the first microbial aggregate comprises microorganisms derived from manure and / or phosphate rock particles. In some embodiments, the container including the product effluent port is a clarifier container configured to separate a portion of the working fluid in the clarifier container into a supernatant portion and a flocculent portion. In some embodiments, the clarifier container includes one or more flocculent baffles configured to agitate the settled flocculent in the clarifier container without resuspending solids in the flocculent portion in the supernatant portion. In some embodiments, the bioreactor system also includes a flocculent return stream flowing from the clarifier to a forward container in a series. In some embodiments, the product effluent stream comprises a supernatant portion. In some embodiments, the product effluent stream comprises at least 1 x 10⁻⁶ CFU / ml of product effluent. 4 CFU / ml of zinc-solubilizing bacterial strains. In some embodiments, the product effluent contains at least 1 x 10⁻⁶ CFU / ml. 2The product effluent contains CFU / ml of zinc-solubilizing bacteria in sporulated form. In some embodiments, the product effluent contains 0.2 to 2.5 mg / ml of total dry weight. In some embodiments, the chemical oxygen demand (COD) of the product effluent is 80 to 500 mg / L. In some embodiments, the conductivity of the product effluent is 0.1 to 1.5 mS / cm. In some embodiments, the first working fluid contains 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 includes microaerobic conditions. In some embodiments, the biostimulant composition can be prepared by the methods or systems described herein. In some embodiments, methods for promoting plant growth may include contacting plants, seeds, or plant growth media with the biostimulant composition. In some embodiments, methods for increasing the amount of solubilized zinc for plant use may include contacting plants, seeds, or plant growth media with the biostimulant composition.

[0009] In another aspect, this disclosure provides a composition comprising: (a) a *Bacillus saffron* strain having one or more of the following: (i) a 16S rRNA gene sequence that is at least 95% identical to that of SEQ ID NO: 1; (ii) a 16S rRNA gene sequence that is at least 95% identical to that of SEQ ID NO: 4. gyrB Gene sequence; and (iii) at least 95% identical to SEQ ID NO: 7. rpoB (a) Gene sequence; and (b) Loading agent. In some embodiments, the *Bacillus salsafoetida* strain is a strain deposited with ATCC accession number PTA-127681 or a clone isolated therefrom. In some embodiments, the composition further comprises the product of *Bacillus salsafoetida* strain digesting an organic substrate. In some embodiments, the loading agent comprises a fertilizer. In some embodiments, the loading agent is a solid coated with *Bacillus salsafoetida* strain. In some embodiments, the loading agent is also coated with a micronutrient. In some embodiments, the micronutrient is zinc oxide, zinc sulfide, zinc carbonate, or zinc phosphate. In some embodiments, the loading agent is a liquid. In some embodiments, the loading agent further comprises an adjuvant selected from wetting agents, spreading agents, dispersants, adhesives, dust control agents, or binders. In some embodiments, the concentration of *Bacillus salsafoetida* strain in the composition ranges from 1 x 10⁻⁶. 3 Up to 1x10 11 CFU / ml. In some embodiments, the concentration of Bacillus saefoetida strain in the composition ranges from 1 x 10⁻⁶. 4 Up to 1x10 6 cfu / ml.

[0010] On the other hand, this disclosure provides an isolated strain of Bacillus safranin, said isolated strain having one or more of the following: (a) a 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 1; (b) a 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 4. gyrB Gene sequence; and (c) at least 95% identical to SEQ ID NO: 7. rpoB Gene sequence. In some embodiments, the *Bacillus saffron* strain is a strain deposited with ATCC accession number PTA-127681 or a clone thereof.

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

[0012] In some embodiments, the *Bacillus megaterium* strain is a strain deposited with ATCC accession number PTA-127683 or an isolated clone thereof. In some embodiments, the isolated strain also comprises the product of *Bacillus megaterium* strain digesting an organic substrate. In some embodiments, the carrier includes a fertilizer. In some embodiments, the carrier is a solid coated with the *Bacillus megaterium* strain. In some embodiments, the carrier is also coated with 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 includes an adjuvant selected from wetting agents, spreading agents, dispersants, adhesives, dust control agents, or binders. In some embodiments, the concentration of the *Bacillus megaterium* strain in the composition ranges from 1 x 10⁻⁶. 3 Up to 1x10 11 CFU / ml. In some embodiments, the concentration of Bacillus megaterium strain in the composition ranges from 1 x 10⁻⁶. 4 Up to 1x10 6 .

[0013] On the other hand, this disclosure provides an isolated strain of Bacillus megaterium, said isolated strain having one or more of the following: (a) a 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 2; (b) a 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 5. gyrB Gene sequence; and (c) at least 95% identical to SEQ ID NO: 8. rpoB Gene sequence.

[0014] In some implementations, the Bacillus megaterium strain is a strain deposited with ATCC accession number PTA-127683 or an isolated clone thereof.

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

[0016] In some embodiments, the *Bacillus megaterium* strain is a strain deposited with ATCC accession number PTA-127682 or an isolated clone thereof. In some embodiments, the composition further comprises the product of *Bacillus megaterium* strain digesting an organic substrate. In some embodiments, the carrier comprises a fertilizer. In some embodiments, the carrier is a solid coated with *Bacillus megaterium* strain. In some embodiments, the carrier is also coated with 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 wetting agents, spreading agents, dispersants, adhesives, dust control agents, or binders. In some embodiments, the concentration of *Bacillus megaterium* strain in the composition ranges from 1 x 10⁻⁶. 3 Up to 1x10 11 CFU / ml. In some embodiments, the concentration of Bacillus megaterium strain in the composition ranges from 1 x 10⁻⁶. 4 Up to 1x10 6 .

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

[0018] In some embodiments, the *Bacillus megaterium* strain is a strain deposited with ATCC accession number PTA-127682 or an isolated clone thereof. In some embodiments, a method for promoting the growth of plants grown in a culture medium may include contacting the plant or the culture medium with a biostimulant, composition, or composition comprising an isolated strain as disclosed herein. In some embodiments, the contact results in an increase of at least 5% in the amount of solubilized zinc available to the plant. In some embodiments, the composition increases plant growth by at least 5% compared to a control. In some embodiments, the composition increases nutrient uptake by the plant by at least 5% compared to a control. In some embodiments, the nutrient is zinc, and wherein the plant's zinc uptake increases by at least 5% 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 culture medium is soil or a hydroponic medium.

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

[0020] This article provides a method for promoting plant growth, the method comprising: (a) contacting a plant and / or a culture medium in which the plant is grown with a composition comprising one or more compounds, said compounds comprising: 1-hexadecyl-2,3-di-o-acetylglycerol, diisodecyl phthalate, allothreonine, (S)-(-)-α-(1-naphthyl)ethylamine, indoleacetic acid (indole-3-acetic acid), salicylaniline, 25-hydroxycholesterol, 4-(2-aminophenyl)-2,4-dioxobutyric acid, 13,14-dihydro-15-one PGD2, 4-ethyloctanoic acid, phthalic acid, α-ketoisovalerate, acorn, Val-Ala N-Methylundec-10-enamide, His-Ile-Lys-Arg, Methylcarbamoyl PAF, Alkylcholine, Ala-Gly-Leu-Val-Ser, Arg-Val-Met, chapters, 2-Phenylethanol, Prostaglandin D3, Ritalin, 5-Hydroxyindoleacetaldehyde, Azithromycin dihydrate, Pro-Ala-Phe, (4R,7S,7aR,12bS)-3-methyl-2,4,4a,7,7a,13-hexahydro-1H-4,12-bridged methylenebenzofurano[3,2-e]isoquinoline-7,9-diol, Glu-Pro-Thr, 2,6- Naphthyldiol, 3-aminoquinoline, acylated phloroglucinol, L-pyrrolidone, tanikolide, Val-Arg-Glu, 3-methyl adipic acid, 10-propoxydecanoic acid, 7-hydroxy-3-(2-methoxyphenyl)-4H-chromene-4-one, N-methylundec-10-enamide, (5Z,9α,11α,13E,15S)-9,11-epidio-15-hydroperoxy-prostaglandin-5,13-diene-1-acid, Ile-Phe-Val-Lys, γ-glutamyl-Se-methylselenocysteine; 5-L-glutamyl-Se-methylselenocysteine, Trp-Ala-L ys, pantothenic acid glucoside, dapoxetine ketone, LPC (O-16:0 / 2:0), sphingosine 1-phosphate, coenzyme Q6, dihydrocortisol, dibutyl ketone hydrochloride, LPE (18:0 / 0:0), nialamidine, androstenedione, 12-(2,3-dihydroxycyclopentyl)-2-dodecanoate, androstenedione-5-en-3,17-dione, hydroxytyrosol, 4-hydroxycinnamic acid, 2,4,5-trihydroxytoluene, isopropylamide, (1R,2R,5R,8R,9S,10R,12S)-12-hydroxy-11-methyl-6-methylene-16-oxo-15-oxapentane [9.3.2.15,8.01,10].[02,8] Heptadecano-9-carboxylic acid, allopurinol nucleoside, 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycerol-3-phosphate ethanolamine, triacetyluridine, JWH 018 7-hydroxyindole metabolite-d9, 1,6-dimethylphenazine, 2-(p-bromophenyl)-8-methyl-8H-thieno(2,3-b)indole, 3,4-dihydroxyphenyl glycol, dodecyl cis-9,10-epoxyoctadecanoate, heptadecanoyl alcohol, lauric acid, lignoceric acid, tetradecane, chromol, one or more derivatives thereof, or combinations thereof. In some embodiments, contact includes contacting a plant with the composition. In some embodiments, contact includes contacting plant seeds with the composition. In some embodiments, contact includes contacting plant leaves with the composition. In some embodiments, the culture medium includes soil, hydroponic medium, turface, or isolite. In some embodiments, contact results in at least a 10% increase in plant growth compared to when the plant or culture medium is not exposed to the composition containing one or more compounds. In some embodiments, the composition further comprises an adjuvant selected from wetting agents, spreading agents, dispersants, adhesives, dust control agents, and binders. In some embodiments, the composition is configured to enhance the availability of zinc in the soil. In some embodiments, the composition is configured to enhance zinc uptake by plant tissues. In some embodiments, the composition is configured to promote efficient zinc utilization.

[0021] This document provides a composition for promoting plant growth, the composition comprising: (a) at least one microbial strain including *Bacillus saffron* or *Bacillus megaterium*; and (b) one or more compounds including 1-hexadecyl-2,3-di-o-acetylglycerol, diisodecyl phthalate, allethreonine, (S)-(-)-α-(1-naphthyl)ethylamine, indoleacetic acid (indole-3-acetic acid), salicylaniline, 25-hydroxycholesterol, 4-(2-aminophenyl)-2,4-dioxobutyric acid, 13,14-dihydro-15-one PGD2, 4-ethyloctanoic acid, phthalic acid, α-ketoisovalerate, acorn, Val -Ala, N-methylundec-10-enamide, His-Ile-Lys-Arg, methylcarbamoyl PAF, homogentisic acid, Ala-Gly-Leu-Val-Ser, Arg-Val-Met, chaps, 2-phenylethanol, prostaglandin D3, methylphenidate, 5-hydroxyindoleacetaldehyde, azithromycin dihydrate, Pro-Ala-Phe, (4R,7S,7aR,12bS)-3-methyl-2,4,4a,7,7a,13-hexahydro-1H-4,12-bridged methylenebenzofurano[3,2-e]isoquinoline-7,9-diol, Glu-Pro-Thr, 2 6-Naphthyldiol, 3-aminoquinoline, acylated phloroglucinol, L-pyrrolidone, tanikolide, Val-Arg-Glu, 3-methyl adipic acid, 10-propoxydecanoic acid, 7-hydroxy-3-(2-methoxyphenyl)-4H-chromene-4-one, N-methylundecyl-10-enamide, (5Z,9α,11α,13E,15S)-9,11-epidio-15-hydroperoxy-prostaglandin-5,13-diene-1-acid, Ile-Phe-Val-Lys, γ-glutamyl-Se-methylselenocysteine; 5-L-glutamyl-Se-methylselenocysteine, Trp-Ala- Lys, pantothenic acid glucoside, dapoxetine ketone, LPC (O-16:0 / 2:0), sphingosine 1-phosphate, coenzyme Q6, dihydrocortisol, dibutyl ketone hydrochloride, LPE (18:0 / 0:0), nialamidine, androstenedione, 12-(2,3-dihydroxycyclopentyl)-2-dodecanoate, androstenedione-5-en-3,17-dione, hydroxytyrosol, 4-hydroxycinnamic acid, 2,4,5-trihydroxytoluene, isopropylamide, (1R,2R,5R,8R,9S,10R,12S)-12-hydroxy-11-methyl-6-methylene-16-oxo-15-oxapentane [9.3.2.15,8.01,10].[02,8] Heptadecano-9-carboxylic acid, allopurinol nucleoside, 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycerol-3-phosphate ethanolamine, triacetyluridine, JWH018 7-hydroxyindole metabolite-d9, 1,6-dimethylphenazine, 2-(p-bromophenyl)-8-methyl-8H-thieno(2,3-b)indole, 3,4-dihydroxyphenyl glycol, dodecyl cis-9,10-epoxyoctadecanoate, heptadecano, lauric acid, lignoceric acid, tetradecane, croterol, one or more of their derivatives or combinations thereof. In some embodiments, at least one microbial strain comprises a *Bacillus safranin* strain, comprising one or more of the following: (i) a 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 1; (ii) a gyrB gene sequence that is at least 95% identical to SEQ ID NO: 4; or (iii) an rpoB gene sequence that is at least 95% identical to SEQ ID NO: 7. In some embodiments, at least one microbial strain comprises a *Bacillus megaterium* strain, comprising one or more of the following: (i) a 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 2; (ii) a gyrB gene sequence that is at least 95% identical to SEQ ID NO: 5; or (iii) an rpoB gene sequence that is at least 95% identical to SEQ ID NO: 8. In some embodiments, at least one microbial strain comprises a *Bacillus megaterium* strain containing one or more of the following: (i) a 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 3; (ii) a gyrB gene sequence that is at least 95% identical to SEQ ID NO: 6; or (iii) an rpoB gene sequence that is 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 plants and / or culture media in which plants grow. In some embodiments, the composition further comprises an adjuvant selected from wetting agents, spreading agents, dispersants, adhesives, dust control agents, and binders. In some embodiments, the composition is configured to enhance the availability of zinc in the soil. In some embodiments, the composition is configured to enhance zinc uptake by plant tissues. In some embodiments, the composition is configured to promote efficient zinc utilization.

[0022] This article provides a composition for promoting plant growth, the composition comprising: (a) two or more compounds including 1-hexadecyl-2,3-di-o-acetylglycerol, diisodecyl phthalate, allothreonine, (S)-(-)-α-(1-naphthyl)ethylamine, indoleacetic acid (indole-3-acetic acid), salicylaniline, 25-hydroxycholesterol, 4-(2-aminophenyl)-2,4-dioxobutyric acid, 13,14-dihydro-15-one PGD2, 4-ethyloctanoic acid, phthalic acid, α-ketoisovaleric acid, acorn, Val-Ala, N-methylundec-10-enamide, His- Ile-Lys-Arg, Methylcarbamoyl PAF, Alkylcholine, Ala-Gly-Leu-Val-Ser, Arg-Val-Met, Chaps, 2-Phenylenic Acid, Prostaglandin D3, Ritalin, 5-Hydroxyindoleacetaldehyde, Azithromycin Dihydrate, Pro-Ala-Phe, (4R,7S,7aR,12bS)-3-Methyl-2,4,4a,7,7a,13-Hexahydro-1H-4,12-Bridged Methylenebenzofurano[3,2-e]isoquinoline-7,9-diol, Glu-Pro-Thr, 2,6-Naphthyldiol, 3-Aminoquinoline, Acylation Phloroglucinol, L-pyrrolidone, tanikolide, Val-Arg-Glu, 3-methyl adipic acid, 10-propoxydecanoic acid, 7-hydroxy-3-(2-methoxyphenyl)-4H-chromene-4-one, N-methylundec-10-enamide, (5Z,9α,11α,13E,15S)-9,11-epidio-15-hydroperoxy-prostaglandin-5,13-diene-1-acid, Ile-Phe-Val-Lys, γ-glutamyl-Se-methylselenocysteine; 5-L-glutamyl-Se-methylselenocysteine, Trp-Ala-Lys, panthenolactone Glucoside, sphingolipidone, LPC (O-16:0 / 2:0), sphingosine 1-phosphate, coenzyme Q6, dihydrocortisol, dibutyl ketone hydrochloride, LPE (18:0 / 0:0), nialamidine, androstenedione, 12-(2,3-dihydroxycyclopentyl)-2-dodecanoate, androstenedione-5-en-3,17-dione, hydroxytyrosol, 4-hydroxycinnamic acid, 2,4,5-trihydroxytoluene, isopropylamide, (1R,2R,5R,8R,9S,10R,12S)-12-hydroxy-11-methyl-6-methylene-16-oxo-15-oxapentane [9.3.2.15,8.01,10].[02,8] Heptadecano-9-carboxylic acid, allopurinol nucleoside, 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycerol-3-phosphate ethanolamine, triacetyluridine, JWH 018 7-hydroxyindole metabolite-d9, 1,6-dimethylphenazine, 2-(p-bromophenyl)-8-methyl-8H-thieno(2,3-b)indole, 3,4-dihydroxyphenyl glycol, dodecyl cis-9,10-epoxyoctadecanoate, heptadecanoylene, lauric acid, lignoceric acid, tetradecane, chromol, one or more derivatives thereof, or combinations thereof; and (ii) a carrier. In some embodiments, the carrier is formulated for application to a plant or a culture medium in which the plant grows. In some embodiments, the carrier includes a fertilizer. In some embodiments, the fertilizer is solid. In some embodiments, the carrier is liquid. In some embodiments, the composition further comprises an adjuvant selected from wetting agents, spreading agents, dispersants, adhesives, dust control agents, and binders. In some embodiments, the composition is configured to enhance the 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 efficient zinc utilization.

[0023] Further aspects and advantages of this disclosure will become apparent to those skilled in the art from the following detailed description (in which only illustrative embodiments of this disclosure are shown and described). As will be appreciated, this disclosure is capable of other and different embodiments, and certain details thereof can be modified in various obvious respects, all without departing from this disclosure. Therefore, the drawings and descriptions should be considered illustrative in nature and not restrictive.

[0024] Incorporation All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent, or patent application is expressly and individually indicated to be incorporated by reference. In the event of any publication, patent, or patent application incorporated by reference that contradicts the disclosure contained in this specification, this specification is intended to supersede and / or give precedence to any such contradictory material. Attached Figure Description

[0025] The novel features of the invention are specifically set forth in the appended claims. A better understanding of the features and advantages of the invention will be obtained by referring to the following detailed description and accompanying drawings (also referred to herein as “drawings” and “figures”) illustrating embodiments utilizing the principles of the invention, wherein: Figure 1 A schematic diagram of the components of a ZST system with a single reactor is shown.

[0026] Figure 2The diagram shows a series of ZST systems characterized by markings on a series of reactors and other components.

[0027] Figure 3 Another series ZST system, characterized by a fully mixed reactor and a series of fluidized bed reactors, is shown.

[0028] Figure 4 Another series of ZST systems, characterized by a fully mixed reactor and a series of packed bed reactors with supports, is shown.

[0029] Figure 5 The graph shows pH, glucose consumption (g / L), and zinc dissolution (mg / L) over time, with glucose added to ZST batch system A.

[0030] Figure 6 The graph shows pH, glucose consumption (g / L), and zinc dissolution (mg / L) over time, with glucose added to the ZST batch system B.

[0031] Figure 7 The graph shows pH, glucose consumption (g / L), and zinc dissolution (mg / L) over time, with glucose added to the ZST batch system C.

[0032] Figure 8 The graph shows pH, glucose consumption (g / L), and zinc dissolution (mg / L) over time, with glucose added to the ZST batch system D.

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

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

[0035] Figure 11 It lists sixteen bacterial isolates (including MS4666) in zinc fertilizer (Circa) TM A table of survival capabilities in Zn.

[0036] Figure 12 The graph shows the Zn solubilizing capacity (mg / L) of target isolates MS4666, MS4687, and MS4689 at different inoculum concentrations when grown using different carbon sources.

[0037] Figures 13A-13B This is a series of graphs showing measurements of Zn uptake and plant growth in common beans treated with MAP fertilizer coated with one of the following four different treatments: water (UTC), Circadian rhythm, and circadian rhythm. TM Zn, MS4666 and MS4666 plus Circa TM Zn. Figure 13A The data shows the zinc intake (µg / plant) of navy beans. Figure 13B The data shows the total biomass, aboveground dry weight, and root dry weight under various conditions.

[0038] Figures 14A-14B This is a series of graphs showing measurements of plant growth in maize treated with MAP fertilizer coated with one of the following four different treatments: water (UTC), Circadian rhythm, and circadian rhythm. TM Zn, MS4666 and MS4666 plus Circa TM Zn. Figure 14A The results show the zinc uptake measurements for each plant under each condition. Figure 14B The data shows the total biomass, aboveground dry weight, and root dry weight under various conditions.

[0039] Figures 15A-15B This is a series of figures showing the evaluation of Zn uptake and plant growth promotion in maize treated with MAP fertilizer coated with one of the following four different treatments: water (UTC), Circa2, and Circa2. TM Zn, MS4666 and MS4666 plus Circa TM Zn. Figure 15A The results show the zinc uptake measurements for each plant under each condition. Figure 15B The data shows the total biomass, aboveground dry weight, and root dry weight under various conditions.

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

[0041] Figure 17 This is a graph showing the promotion of plant growth (measured in dry weight (g)) in maize fertilized with one of the following four different treatments: water (UTC), Circa TM Zn, MS4666 and MS4666 plus Circa TMZn. Dry weight was measured for total biomass, aboveground parts, and roots. For each treatment condition, the columns from left to right are: roots, stems, and total biomass.

[0042] Figures 18A-18D Measurements of maize growth treated with various microbial isolates applied via furrow at planting time are shown. Measurements were performed across the control, MS4666, and other isolates (isolated concentration 1E x 10⁻⁶). 4 Corn height (cfu / ml) Figure 18A Fresh weight () Figure 18B ) and dry weight ( Figure 18C ). Figure 18D It shows Figures 18A-18C The table shows the results of the dry weight analysis. For each isolate condition, the bars from left to right are: root, stem, and total.

[0043] Figure 19 The in vitro zinc solubilization of solutions from three ZST systems was demonstrated at different zinc oxide (ZnO) concentrations.

[0044] Figure 20 The zinc solubilization capacity of the complete solution from the ZST system and its filtered and sterilized (metabolite-only) counterparts is shown at a fixed ZnO concentration of 0.1%.

[0045] Figure 21 The available zinc (mg / L) from MESZ fertilizer granules coated with ZST 2.0 FBR is shown at different application ratios.

[0046] Figure 22 The percentage of zinc that became soluble in ZnO-fertilized soils after treatment with different concentrations of ZST 1.5 is shown.

[0047] Figure 23 The growth of Arabidopsis thaliana (mean leaf area (cm²)) in both intact and filtered sterilized (F / S) solutions was shown for all ZST systems. 2 The differences between UTC and Accomplish LM are shown. UTC and Accomplish LM are used as controls.

[0048] Figures 24A-24C The effects of different treatments on maize dry biomass are shown. The percentages above each bar represent the percentage change relative to UTC. An asterisk indicates a significant difference relative to UTC. Figure 24A Shows the percentage change in total dry biomass relative to UTC across multiple treatments. Figure 24B The average aboveground dry biomass is shown. Figure 24C The average root trunk biomass is shown.

[0049] Figures 25A-25C This is a series of figures showing the effects of loading input components and supernatant (SPN) on maize plant growth and yield in the ZST system. Figure 25A The total biomass is shown using the loaded input and ZST 1.5 SPN treatment. Figure 25B The display shows the input of fertilizer and loading Circa. TM Zn and ZST 1.5 SPN plus Circa TM Total biomass under Zn. Figure 25C Shown in a separate Circa TM Zn, ZST 1.5 SPN alone and ZST 1.5 SPN with Circa TM Total biomass under Zn.

[0050] Figures 26A-26B It is a series of figures showing the relationship with UTC and Circa TM Zinc uptake (CZN) in maize compared to zinc uptake (µg / plant) after treatment with ZST SPN or isolate input (MS4666). Figure 26A The zinc uptake of inputs into the ZST system (MS4666) and ZST SPN is shown compared to UTC and CZN alone. Figure 26B The zinc uptake of isolates entering the ZST system with the combination of isolate input and CZN (MS4666 CZN) and ZST 1.5 SPN plus CZN is shown.

[0051] Figure 27 A series of figures are shown, illustrating different ZST systems, untreated control (UTC), and individual Circa. TM Total corn kernel yield (g) in Zn (CZN) fertilizer.

[0052] Figure 28 The figures show different ZST systems, untreated control (UTC), and individual Circa. TM Total zinc (ug) measured per ear of maize using Zn (CZN) fertilizer and in combination with the ZST system.

[0053] Figures 29A-29B It is a series of figures showing different ZST systems, untreated control (UTC), and individual Circa TM Total macronutrients (nitrogen, phosphorus, and potassium) (mg) measured per ear of maize under Zn (CZN) fertilizer. Figure 29A The results for the ZST system without CZn are shown. Figure 29BThe results are shown for CZN and ZST systems with CZn alone. For each condition, the bars from left to right are: UTC, ZST 1.5, ZST 2.0 FBR, and ZST 2.0 PBR.

[0054] Figures 30A-30B It shows the control (UTC) being used (unprocessed). Figure 30A ) and the use of CZN fertilizer ( Figure 30B The table shows the macronutrient and micronutrient content of corn kernels in the ZST processing system.

[0055] Figures 31A-31C These are a series of graphs showing the harvest yield of three vegetables, measured in boxes per acre: broccoli ( Figure 31A ), cabbage ( Figure 31B ) and lettuce ( Figure 31C ).

[0056] Figures 32A-32C A series of graphs show the plant growth-promoting effects of MS4666. Treatment with MS4666 showed superior maize yield compared to the untreated control. Figures 32A-32B Treatment with MS4666 at a rate of 2.0 quarts / acre showed superior soybean yield compared to the untreated control. Figure 32C ).

[0057] Figure 33 The count of isolate MS4666 over time is shown after inoculation at a high concentration and flocculant recycling.

[0058] Figure 34 A distance-based neighbor-joining phylogenetic tree based on the 16S rRNA gene is shown.

[0059] Figure 35 Results of the in vitro determination of zinc solubilization capacity between target isolate MS4666, PST flocculant and ZST-1.0 batch solution using PST flocculant (batch-A) or PST flocculant plus MS4666 (batch-B) are shown.

[0060] Figure 36 Soil analysis results show the bioavailability of Zn Mehlich-3 extractable Zn (Zn-bioavailability) between the target isolates (at 1.00E+4 CFU / mL and 1.00E+6 CFU / mL) and the control treatment.

[0061] Figure 37 The graph shows the concentrations of total bacteria and Zn-solvent in the ZST-1.5 method and base product.

[0062] Figure 38The measurements of soluble Zn2+ from ZST-1.5 reactor 1 (R1) and the ZST-1.5 base product are shown. This means p < 0.0001.

[0063] Figure 39 The concentrations of Zn2+ present in the ZST-2.0 FBR and ZST-2.5 reactors and the base product are shown.

[0064] Figure 40A The results show the measurements of Zn2+ that became soluble in ZET experiments under each treatment condition (ZST-2.5 BP, ZST-2.5 BP+MS4666 and ZST-2.0 FBR BP). Figure 40B The results show the measurements of soluble Zn2+ in ZST experiments (UTC, ZST-2.5 BP, ZST-2.5 BP+MS4666 and ZST-2.0 FBR BP) under each treatment condition.

[0065] Figure 41A The average leaf area (cm²) of Arabidopsis thaliana used for plant growth promotion is shown. 2 ). Figure 41B The average leaf area (cm²) of Arabidopsis thaliana used for abiotic (salt) stress relief is shown. 2 ). Figure 41C The average leaf area (cm²) of Arabidopsis thaliana used for abiotic (cold) stress relief is shown. 2 ).

[0066] Figure 42A and Figure 42B The complete sample was displayed. Figure 42A ) and filtered sterilized samples ( Figure 42B Principal component analysis (PCA) of ZST-sIP and ZST-non-sIP base products based on gas chromatography-mass spectrometry (GC-MS). Figure 42C The diagram shows the number of upregulated compounds in whole and filtered sterilized samples of ZST-sIP relative to ZST-non-sIP, based on GC-MS analysis. Figure 42D and Figure 42E The complete sample was displayed. Figure 42D ) and filtered sterilized samples ( Figure 42E PCA based on LC-MS analysis of ZST-sIP and ZST-non-sIP base products. Figure 42F The diagram shows the number of upregulated compounds in whole and filtered sterilized samples of ZST-sIP relative to ZST-non-sIP, based on LC-MS analysis. Detailed Implementation

[0067] Although various embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many changes, modifications, and substitutions can be made by 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] The systems and methods described herein employ microbial digestion of a variety of feedstocks. The systems disclosed herein may comprise continuous systems (e.g., sIP systems) capable of producing isolates in a tandem series. Isolate production in the sIP system can be carried out in a mixed aggregate of microorganisms. Target isolates from the sIP digestion system can be enriched in a microbial environment and can exhibit improved efficacy and function. Primary targeted functionalities may include zinc solubilization of bound zinc from soil or fertilizer and improved nutrient uptake in plants. Target isolates may possess commercially valuable properties and can be incorporated into continuous (e.g., tandem) reactor systems comprising complex microbial aggregates functionally modified (e.g., modified for zinc solubilization). Without wishing to be bound by theory, target isolates may contribute performance gains to the microbial community of the digestion system described herein, thereby providing chemical and / or functional synergies as they grow in the system. The digestion systems described herein may include zinc solubilization technology (ZST) systems with targeted functions to enrich one or more populations, metabolites, or any combination thereof of zinc-soothing microorganisms.

[0069] The products of the digestion methods and systems described herein may include microorganisms and metabolites produced by the microorganisms during digestion of a substrate. In some embodiments, the products of the digestion methods and systems described herein may include a biostimulant composition that, when applied to a plant or a culture medium in which the plant grows (e.g., soil), has plant-growth-promoting properties. In some embodiments, the methods and systems described herein are arranged to selectively promote the growth of microorganisms that themselves possess the desired plant-growth-promoting properties or produce metabolites with the desired plant-growth-promoting properties, such that the biostimulant product possesses the desired plant-growth-promoting properties. The application of the products of the digestion systems described herein may be applied to a dry fertilizer, in conjunction with fertilizer application, in a formulation having additional components (including liquid fertilizer) or a micronutrient coating formulation, or on a foliar surface. The application of the products of the digestion systems described herein may be applied to a portion of a plant, such as aboveground parts, stems, leaves, lateral buds, terminal buds, flowers, leaf axils, roots (e.g., taproot, lateral roots, root hairs, root cap), or any combination thereof. These and other features of the embodiments disclosed herein are described in more detail below.

[0070] A. Definition In the following description, certain specific details are set forth for a comprehensive understanding of the various embodiments. However, those skilled in the art will understand that the provided embodiments can be practiced without these details. Unless the context otherwise requires, throughout this specification and the appended claims, the word “comprise” and its variations, such as “comprises” and “comprising”, shall be interpreted in an open-ended, inclusive sense, meaning “including but not limited to”. Unless otherwise expressly stated, the singular forms “a / an,” “a / an,” and “the” as used in this specification and the appended claims include plural indicators. It should also be noted that unless otherwise expressly stated, the term “or” is generally used in its meaning, including “and / or”. Furthermore, the headings provided herein are for convenience only and do not explain the scope or meaning of the claimed embodiments.

[0071] The terms "about" or "approximately" refer to a specific value within an acceptable range of error as determined by a person skilled in the art, the acceptable range of error being partly dependent on how the value is measured or determined, such as limitations of the measurement system. For example, "about" may conventionally refer to a value within one standard deviation or greater than one standard deviation. When describing a specific value in this application and claims, unless otherwise stated, the term "about" should be considered to refer to an acceptable range of error for that specific value.

[0072] As used herein, the term "culture" can refer to the propagation of an organism on or in a variety of culture media. Non-limiting examples of suitable culture media include trypsin-soy agar (TSA), zinc agar, nutrient medium, lysozyme broth (LB medium), and / or plate counting agar.

[0073] As used herein, the term "enriched culture" of isolated microbial strains can refer to a microbial culture in which the total microbial population of the culture contains a certain percentage of the target isolated strain. An enriched culture may contain a certain percentage of the target isolated strain and a microbial population enriched for a specific function (e.g., zinc solubilization). In some embodiments, an enriched culture may contain a certain percentage of the target isolated strain, a microbial population enriched for a specific function, and metabolites enriched for a specific function (e.g., zinc solubilization). An enriched culture may contain a certain percentage of the total bacterial population in the container of the digestive system described herein. An enriched culture may contain a certain percentage of the total bacterial population in the output products described herein (e.g., biostimulants). An enriched culture may contain an increased amount of the target isolated strain and / or the target population of microorganisms compared to the total microbial population of the culture. An enriched culture may contain a growing population of the target isolated strain and a microbial population enriched for a specific function (e.g., zinc solubilization) over a time period. In some embodiments, an enrichment culture may refer to a microbial culture in which the total microbial population 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 the target isolate, the microbial population enriched for a specific function, the metabolite enriched for a specific function, or any combination thereof. In some embodiments, the enrichment culture may refer to a microbial culture in which the total microbial population contains up to about 75%, up to about 50%, up to about 25%, up to about 20%, up to about 15%, up to about 10%, up to about 5%, up to about 2.5%, up to about 2%, up to about 1.5%, up to about 1%, or up to about 0.5% of the target isolate, a microbial population enriched for a specific function, a metabolite enriched for a specific function, or any combination thereof. In some embodiments, the enrichment culture may refer to a microbial culture in which the total microbial population contains about 0.5% to about 75% of the target isolate, a microbial population enriched for a specific function, a metabolite enriched for a specific function, or any combination thereof.In some embodiments, the enrichment culture may refer to a microbial culture, wherein the total microbial population of the culture contains approximately 0.5% to approximately 1%, approximately 0.5% to approximately 2%, approximately 0.5% to approximately 3%, approximately 0.5% to approximately 5%, approximately 0.5% to approximately 10%, approximately 0.5% to approximately 15%, approximately 0.5% to approximately 20%, approximately 0.5% to approximately 25%, approximately 0.5% to approximately 50%, approximately 0.5% to approximately 60%, approximately 0.5% to approximately 75%, approximately 1% to approximately 2%, approximately 1% to approximately 3%, approximately 1% to approximately 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 2 0%, 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 The target isolates, microbial populations enriched for a specific function, metabolites enriched for a specific function, or any combination thereof, are approximately 25%, approximately 15% to approximately 50%, approximately 15% to approximately 60%, approximately 15% to approximately 75%, approximately 20% to approximately 25%, approximately 20% to approximately 50%, approximately 20% to approximately 60%, approximately 20% to approximately 75%, approximately 25% to approximately 50%, approximately 25% to approximately 60%, approximately 25% to approximately 75%, approximately 50% to approximately 60%, approximately 50% to approximately 75%, or approximately 60% to approximately 75%.

[0074] As used herein, the term "composition" can refer to a combination of an active agent (e.g., the microbial strain described herein) with at least one other compound, carrier, or composition, which may be inert (e.g., a detectable agent or liquid carrier) or active, such as, but not limited to, fertilizers, nutrients, or pesticides. A microbial composition is a composition comprising at least one microbial species. The composition may contain microbial metabolites produced in the microbial aggregates of the digestive system described herein.

[0075] As used herein, “effective amount” can refer to an amount sufficient to produce a beneficial and / or desired effect. An effective amount can be applied once or multiple times. The term “effective microbial” as used herein when referring to microorganisms is intended to mean a target strain that, at a statistically significant level, promotes plant health, growth, and / or yield relative to an untreated control. In some cases, the term “effective amount” is used herein to refer to the quantity of microbial treatments that can be used to obtain beneficial or desired results relative to an untreated control under appropriate treatment conditions as described herein. For example, the term “agricultural effective amount” is used herein to refer to the quantity of microbial treatments that can be used to obtain agriculturally beneficial or desired results relative to an untreated control under appropriate treatment conditions as described herein. The effective amount of agricultural agents or compositions that can be applied to improve plant health, growth, and / or yield can be readily determined.

[0076] As used herein, "carrier" can refer to a substance or composition that supports the survival of microorganisms. Such carriers can be organic or non-organic.

[0077] As used herein, the “sequence identity percentage” can be determined by comparing two optimally aligned local sequences within a comparison window defined by the length of the local alignment between the two sequences. For optimal alignment of the two sequences, the amino acid sequence in the comparison window may contain additions or deletions (e.g., vacancies or overhangs) compared to the reference sequence (excluding additions or deletions).

[0078] Local alignment between two sequences can include regions in each sequence deemed sufficiently similar according to criteria determined by the algorithm used to perform the alignment (e.g., BLAST). The percentage of sequence identity is calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue appears, generating a matching position count, dividing the matching position count by the total number of positions in the comparison window, and multiplying the result by 100. Optimal alignments for the sequences used for comparison can be performed using: Smith and Waterman's local homology algorithm (Add. APL. Math. 2:482, 1981); Needleman and Wunsch's global homology alignment algorithm (J Mol. Biol. 48:443, 1970); Pearson and Lipman's similarity search method (Proc. Natl. Acad. Sci. USA 85:2444, 1988); heuristic implementations of these algorithms (NCBI BLAST, WU-BLAST, BLAT, SIM, BLASTZ); or by inspection. When two sequences have been identified for comparison, GAP and BESTFIT can be used to determine their optimal alignment. Typically, the default values ​​of 5.00 for vacancy weight and 0.30 for vacancy weight length are used. The term "significant sequence identity" between polynucleotide or polypeptide sequences refers to a polynucleotide or polypeptide containing a sequence that, when compared with a reference sequence using these procedures, has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity. Furthermore, pairwise sequence homology or sequence similarity, as used, refers to the percentage of similar residues between the two aligned sequences. Families of amino acid residues with similar side chains are well-defined in the art. These families include basic side-chain amino acids (e.g., lysine, arginine, histidine), acidic side-chain amino acids (e.g., aspartic acid, glutamic acid), uncharged polar side-chain amino acids (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side-chain amino acids (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side-chain amino acids (e.g., threonine, valine, isoleucine), and aromatic side-chain amino acids (e.g., tyrosine, phenylalanine, tryptophan, histidine). Nucleic acid and amino acid sequences can be searched within target nucleic acid or amino acid sequences existing in public or proprietary databases.These types of searches can be performed using the NCBI BLAST v2.18 program, a basic local alignment search tool from the National Center for Biotechnology Information (NCBI). The NCBI BLAST program is available online from the NCBI (blast.ncbi.nlm.nih.gov / Blast.cgi). The following NCBI BLAST parameters are typically used: filter options set to "default"; comparison matrix set to "BLOSUM62"; empty space cost set to "existence: 11, extension: 1"; word length set to 3; expected value (E threshold) set to 1e-3; and minimum length of local alignment set to 50% of the query sequence length. Sequence identity and similarity can also be performed using GenomeQuest. TM The software (Gene-IT, Worcester Mass. USA) was used for identification.

[0079] The term "plant growth promotion" (e.g., "PGP") can refer to processes that promote plant health, growth, and / or yield. In some embodiments, PGP can encompass improvements in a variety of plant characteristics, including but not limited to improved nitrogen fixation, improved phosphate uptake, improved zinc uptake, improved root development, increased leaf area, increased plant yield, increased macronutrient uptake, increased micronutrient uptake, increased seed germination, enhanced seed germination, enhanced early plant development, improved root growth, improved aboveground growth, improved plant height, increased nutrient uptake, relief of transplant shock, improved plant reproduction, improved soil microbial activity, increased photosynthesis, increased abundance of functional enzymes, increased dry biomass, or increased cumulative plant biomass. In some embodiments, the microbial strains, isolates, cultures, compositions, or synthetic aggregates described herein improve nutrient uptake, plant health and vigor, improved root development, increased leaf area, increased plant yield, increased macronutrient uptake, increased micronutrient uptake, increased seed germination, increased abundance of functional enzymes, increased dry biomass, or increased cumulative plant biomass. In some embodiments, microbial strains, isolates, cultures, or compositions described herein increase the size or mass of a plant or its parts compared to control plants or untreated plants or parts thereof, or compared to predetermined standards. In some embodiments, microbial strains, isolates, cultures, compositions, or synthetic aggregates described herein improve plant health, vigor, and yield compared to control plants or untreated plants, and may also survive and proliferate in the microenvironment associated with the root surface.

[0080] As used in this article, the term “yield” can refer to the amount of harvestable plant material or products derived from plants, and is generally defined as the economically measurable outcome of a crop.

[0081] For crops, "yield" can also refer to the amount of harvested material per acre or per unit of production. Yield can be defined in terms of both quantity and quality. The harvested material can vary from crop to crop; for example, harvested material can be seeds, aboveground biomass, roots, fruit, cotton fibers, any other part of the plant, or any economically valuable product derived from the plant.

[0082] In some embodiments, the microbial strains, isolates, cultures, and compositions and biostimulant compositions described herein can be used to promote improved crop or product quality, including food quality. Improving crop quality can include improving characteristics that make the crop or product more marketable, such as desired color, size, or shape. Improving crop quality can also include giving the food desired nutritional, nutraceutical, and / or pharmaceutical characteristics. Improving crop or product quality can include increasing valuable attributes that can increase prices. Improving crop or product quality can include increasing value / harvest unit, 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 improvement, increasing the measured characteristic by at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, or at least 100%. In some embodiments, the microbial strains, isolates, cultures, and compositions according to the embodiments of this application lead to plant growth promotion or improvement, increasing the measured characteristic by 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%. In some embodiments, the microbial strains, isolates, cultures, and compositions of this application can produce the percentage increase in zinc uptake described above, the increase in zinc solubility described above, the increase in total root weight or leaf area or plant product yield described above (e.g., the percentage increase in plant product weight described above).

[0084] As used herein, a “control plant” can serve as a reference point for measuring phenotypic changes in a target plant and can be any suitable plant cell, seed, plant component, plant tissue, plant organ, or whole plant. A control plant can include, but is not limited to, (a) a plant genetically identical to the target plant but not exposed to the same treatment (e.g., inoculum treatment) as the target plant, or (b) the target plant itself under conditions that have not been exposed to a specific treatment (e.g., an inoculum or a combination of inoculum and / or other chemicals). A treated plant can include a plant to which any part of the plant (e.g., seeds, stem, roots, aboveground parts, leaves, or combinations thereof) has been applied with an inoculum or biostimulant composition of microorganisms as described herein. A treated plant can include a plant to which an inoculum or biostimulant composition of microorganisms as described herein has been applied using furrow application. A treated plant can include a plant to which an inoculum or biostimulant composition of microorganisms as described herein has been applied using lateral application, broadcast application, y-drop application, strip application, or irrigation fertilization. A treated plant can include a plant to which an inoculum or biostimulant composition of microorganisms as described herein has been applied to the soil. Untreated plants may include plants that have not been directly or indirectly inoculated with a composition of microorganisms or biostimulants as described herein.

[0085] As used herein, “inoculum” can refer to any culture or formulation containing at least one microorganism. In some embodiments, an inoculum (sometimes referred to as a microbial inoculum or soil inoculum) is an agricultural additive that utilizes beneficial microorganisms (including, but not limited to, endophytes) to promote plant health, growth, and / or yield. Many microorganisms suitable for use in inoculum form a symbiotic relationship with the target crop, where both parties benefit (mutualism). For example, the isolated microbial strains described herein may benefit from a carbon source in the plant roots, and the plant may benefit from metabolites produced by the metabolism of the microorganisms. Not wishing to be bound by theory, isolates may colonize plants, and root colonization may inhibit the entry of plant pathogens into the roots and / or may provide plant growth benefits. Inoculums (e.g., microorganism / microbial strain inoculum) may be added at one point in time during the digestive system process. Inoculums (e.g., microorganism / microbial strain inoculum) may be added at multiple points in time during the digestive system process.

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

[0087] The term "flocculation" can refer to clumps formed by the aggregation of a large number of fine suspended particles. For example, flocs can contain organic material recovered from feedstocks, waste, wastewater, and / or sludge materials used in digestive systems. Flocs can contain biological solids and / or particles in the digestion products of organic material. Flocs can contain aggregates of microorganisms (e.g., bacteria).

[0088] The term "whole broth" (e.g., WB) can refer to a mixture of supernatant and flocculants in a specific proportion used in the techniques described herein. Whole broth may contain microbial communities (e.g., zinc-solubilizing microorganisms), enzymes, fungi, biosolids, or any combination thereof. For example, the bacterial genera of whole broth may include *Bacillus*, *Lymans*, *Thermophyton*, *Geocystis*, *Acidobacter*, *Lymans*, *Synthia*, *Mesenis*, *Solitaria*, *Nitrospis*, or any combination thereof. Whole broth may possess plant growth-promoting properties. For example, whole broth may have nitrogen-fixing capabilities.

[0089] The term "microbial aggregate" or "microbial community" can refer to a group of microorganisms in a given environment. An aggregate can be an endosymbiotic aggregate or an ectosymbiotic aggregate. The microorganisms in a microbial aggregate can include, but are not limited to, bacteria, fungi, yeasts, lichens, algae, protozoa, archaea, and / or molds.

[0090] The term "supernatant" (e.g., "basal product") can refer to the final product of a digestive system. The amount of microbial isolates, the number of members in microbial aggregates, or the type and amount of microbial metabolites with plant growth-promoting properties can be measured in the supernatant.

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

[0092] The term "internal recycling rate" can refer to the rate at which the working fluid is recycled within the phase space.

[0093] The term "hydraulic feed rate" can refer to the rate at which the working fluid transfers between phase spaces.

[0094] The term "hydraulic residence time" can refer to the duration of time that a working fluid exists in phase space.

[0095] The term "working fluid" can refer to a fluid substance that carries and delivers organisms and nutrients through a container system. For example, a working fluid can contain organic material, microbial cells (e.g., microorganisms and / or metabolites), biological solids, macronutrients, micronutrients, organic nutrients, inorganic nutrients, or any combination thereof. A working fluid can also comprise a solution that flows through the digestive system and can provide an enrichment environment for microorganisms within the digestive system.

[0096] When the terms “at least,” “greater than,” or “greater than or equal to” appear before the first value in a series of two or more values, the terms “at least,” “greater than,” or “greater than or equal to” can be applied to each value in that series. 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] When the terms “not exceeding,” “less than,” or “less than or equal to” appear before the first value in a series of two or more values, the terms “not exceeding,” “less than,” or “less than or equal to” can be applied to each value in that series. 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.

[0098] B. Microbial digestion methods and systems Some embodiments disclosed herein include methods and systems in which microorganisms within a microbial aggregate digest substances provided in a feedstock. The digestion system may consist of a series of separate, fluidly connected containers (also referred to herein as “reactors”). In each reactor, different microbial aggregates can be established and maintained throughout the continuous operation of the digestion system. The unique microbial aggregates present in each reactor can provide different physiological activities in different reactors. Therefore, different steps in the digestion of the feedstock can be carried out in different reactors, thereby potentially leading to (1) more complete digestion, i.e., more complete breakdown of macromolecules in the feedstock (compared to other types of digestion systems), and / or (2) the production of various microbial digestion products with plant growth-promoting properties (e.g., enhancing or improving the soil’s ability to utilize zinc by zinc solubilization, zinc uptake by plant tissues, and / or otherwise promoting zinc utilization efficiency). In some embodiments, conditions that may allow the enrichment of microbial communities with targeted functionalities (e.g., zinc solubilization) may include: residence time, selective pressure addition, hydraulic flow rate, one or more carbon sources, one or more nitrogen sources, pH, nutrient mixing, microaerobic conditions, recirculation of the working fluid, or any combination thereof.

[0099] In some embodiments, the bioreactor system (e.g., a digestion system) includes an established population of one or more zinc-solubilizing microbial strains in one or more containers of the system. An “established population” of a particular microbial strain is a population that remains within the operating bioreactor system without being replenished from outside the bioreactor system. In some embodiments, an established population is a population that has not decreased by more than about 1%, 3%, 5%, 10%, 15%, 20%, or 25% during at least about 5, 10, 15, 20, 25, 30, 60, or 90 days of continuous operation of the bioreactor system without being added 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, respectively. In some embodiments, the established population of the microbial strain has been established by inoculating the microbial strain into one or more containers of the bioreactor system once or multiple times. In some embodiments, the digestive system can be measured using an established population (e.g., concentration or bacterial count). In some embodiments, the established population is derived from a population of microorganisms inoculated into the system at least 5, 10, 20, 30, 40, 50, 60, or 90 days prior to the measurement of the established population.

[0100] In some embodiments, the bioreactor system includes at least one microbial strain. In some embodiments, the bioreactor system includes at least one zinc-solubilizing microbial strain. In some embodiments, the bioreactor system includes 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 also includes an established population of a third zinc-solubilizing microbial strain. The established populations of the respective microbial strains can be established in the bioreactor system by individual or combined inoculation. Individual inoculation can include an inoculum containing one microbial strain (e.g., a microorganism). Combined inoculation can include an inoculum containing at least two microbial strains. Combined inoculation can include the same isolated microbial strain. Combined inoculation can include isolated and non-isolated microbial strains. Combined inoculation can include two or more isolated microbial strains.

[0101] In some embodiments, the reactor or reactor series (e.g., a series assembly of reactors in tandem) functions to facilitate the growth of one or more microorganisms with desired plant growth-promoting properties and / or the production of digestible products with plant growth-promoting properties. The system may include 1, 2, 3, 4, 5, 6, or more reactors. In some embodiments, operation of the digestion system may result in the growth of one or more microorganisms with desired plant growth-promoting effects. The one or more microorganisms may be one or more isolated microorganisms added separately to the digestion system as inoculum. The one or more microorganisms may also be introduced into the system as part of a feed material comprising a mixture of microorganisms. The one or more microorganisms may be endogenous to organic materials (e.g., manure, plants, lignocellulosic materials, or algae). The one or more microorganisms may also be endogenous to other types of feed materials such as phosphate rock. In some embodiments, endogenous microorganisms are those microorganisms naturally present in the feed material (e.g., manure, plants, lignocellulosic materials, or algae). These microorganisms may be naturally present in a closed system and / or in the ecosystem of the feed material.

[0102] The inputs to the digestive system may include one or more of water, microbial inoculum (e.g., inoculum of microorganisms or inoculum of microbial strains), nutrients (e.g., one or more sources of carbon, nitrogen, phosphorus, etc.), and digestible substrates. The fluid within the reactor of the digestive system may be referred to herein as the "working fluid." In continuous operation, each reactor may have a constant volume of working fluid therein, with the inflow rate matching the outflow rate. Because each reactor may include different microbial aggregates and have conditions different from other reactors, the working fluid within each reactor may be considered different from the working fluid within other reactors. The total volume of working fluid within the digestive system may be referred to herein as the "total working volume" of the digestive system.

[0103] The digestible substrate included in the input stream entering the digestive system may include, for example, organic material that can be digested by microorganisms in the digestive system. Such organic material may include, for example, manure, lignocellulosic material, wastewater biosolids, food waste, energy crops, yeast, guano, agricultural waste, algae, or any combination thereof. 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 types of manure. The digestible substrate entering the digestive system may have been partially digested before being introduced into the system. Therefore, the input entering the system may include the products of the digestion of the original digestible substrate by microorganisms endogenously derived from the original digestible substrate, as well as any digestible material still present in the input. In some embodiments, the digestible substrate included in the input stream may include inorganic substrate. Inorganic substrate may include, for example, sand, vermiculite, perlite, and / or pumice. In some embodiments, the inorganic substrate includes minerals. In some embodiments, the inorganic substrate includes phosphate rock. In some implementations, the input stream may include a zinc source, such as an insoluble form of zinc.

[0104] In some embodiments, the microbial inoculum comprises a single isolated microorganism. The isolated microorganism may include microorganisms generated outside the natural environment (e.g., via a plate inoculation method or culture medium). In some embodiments, the microbial inoculum may include 1 to 5 isolated microorganisms (e.g., microbial strains). In some embodiments, the inoculum may include 1, 2, 3, 4, or 5 isolated microorganisms. In some embodiments, the inoculum may include more than 5 isolated microorganisms. In some embodiments, in addition to one or more isolated microorganisms, the microbial inoculum introduced into the digestive system may also comprise a complex mixture of microorganisms, which may include at least 5, 10, 20, 25, 50, 100, 200, 225, 250, 275, 300, 350, 400, or more microbial species.

[0105] The inoculum of the microorganisms described herein may possess at least one plant growth-promoting property (e.g., plant growth characteristics). Plant growth-promoting properties may include: aboveground biomass, root biomass, nutrient uptake, crop yield, leaf area, chlorophyll content, photosynthetic activity, zinc uptake, zinc solubilization, micronutrient uptake, or total biomass. The digestive system may be configured to enhance the production of the microbial inoculum. The microorganisms may be bacterial, fungal, or algal species.

[0106] In some embodiments, the inoculum may include at least two isolated microorganisms. In some embodiments, the inoculum may include at least one isolated microorganism and at least one non-isolated microorganism. In some embodiments, the inoculum may include 1, 2, 3, 4, 5 or more microorganisms. The inoculum may be transferred to a first container (e.g., a reactor) of the digestive system once, twice, three times, four times, five times or more. The inoculum may be provided to a second, third, fourth, fifth, sixth or any other container of the system described herein.

[0107] Before being transferred to the first container in the digestive system, the concentration of the microbial inoculum can be at least about, at most about, or about 1.0 x 10⁻⁶. 2 cfu / ml, 1.0 x 10 3 cfu / ml, 1.0 x 10 4 cfu / ml, 1.0 x 10 5 cfu / ml, 1.0 x 10 6 cfu / ml, 1.0 x 10 7 cfu / ml, 1.0 x 10 8 cfu / ml, 1.0 x 10 9 cfu / ml, 1.0 x 10 10 cfu / ml, 1.0 x 10 11 cfu / ml or 1.0 x 10 12 The concentration of the inoculum, after incubation in the digestive system described herein, may be at least about, and at most about, or about 1.0 x 10⁻⁶ cfu / ml, or a range between two of these values. 2 cfu / ml, 1.0 x 10 3 cfu / ml, 1.0 x 10 4 cfu / ml, 1.0 x 10 5 cfu / ml, 1.0 x 10 6 cfu / ml, 1.0 x 10 7 cfu / ml, 1.0 x 10 8 cfu / ml, 1.0 x 10 9 cfu / ml, 1.0 x 10 10 cfu / ml, 1.0 x 10 11 cfu / ml or 1.0 x 10 12 cfu / ml, or a range between two of these values.

[0108] In some embodiments, the inoculum contains a single microorganism. In some embodiments, the inoculum may contain 1 to 5 microorganisms. In some embodiments, the inoculum may contain at least 1, at least 2, at least 3, at least 4, at least 5 or more microorganisms. In some embodiments, the inoculum may contain at most 5, at most 4, at most 3, at most 2 or at most 1 microorganism.

[0109] In some embodiments, the raw material (e.g., an aqueous organic feedstock) and the microbial inoculum may be transferred to the first reactor separately. In some embodiments, the raw material (e.g., an 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 raw material (e.g., an aqueous organic feedstock). In some embodiments, the raw material (e.g., an aqueous organic feedstock) and the 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 microorganism). For example, the aqueous feedstock may not contain the target isolate strain before being transferred to the first container. The concentration of the target isolate strain may be 0 cfu / ml. In some embodiments, the aqueous feedstock may contain the target isolate strain before being transferred to the first container. In some embodiments, the aqueous feedstock may contain up to about 5%, up to about 4%, up to about 3%, up to about 2%, up to about 1%, up to about 0.5%, up to about 0.1%, up to about 0.05%, up to about 0.04%, up to about 0.03%, up to about 0.02%, up to about 0.01%, up to about 0.008%, up to about 0.005%, up to about 0.004%, up to about 0.003%, up to about 0.002%, up to about 0.001%, up to about 0.0001%, or less than about 0.0001% of the target isolate strain. In some embodiments, the aqueous feedstock may not contain microbial strains (e.g., zinc-solubilizing microorganisms) at concentrations 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.

[0111] In some embodiments, the digestion system includes a clarifier chamber or clarifier tank (CLF). The clarifier may include a single inlet port and a single outlet port. Alternatively, the clarifier may include a single inlet port and multiple outlet ports. In some embodiments, the clarifier includes a flocculant folding scraper that rotates and releases microorganisms immobilized in the flocculant without introducing solids into the supernatant. The flocculant folding scraper can move the working fluid within the clarifier to resuspend the microorganisms within the working fluid. In some embodiments, microorganisms and / or a certain amount of target isolate strains can be resuspended in the solution in the clarifier and transferred to the supernatant (e.g., the base product). In some embodiments, the clarifier also includes a flow path to return the flocculant to the first reactor. The flow path may include a conduit from the clarifier to a container or combination of containers in 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 can be refluxed to any container in the digestive 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) to provide recirculation of the working fluid. The working fluid recirculated from the clarifier can contain a different microbial community (e.g., a different amount of microorganisms) than the working fluid in another container in the digestive system (e.g., the first working fluid, the second working fluid, the third working fluid, the fourth working fluid, the fifth working fluid, and / or the sixth working fluid). Not wishing to be bound by theory, recirculation of the working fluid from the clarifier to containers in the digestive system can contribute to the enrichment of the microbial community of microbial aggregates in the digestive system by providing the working fluid from the clarifier to different points in the system (e.g., containers). The recirculated working fluid can contain organic material, microorganisms of microbial aggregates, target isolates, metabolites, or any combination thereof.

[0112] In a clarifier, the flocculated portion of the working fluid (e.g., the clarifier working fluid) can be separated from the supernatant portion of the working fluid. Flocculation scrapers in the clarifier can facilitate the separation of the clarifier's working fluid. In some embodiments, the separation may include gravity separation. The flocs may settle to the bottom of the clarifier, and the supernatant may be collected.

[0113] The biostimulant composition produced by the digestive process described herein can be used as is or may be further processed prior to use. For example, effluent from the digestive system (referred to herein as the “base product”) may be concentrated, sterilized, filtered, pasteurized, dehydrated, or any combination thereof prior to use. In some embodiments, the base product may be concentrated to 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, or more. In some embodiments, the base product may be filtered and sterilized to remove any bacteria or other microorganisms from the composition.

[0114] In one aspect, this document provides a method comprising transferring a raw material (e.g., an aqueous organic raw material) into a first container. An inoculum of microorganisms may be transferred into the first container. The raw material (e.g., an aqueous organic raw material) and the inoculum of microorganisms may be transferred into the first container. The first container may include a volume of a first working fluid. The raw material (e.g., an aqueous organic raw material) may contain microbial aggregates. The raw material (e.g., an aqueous organic raw material) may contain digestion products generated by digesting organic material. The raw material (e.g., an aqueous organic raw material) may contain microbial aggregates and digestion products generated by digesting organic material. The organic material may be digested by one or more microorganisms in the microbial aggregates. The organic material may be digested by a population of microorganisms of the microbial inoculum (such as zinc-solubilized target isolates). The digestion products described herein may include sugars (e.g., xylose, mannose, glucose, or any combination thereof), metabolites produced by microorganisms in the working fluid, fatty acids, dead microbial cells, fragments of dead microbial cells, microbial fermentation products, enzymes, bio-plant growth regulators, organic acids, chelating agents, or any combination thereof. The method may also include incubating the microbial inoculum under conditions that selectively promote microbial growth and increase the microbial population. The method may also include incubating the microbial inoculum under conditions that selectively promote the growth of at least a portion of the microorganisms in a microbial aggregate. The terms "microbial digestion" and "digestion" are used interchangeably.

[0115] In some embodiments, digestion is anaerobic digestion. In some embodiments, digestion is aerobic digestion. In some embodiments, digestion is microaerobic digestion. In some embodiments, digestion is aerobic digestion, microaerobic digestion, anaerobic digestion, or some combination thereof. Not wishing to be bound by theory, it is believed that during the digestion process, microorganisms digest biomolecules and other nutrients present in manure, yeast, or kelp, and / or produce digestion products including compounds that promote plant growth and soil health. In some embodiments of the digestion process, the feedstock (e.g., organic feedstock) may be mixed with water to prepare a feedstock (e.g., organic feedstock) for use in the anaerobic digestion system. The anaerobic digestion system may include a mixing tank in which feedstock (e.g., organic feedstock) are mixed to prepare a fluid feed mixture or working fluid. In some embodiments, the fluid feed mixture may include manure, water, and brewer's yeast (…). Saccharomyces cerevisiae In some embodiments, anaerobic digestion includes the process of bacteria breaking down organic biological material in the absence of oxygen. Biostimulants may also contain microorganisms that promote beneficial plant properties of the biostimulant product. The microorganisms in the biostimulant product may be derived from the microbial community present in the raw material (e.g., organic raw material).

[0116] In the exemplary systems disclosed herein, a series of reactors functions to facilitate the growth of inoculums (e.g., isolates) of microorganisms with desired plant growth-promoting properties. The series of reactors (e.g., tandem assembly of reactors) can also function to facilitate the production of microbial metabolites with desired plant growth-promoting properties. The digestion system described herein can enrich inoculums of target microorganisms, populations of microorganisms within microbial aggregates with plant growth-promoting properties (e.g., zinc solubilization), metabolites produced by enriched aggregates of microbial aggregates with plant growth-promoting properties (e.g., zinc solubilization), or any combination thereof. This system offers additional benefits compared to other digestion systems because it can target functional communities of microorganisms and / or metabolites with specific functions and enrich and / or maintain such communities within the digestion system. The system may include two, three, four, five, or more reactor chambers (e.g., containers or compartments). Not wishing to be bound by theory, tandem reactor series enable the growth and enrichment of proprietary, expert-specific target microorganisms with optimal plant growth-promoting properties. This system can guide the flow of a working fluid containing 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 the reactor through an inlet port to contain the first working fluid in the reactor tank. A substance providing selective pressure can flow into the reactor through an inlet port to contain the first working fluid in the reactor tank. Both the hydraulic source and the substance providing selective pressure can flow into the reactor through an inlet port to contain the first working fluid in the reactor tank. The hydraulic source can be input into the system's first reactor or any reactor. In some embodiments, the hydraulic source can be input (e.g., flow into) a tank or container preceding the first reactor. In some embodiments, the container may include a "completely mixed reactor" (CMR). Other inputs entering the system described herein can flow into any reactor of the system, including but not limited to the first reactor, second reactor, third reactor, or any other reactor following the first reactor.

[0117] Microbial inoculum (e.g., target microbial strains) can be incubated in a reactor (e.g., container) of the digestive system described herein. In some embodiments, the microbial inoculum can be incubated under conditions that selectively enrich and / or maintain the concentration of microbial strains in the digestive system. These conditions for selectively enriching and / or maintaining the population of microbial strains may include the addition of a selective pressure source. In some cases, the selective pressure may be a zinc source. In some cases, the zinc source may be in the form of insoluble zinc. Upon addition of selective pressure (e.g., a zinc source), the inoculation of zinc-solubilizing microbial strains can solubilize insoluble zinc and produce a high zinc ion concentration. This high zinc ion concentration environment can further enrich the microbial population with the target functionality (e.g., zinc solubilization). In some cases, the microbial strain population can survive in the digestive system in a vegetative or sporulated state (e.g., a dormant state in the system). In some cases, a selective pressure source (e.g., selective pressure) can establish an environment that enhances the survival of the microbial strain population. Without being bound by theory, selective stressors can transform complex microbial aggregates in the digestive system to enrich microorganisms within these aggregates that possess at least a portion of plant growth-promoting properties (e.g., zinc solubilization). Incubation of inoculum and / or portions of microorganisms with plant growth-promoting properties within the microbial aggregates can further generate metabolites with plant growth-promoting properties (e.g., zinc solubilization).

[0118] The microbial inoculum may include zinc-solubilizing microorganisms that can be maintained in the working fluid at a higher ion concentration caused by the addition of selective pressure. Survival of the microbial inoculum may include an inoculum configured to maintain its initial quantity of microorganisms in an environment caused by the addition of selective pressure. Survival of the microbial inoculum may include the condition that a certain quantity of the microbial inoculum is viable (e.g., maintained) at the end of the residence time in the digestion system (e.g., in a reactor or clarifier chamber). Not wishing to be bound by theory, the microbial inoculum may be insensitive to changes in ion concentration, or may have solubilizing properties for ions that allow its survival in the working fluid. After the addition of selective pressure, at least a portion of the microorganisms in the microbial aggregate may be enriched (e.g., grow or increase in number). These microorganisms in the microbial portion of the microbial aggregate may have zinc-solubilizing capacity. After the addition of selective pressure, other microorganisms in the working fluid may decrease in number due to the inability of the microorganisms in the microbial aggregate to survive in the ion environment (e.g., susceptible). The proportion of zinc-solubilizing microbial strains relative to at least a portion of the microorganisms in the microbial aggregate can be maintained in the first container of the digestion system. The proportion of zinc-solubilizing microbial strains relative to at least a portion of the microorganisms in the microbial aggregate can be maintained in the second, third, fourth, fifth, sixth, seventh, or eighth container of the digestive system. During the measurement period, the variation in the amount of maintained zinc-solubilizing microbial strains in the working fluid of the digestive system can be 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%.

[0119] In some cases, changes in ion concentration caused by the addition of selective pressure can promote the growth of microorganisms or at least a portion of microorganisms in a microbial aggregate. These microorganisms may be zinc-solubilizing microorganisms. After the addition of selective pressure, the amount of microorganisms or at least a portion of microorganisms in the microbial aggregate may increase by at least about, and at most about or about 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 30%, or any range between these values. In some cases, at least a portion of the zinc-solubilizing microorganisms in the microbial aggregate may accumulate and / or grow in the system without the addition of an inoculum of microbial strains.

[0120] Selective stressors can be added to the digestive system on the first day of the digestive process. In some embodiments, selective stressors can be added to the digestive system daily. In some embodiments, selective stressors can be added to the digestive system every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, and / or weekly.

[0121] It is not desirable to be bound by theory, but high zinc concentrations can inhibit the growth of microorganisms and / or microbial communities unless zinc solubility is achieved. Nutrients (e.g., macronutrients, micronutrients, inorganic nutrients, or any combination thereof) can be present in the digestive system to provide an environment for bacterial growth. The addition of selective stress (e.g., increased zinc ion concentration), along with inputs that promote microbial growth, can allow the enrichment of zinc solubilizers in the working fluid of the digestive system described herein. Zinc solubilizers may include microbial inoculum (e.g., a population of microbial inoculum), zinc-soothing microorganisms of microbial aggregates, zinc-soothing metabolites produced by microbial inoculum and / or zinc-soothing microorganisms of microbial aggregates, or any combination thereof.

[0122] The inoculum of the microorganisms described herein can contact (e.g., be applied to) plants. In some embodiments, contact between the inoculum and the plant can enhance at least one plant growth-promoting property of the plant. In some embodiments, one, two, three, four, or more inoculums of the microorganisms can be transferred to the digestive system. The inoculum of the microorganisms and another inoculum of the microorganisms can be the same. The inoculum of the microorganisms and another inoculum of the microorganisms can be different. In some embodiments, the inoculum of the microorganisms and the raw material (e.g., aqueous organic raw material) are transferred simultaneously to the container of the digestive system. In some embodiments, the inoculum of the microorganisms and the raw material (e.g., aqueous organic raw material) are not transferred simultaneously to the container of the digestive system. In some embodiments, the inoculum of the microorganisms is transferred to the container of the digestive system before the raw material (e.g., aqueous organic raw material). In some embodiments, the inoculum of the microorganisms is transferred to the container of the digestive system after the raw material (e.g., aqueous organic raw material).

[0123] In some embodiments, selective pressure can enrich a population of zinc-solubilizing microorganisms (e.g., zinc solubilizers) in one or more containers of the digestive system. The population of zinc-solubilizing microorganisms may include inoculums of target zinc-solubilizing microorganisms, zinc-solubilizing microorganisms in microbial aggregates, zinc-solubilizing metabolites, or any combination thereof.

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

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

[0126] Microbial inoculants can produce metabolites in the digestive system described herein. Microorganisms in a microbial aggregate and / or microbial inoculants can be metabolized by catalytic enzymes to produce metabolites. Metabolites can be produced through microbial metabolism. Metabolites can be produced by enzymatically catalytic biochemical reactions of organic substrates (e.g., aqueous organic feedstocks) in the working fluid of the digestive system described herein. Metabolites produced by microbial inoculants can have plant growth-promoting properties. Metabolites produced by microbial inoculants can have two or more plant growth-promoting properties. Plant growth-promoting properties may include: aboveground 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 can be used in biostimulant compositions and / or can be applied to plants.

[0127] In some embodiments, the raw material (e.g., an aqueous organic raw material) includes metabolites. In some embodiments, the raw material (e.g., an aqueous organic raw material) includes metabolites produced by endogenous microorganisms of the raw material (e.g., an organic raw material). Primary metabolites may include carbohydrates, proteins, fats, vitamins, and nucleic acid components. Metabolites may also include alkaloids, amino acids, biogenic amines, carboxylic acids, cresols, terpenoids, phenols (e.g., flavonoids, coumarins, tannins, lignans, arbutin compounds, or chromones), polyketides, eicosanoic acid-like acids, 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 include sugars and / or fatty acids. Sugars may include fructose, hexose, galactose, glucose, lactose, maltose, sucrose, and / or xylose. Fatty acids may include stearic acid, lauric acid, myristic acid, palmitic acid, octadecenoic acid, octadecadienoic acid, oleic acid, arachidic acid, behenic acid, erucic acid, adrenic acid, trisaccharide, ceramide, nervonic acid, nonadecanoic acid, arachidic acid, myristolic acid, or hydroxylated myristic acid.

[0128] Metabolites produced by inoculum of microorganisms or by at least a portion of microorganisms in a microbial aggregate may be present in the supernatant of the digestive system (e.g., the basal product). In some embodiments, the metabolites may be present by weight in a volume of solution (e.g., mg / 100 ml). In some embodiments, the weight of the metabolites per 100 ml of basal product solution may 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, the weight of the metabolite per 100 ml of basal product solution can be up to about 500 mg, up to about 400 mg, up to about 300 mg, up to about 280 mg, up to about 260 mg, up to about 240 mg, up to about 220 mg, up to about 200 mg, up to about 180 mg, up to about 160 mg, up to about 140 mg, up to about 120 mg, up to about 100 mg, up to about 90 mg, up to about 80 mg, up to about 70 mg, up to about 60 mg, up to about 50 mg, up to about 40 mg, up to about 30 mg, up to about 20 mg, up to about 10 mg, or less than about 10 mg. In some embodiments, the weight of the metabolite per 100 ml of basal product solution can be from about 10 mg to about 500 mg. In some embodiments, the weight of the metabolite in each 100 ml of the base product solution may be 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.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 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 "raw material" (e.g., "organic raw material") as used herein can refer to primary biological materials such as carbon compounds, proteins, and / or carbohydrates. Raw materials can be organic. Raw materials can be inorganic. Organic raw materials can be aqueous organic raw materials. In some cases, organic raw materials may contain one or more inorganic nutrients. In some embodiments, raw materials (e.g., organic raw materials) may contain organic substrates, including cottonseed, algae, neem, orange seeds, flaxseed, jojoba, kusum, rubber seeds, alfalfa, sugarcane, cactus, coffee, Deccanhemp, or any combination thereof. In some embodiments, raw materials may contain inorganic raw materials. In some embodiments, raw materials (e.g., organic raw materials) may include, but are not limited to, manure, giant kelp, lignocellulose, wastewater biosolids, food waste, energy crops, glucose solution, ammonium sulfate, oil, fat, grease, or any combination thereof. In some embodiments, raw materials are added at the beginning of the system (e.g., to the first reactor and / or CMR). In some embodiments, raw materials are added to intermediate reactors of the system (e.g., not in the first or last reactor of the system). In some embodiments, the raw material is added to the system once. In some embodiments, the raw material is added to the system two, three, four, or more times. In some embodiments, the raw material is added to the system continuously. In some embodiments, the raw material (e.g., an organic raw material) is a composition of a single original biological material. In some embodiments, the raw material (e.g., an organic raw material) is a mixture of two, three, four, five, six, seven, eight, nine, ten, or more biological materials.

[0130] Raw materials containing carbon and nitrogen sources (e.g., organic feedstock) can flow into the reactor tank. Raw materials containing carbon and nitrogen sources (e.g., organic feedstock) can flow into the reactor tank via conduits (e.g., pipes). The raw materials (e.g., organic feedstock) may also contain microbial strains with desired plant growth-promoting properties (e.g., zinc-solubilizing microbial strains). In some embodiments, the raw material (e.g., aqueous raw material or aqueous organic raw material) may contain microbial strains (e.g., zinc-solubilizing microbial strains) at concentrations of up to about 15 CFU / ml, up to about 14 CFU / ml, up to about 13 CFU / ml, up to about 12 CFU / ml, up to about 11 CFU / ml, up to about 10 CFU / ml, up to about 9 CFU / ml, up to about 8 CFU / ml, up to about 7 CFU / ml, up to about 6 CFU / ml, up to about 5 CFU / ml, up to about 4 CFU / ml, up to about 3 CFU / ml, up to about 2 CFU / ml, up to about 1 CFU / ml, or less than about 1 CFU / ml.

[0131] In some embodiments, the reactor tanks circulate their internal working fluid for recycling, wherein the reactor tanks may include outflow conduits for circulating and recycling the working fluid within each tank. Ports and conduits between tanks can facilitate the transfer of working fluid to adjacent reactor tanks. Working fluid within the final clarifier of the system can be transferred from the reactor tanks to the clarifier, producing a supernatant (e.g., a base product). The working fluid flows through a series of reactor systems, which can facilitate the selective growth of added isolates and other microorganisms present with similar characteristics to the added isolates. The base product from the clarifier can be obtained and further analyzed for microbial composition. In the digestion system described herein, the working fluid may exit from the mixing chamber, flow through at least one reaction, and flow into the clarifier chamber.

[0132] The feedstock (e.g., organic feedstock) may comprise digestion products derived from digesting organic substrates present in the feedstock (e.g., organic feedstock). Organic substrates can improve the stability of the fluid feed mixture. Organic substrates may include coconut palm, peat moss, hemp, and / or lignocellulose. In some embodiments, the organic substrate comprises raw biological material present in the feedstock (e.g., aqueous organic feedstock). The digestion system may include a variety of microorganisms and / or microbial cells derived from the digestion of organic substrates in the feedstock (e.g., aqueous organic feedstock).

[0133] The raw materials described herein (e.g., organic raw materials) may comprise a variety of organic and / or biological materials. In some embodiments, the raw materials (e.g., organic raw materials) also include brewer's yeast (Saccharomyces cerevisiae). Saccharomyces cerevisiae Tree yeast () Saccharomyces arboricola ), Micarta yeast ( Saccharomyces mikatae ), Zhu's yeast ( Saccharomyces jurei ), Zhenbei yeast ( Saccharomyces eubayanus ), Kudriazwiel yeast ( Saccharomyces kudriavzevii ), grape juice yeast ( Saccharomyces uvarum The raw material may be an aqueous mixture of at least one raw material and water, or any combination thereof. In some embodiments, the raw material (e.g., organic raw material) may be an aqueous mixture of cow manure, brewer's yeast, water, or any combination thereof. The raw material may be an organic raw material. The organic raw material may be an aqueous organic raw material (e.g., an organic raw material containing water).

[0134] Parameters of the digestive system (e.g., flow rate and solids content of the feedstock (e.g., organic feedstock)) can be altered to achieve the desired characteristics of the effluent 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 digestive system to prepare an aqueous organic feedstock.

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

[0136] In some embodiments, the loading input into the reactor may comprise a carbon source, a nitrogen source, flour, isolates, or any combination thereof. In some embodiments, the loading input includes reclaimed flocculants from the system. In some embodiments, the loading input comprises whole broth (WB). The inoculum of the microorganisms described herein can metabolize the carbon source. The metabolism of carbon by the microbial inoculum may include transferring a carbon-based portion of the carbon source to a substrate in the working fluid.

[0137] The inoculum of the microorganisms described herein can metabolize a nitrogen source. The metabolism of nitrogen by the microbial inoculum may include transferring a nitrogen-based portion of the nitrogen source to a substrate in the working fluid. In some embodiments, a carbon source may be transferred to a first container of the digestive system. In some embodiments, a carbon source may be transferred to a second, third, fourth, fifth, or sixth container of the digestive system. In some embodiments, a nitrogen source may be transferred to a first container of the digestive system. In some embodiments, a nitrogen source may be transferred to a second, third, fourth, fifth, or sixth container of the digestive system.

[0138] In some embodiments, the raw materials (e.g., organic raw materials) are mixed inside the reactor. In some embodiments, the raw materials (e.g., organic raw materials) are mixed outside the reactor. In some embodiments, the raw materials (e.g., organic raw materials) are mixed between one, two, three, or more reactors. In some embodiments, the raw materials (e.g., organic raw materials) are a homogeneous mixture.

[0139] In some embodiments, the raw material (e.g., organic raw material) also comprises microbial aggregates. The terms "microorganism," "microbial strain," and "microbial body" can refer to a microscopic organism, including bacteria, fungi, lichens, algae, protozoa, archaea, molds, or any combination thereof. The terms "microorganism" and "microbial body" are used interchangeably herein. The raw material (e.g., organic raw material) may comprise microbial aggregates having 2, 3, 4, 5, 6, 7, 8, 9, 10, or more types of microorganisms. The raw material (e.g., organic raw material) may comprise microbial aggregates having 2, 3, 4, 5, 6, 7, 8, 9, 10, or more groups of microorganisms. The raw material (e.g., organic raw material) may comprise microbial aggregates having a single group of microorganisms. Microbial aggregates may comprise different microorganisms. Microbial aggregates may comprise the same microorganisms. The microorganisms in the aggregate may be derived from microorganisms originally present in the raw material (e.g., organic raw material). The microorganisms may digest manure, yeast, other organic raw materials, or any combination thereof to produce digestive products.

[0140] In some embodiments, the microorganisms may be added at the beginning of the system (e.g., in the first reactor). In some embodiments, the microorganisms may be added in the middle of the system (e.g., in a reactor that is not the first or last reactor of the system), or the microorganisms may be added at the end of the system (e.g., in the last reactor). The microorganisms may be added simultaneously with the feedstock (e.g., organic feedstock). The microorganisms may be added separately from the feedstock (e.g., organic feedstock). In some embodiments, the microorganisms may be added to the system together with the feedstock (e.g., organic feedstock) in the same reactor. In some embodiments, the microorganisms may be added to the system together with the feedstock (e.g., organic feedstock) in a different reactor. In some embodiments, the microorganisms may be added to the system before the feedstock (e.g., organic feedstock). In some embodiments, the microorganisms may be added to the system after the feedstock (e.g., organic feedstock). In some embodiments, the time interval between adding the microorganisms to the system and adding the feedstock (e.g., organic feedstock) to the system may 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 hour. In some implementations, the time interval between adding microorganisms to the system and adding raw materials (e.g., organic raw materials) to the system can be up to about 2 hours, up to about 1 hour, up to about 45 minutes, up to about 30 minutes, up to about 15 minutes, up to about 10 minutes, up to about 5 minutes, up to about 1 minute, or up to about 30 seconds.

[0141] Microorganisms can possess nutrient solubilizing properties and / or plant growth promoting properties. For example, microorganisms can enhance plant growth, increase aboveground and / or root biomass, increase crop yield, increase soil enzyme activity, increase photosynthetic efficiency, reduce heavy metal uptake, and / or lower soil pH. Microorganisms can enhance plant growth in high-salinity soils, in heavy metal-contaminated soils, or in arid conditions. The microorganisms described herein (e.g., isolates) can possess zinc solubilizing properties.

[0142] In some implementations, the digestive system may include a residence time. Residence time may include the time the inoculum of the microorganism is consumed within the digestive system, or the time from transfer to the first container until collection from the digestive system. A longer residence time may favor the growth or enrichment of the inoculum of the microorganism in the digestive system. A shorter residence time may favor the growth or enrichment of the inoculum of the microorganism in the digestive system. The residence time in the digestive system may include at least about, and 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 can be agitated at a rate that allows heavier or undigested solids to settle to the bottom. An outlet at the top of the first reactor allows fluid to flow into a second reactor. An outlet at the bottom of the first reactor transfers settled solids back into the reactor tank. Each reactor in the reactor series can have an immersed support providing a surface for biofilm growth. This support can be referred to as a “fixed media matrix.” Fluid flow between reactors can include a plug flow model, where the particles of the input fluid have the same velocity and direction of motion. In some embodiments, fluid flow in the digestion system is gravity-driven. In some embodiments, fluid flow in the digestion system is pump-driven. The effluent from the top of the final reactor can be used to produce a product. The product of the methods and systems described herein can be a biostimulant. Biostimulants can promote plant growth, improve plant quality, or improve soil quality.

[0144] C. Reactor Digestion methods for producing biostimulants can be carried out in digestion systems comprising tanks, containers, vessels (e.g., reactors), or series of tanks, containers, or vessels (e.g., reactors) through which feedstock flows continuously. Reactors can be fluidly connected containers, systems, vessels, or tanks in which microbial aggregates containing the microorganisms, isolates, and / or microbial bodies described herein can be grown. Reactors can be discrete or continuous. Reactors can be physically contained systems arranged in a discrete sequence to favor the growth of specific microorganisms. Reactor types can include, but are not limited to, fluidized bed reactors (FBRs) or packed bed reactors (PBRs).

[0145] In some embodiments, the reactor can be arranged such that fluid can flow from the reactor's outlet port into an adjacent reactor or tank. Fluid from near the top of the working fluid in the reactor can continuously flow into the next reactor. Fluid from the middle of the reactor can continuously flow into the next reactor. Fluid from the bottom of the reactor can continuously flow into the next reactor. Fluid can also be reintroduced into the same reactor from any outlet source. In some embodiments, the outlet port is located 0.1 to 35 inches below the top of the working fluid within the reactor. In some embodiments, the outlet port is located 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 the reactor. In some embodiments, the effluent port is located at a distance of up to approximately 10,000 inches, 7,500 inches, 5,000 inches, 2,500 inches, 1,000 inches, 750 inches, 500 inches, 250 inches, 100 inches, 50 inches, 20 inches, 10 inches, 5 inches, 2 inches, or 1 inch below the top of the working fluid within the reactor. In some embodiments, the product effluent rate from the digestion system can be matched to the feed inflow rate, thereby providing hydraulically balanced flow throughout the system. Reactors within the system may have unique, stable microbial aggregates with different physiological characteristics and digestive capacities compared to aggregates in other tanks within the system. Reactors within the system may have the same microbial aggregates with similar physiological characteristics and digestive capacities to another reactor within the system. Each reactor within the system may have the same capacity. Each reactor within the system may have a different capacity. A digestion system may include at least two reactors. A digestion system may include 2, 3, 4, 5, 6, 7, 8, 9, 10, or more reactors. The reactors of the digestion system may be arranged as a series assembly of reactors. The series assembly of reactors may have conduits (e.g., ports or outlets) connecting each reactor to adjacent reactors and / or vessels. The series assembly of reactors may have continuous flow of working fluid through each reactor to adjacent reactors.

[0146] In some embodiments, the reactor may have a single inlet port and a single outlet port. In some embodiments, the reactor may have multiple inlet ports and outlet ports. In some embodiments, the reactor may have a single inlet port and multiple outlet ports. In some embodiments, the reactor may have multiple inlet ports and a single outlet port. The reactor may have an additional inlet port to provide a carbon source and / or polymer inoculum. The inlet port may be located anywhere within the reactor of the digestion system. The inlet port may be located at the top or bottom of the reactor. The outlet port may be located at the top or bottom of the reactor. In some embodiments, the outlet port or inlet port described herein includes pipes, pumps, venting devices, or other systems known to those skilled in the art for transferring fluid from one vessel to another.

[0147] The reactor may have a single fluid connection. The reactor may have multiple fluid connections. These fluid connections may be located at the top of or near the top of the working fluid in each reactor. In some embodiments, the reactor may have a flow returning from the bottom to the top of the vessel to prevent sludge accumulation at the bottom of the reactor. In some embodiments, the reactor may have an agitator at the bottom of the vessel. In some embodiments, the reactor may have a scraper at the bottom of the vessel. The scraper can agitate the feed and prevent clogging within the reactor. The scraper can fold up flocs and facilitate their return.

[0148] In some embodiments, the reactor may include a packed bed reactor. In some embodiments, each packed bed reactor has an open design to allow free movement of the working fluid. A stationary medium (e.g., a support) may be secured to the inside of each packed bed reactor. The stationary medium contains materials that increase the contact surface area between the microbial community and the working fluid. The stationary medium also provides a stabilizing platform for anchoring the biofilm. The packed bed reactor may be filled with supports to increase the surface area within the reactor. The supports within the reactor can increase the biofilm. The packed bed reactor can improve the contact between the biofilm and the substrate within the reactor. The stationary medium can be of several types, including durable plastics, polyvinyl chloride (PVC), metals, metal alloys, glass, glass compounds, glass fibers, or any suitable robust inert material. The design and configuration of the stationary medium can present a variety of geometries, thereby allowing the working fluid to move freely through each packed bed reactor and preventing fouling. Free flow supports controlled hydraulic shear, thereby promptly promoting uniform distribution of the working fluid. In this embodiment, the stationary medium is distributed throughout the cross-section of each packed bed reactor.

[0149] The support may include tubes, rings, or other packing materials. In some embodiments, the packed bed reactor provided herein may include tube bundles or columns. In some embodiments, the support may include hexagonal, mesh-like, perforated tubes, or any combination thereof. Without being bound by theory, hexagonal, mesh-like, and / or perforated supports can increase surface area within the vessel and flow through the column. In some embodiments, the tubes or columns of the support may contain diameters from 0.25 to 50 inches. In some embodiments, the support may contain diameters 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 support may have a diameter of up to about 75 inches, at least about 60 inches, up to about 50 inches, up to about 40 inches, up to about 30 inches, up to about 25 inches, up to about 20 inches, up to about 15 inches, up to about 10 inches, up to about 5 inches, up to about 4 inches, up to about 3 inches, up to about 2 inches, up to about 1 inch, up to about 0.9 inches, up to about 0.8 inches, up to about 0.7 inches, up to about 0.6 inches, or up to about 0.5 inches. Not wishing to be bound by theory, systems with packed-bed reactors can improve the production of bacterial isolates or other microorganisms. In some embodiments, a supportless reactor (i.e., a reactor that is not a packed-bed reactor) improves the production of bacterial isolates or other microorganisms or improves the digestion of digestible substrates.

[0150] In some embodiments, the reactor may include a fluidized bed reactor. In a fluidized bed reactor, solid particles can circulate within the working fluid of the reactor, thereby providing a surface for microbial colonization. Such particles may include, for example, particles of inorganic substrates such as phosphate rock particles. In some embodiments, the fluidized bed reactor may be of uniform volume. In some embodiments, the fluidized bed reactor may be of varying volumes. In some embodiments, the fluidized bed reactor increases the homogeneity of particles mixed within the digestion system. The solid material in the fluidized bed reactor may have inherent fluid-like properties and allow for more complete mixing. Reducing or eliminating radial and axial concentration gradients can provide better fluid-solid contact and achieve better homogeneity of particle mixing. In some embodiments, the fluidized bed reactor increases the homogeneity of temperature gradients within the digestion system. Without being bound by theory, the open container of the fluidized bed reactor can provide a reduction in isolated hot or cold spots that may occur during mixing of the working fluid within the container. Hot spots can be areas of localized temperature variation during heating of the working fluid. Cold spots can be areas of localized temperature variation during cooling of the working fluid. The reduction in isolated temperature variations due to the configuration of the fluidized bed reactor can provide a more uniform temperature distribution of the fluid.

[0151] The flow rate of the digestion system can be selected to allow sufficient residence time in each reactor for the formation of stable and unique microbial aggregates within each reactor. In some embodiments, the working fluid in each reactor is continuously recycled at a rate ratio in the range of approximately 25:1 to 35:1, 25 to 35 gallons / minute, compared to a hydraulic feed rate of 1 gallon / minute. In some embodiments, the 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 two of these values. The working fluid can be recycled via pump to prevent solid sedimentation, and sufficient flow rate and hydraulic shearing are provided to prevent excessive accumulation and detachment of biofilm. The digestion system described herein may include a first flow rate, a second flow rate, a third flow rate, a fourth flow rate, a fifth flow rate, a sixth flow rate, or a seventh flow rate.

[0152] The reactor can be maintained at a specific temperature, which can facilitate the digestion and growth of microbial aggregates within the system. In some embodiments, the reactor temperature 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, the reactor temperature 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, the reactor temperature is from about 15°C to about 45°C. In some embodiments, the reactor temperature is from about 15°C to about 20°C, from about 15°C to about 22°C, from about 15°C to about 24°C, from about 15°C to about 26°C, from about 15°C to about 28°C, from about 15°C to about 30°C, from about 15°C to about 32°C, from about 15°C to about 34°C, from about 15°C to about 36°C, from about 15°C to about 40°C, from about 15°C to about 45°C, from about 20°C to about 22°C, from about 20°C to about 24°C, from about 20°C to about 26°C, from about 20°C to about 28°C, from about 20°C to about 20°C. ℃ to about 30℃, about 20℃ to about 32℃, about 20℃ to about 34℃, about 20℃ to about 36℃, about 20℃ to about 40℃, about 20℃ to about 45℃, about 22℃ to about 24℃, about 22℃ to about 26℃, about 22℃ to about 28℃, about 22℃ to about 30℃, about 22℃ to about 32℃, about 22℃ to about 34℃, about 22℃ to about 36℃, about 22℃ to about 40℃, about 22℃ to about 45℃, about 24℃ to about 26℃, about 24℃ to about 28℃, about 2 4°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] The reactor can be maintained under aerobic, microaerobic, or anaerobic conditions. A series of reactors in a digestion system can have different aerobic conditions. A series of reactors in a digestion system can have the same aerobic conditions. In some embodiments, the reactor can have the same aerobic conditions as adjacent reactors. In some embodiments, the reactor can have different aerobic conditions than adjacent reactors. In some embodiments, the digestion system can have aerobic, microaerobic, anaerobic conditions, or any combination thereof.

[0155] In some embodiments, the aerobic conditions include dissolved oxygen measurements greater than 2 mg / L. In some embodiments, the aerobic conditions include dissolved oxygen measurements of at least about 2 mg / L, at least about 3 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, the aerobic conditions include dissolved oxygen measurements of about 2 mg / L to about 15 mg / L. In some embodiments, the aerobic conditions include 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. 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 4 mg / 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 7 mg / L, about 5 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 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 12 mg / L, about 6 mg / L to about 14 mg / L, about 6 mg / L to about 15 mg / L, about 7 mg / Lmg / 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 Conditions with dissolved oxygen measurements ranging from approximately 15 mg / L to approximately 10 mg / L. In some embodiments, aerobic conditions include dissolved oxygen measurements ranging from 2 mg / L to 10 mg / L. In some embodiments, microaerobic conditions include dissolved oxygen measurements less than 2 mg / L. In some embodiments, micro-oxygen conditions include dissolved oxygen measurements of up to about 1.99 mg / L, up to about 1.8 mg / L, up to about 1.6 mg / L, up to about 1.5 mg / L, up to about 1.4 mg / L, up to about 1.3 mg / L, up to about 1.2 mg / L, up to about 1.1 mg / L, up to about 1 mg / L, up to about 0.9 mg / L, up to about 0.8 mg / L, up to about 0.7 mg / L, up to about 0.6 mg / L, up to about 0.5 mg / L, up to about 0.4 mg / L, up to about 0.3 mg / L, up to about 0.2 mg / L, up to about 0.1 mg / L, or less than about 0.1 mg / L but not 0 mg / L. In some embodiments, micro-oxygen conditions include dissolved oxygen measurements of about 0.1 mg / L to about 1.99 mg / L. In some embodiments, microaerobic conditions include 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, and about 0.2 mg / L to about 0.3 mg / L.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 1 mg / 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. 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. 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. mg / L, approximately 1.4The anaerobic conditions include dissolved oxygen measurements of approximately 1.6 mg / L to approximately 1.8 mg / L, approximately 1.6 mg / L to approximately 1.99 mg / L, or approximately 1.8 mg / L to approximately 1.99 mg / L. In some embodiments, the anaerobic conditions include dissolved oxygen measurements of 0 mg / L.

[0156] The reactor can be maintained at different pH levels within the digestive system. The reactor can also be maintained at the same pH level within the digestive system. The pH of the reactor within the digestive system can be 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 of the reactor within the digestive system can 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, or at most about 4.0.

[0157] In some embodiments, the pH of the reactor in the digestive system can be from about 3 to about 9. In some embodiments, the pH of the reactor in the digestive system can be from about 3 to about 3.5, from about 3 to about 4, from about 3 to about 4.5, from about 3 to about 5, from about 3 to about 5.5, from about 3 to about 6, from about 3 to about 6.5, from about 3 to about 7, from about 3 to about 7.5, from about 3 to about 8, from about 3 to about 9, from about 3.5 to about 4, from about 3.5 to about 4.5, from about 3.5 to about 5, from about 3.5 to about 5.5, from about 3.5 to about 6, from about 3.5 to about 6.5, from about 3.5 to about 7, from about 3.5 to about 7.5, from about 4 to about 8, from about 4 to about 9, from about 4.5 to about 5, from 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 reactor may include a distribution component (e.g., a distribution ring) beneath the surface of the discharge volume. The distribution component may reduce the amount of surface damage and / or maintain an anaerobic environment within the reactor.

[0159] D. Working fluids and microbial aggregates The working fluid may include a fluid substance that moves through the digestive system described herein. The working fluid may contain solid components, liquid components, gaseous components, or any combination thereof. The working fluid may contain microbial aggregates, isolated microorganisms or inoculum of microbial strains (e.g., target isolates), additional organic and / or materials, or any combination thereof. The mixture of microbial aggregates, isolated microorganisms (e.g., target isolates), and additional organic and / or materials within the working fluid may allow for the amplification of microorganisms or serve as a growth medium for inoculum of microorganisms. The working fluid may include pH, viscosity, temperature, surface tension, adhesion, and / or volume that enhance the growth and / or function of microorganisms or microbial cells. In some embodiments, the volume of working fluid in each reactor may be continuously replenished and withdrawn. In some embodiments, the volume of working fluid in each reactor may be replenished and withdrawn in batches (e.g., intermittently). In some embodiments, the working fluid in a first reactor may include a first working fluid. In some embodiments, the working fluid in a second reactor may include a second working fluid. In some embodiments, the working fluid in a third reactor may include a third working fluid. In some embodiments, the working fluid in a fourth reactor may include a fourth working fluid. In some embodiments, the working fluid in the fifth reactor may include 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 transferred to the fifth reactor. In some embodiments, the working fluids may be mixed in a reactor (e.g., a chamber or vessel) preceding the first reactor. In some embodiments, the working fluid in each reactor may be different from the working fluids in other reactors within the digestion system. Different working fluids may contain different microbial communities. Different microbial communities may include different microorganisms (e.g., bacteria, fungi, and / or algae). Different working fluids may contain different concentrations of target isolates. Different working fluids may contain different concentrations of carbon and / or nitrogen sources. Different working fluids may contain different microbial communities, different concentrations of target isolates, different concentrations of carbon sources, different combinations of nitrogen sources, or any combination thereof. The working fluids in the reactors of the digestion system may be similar to the working fluids in the different reactors of the digestion system.

[0160] The working fluid within each reactor can contain different enzymes, which can be produced by microorganisms within the working fluid. Enzymes in the working fluid may include: dehydrogenases, hydrogenases, oxidases, catalases, peroxidases, phenol hydroxylases, glucan sucrase, aminotransferases, thiocyanates, carboxylesterases, lipases, phosphatases, nucleases, phytases, arylsulfatases, amylases, cellulases, inulinases, xylanases, glucanases, fructanases, polygalacturonases, glucosidases, galactosidases, invertases, peptidases, asparaginases, glutaminases, amidases, ureases, aspartate decarboxylases, glutamate decarboxylases, aromatic amino acid decarboxylases, or any combination thereof. The enzymes in the working fluid may include nitrogenase, 1-aminocyclopropane-1-carboxylic acid deaminase (e.g., ACC-deaminase), quinone protein glucose dehydrogenase (e.g., PQQ or quinone), gluconic acid 2-dehydrogenase, cellulase, endo-1,3(4)-β-glucanase, and / or pectin lyase. The working fluid in each reactor may contain different concentrations of enzymes. The working fluid in each reactor may contain enzymes with different average abundances. Enzymes may be present at an average abundance of 0.001% to 1%. Enzymes may be present at an average abundance of less than 0.001%. Enzymes may be present at an average abundance of greater than 1%. In some embodiments, the enzyme may be present at an average abundance of at least about, and 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 any two of these values. The working fluid within each reactor may contain enzymes with different enzymatic activities. Enzyme activities may include, but are not limited to, nitrogen fixation, ammonia production, phosphate solubilization, zinc solubilization, and / or cell wall lysis.

[0161] The working fluid may contain different digestion products from working fluids in other reactors within the system. In some embodiments, the working fluid may contain digestion products from feedstocks (e.g., aqueous organic feedstocks) and microbial aggregates at least partially derived from previous working fluids.

[0162] The pH of the working fluid in each reactor may differ from that in the other reactors. The pH of the working fluid in each reactor may be the same. The pH of the working fluid may be less than 6. The pH of the working fluid may be greater than 6. The pH of the working fluid may range from 2 to 11. The pH of the working fluid may be at least about, and 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 two of these values.

[0163] In some embodiments, a first microbial aggregate may be established in a mixing chamber, wherein various inputs can be mixed into a homogeneous aqueous mixture before being fed into the digestion reactor. In some embodiments, the digestion system described herein may include one or more mixing chambers in which microbial aggregates can be established. The mixing chamber may be supplied with an input or a series of inputs (e.g., a target microbial strain, aqueous feedstock, carbon source, nitrogen source, nutrients, water, or any combination thereof), which may be mixed before being fed into a first container (e.g., a first reactor). The first microbial aggregate may be derived from microorganisms originally present in one or more digestion substrates and / or from other inputs entering the mixing chamber. The first microbial aggregate may be derived from inputs fed into the reactor of the digestion system described herein. Inputs may come from whole broth (WB) of another digestion system (e.g., water-based phosphate solubilization technology (PwST) or phosphate solubilization technology (PST)). In some embodiments, a second microbial aggregate is established in the first reactor. The second microbial aggregate may be derived from microorganisms in the mixing chamber. In some embodiments, a third microbial aggregate is established in the second reactor. A third microbial aggregate may be derived from a first working fluid present in the first reactor and transferred to the second reactor. In some embodiments, a fourth microbial aggregate is established in the third reactor. A fourth microbial aggregate may be derived from a second working fluid present in the second reactor and transferred to the third reactor. In some embodiments, a fifth microbial aggregate is established in the fourth reactor. A fifth microbial aggregate may be derived from a fourth working fluid present in the fourth reactor and transferred to the fifth reactor. Microbial aggregates may be present in any reactor of the digestive system described herein. Microbial aggregates may be derived from the working fluid of the reactor of the digestive system described herein. A first microbial aggregate may be derived from the input to the first reactor and may be present in the basic products of the digestive system. A first microbial aggregate may be present in the first reactor, the second reactor, the third reactor, or a clarifier. Without being bound by theory, a first microbial aggregate in the working fluid may transform its microbial community and form a second microbial aggregate. A second microbial aggregate may be present in the first reactor, the second reactor, the third reactor, or a clarifier. Without being bound by theory, a second microbial aggregate in the working fluid may transform its microbial community and form a third microbial aggregate. A third microbial aggregate can exist in the first, second, and third reactors, or in the clarifier. Without being bound by theory, the third microbial aggregate in the working fluid can transform its microbial community and form a fourth microbial aggregate. The fourth microbial aggregate can exist in the first, second, and third reactors, or in the clarifier.Without being bound by theory, a fourth microbial aggregate in the working fluid can transform its microbial community and form a fifth microbial aggregate. This fifth microbial aggregate can exist in the first, second, third, or clarifier reactors. The microbial aggregates of the digestive system described herein can undergo microbial community transformation based on the reactor and working fluid conditions of the system (e.g., nutrients, residence time, flow rate, pH, oxygen content, digestion products). Without being bound by theory, as the working fluid incubates in the reactors of the bioreactor system described herein, the microorganisms in the microbial aggregates can become enriched, maintained, or die, thereby transforming the microbial community in the working fluid and establishing new microbial aggregates.

[0164] A portion of the first working fluid can be transferred to the second container of the tandem series container assembly of the digestive system described herein. The working fluid in the second container may contain the second working fluid. A portion of the second working fluid can be transferred to the third container of the tandem series container assembly of the digestive system described herein. The working fluid in the third container may contain the third working fluid. A portion of the third working fluid can be transferred to the fourth container of the tandem series container assembly of the digestive system described herein. The working fluid in the fourth container may include the fourth working fluid. A portion of the fourth working fluid can be transferred to the fifth container of the tandem series container assembly of the digestive system described herein. The working fluid in the fifth container may include the fifth working fluid. A portion of the fifth working fluid can be transferred to the sixth container of the tandem series container assembly of the digestive system described herein. The working fluid in the sixth container may include the sixth working fluid.

[0165] The working fluid in the containers of the digestive system can be incubated within those containers. The flow rate of the digestive system can increase or decrease the volume of the working fluid. The volume of the working fluid in the first, second, third, fourth, fifth, or sixth containers can increase over a time period. The volume of the working fluid in the first, second, third, fourth, fifth, or sixth containers can decrease over a time period. The volume of the working fluid in the first, second, third, fourth, fifth, or sixth containers can remain unchanged over a time period. The volume of the working fluid in each container of the digestive system can be the same. The volume of the working fluid in each container of the digestive system can be different. The volume of the first, second, third, fourth, fifth, and / or sixth working fluid can be constant (e.g., does not change over a time period). A constant volume can include a volume that does not increase or decrease over 1 minute, 5 minutes, 10 minutes, 30 minutes, 1 hour, 5 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, or 1 week.

[0166] As the working fluid flows from the various containers of the digestion system, the working fluid may include flow rates. A first flow rate may include the flow rate of the aqueous feedstock input from an external source into the first container. A second flow rate may include the flow rate of the working fluid flowing from the first container into the second container. A third flow rate may include the flow rate of the working fluid flowing from the second container into the third container. A fourth flow rate may include the flow rate of the working fluid flowing from the third container into the fourth container. A fifth flow rate may include the flow rate of the working fluid flowing from the fourth container into the fifth container. The flow rate (e.g., first flow rate, second flow rate, third flow rate, fourth flow rate, fifth flow rate) can 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] Microbial aggregates can contain communities of microorganisms. Microbial communities can be generated from inputs to a digestive system. Aqueous feedstocks input into a digestive system can contain microbial aggregates. Incubation with working fluids within the digestive system can promote the growth of microorganisms within the microbial aggregate (e.g., a first, second, third, fourth, fifth, or sixth microbial aggregate). The microorganisms in the microbial aggregate, or at least a portion of the microorganisms within the microbial aggregate, can possess desired plant growth-promoting properties. Plant growth-promoting properties can include aboveground biomass, root biomass, nutrient uptake, crop yield, zinc solubilization, acid production, leaf area, chlorophyll content, photosynthetic activity, or total biomass.

[0168] The reactors of the digestion system can be fluidly connected. A portion of the working fluid in a first container can be transferred to a second container, which is also fluidly connected. A portion of the working fluid in a second container can be transferred to a third container, which is also fluidly connected. A portion of the working fluid in a third container can be transferred to a fourth container, which is also fluidly connected. A portion of the working fluid in a fourth container can be transferred to a fifth container, which is also fluidly connected. A portion of the working fluid in a fifth container can be transferred to a sixth container, which is also fluidly connected. In some embodiments, the transfer of working fluid between containers of the digestion system described herein can be continuous. Continuous flow of the working fluid can include uninterrupted flow of the working fluid, or flow with interruptions of 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. Continuous flow of the working fluid within the digestion system described herein (e.g., between containers of the digestion system) can have a first flow rate. Continuous flow of the working fluid within the digestion system described herein (e.g., between containers of the digestion system) can have a second flow rate. In some embodiments, the first and second flow rates are equal. The first flow rate may include the flow rate of fluid transferred from an external source of the digestion system into the first container. The second flow rate may include the flow rate of fluid flowing from the first container into the second container. In some embodiments, the amount of working fluid and / or feedstock (e.g., aqueous organic feedstock) transferred into the first container during a time period is equal to the amount of working fluid and / or feedstock (e.g., aqueous organic feedstock) transferred into the fluidly connected second container during the same time period. In some embodiments, the first flow rate and the second flow rate are different. In some embodiments, the first flow rate may be faster than the second flow rate. In some embodiments, the first flow rate may be slower than the second flow rate. For example, the flow rates may be different if the input flow entering the first reactor is faster or slower than the working fluid flow transferred from the first reactor to the second reactor. This difference in flow rate can result in different volumes of working fluid in the containers of the digestion system.

[0169] In some embodiments, the first container of the digestive system comprises a constant volume. In some embodiments, the volume of the first container of the digestive system varies over time. In some embodiments, the flow rate between the containers of the digestive system can maintain a constant volume in each container. The first, second, third, fourth, fifth, and / or sixth containers can be maintained at a constant volume. A constant volume can be maintained by continuous flow of fluid via the digestive system described herein. For example, similar flow rates of working fluid transferred between containers of the digestive system can result in a constant volume of working fluid in the containers. This continuous flow in the bioreactor system can facilitate the enrichment of microbial communities with targeted functionalities (e.g., zinc solubilization) by preventing the working fluid in the containers of the system from overflowing or being depleted.

[0170] In some embodiments, the digestive system may be inoculated with an inoculum of microorganisms (e.g., an inoculum of a microbial strain). The inoculum of a microbial strain may be an isolated microorganism. Isolated microorganisms may include microorganisms that grow or accumulate outside the natural environment (e.g., in a culture medium or streak plate). In some embodiments, the inoculum of microorganisms may include a mixture of multiple isolated microorganisms. The inoculum of microorganisms may include a mixture of at least about two, at least three, at least four, at least five, at least six, at least seven, at least eight, or more isolated microorganisms.

[0171] In some cases, the digestive system may not need to be inoculated with isolates (e.g., microbial strains) or combinations of isolates after the initial inoculation. Re-inoculation of the digestive system may include providing microbial strains after the previous inoculation. Re-inoculation of the digestive system may include introducing the target isolate (e.g., microbial strain) into the container of the digestive system at a point in time during the operation of the digestive system. In some cases, the digestive system may be re-inoculated with isolates or combinations 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, the digestive system may be inoculated with isolates or combinations of isolates on day 1 of the digestion process, and then inoculated 1, 2, 3, 4, 5, or more times after day 1 of the digestion process. In some cases, the digestive system may be re-inoculated with the microbial strains described herein after the digestive system has been operated on during a time period. For example, after operating the digestive system for at least about, at most about, or for a duration of 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 two of these values, the digestive system may be re-inoculated with the microbial strains described herein.

[0172] In some implementation schemes, the vaccination concentration is at least, at most, or about 1x10⁻⁶. 2 0.5x10 3 1x10 3 0.5x10 4 1x10 4 0.5x10 5 1x10 5 0.5x10 6 The initial population of target isolates, CFU / ml, or any two of these values ​​within a range.

[0173] In some embodiments, the population of microbial strains (e.g., the concentration of microbial strains) is maintained (e.g., retained) at least about 0.00001%, at least about 0.0001%, 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%. Biosolids (e.g., flocculants) may contain small particles from the working fluid of the digestive system. Biosolids (e.g., flocculants) may accumulate in the clarifier chamber over time and separate from the supernatant (e.g., the base product). In some embodiments, the population of microbial strains may be retained in the flocculants (e.g., biosolids) of the digestive system. In some embodiments, a major portion (95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%) of the microbial inoculum may be retained in the flocculants (e.g., biosolids) of the digestive system. The flocculants may be generated at any point during operation of the bioreactor system described herein. For example, the flocculants may be generated in the reactor of the bioreactor system (e.g., the first, second, third, fourth, fifth, sixth, or any other container of the system). For example, the flocculants may be generated in the clarifier chamber of the bioreactor system. In some embodiments, the flocculants may contain at least a portion of the zinc-solubilizing microorganisms generated in the digestive system described herein.

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

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

[0176] Incubation of microbial aggregates in a digestive system under selective pressure (e.g., a zinc source) can enrich microbial communities with plant growth-promoting properties (e.g., zinc-solubilizing microorganisms). Incubation of the inoculum and / or the zinc-solubilizing microorganisms of the microbial aggregates can produce metabolites with desired plant growth-promoting properties (e.g., zinc-solubilizing metabolites). The inoculum, the zinc-solubilizing microorganisms of the microbial aggregates, the zinc-solubilizing metabolites, or any combination thereof can be a zinc solubilizer in the working fluid of the system and / or in the output products (e.g., basal products) of the digestive system. The concentration of the zinc solubilizer can be increased throughout the residence time of the digestive system described herein. The concentration of the zinc solubilizer can be increased from the first container of the digestive system to the second container. The concentration of the zinc solubilizer can be increased from the first container of the digestive system to the third container. The concentration of the zinc solubilizer can be increased from the first container of the digestive system to the fourth container. The concentration of the zinc solubilizer can be increased from the first container of the digestive system to the fifth container. The concentration of the zinc solubilizer can be increased from the first container of the digestive system to the sixth container. In some implementations, the output product (e.g., the base product) may have a higher concentration of zinc solubilizer than the concentration of zinc solubilizer in the first container of the digestion system.

[0177] In some embodiments, the concentration of the zinc solubilizer can be increased from the first container of the digestive system described herein to the second, third, fourth, fifth, and / or sixth containers. In some embodiments, the concentration of the zinc solubilizer in the second, third, fourth, fifth, and / or sixth containers can be at least about 50x, at least about 100x, 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 800x, at least about 900x, at least about 1000x, at least about 1250x, at least about 1500x, at least about 2000x, or greater than about 2000x, the concentration of the zinc solubilizer in the first container of the digestive system described herein. In some embodiments, the concentration of zinc solubilizer in the second, third, fourth, fifth, and / or sixth containers may be up to about 2000x, up to about 1500x, up to about 1250x, up to about 1000x, up to about 900x, up to about 800x, up to about 700x, up to about 600x, up to about 500x, up to about 400x, up to about 300x, up to about 250x, up to about 200x, up to about 100x, up to about 50x, or less than about 50x, the concentration of zinc solubilizer in the first container of the digestive system described herein.

[0178] In some embodiments, the concentration of zinc solubilizer in the second, third, fourth, fifth, and / or sixth containers may be about 25x to about 2,500x the concentration of zinc solubilizer in the first container of the digestive system described herein.In some embodiments, the concentration of the zinc solubilizer in the second, third, fourth, fifth, and / or sixth containers may be approximately 25x to 50x, approximately 25x to 100x, approximately 25x to 150x, approximately 25x to 200x, approximately 25x to 250x, approximately 25x to 500x, approximately 25x to 750x, approximately 25x to 1,000x, approximately 25x to 1,500x, approximately 25x to 2,000x, approximately 25x to 2,500x, approximately 50x to 100x, approximately 50x to 150x, approximately 50x to 200x, or approximately 50x to 200x, the concentration of the zinc solubilizer in the first container of the digestive system described herein. About 50x to about 250x, about 50x to about 500x, about 50x to about 750x, about 50x to about 1,000x, about 50x to about 1,500x, about 50x to about 2,000x, about 50x to about 2,500x, about 100x to about 150x, about 100x to about 200x, about 100x to about 250x, about 100x to about 500x, about 100x to about 750x, about 100x to about 1,000x, about 100x to about 1,500x, about 100x to about 2,000x, about 100x to about 2,500x, about 150x to about 200x, about 150x to about 250x, about 150x to about 5 00x, about 150x to about 750x, about 150x to about 1,000x, about 150x to about 1,500x, 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 1,000x, about 200x to about 1,500x, 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 1,000x, about 250x to about 1,500x, about 250x to about 2,000x, about 2 50x to about 2,500x, about 500x to about 750x, about 500x to about 1,000x, about 500x to about 1,500x, about 500x to about 2,000x, about 500x to about 2,500x, about 750x to about 1,000x, about 750x to about 1,500x, about 750x to about 2,000x, about 750x to about 2,500x, about 1,000x to about 1,500x, about 1,000x to about 2,000x, about 1,000x to about 2,500x, about 1,500x to about 2,000x, or about 2,000x to about 2,500x.

[0179] In some implementations, after incubation, the absolute population of the microbial inoculum of the digestive 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%. In some implementations, after the residence time incubated in the digestive 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), the absolute population of the microbial inoculum in the digestive 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%). In some implementations, after incubation, without the inoculation of additional microorganisms, the absolute population of the microbial inoculum for the digestive 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%.

[0180] In some embodiments, the concentration of the zinc solubilizer relative to the total bacterial count can be increased between containers of the fluidly connected digestion system. In some embodiments, the concentration of the zinc solubilizer relative to the total bacterial count can be increased from a first container of the digestion system described herein to a second container. In some embodiments, the concentration of the zinc solubilizer relative to the total bacterial count can be increased from a first container of the digestion system described herein to a third container. In some embodiments, the concentration of the zinc solubilizer relative to the total bacterial count can be increased from a first container of the digestion system described herein to a fourth container. In some embodiments, the concentration of the zinc solubilizer relative to the total bacterial count can be increased from a first container of the digestion system described herein to a fifth container. In some embodiments, the concentration of the zinc solubilizer relative to the total bacterial count can be increased from a first container of the digestion system described herein to a sixth container.

[0181] Since the microorganisms described in this article solubilize insoluble zinc sources, zinc solubilization can lead to zinc ion (Zn) formation. 2+ The production of zinc. Measurement of zinc solubilization and the zinc solubilization capacity of microorganisms can be achieved by increasing the amount of zinc ions (Zn) that become soluble. 2+ The amount of zinc in the working solution. In some embodiments, the zinc solubilizing ability of the working solution (e.g., as the amount of Zn that becomes soluble)2+ (mg / L) can be increased in the digestive system provided herein. In some embodiments, the zinc solubilizing capacity of the working solution in the second, third, or fourth container of the digestive system (e.g., as Zn becomes soluble) 2+ The zinc solubilization capacity (mg / L) of the working solution in the first container can be at least about 50x, at least about 75x, at least about 100x, at least about 150x, at least about 200x, at least about 250x, at least about 300x, at least about 350x, at least about 400x, at least about 450x, at least about 500x, at least about 600x, at least about 700x, at least about 800x, at least about 900x, at least about 1000x, or greater than about 1000x. In some embodiments, the zinc solubilization capacity (e.g., as Zn becomes soluble) of the working solution in the second, third, or fourth container of the digestive system... 2+ The zinc solubilization capacity (mg / L) of the working solution in the first container can be up to about 1,000x, up to about 900x, up to about 800x, up to about 700x, up to about 600x, up to about 500x, up to about 450x, up to about 400x, up to about 350x, up to about 300x, up to about 250x, up to about 200x, up to about 150x, up to about 100x, up to about 75x, up to about 50x, or less than about 50x.

[0182] In some implementations, the working solution can be obtained from the reactor of the digestion system and subjected to zinc solubilization (e.g., quantified as Zn becoming soluble). 2+ (mg / L) test. In some embodiments, the working solution of the digestive system described herein may have 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 of soluble Zn. 2+Measured values. In some embodiments, the working solution of the digestive system described herein may have up to about 800 mg / L, up to about 700 mg / L, up to about 600 mg / L, up to about 500 mg / L, up to about 450 mg / L, up to about 400 mg / L, up to about 350 mg / L, up to about 300 mg / L, up to about 250 mg / L, up to about 200 mg / L, up to about 150 mg / L, up to about 100 mg / L, up to about 50 mg / L, or less than about 50 mg / L of soluble Zn. 2+Measurement values. In some embodiments, the working solution of the digestive system described herein may have a measured value of soluble Zn2+ ranging from about 20 mg / L to about 750 mg / L. In some embodiments, the working solution for the digestive system described herein may have a concentration of 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. 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 / L to about 250 mg / L, about 150 mg / L to about 30 ...150 mg / L to about 300 mg / L, about 150 mg / L to about 150 mg / L to about 300 mg / L, about 150 mg / 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.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 Measured values ​​of soluble Zn2+ at approximately 450 mg / L to approximately 500 mg / L, approximately 450 mg / L to approximately 750 mg / L, or approximately 500 mg / L to approximately 750 mg / L.

[0183] Microbial populations can be measured using methods including, but not limited to, spectrophotometers, cell counting, turbidity measurement, hemocytometers, electronic enumeration, cell mass measurement, cell viability measurement, CFU morphology on universal nutrient media or selective and / or differentiation media, PCR, semi-quantitative PCR, and qPCR.

[0184] Zinc-solubilizing microorganisms can be identified using either agar plates or liquid test solutions. Microorganisms can be tested on agar plates containing insoluble zinc, and their zinc-solubilizing capacity (e.g., the amount of zinc that becomes soluble) is typically measured by the diameter of the clear zone surrounding the microbial colony on the agar plate after incubation, classifying microorganisms as capable of zinc solubilization. The larger this diameter, the more zinc is solubilized by the microbial colony. Microorganisms can also be tested in liquid solutions containing insoluble zinc to measure their zinc-solubilizing capacity (by measuring the amount of zinc that becomes soluble after incubation). Microorganisms are inoculated into a liquid zinc medium containing an insoluble zinc source. After incubation, the solubilized zinc is measured as Zn²⁺ using instruments such as inductively coupled plasma (ICP) atomic emission spectrometry.

[0185] The addition of a target isolate (e.g., a microbial strain / inoculum) can have the additional benefit of improving the target functionality of the working solution in the reactor of the digestion system and / or the output base product of the digestion system. In some embodiments, the addition of a microbial strain inoculum can improve the zinc solubility of the working fluid and base product of the digestion system compared to the working fluid and base product of a digestion system without a microbial strain inoculum.

[0186] In some embodiments, compared to a working solution and / or basal product of the digestive system without inoculation of the target isolate, the working solution and / or basal product of the digestive system inoculated with the target isolate may have 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% Zn. 2+ Increased concentration. In some embodiments, compared to the working solution and / or basal product of the target isolate inoculated in the digestive system, the working solution and / or basal product of the target isolate inoculated in the digestive system may have up to about 80%, up to about 75%, up to about 70%, up to about 65%, up to about 60%, up to about 55%, up to about 50%, up to about 45%, up to about 40%, up to about 35%, up to about 30%, up to about 25%, up to about 20%, up to about 15%, or less than about 15% Zn. 2+ Concentration increases.

[0187] In some embodiments, the working solution and / or basal product of the target isolate inoculated into the digestive system may have about 10% to about 80% Zn content, compared to a working solution and / or basal product of the digestive system without inoculation of the target isolate. 2+Increased concentration. In some embodiments, compared to the working solution and / or basal product of the target isolate inoculated in the digestive system, the working solution and / or basal product of the target isolate inoculated in the digestive system may have a concentration of 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 Approximately 20%, approximately 15% to approximately 25%, approximately 15% to approximately 30%, approximately 15% to approximately 35%, approximately 15% to approximately 40%, approximately 15% to approximately 45%, approximately 15% to approximately 50%, approximately 15% to approximately 60%, approximately 15% to approximately 70%, approximately 15% to approximately 80%, approximately 20% to approximately 25%, approximately 20% to approximately 30%, approximately 20% to approximately 35%, approximately 20% to approximately 40%, approximately 20% to approximately 45%, approximately 20% to approximately 50%, approximately 20% to approximately 60%, approximately 20% to approximately 70%, approximately 20% to approximately 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%, Zn of approximately 35% to approximately 60%, approximately 35% to approximately 70%, approximately 35% to approximately 80%, approximately 40% to approximately 45%, approximately 40% to approximately 50%, approximately 40% to approximately 60%, approximately 40% to approximately 70%, approximately 40% to approximately 80%, approximately 45% to approximately 50%, approximately 45% to approximately 60%, approximately 45% to approximately 70%, approximately 45% to approximately 80%, approximately 50% to approximately 60%, approximately 50% to approximately 70%, approximately 50% to approximately 80%, approximately 60% to approximately 70%, approximately 60% to approximately 80%, or approximately 70% to approximately 80% 2+ Concentration increases.

[0188] A digestive system can be inoculated with microbial strains at the beginning of the digestive system, thereby allowing the microbial strains to flow through the working fluid of the system and reactor. Without being bound by theory, a digestive system inoculated with microbial strains may increase the zinc solubility of the working fluid and / or base product compared to a digestive system with the same aspects as others, without inoculation of microbial strains. Without being bound by theory, a digestive system inoculated with microbial strains may increase the zinc solubility of the working fluid and / or base product compared to a digestive system with the same aspects as others, with microbial strains added (e.g., co-added) at the end of the system. Without being bound by theory, a digestive system inoculated with microbial strains may increase the zinc solubility of the base product compared to a digestive system with the same aspects as others, with microbial strains added (e.g., co-added) at the end of the system. Incubation of microbial strains in the digestive system may enrich the working fluid with zinc-soothing microorganisms and / or produce zinc-soothing metabolites.

[0189] E. Microbial isolates Certain microorganisms disclosed herein possess all the identification characteristics of deposited strains, specifically those characteristics that promote plant growth and / or yield, as described herein. Specifically, certain microorganisms of this disclosure may refer to the deposited microorganisms described herein and strains derived therefrom.

[0190] Zinc is an essential micronutrient for plant growth and development. It acts as an antioxidant in plants and is important in carbohydrate metabolism, auxin metabolism, and other plant growth-promoting properties. Small amounts of zinc exist in the soil as soluble zinc, while the remainder exists as insoluble minerals and other complexes. Zinc-solubilizing microorganisms can solubilize zinc through acidification, secretion of organic acids, vitamins, plant hormones and amino acids, redox systems, chelate production, proton expulsion, or other biofortification mechanisms. In some embodiments, zinc-solubilizing microbial strains (including compositions containing such strains) and methods for promoting plant growth using such strains are disclosed herein.

[0191] In some embodiments, the microbial strain is derived from the genus *Bacillus*. In some embodiments, the microbial strain is derived from the species *Bacillus safranin*. In some embodiments, the microbial strain is derived from a bacterial species other than *Bacillus safranin*. In some embodiments, the *Bacillus safranin* strain is a clone deposited with or isolated from ATCC accession number PTA-127681. In some embodiments, the 16S rRNA gene of the microbial strain includes the nucleotide sequence of SEQ ID NO.: 1. In some embodiments, the microbial strain includes a 16S rRNA gene comprising a nucleotide sequence exhibiting 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 with the nucleotide sequence shown in SEQ ID NO.: 1. In some embodiments, the 16S rRNA gene of the microbial strain comprises a nucleotide sequence exhibiting 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 with the nucleotide sequence shown 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 the gyrB gene, which comprises a nucleotide sequence exhibiting 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 with the nucleotide sequence shown in SEQ ID NO: 4. In some embodiments, the rpoB gene of the microbial strain includes the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the microbial strain includes the rpoB gene, which comprises a nucleotide sequence exhibiting 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 with the nucleotide sequence shown in SEQ ID NO: 7.Some implementations provide microbial genera that promote plant growth, including any of the DNA sequences described herein, and enhance plant health, growth, and / or yield, as described herein.

[0192] In some embodiments, the microbial strain is derived from the *Bacillus megaterium* species. In some embodiments, the microbial strain is derived from a bacterial species other than *Bacillus megaterium*. In some embodiments, the *Bacillus megaterium* strain is a clone deposited with or isolated from ATCC accession number PTA-127683. In some embodiments, the 16S rRNA gene of the microbial strain includes the nucleotide sequence of SEQ ID NO.: 2. In some embodiments, the microbial strain includes a 16S rRNA gene comprising a nucleotide sequence exhibiting 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 with the nucleotide sequence shown in SEQ ID NO.: 2. In some embodiments, the 16S rRNA gene of the microbial strain comprises a nucleotide sequence exhibiting 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 with the nucleotide sequence shown 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 the gyrB gene, which comprises a nucleotide sequence exhibiting 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 with the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the rpoB gene of the microbial strain includes the nucleotide sequence of SEQ ID NO: 8. In some embodiments, the microbial strain includes the rpoB gene, which comprises a nucleotide sequence exhibiting 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 with the nucleotide sequence shown in SEQ ID NO: 8.Some implementations provide microbial genera that promote plant growth, including any of the DNA sequences described herein, and enhance plant health, growth, and / or yield, as described herein.

[0193] In some embodiments, the microbial strain is derived from the Bacillus megaterium species. In some embodiments, the microbial strain is derived from a bacterial species other than Bacillus megaterium. In some embodiments, the Bacillus megaterium strain is a clone deposited with or isolated from ATCC accession number PTA-127682. In some embodiments, the 16S rRNA gene of the microbial strain includes the nucleotide sequence of SEQ ID NO.: 3. In some embodiments, the microbial strain includes a 16S rRNA gene comprising a nucleotide sequence exhibiting 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 with the nucleotide sequence shown in SEQ ID NO.: 3. In some embodiments, the 16S rRNA gene of the microbial strain comprises a nucleotide sequence exhibiting 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 with the nucleotide sequence shown 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 that includes a nucleotide sequence exhibiting 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 with the nucleotide sequence shown in SEQ ID NO: 6. In some embodiments, the rpoB gene of the microbial strain includes the nucleotide sequence of SEQ ID NO: 9. In some embodiments, the microbial strain includes the rpoB gene, which comprises a nucleotide sequence exhibiting 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 with the nucleotide sequence shown in SEQ ID NO: 9.Some implementations provide microbial genera that promote plant growth, including any of the DNA sequences described herein, and enhance plant health, growth, and / or yield, as described herein.

[0194] F. An exemplary digestive system for producing isolates This disclosure provides a system having conditions for producing a biostimulant product that has the ability to promote plant growth (e.g., improved zinc solubility).

[0195] Zinc Solubilization Technology (ZST) System Embodiments of the systems and methods described herein produce biostimulant products that can have a multimodal approach to promoting zinc uptake in plants. The biostimulant products produced by the embodiments described herein can be used to promote plant growth by applying the product to plants and / or plant growth substrates (e.g., soil, fertilizer). In some embodiments, one mode of action of the product produced can enhance the uptake of zinc included in the product. Another mode of action can increase soil zinc and zinc uptake stimulated by microorganisms and / or microbial metabolites present in the product produced by the embodiments described herein.

[0196] In one aspect, this disclosure provides a method comprising: transferring a raw material (e.g., an aqueous organic raw material) and an inoculum of microorganisms capable of promoting zinc solubility into a first container containing a volume of a first working fluid. The aqueous organic raw material may comprise: (i) a first microbial aggregate; and / or (ii) digestion products generated by microorganisms in the first microbial aggregate digesting the organic material. In some cases, the inoculum may be incubated at a zinc ion concentration that selectively promotes microbial growth. The microbial population may increase. The microbial population may decrease.

[0197] In one aspect, this disclosure provides a method comprising (a) transferring a raw material (e.g., an aqueous organic raw material) and an inoculum of microorganisms capable of promoting zinc solubility into a first container containing a first working fluid of a given volume, wherein the raw material (e.g., the aqueous organic raw material) comprises: (i) a first microbial aggregate; and (ii) digestion products generated by the digestion of the organic material by microorganisms in the first microbial aggregate; and (b) incubating the inoculum at a zinc ion concentration that selectively promotes microbial growth, thereby increasing the microbial population. Incubation of the inoculum with respect to one or more microorganisms in the microbial aggregate of the reactor in the digestion system can increase or maintain the microbial population.

[0198] In some embodiments, incubating zinc-solubilizing microbial strains at zinc ion concentrations can selectively enhance the survival of zinc-solubilizing microorganisms. In some embodiments, incubating microbial aggregates at zinc ion concentrations can increase the proportion of zinc-tolerant microorganisms (e.g., zinc solubilizers or zinc-solubilizing microorganisms). Without being bound by theory, the addition of selective pressure with zinc ion concentrations can induce a shift in the microbial population towards microorganisms with zinc-solubilizing properties.

[0199] In some embodiments, zinc-solubilizing microbial strains (e.g., microorganisms) are capable of enhancing zinc uptake in plants or promoting soil zinc content and zinc uptake from the soil. In some embodiments, digestive system conditions (e.g., pH, nutrients, flow rate, residence time, selective pressure, carbon source, nitrogen source, or any combination thereof) can promote the growth of one or more microorganisms in a first microbial aggregate, which may be capable of promoting plant growth or zinc uptake in plants, or capable of producing metabolites that can promote plant growth or zinc uptake in plants. In some embodiments, during incubation of microorganisms or one or more microorganisms in the first microbial aggregate, metabolites capable of promoting plant growth and zinc solubilization may be produced. Metabolites may have zinc solubilizing capacity and increase the amount of zinc available to plants. Metabolites may enhance the presence of biologically available zinc ions. The increase in zinc ions after incubation with zinc-solubilizing metabolites may be due to zinc solubilization or bacterial stimulation of a zinc source (e.g., ZnO). Plant-available zinc can promote plant growth, vigor, or yield. In some embodiments, the incubation increases the population of one or more microorganisms in the microbial aggregate capable of promoting plant growth. In some embodiments, the feedstock (e.g., an aqueous organic feedstock) also includes an inorganic substrate. In some embodiments, the first microbial aggregate further comprises microorganisms derived from the inorganic substrate. In some embodiments, the inorganic substrate includes phosphate rock. In some embodiments, the one or more microorganisms may belong to the following species: *Bacillus saefoetida*, *Bacillus amyloliquefaciens*, etc. Bacillus amyloliquefaciens ), Bacillus megaterium, Pseudomonas spp. Pseudomonas spp.), Agrobacterium ( Agrobacterium spp.), Bacillus circulatoryus ( Bacillus circulans Burkholderia spp. ( ) Burkholderia lata ), Lysine Bacillus spp. Lysinibacillus spp.), Burkholderia latentis ( Burkholderia latens ), Bacillus cereus ( Bacillus cereus ), Bacillus aureus ( B. aryabhattai ), Taiwan Pseudomonas ( Pseudomonas taiwenensis Acinetobacter spp. Acinetobacter spp.), Bacillus cereus ( Paenibacillus sonchi), Chitinophytes ( Chitinophaga spp.), Staphylococcus spp. Gluconobacter spp.), genus of nitrogen-fixing bacteria ( Diazotrophicus spp.), Klebsiella spp. Klebsiella spp.), Serratia ( Serratia spp.), Thiobacillus spp. Thiobacillus(spp.) or any combination thereof. In some embodiments, the microorganism is a strain of *Bacillus sabolicus* deposited with ATCC accession number PTA-127681 (MS4666), a strain of *Bacillus megaterium* deposited with ATCC accession number PTA-127683 (MS4689), or a strain of *Bacillus megaterium* deposited with ATCC accession number PTA-127682 (MS4687). In some embodiments, the first working fluid comprises: (a) a second microbial aggregate derived from the raw material (e.g., an aqueous organic raw material), and / or (b) a digestion product produced by digestion of a substance present in the organic raw material by the first microbial aggregate and microorganisms. In some embodiments, the method further comprises transferring at least a portion of the first working fluid to a second container containing 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 aggregate derived from the first working fluid, and (b) a digestion product produced by digestion of a substance present in the first working fluid by the third microbial aggregate and microorganisms. In some embodiments, the amount of raw material (e.g., aqueous organic raw material) transferred to the first container during a time period is equal to the amount of first working fluid transferred to the second container during the same time period. In some embodiments, the amount of raw material (e.g., aqueous organic raw material) transferred to the first container during a time period is equal to the amount of first working fluid transferred to the second container during different time periods. In some embodiments, the amount of raw material (e.g., aqueous organic raw material) transferred to the first container during a time period is not equal to the amount of first working fluid transferred to the second container during the same time period. In some embodiments, the amount of raw material (e.g., aqueous organic raw material) transferred to the first container during a time period is not equal to the amount of first working fluid transferred to the second container during different time periods. In some embodiments, a constant volume of first working fluid is maintained in the first container. In some embodiments, transferring the raw material (e.g., an aqueous organic raw material) into a first container includes continuously flowing the raw material (e.g., an aqueous organic raw material) into the first container at a first flow rate, and transferring a portion of the first working fluid into a second container includes continuously flowing a portion of the first working fluid into the second container at a second flow rate, wherein the first flow rate and the second flow rate may be equal. In some embodiments, the method further includes transferring at least a portion of the second working fluid into a third container containing a third working fluid, and incubating the third working fluid in the third container. In some embodiments, the method further includes transferring at least a portion of the third working fluid into a fourth container containing a fourth working fluid, and incubating the fourth working fluid in the fourth container. In some embodiments, the first working fluid, second working fluid, third working fluid, and fourth working fluid may be maintained at a constant volume.In some embodiments, the first, second, third, and fourth working fluids may not be maintained at a constant volume. In some embodiments, the plant growth-promoting product is prepared by the method described above. In some embodiments, a method for promoting zinc solubility, which increases the bioavailable zinc in plants, includes contacting the plant and / or the culture medium in which the plant is grown with the product.

[0200] The digestion process in zinc solubilization technology (ZST) can be anaerobic. The digestion process in a ZST system can incorporate an aerobic, microaerobic, or anaerobic phase within any container of the digestion system. The digestion systems described herein can include aerobic, microaerobic, anaerobic processes, or any combination thereof.

[0201] In some embodiments, a series of reaction chambers can be used in methods for producing biostimulant products, as described herein. In some embodiments, conditions within the reactor chamber can be established to selectively promote the production of one or more microorganisms with specific desired plant growth-promoting effects.

[0202] ZST 1.0 system Figure 1 An exemplary batch digestion system 100 is illustrated with conditions for producing biostimulant products (e.g., microorganisms), which may have a multimodal mode of promoting zinc solubilization. System 100 may include at least one reactor 110, and reactor 110 may be a fluidized bed reactor or a packed bed reactor. Water may act as a hydraulic source. The reactor may include input channels or a series of input channels for flowing inputs 130, 140, 150, 160, 170, 180 into the digestion system. In some cases, inputs may be added to reactor 110 individually. In some cases, inputs may be added to reactor 110 in combination with each other. Inputs described herein may include water, carbon source 130, nitrogen source 140, micronutrient 150, microbial products or intermediates from another digestion system (e.g., PST WB 160), microbial isolate 170, zinc source 180, or any combination thereof. Inputs may include other organic materials. The effluent port can extend from the reactor and can be used to collect biostimulant products or digestion products (e.g., basic products).

[0203] System 100 may include a pH sensor, pH controller, or similar mechanism to monitor and / or control the pH of the reactor and / or the working fluid within the reactor to maintain the pH at a threshold. The pH sensor or controller may be automatic. In some embodiments, the pH in reactor 110 may be 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 can be up to about 10.0, up to about 9.5, up to about 9.0, up to about 8.5, up to about 8.0, up to about 7.5, up to about 7.0, up to about 6.5, up to about 6.0, up to about 5.5, up to about 5.0, up to about 4.5, up to about 4.0, up to about 3.5, or up to about 3.0. In some embodiments, the pH in the reactor can be from about 2 to about 10. In some embodiments, the pH in the reactor can be 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 about 10, 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] The effluent port may be located at the top, middle, and / or bottom of reactor 110. Additionally, the reactor may include another effluent port 180 to reintroduce the working fluid back into the same reactor and may be pumped back just below the surface of the same reactor to maintain homogeneous conditions within the working solution. In this way, the working fluid can be recycled within the digestion system 100. For example, the working fluid from reactor 110 can be reintroduced into reactor 110. Biosolids (e.g., flocculants) may be generated by the process. Biosolids may comprise organic materials recovered from the feedstock, waste, wastewater, and / or sludge material of the working fluid of the digestion system described herein. In some embodiments, the flocculant may comprise biosolids. In some embodiments, the flocculant may comprise biosolids and / or other solid particles from the digestion products of organic materials. In some embodiments, the flocculant may be flocculant material of microorganisms, extracellular polymeric substances (EPS), and adsorbed organic and inorganic materials. The flocculant material may comprise aggregated material of microorganisms, extracellular polymeric substances (EPS), and adsorbed organic and inorganic materials. Suspended particles of organic substrate from the working fluid can aggregate to form flocs. Flocs can be generated throughout the fermentation process in a digestive system.

[0205] In some embodiments, the input composition described herein may include selective pressure or a zinc source. Without wishing to be bound by theory, a selective pressure source (e.g., a zinc source) can transform complex microbial aggregates of the digestive system 100 into increased zinc-solubilizing microorganisms. A selective pressure source can alter the ion concentration of the working fluid. The transformation of complex microbial aggregates can occur in favor of microorganisms with zinc-solubilizing properties within the microbial aggregates. In some embodiments, selective pressure can produce high ion concentrations, thereby selectively inhibiting the growth of some microorganisms within the working fluid while promoting the growth or survival of other microorganisms in the working fluid (e.g., inoculum of microorganisms and / or zinc-solubilizing microorganisms of the microbial aggregates). Zinc-solubilizing microorganisms may include microorganisms that can survive at higher zinc ion concentrations in the working fluid resulting from the addition of selective pressure. In some embodiments, the inoculum of microorganisms may comprise zinc-solubilizing microorganisms. Adding selective pressure to the digestive system 100 can promote the survival of the inoculum of microorganisms. The terms "selective pressure source" and "selective pressure" are used interchangeably. The addition of a selective pressure source (e.g., a zinc source) can increase the zinc concentration within the working fluid of the digestive system 100.

[0206] Inoculum of microbial strains in digestive system 100 can be configured to survive as a population of microbial strains at increased zinc concentrations. Survival of microbial strains can include microbial strains that do not die in the presence of high zinc concentrations. Survival (e.g., maintenance) of microbial strains can include microbial strains that grow (e.g., increase in number) or whose concentration does not fluctuate in the presence of high zinc concentrations. Population survival (e.g., maintenance) of microbial strains can include a population of microbial strains that die at a slower rate relative to the death rate of other microorganisms in the presence of high zinc concentrations. Maintenance of microbial strains added to the digestive system herein can include the percentage of the concentration of microbial strains present in the system's base product (e.g., supernatant) relative to the initial concentration added to the digestive system. The survival (e.g., maintenance) of the microbial strain population after the addition of selective pressure may include 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 the initial concentration of the microbial strain population present in the basal products of the digestive system. The initial concentration may include the concentration of the microbial strain population added to the first container of the digestive system. The survival (e.g., maintenance) of a population of microbial strains after the addition of selective stress can include a concentration of up to about 75%, up to about 70%, up to about 60%, up to about 50%, up to about 45%, up to about 40%, up to about 35%, up to about 30%, up to about 25%, up to about 20%, or up to about 10% of the concentration of the microbial strains at the start of operation on the digestive system. The survival (e.g., maintenance) of a population of microbial strains after the addition of selective stress can include a concentration of about 5% to about 80% of the concentration of the microbial strains at the start of operation on the digestive system.The survival (e.g., maintenance) of the microbial strain population after the addition of selective pressure can include approximately 5% to approximately 10%, approximately 5% to approximately 15%, approximately 5% to approximately 20%, approximately 5% to approximately 25%, approximately 5% to approximately 30%, approximately 5% to approximately 35%, approximately 5% to approximately 40%, approximately 5% to approximately 50%, approximately 5% to approximately 60%, approximately 5% to approximately 70%, approximately 5% to approximately 80%, approximately 10% to approximately 15%, approximately 10% to approximately 20%, and approximately 10% to approximately 25% of the concentration of the microbial strain at the start of digestive system operation. %, 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 Approximately 30%, approximately 20% to approximately 35%, approximately 20% to approximately 40%, approximately 20% to approximately 50%, approximately 20% to approximately 60%, approximately 20% to approximately 70%, approximately 20% to approximately 80%, approximately 25% to approximately 30%, approximately 25% to approximately 35%, approximately 25% to approximately 40%, approximately 25% to approximately 50%, approximately 25% to approximately 60%, approximately 25% to approximately 70%, approximately 25% to approximately 80%, approximately 30% to approximately 35%, approximately 30% to approximately 40%, approximately 30% to approximately 50%, approximately 30% to approximately 60%, approximately 3 Concentrations of microbial strains of 0% to 70%, about 30% to 80%, about 35% to 40%, about 35% to 50%, about 35% to 60%, about 35% to 70%, about 35% to 80%, about 40% to 50%, about 40% to 60%, about 40% to 70%, about 40% to 80%, about 50% to 60%, about 50% to 70%, about 50% to 80%, about 60% to 70%, about 60% to 80%, or about 70% to 80%.

[0207] One or more microorganisms in a microbial aggregate in digestive system 100 may not survive at increased zinc concentrations. Incubating a working fluid containing a microbial aggregate with a selective pressure source (e.g., a zinc source) can increase the zinc ion concentration in the working fluid. The population of microbial strains may tolerate the increased zinc ion concentration. The increased zinc ion concentration may be toxic to some of the microorganisms in the microbial aggregate. Some of the microorganisms in the microbial aggregate may tolerate the increased zinc ion concentration. Incubating a working fluid containing a microbial aggregate with a selective pressure source (e.g., a zinc source) can increase or maintain the population of microbial strains (e.g., target isolates or isolated microorganisms) and reduce other microorganisms in digestive system 100. Incubating a working fluid containing a microbial aggregate with a selective pressure source (e.g., a zinc source) can increase or maintain the amount of zinc-solubilizing microorganisms in the microbial aggregate of the working fluid. The maintained population of microbial strains may include a constant amount of microorganisms throughout the duration. The amount of maintained microbial inoculum can vary in the working fluid of the digestive system 100 by less than 0.1%, less than 0.5%, less than 1%, less than 5%, or less than 10% during the duration. The duration can be at least about 1 hour, at most about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 12 hours, 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. Not wishing to be bound by theory, the addition of a selective stress source (e.g., a zinc source) can increase the proportion of the microbial strain population relative to the microorganisms or at least a portion of the microbial community in the digestive system 100. Without being bound by theory, in the digestive system 100, incubating microbial strains with a selective stressor (e.g., a zinc source) can reduce the population size of the microbial strains by less than the reduction of at least a portion of the microorganisms in the microbial aggregate.

[0208] The zinc source may comprise zinc oxide (ZnO) and may be added to digestion system 100 on the first day of the digestion process. In some cases, the zinc source (e.g., ZnO) may be added to digestion system 100 daily. In some cases, the zinc source (e.g., ZnO) may be added to 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 of 0.1–1.0% w / v. The concentration of the zinc source may be based on the concentration of all components of the working fluid of the system described herein.

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

[0210] In some implementations, a 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 in the form 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, or 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 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 The concentrations of approximately 0.7% w / v to approximately 0.8% w / v, approximately 0.7% w / v to approximately 0.9% w / v, approximately 0.7% w / v to approximately 1% w / v, approximately 0.8% w / v to approximately 0.9% w / v, approximately 0.8% w / v to approximately 1% w / v, or approximately 0.9% w / v to approximately 1% w / v are added to the reactor.

[0211] In some embodiments, the composition fed into digestion system 100 may include carbon source 130. The carbon source may be glucose, malic acid, gluconic acid, lactose, sucrose, pyruvate, other monosaccharides, or any combination thereof. In some cases, the carbon source may be added to the digestion system on the first day of the digestion process. In some cases, the carbon source may be added to the digestion system on the second, third, fourth, or any day after the first day of the digestion process. The carbon source (e.g., glucose) may be added to the reactor to maintain a carbon concentration range of 0.5%–3.0% w / v based on the total volume of the system's working fluid. In some embodiments, the carbon concentration that can be maintained may be based on the total concentration of all components of the working fluid at a certain point in time during the digestion process of digestion system 100.

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

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

[0214] In some embodiments, the composition fed into the digestion system 100 may include 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 the first day of the digestion process. In some cases, the nitrogen source may be added to the digestion system on the second, third, fourth, or any day after the first day of the digestion process. The nitrogen source (e.g., ammonium sulfate) may be added to the reactor to maintain a nitrogen concentration in the range of 0.05-0.2% w / v based on the total volume of the working fluid in the system. In some embodiments, the nitrogen concentration that can be maintained may be based on the total concentration of all components of the working fluid at a certain point in time during the digestion process of the digestion system 100.

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

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

[0217] In some embodiments, the composition introduced into the digestive system 100 may comprise micronutrients 150. Micronutrients may include elements essential for supporting the physiological functions of the microbiome. 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 the plant's dry weight. Elemental micronutrients may include boron, zinc, manganese, copper, chlorine, molybdenum, or any combination thereof. The addition of micronutrients or compounds providing micronutrients may enrich the microbial populations and / or microbial aggregates of the working fluid of the digestive system described herein. The addition of inorganic nutrients may enrich the microbial populations and / or microbial aggregates of the working fluid of the digestive system described herein. Inorganic nutrients may include potassium chloride (KCl), dipotassium hydrogen phosphate (K₂HPO₄), magnesium sulfate (MgSO₄), or any combination thereof. Potassium chloride may be added to the reactor at a concentration range of at least about, and 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 any two of these values. Dipotassium hydrogen phosphate may be added to the reactor at a concentration range of at least about, and 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 any two of these values. Magnesium sulfate may be added to the reactor at a concentration range of at least about, and 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 any two of these values.In some implementations, micronutrients may be added on the first, second, third, fourth and / or later day of the digestive process using the digestive system 100, based on the total volume of the system’s working fluid.

[0218] In some embodiments, phosphate solubilization technology (PST / PwST) flocculants (PST WB) with a concentration range of 0.1–5.0% v / v may also be added to the reactor, within the hydraulic retention time range of the digestion system. The hydraulic retention time may include the amount of time the working fluid is maintained (e.g., not transferred out) in the vessel of the digestion system described herein. In some implementations, the concentration range of the PST WB input to the system can be at least about, and 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. v / v, or a range between any two of these values. In some cases, the PST WB provides the phosphorus source from the phosphate rock to the working fluid of the digestion system 100. The PST WB may provide the first aggregate to the working fluid of the digestion system 100 to aggregate the first reactor.

[0219] In some embodiments, the digestive system 100 may be inoculated with isolates MS4666, MS4689, MS4687, or any combination thereof. The digestive system 100 may be inoculated with zinc-soluble spore-forming materials. The digestive system 100 may be inoculated with zinc-soluble rhizosphere bacteria. In some cases, the digestive system 100 may be inoculated with an isolate or combination of isolates only once. In some cases, after the first inoculation, the digestive system 100 may not be inoculated with isolates or combinations of isolates again. In some cases, the digestive system 100 may be re-inoculated with isolates or combinations 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, the digestive system 100 may be re-inoculated with isolates or combinations 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 fewer. In some cases, the digestive system 100 may be inoculated with isolates or combinations of isolates on day 1 of the digestion process, and then inoculated 1, 2, 3, 4, or 5 more times after day 1 of the digestion process. In some embodiments, between the first and second transfers of the isolate (e.g., microorganisms), the population of microorganisms does not decrease by more than 60%, more than 50%, more than 45%, more than 40%, more than 35%, more than 30%, more than 25%, more than 20%, more than 15%, more than 10%, or more than 5%. In some implementations, the microbial population is maintained at 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 for the microorganisms can be isolated microorganisms. Isolated microorganisms may include microorganisms that grow or accumulate outside the natural environment (e.g., in a culture medium or streak plate). In some embodiments, the inoculum for the microorganisms may include a mixture of multiple isolated microorganisms. The inoculum for the microorganisms may include 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 microorganisms.

[0221] Refer again Figure 1The working fluid can be maintained within reactor 110. The maintained working fluid can be the same as when it is stagnant within reactor 110 of the digestion system. Without being bound by theory, the maintenance of the working fluid can support the population of the microbial strains described herein or the enrichment and / or growth of another component of the working fluid. In some embodiments, the volume of the working fluid within reactor 110 can be maintained at 5 gallons. In some implementations, the volume of the working fluid within the reactor can be maintained at 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 implementations, the volume of the working fluid within the reactor can be maintained at up to about 10,000 gallons, up to about 5,000 gallons, up to about 2,500 gallons, up to about 1,000 gallons, up to about 500 gallons, up to about 250 gallons, up to about 100 gallons, up to about 75 gallons, up to about 50 gallons, up to about 30 gallons, up to about 25 gallons, up to about 20 gallons, up to about 15 gallons, up to about 10 gallons, up to about 7 gallons, up to about 6 gallons, up to about 5 gallons, up to about 4 gallons, up to about 3 gallons, up to about 2 gallons, up to about 1 gallon, or less than about 1 gallon.

[0222] In some implementations, the volume of the working fluid within the reactor can be maintained at approximately 5 gallons to approximately 10,000 gallons.In some embodiments, the volume of the working fluid within the reactor can be maintained at approximately 5 gallons to 10 gallons, approximately 5 gallons to 25 gallons, approximately 5 gallons to 50 gallons, approximately 5 gallons to 75 gallons, approximately 5 gallons to 100 gallons, approximately 5 gallons to 150 gallons, approximately 5 gallons to 500 gallons, approximately 5 gallons to 1,000 gallons, approximately 5 gallons to 2,500 gallons, approximately 5 gallons to 5,000 gallons, approximately 5 gallons to 10,000 gallons, approximately 10 gallons to 25 gallons, approximately 10 gallons to 50 gallons, approximately 10 gallons to 75 gallons, approximately 10 gallons to 100 gallons, approximately 10 gallons to 150 gallons, approximately 10 gallons to 500 gallons, approximately 10 gallons to 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 150 gallons, about 50 gallons to about 500 gallons, about 50 gallons to about 1,000 gallons, about 50 Approximately 2,500 gallons, approximately 50 gallons to approximately 5,000 gallons, approximately 50 gallons to approximately 10,000 gallons, approximately 75 gallons to approximately 100 gallons, approximately 75 gallons to approximately 150 gallons, approximately 75 gallons to approximately 500 gallons, approximately 75 gallons to approximately 1,000 gallons, approximately 75 gallons to approximately 2,500 gallons, approximately 75 gallons to approximately 5,000 gallons, approximately 75 gallons to approximately 10,000 gallons, approximately 100 gallons to approximately 150 gallons, approximately 100 gallons to approximately 500 gallons, approximately 100 gallons to approximately 1,000 gallons, approximately 100 gallons to approximately 2,500 gallons, approximately 100 gallons to approximately 5,000 gallons, approximately 100 gallons to approximately 10,000 gallons, approximately 150 gallons to approximately 500 gallons. 0 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 reactor 110 can be recirculated (e.g., recycled) within the reactor of digestion system 100. The working fluid can be recirculated through conduits or the reactor outlet and reintroduced into the same reactor. Not wishing to be bound by theory, recirculation of the working fluid within the reactor can support the enrichment and / or growth of a population of the microbial strains described herein or another component of the working fluid. In some embodiments, the recirculation rate within the reactor can be 4-9 gallons / min. In some embodiments, the rate of recirculation within reactor 110 may be at least about 0.5 gallons / 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 the working fluid remains in the digestive system 100 before the product is removed from the digestive system 100, may be 7 days. In some embodiments, the incubation time of the digestive system may be at least 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 implementations, the incubation time of the digestive system 100 may be up to about 50 days, up to about 30 days, up to about 25 days, up to about 20 days, up to about 19 days, up to about 18 days, up to about 17 days, up to about 16 days, up to about 15 days, up to about 14 days, up to about 13 days, up to about 12 days, up to about 11 days, up to about 10 days, up to about 9 days, up to about 8 days, up to about 7 days, up to about 6 days, up to about 5 days, up to about 4 days, up to about 3 days, up to about 2 days, or up to about 1 day.

[0225] In some embodiments, the incubation time of the digestive system can be from about 1 day to about 30 days. In some embodiments, the incubation time of the digestive system can be from about 1 day to about 3 days, from about 1 day to about 5 days, from about 1 day to about 8 days, from about 1 day to about 10 days, from about 1 day to about 12 days, from about 1 day to about 14 days, from about 1 day to about 16 days, from about 1 day to about 18 days, from about 1 day to about 20 days, from about 1 day to about 25 days, from about 1 day to about 30 days, from about 3 days to about 5 days, from about 3 days to about 8 days, from about 3 days to about 10 days, from about 3 days to about 12 days, or from about 3 days to about 14 days. Approximately 3 days to approximately 16 days, approximately 3 days to approximately 18 days, approximately 3 days to approximately 20 days, approximately 3 days to approximately 25 days, approximately 3 days to approximately 30 days, approximately 5 days to approximately 8 days, approximately 5 days to approximately 10 days, approximately 5 days to approximately 12 days, approximately 5 days to approximately 14 days, approximately 5 days to approximately 16 days, approximately 5 days to approximately 18 days, approximately 5 days to approximately 20 days, approximately 5 days to approximately 25 days, approximately 5 days to approximately 30 days, approximately 8 days to approximately 10 days, approximately 8 days to approximately 12 days, approximately 8 days to approximately 14 days, approximately 8 days to approximately 16 days days, approximately 8 to 18 days, approximately 8 to 20 days, approximately 8 to 25 days, approximately 8 to 30 days, approximately 10 to 12 days, approximately 10 to 14 days, approximately 10 to 16 days, approximately 10 to 18 days, approximately 10 to 20 days, approximately 10 to 25 days, approximately 10 to 30 days, approximately 12 to 14 days, approximately 12 to 16 days, approximately 12 to 18 days, approximately 12 to 20 days, approximately 12 to 25 days, approximately 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, the digestion system may include multiple digestion systems 100, each of which produces a base product. Each reactor can operate independently and produce a base product.

[0227] ZST 1.5 / 1.5+ system Figure 2An example of a digestive system 300 with conditions (e.g., microorganisms) for producing biostimulant products may be illustrated, these biostimulant products having a multimodal nature that promotes plant access to bound zinc sources and / or enhances the uptake of zinc and other nutrients. System 300 may include a first reactor 310, a second reactor 315, a third reactor 320, a fourth reactor 325, and / or a clarifier chamber 330 connected in sequence, wherein feedstocks can flow continuously and the microbial aggregates described herein can grow. The first, second, third, and / or fourth reactors may be supportless fluidized bed reactors or packed bed reactors with internal support structures. Water may act as a hydraulic source 340 and may be coupled to the first reactor 310 to provide a continuous flow of water to the reactor. System 300 may also include input channels or a series of input channels for allowing inputs 350, 360, 370, 380 to flow into the digestive system. In some cases, inputs may be added to the digestive system 300 individually. In some cases, inputs may be added to the digestive system in combination with each other. The input described herein may include one or more of the following: water, microbial inoculum 350, microbial products or intermediates of another digestion system 360, carbon source 370, nitrogen source 380, micronutrients, and zinc source. The input may include other organic materials. In some cases, the input entering the digestion system may flow into the first reactor via a water tank. In other cases, the input composition may be added to the first reactor. In still other cases, the input composition may be added to the second reactor.

[0228] System 300 may include a pH sensor, pH controller, or similar mechanism to monitor and / or control the pH of the reactor and / or the working fluid within the reactor to maintain the pH 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 a threshold, an alkali (e.g., 3M NaOH) may be automatically added to the first reactor until the pH reaches the threshold (e.g., at least about 7). In some embodiments, the pH in the reactor of the digestion system 300 may be 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.

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

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

[0231] The effluent port can be located at the top, middle, and / or bottom of the reactor. Furthermore, each reactor or clarifier chamber may include another effluent port to reintroduce fluid into the same reactor or clarifier chamber and may be pumped back just below the surface of the same reactor to maintain homogeneous conditions within the working solution. Reactor 1 may include another effluent port 395 to reintroduce fluid into reactor 1. Reactor 2 may include another effluent port 396 to reintroduce fluid into reactor 2. Reactor 3 may include another effluent port 397 to reintroduce fluid into reactor 3. Reactor 4 may include another effluent port 398 to reintroduce fluid into reactor 4. In some cases, the working fluid from each reactor is recirculated from the bottom of each reactor back to just below the surface of the working solution within each reactor to maintain a homogeneous fermentation environment. For example, fluid from the first reactor 310 may be reintroduced into the first reactor 310. Biosolids (e.g., flocculants) may be generated through this process. Biosolids may comprise organic materials recovered from the feedstock, waste, wastewater, and / or sludge material of the working fluid of the digestion system described herein. In some embodiments, flocculants may comprise biosolids. Flocculants may be generated throughout the fermentation process in the digestion system. Flocculants may be collected in a clarifier chamber and reintroduced into the system's container (e.g., a reactor) to serve as seed for microorganisms. In some embodiments, flocculants may be flocculants of microbial cells, extracellular polymeric substances (EPS), and adsorbed organic and inorganic materials. Flocculants may comprise aggregates of microbial cells, extracellular polymeric substances (EPS), and adsorbed organic and inorganic materials.

[0232] In some cases, the clarifier chamber 330 may include an effluent port to reintroduce fluid into 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 flocculent at the bottom of the clarifier may be returned to the first reactor. In some cases, the flocculent may be manually returned to the first reactor 310. In some cases, the flocculent may be returned to any container of the digestion system 300.

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

[0234] In some embodiments, water can act as a hydraulic source 340 and, when coupled to the first reactor 310, provide a continuous flow of water to the first reactor 310 with a conductivity of about 200 µS / cm to about 1,200 µS / cm. In some embodiments, water can act as a hydraulic source 340, and when coupled to the first reactor 310, at speeds of approximately 200 µS / cm to approximately 300 µS / cm, approximately 200 µS / cm to approximately 400 µS / cm, approximately 200 µS / cm to approximately 450 µS / cm, approximately 200 µS / cm to approximately 500 µS / cm, approximately 200 µS / cm to approximately 550 µS / cm, approximately 200 µS / cm to approximately 600 µS / cm, approximately 200 µS / cm to approximately 700 µS / cm, approximately 200 µS / cm to approximately 800 µS / cm, approximately 200 µS / cm to approximately 900 µS / cm, approximately 200 µS / cm to approximately 1,000 µS / cm, approximately 200 µS / cm to approximately 1,200 µS / cm, approximately 300 µS / cm to approximately 400 µS / cm, and approximately 300 µS / cm to approximately 450 µS / cm. µS / cm, about 300 µS / cm to about 500 µS / cm, about 300 µS / cm to about 550 µS / cm, about 300 µS / cm to about 600 µS / cm, about 300 µS / cm to about 700 µS / cm, about 300 µS / cm to about 800 µS / cm, about 300 µS / cm to about 900 µS / cm, about 300 µS / cm to about 1,000 µS / cm, about 300 µS / cm to about 1,200 µS / cm, about 400 µS / cm to about 450 µS / cm, about 400 µS / cm to about 500 µS / cm, about 400 µS / cm to about 550 µS / cm, about 400 µS / cm to about 600 µS / cm, about 400 µS / cm to about 700 µS / cm, about 400 µS / cm to about 800 µS / cm µS / cm, about 400 µS / cm to about 900 µS / cm, about 400 µS / cm to about 1,000 µS / cm, about 400 µS / cm to about 1,200 µS / cm, about 450 µS / cm to about 500 µS / cm, about 450 µS / cm to about 550 µS / cm, about 450 µS / cm to about 600 µS / cm, about 450 µS / cm to about 700 µS / cm, about 450 µS / cm to about 800 µS / cm, about 450 µS / cm to about 900 µS / cm, about 450 µS / cm to about 1,000 µS / cm, about 450 µS / cm to about 1,200 µS / cm, about 500 µS / cm to about 550 µS / cm, about 500 µS / cm to about 600 µS / cm, about 500 µS / cm to about 700 µS / cm, about 500 µS / cm to about 800 µS / cm, about 500 µS / cm to about 900 µS / cm, about 500 µS / cm to about 1,000 µS / cm, about 500 µS / cm to about 1,200 µS / cm, about 550 µS / cm to about 600 µS / cm, about 550 µS / cm to about 700 µS / cm, about 550 µS / cm to about 800 µS / cm, about 550 µS / cm to about 900 µS / cm, about 550 µS / cm to about 1,000 µS / cm, about 550 µS / cm to about 1,200 µS / cm, about 600 µS / cm µS / cm to about 700 µS / cm, about 600 µS / cm to about 800 µS / cm, about 600 µS / cm to about 900 µS / cm, about 600 µS / cm to about 1,000 µS / cm, about 600 µS / cm to about 1,200 µS / cm, about 700 µS / cm to about 800 µS / cm, about 700 µS / cm to about 900 µS / cm, about 700 µS / cm to about 1,000 µS / cm, about 700 µS / cm to about 1,200 µS / cm, about 800 µS / cm to about 900 µS / cm, about 800 µS / cm to about 1,000 µS / cm, about 800 µS / cm to about 1,200 µS / cm, about 900 µS / cm to about 1,000 µS / cm, about 900 µS / cm to about 1,200 µS / cm A continuous flow of water is supplied to the first reactor 310 with a conductivity of µS / cm or approximately 1,000 µS / cm to approximately 1,200 µS / cm.

[0235] A certain amount of biosolids can be maintained in the digestive system. Maintaining biosolids can support the enrichment and / or growth of the microbial community, microbial aggregates, nutrients, or additional components of the digestive system described herein. In some embodiments, at least about 20-25% biosolids v / v can be maintained in the digestive system as biosolids accumulate over time in the clarifier chamber 330. In some embodiments, additional flocculants can be harvested from and removed from the digestive system. 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, and at least about 23% biosolids v / v can be maintained in the digestive system. % 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.

[0236] In some implementations, up to about 50% biosolids v / v, up to about 45% biosolids v / v, up to about 40% biosolids v / v, up to about 35% biosolids v / v, up to about 30% biosolids v / v, up to about 29% biosolids v / v, up to about 28% biosolids v / v, up to about 27% biosolids v / v, up to about 26% biosolids v / v, up to about 25% biosolids v / v, up to about 24% biosolids v / v, up to about 23% biosolids v / v, up to about 2 2% biosolids v / v, up to about 21% biosolids v / v, up to about 20% biosolids v / v, up to about 19% biosolids v / v, up to about 18% biosolids v / v, up to about 17% biosolids v / v, up to about 16% biosolids v / v, up to about 15% biosolids v / v, up to about 14% biosolids v / v, up to about 13% biosolids v / v, up to about 12% biosolids v / v, up to about 11% biosolids v / v, up to about 10% biosolids v / v, or up to about 5% biosolids v / v.

[0237] In some embodiments, approximately 0.1% biosolids v / v to approximately 60% biosolids v / v can be maintained in the digestive system. In some embodiments, approximately 0.1% biosolids v / v to approximately 1% biosolids v / v, approximately 0.1% biosolids v / v to approximately 5% biosolids v / v, approximately 0.1% biosolids v / v to approximately 10% biosolids v / v, approximately 0.1% biosolids v / v to approximately 15% biosolids v / v, approximately 0.1% biosolids v / v to approximately 20% biosolids v / v, approximately 0.1% biosolids v / v to approximately 25% biosolids v / v, approximately 0.1% biosolids v / v to approximately 30% biosolids v / v, approximately 0.1% biosolids v / v to approximately 35% biosolids v / v, and approximately 0.1% biosolids v / v to approximately 40% biosolids v / v can be maintained in the digestive system. Solids 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 / 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 / vApproximately 15% biosolids v / v to approximately 25% biosolids v / v, approximately 15% biosolids v / v to approximately 30% biosolids v / v, approximately 15% biosolids v / v to approximately 35% biosolids v / v, approximately 15% biosolids v / v to approximately 40% biosolids v / v, approximately 15% biosolids v / v to approximately 50% biosolids v / v, approximately 15% biosolids v / v to approximately 60% biosolids v / v, approximately 20% biosolids v / v to approximately 25% biosolids v / v Solids 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 Approximately 35% biosolids v / v, approximately 25% biosolids v / v to approximately 40% biosolids v / v, approximately 25% biosolids v / v to approximately 50% biosolids v / v, approximately 25% biosolids v / v to approximately 60% biosolids v / v, approximately 30% biosolids v / v to approximately 35% biosolids v / v, approximately 30% biosolids v / v to approximately 40% biosolids v / v, approximately 30% biosolids v / v to approximately 50% biosolids v / v, approximately 30% biosolids v / v Solids 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.

[0238] In some embodiments, the input composition described herein may comprise selective pressure or a zinc source. Without wishing to be bound by theory, a selective pressure source (e.g., a zinc source) can transform a complex microbial aggregate of digestive system 300 into increased zinc-solubilizing microorganisms. Zinc-solubilizing microorganisms may include microorganisms that can survive at higher zinc ion concentrations in the working fluid resulting from the addition of selective pressure. In some embodiments, the inoculum of microbial strains may comprise zinc-solubilizing microorganisms. Adding selective pressure to digestive system 300 can promote (e.g., maintain) the survival of the microbial inoculum. The terms "selective pressure source" and "selective pressure" are used interchangeably. The addition of a selective pressure source (e.g., a zinc source) can increase the zinc concentration within the working fluid of digestive system 300. The population of microbial strains in digestive system 300 can be configured to survive at the increased zinc concentration. At least a portion of the microorganisms in the microbial aggregate of digestive system 300 may not survive at the increased zinc concentration. The transformation of complex microbial aggregates can occur to enrich the microorganisms in the microbial aggregates with zinc-solubilizing properties. In some embodiments, selective pressure can generate high ion concentrations, thereby selectively inhibiting the growth of some microorganisms within the working fluid while promoting the growth or survival of other microorganisms (e.g., populations of microbial strains and / or zinc-solubilizing microorganisms in microbial aggregates). The addition of a selective pressure source (e.g., a zinc source) can increase or maintain the population size of microbial strains (e.g., target isolates or isolated microorganisms) and reduce other microorganisms in the digestive system 300. Maintaining the population size of microbial strains may include no change in the amount of microorganisms over two time periods. The maintained population size of microbial strains may vary in the working fluid of the digestive system 300 by less than 0.1%, less than 0.5%, less than 1%, less than 5%, or less than 10% over two time periods. It is not desirable to be bound by theory, but the addition of a selective pressure source (e.g., a zinc source) can increase the proportion of microbial strains relative to the microorganisms or at least a portion of the microorganisms in the microbial aggregates of the digestive system 300. Without being bound by theory, in the digestive system 300, the addition of a selective stressor (e.g., a zinc source) can reduce the population size of microbial strains by less than the reduction in microorganisms or at least a portion of microorganisms in a microbial aggregate. The zinc source may comprise zinc oxide (ZnO) and may be added to the digestive system 300 on the first day of the digestion process. In some cases, the zinc source (e.g., ZnO) may be added to the digestive system 300 daily. In some cases, the zinc source (e.g., ZnO) may be added to the digestive 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.A zinc source (e.g., zinc oxide) may be added to the first reactor 310, the second reactor 320, or another reactor in the digestion system 300. The zinc source (e.g., zinc oxide) may be added to the reactor at a concentration of 0.05-0.5% w / v. The concentration of the zinc source may be based on the total concentration of the components of the working fluid in the 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 residence time of the system. In some embodiments, a 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, a zinc source (e.g., zinc oxide) may be added to the reactor at a concentration of up to about 1.5% w / v, up to about 1.4% w / v, up to about 1.3% w / v, up to about 1.2% w / v, up to about 1.1% w / v, up to about 1.0% w / v, up to about 0.9% w / v, up to about 0.8% w / v, up to about 0.7% w / v, up to about 0.6% w / v, up to about 0.5% w / v, up to about 0.4% w / v, up to about 0.3% w / v, up to about 0.2% w / v, up to about 0.1% w / v, up to about 0.05% w / v, up to about 0.04% w / v, up to about 0.03% w / v, up to about 0.02% w / v, or up to about 0.01% w / v.

[0239] In some implementations, a 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 in the form 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, or about 0.025% w / v. 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 The following concentrations are added to the reactor: approximately 0.3% w / v to approximately 0.5% w / v, approximately 0.3% w / v to approximately 0.75% w / v, approximately 0.3% w / v to approximately 1% w / v, approximately 0.4% w / v to approximately 0.5% w / v, approximately 0.4% w / v to approximately 0.75% w / v, approximately 0.4% w / v to approximately 1% w / v, approximately 0.5% w / v to approximately 0.75% w / v, approximately 0.5% w / v to approximately 1% w / v, or approximately 0.75% w / v to approximately 1% w / v.

[0240] In some embodiments, the composition fed into the digestion system 300 may include a carbon source 370. The carbon source may be glucose, malic acid, gluconic acid, lactose, sucrose, pyruvate, other monosaccharides, or any combination thereof. In some cases, the carbon source may be added to the digestion system on the first day of the digestion process. In some cases, the carbon source may be added to the digestion system on the second, third, fourth, and / or any day after the first day of the 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 a 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. Based on the hydraulic residence time span of the digestion system, the carbon source (e.g., glucose) may be added to the reactor to maintain a concentration range of 0.5%–3.0% w / v. In some embodiments, the carbon concentration that can be maintained may be based on the total concentration of all components of the working fluid at a certain point in time during the digestion process of the digestion system 300.

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

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

[0243] In some embodiments, the composition fed into the digestion system 300 may include 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 the first day of the digestion process. In some cases, the nitrogen source may be added to the digestion system on the second, third, fourth, and / or any day after the first day of the digestion process. In some cases, the nitrogen source may be added to the digestion system daily. In some embo...

Claims

1. A method for preparing a biostimulant composition, the method comprising: (a) Providing a bioreactor system comprising two or more containers arranged in series, each of the two or more containers comprising a volume of working fluid, wherein a first container comprises a first working fluid containing a first microbial aggregate and a population of established zinc-solubilizing bacterial strains; (b) Operate the bioreactor system during the duration by the following manner: (i) Transferring the aqueous raw material containing the second microbial aggregate into the first container; (ii) transferring a portion of the working fluid from each of the two or more containers to a subsequent container of the bioreactor system or to the product effluent stream; (iii) Maintaining, at least in the first container, the concentration of the zinc-solubilizing bacteria strain during the duration at least 80% of the concentration of the zinc-solubilizing bacteria strain at the start of the duration; and (iv) Collect at least a portion of the product effluent as the biostimulant composition; The duration is at least 5 days; and during the duration, the concentration of zinc-solubilizing bacterial strains present in the aqueous feedstock or any other input into the bioreactor system is not higher than 1% of the concentration of zinc-solubilizing microorganisms in the first container.

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

3. The method of claim 1, wherein the first microbial aggregate comprises a first portion of microorganisms and a second portion of microorganisms, and wherein the method further comprises maintaining at least the concentration of zinc molecules in the first container at a concentration that inhibits the growth of the first portion of microorganisms relative to the zinc-solubilizing bacterial strain.

4. The method of claim 3, wherein the first portion of microorganisms comprises non-zinc solubilizers and / or non-zinc tolerant microorganisms.

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

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

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

8. The method according to any one of claims 1-7, wherein the zinc-solubilizing bacterial strain belongs to Bacillus safortus or Bacillus megaterium.

9. The method according to any one of claims 1-8, wherein the zinc-solubilizing bacteria strain is one of the following strains: (a) A strain of Bacillus safranin with one or more of the following characteristics: (i) A 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 1; (ii) At least 95% identical to SEQ ID NO: 4 gyrB Gene sequence; as well as (iii) At least 95% identical to SEQ ID NO: 7 rpoB Gene sequence; (b) A *Bacillus megaterium* strain that has one or more of the following characteristics: (i) A 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 2; (ii) At least 95% identical to SEQ ID NO: 5 gyrB Gene sequence; as well as (iii) At least 95% identical to SEQ ID NO: 8 rpoB Gene sequence; or (b) A *Bacillus megaterium* strain that has one or more of the following characteristics: (i) A 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 3; (ii) At least 95% identical to SEQ ID NO: 6 gyrB Gene sequence; as well as (iii) At least 95% identical to SEQ ID NO: 9 rpoB Gene sequence.

10. The method according to any one of claims 1-9, wherein the zinc-solubilizing bacterial strain is a *Bacillus saffron* strain deposited with ATCC accession number PTA-127681, a *Bacillus megaterium* strain deposited with ATCC accession number PTA-127683, or a *Bacillus megaterium* strain deposited with ATCC accession number PTA-127682.

11. The method of any one of claims 1-10, wherein during the duration, the concentration of zinc-solubilizing bacterial strains present in the aqueous feedstock or any other input into the bioreactor system is not 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 the duration.

13. The method of any one of claims 1-12, wherein the maintenance in step (b)(iii) comprises maintaining the concentration of the zinc-solubilizing bacterial strain at at least 1 x 10⁻⁶. 3 CFU / ml.

14. The method of any one of claims 1-13, wherein prior to step (b), the first container further comprises an established population of other zinc-solubilizing microorganisms that are not the zinc-solubilizing bacterial strain, and wherein step (b)(iii) further comprises maintaining the concentration of the other zinc-solubilizing microorganisms in at least the first container at at least 1 x 10⁻⁶ during the duration. 4 CFU / ml or at least 80% of the concentration of the other zinc-solubilizing microorganisms at the start of the duration, wherein during the duration, the concentration of the other zinc-solubilizing microorganisms added to the bioreactor system is not higher than 1% of the concentration of the other zinc-solubilizing microorganisms in the first container.

15. The method of claim 14, wherein the concentration of other zinc-solubilizing microorganisms present in the aqueous feedstock or any other input into the bioreactor system is not greater than 10. 4 CFU / ml.

16. The method of claim 14 or 15, wherein at the start of the duration, the population of the other zinc-solubilizing microorganisms in the first container is at least 1 x 102 3 CFU / ml.

17. The method of any one of claims 1-16, further comprising, prior to 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 at least 0.5 x 10⁻⁶ ppm in at least one container. 4 The initial population of zinc-solubilizing bacterial strains at CFU / ml.

18. The method of claim 17, wherein the concentration of the zinc-solubilizing bacteria strain is less than 1 x 10⁻⁶ before adding the inoculum. 2 CFU / ml.

19. The method of any one of claims 1-18, wherein the aqueous raw material further comprises organic material that can be digested at least partially by microorganisms present in at least one of the containers.

20. The method of claim 19, wherein prior to the transfer in step (b)(i), the organic material has been partially digested by microorganisms endogenous to the organic material.

21. The method of claim 19, further comprising digesting the organic material in two or more containers connected in series prior to the transfer in 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 raw material further comprises inorganic materials.

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

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

26. The method of claim 24, further comprising, prior to the transfer in step (b)(i), partially digesting the phosphate rock particles in two or more containers connected in series.

27. The method of any one of claims 1-26, wherein the second microbial aggregate comprises at least 1 x 102 5 CFU / ml.

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

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

30. The method of any one of claims 1-29, wherein the transfer in step (b)(i), the transfer in step (b)(ii), and the collection in step (b)(iv) are performed continuously during the duration.

31. The method of any one of claims 1-30, wherein the transfer in step (b)(i), the transfer in step (b)(ii), and the collection in step (b)(iv) are performed periodically during the duration.

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 contained in the aqueous feedstock.

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

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 containing clarifier working fluid.

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

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

40. The method of claim 38 or 39, further comprising folding the flocculent 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 flocculent solids into the supernatant portion.

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

43. The method of any one of claims 38-42, wherein the operation further comprises transferring a portion of the flocculent from the clarifier container to a forward container in the bioreactor system.

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

45. The method of any one of claims 1-44, wherein the method further comprises generating at least 1x10 in the product effluent. 4 Zinc-solubilizing bacterial strains at CFU / ml.

46. ​​The method of any one of claims 1-45, wherein the bioreactor system comprises: The first container contains a certain volume of first working fluid; The second container contains a certain volume of the second working fluid; And a third container, which contains a certain volume of a third working fluid.

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

48. The method of claim 47, wherein the third container includes an outlet port that is fluidly connected to the clarifier container.

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

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 transfer in step (b)(i), the transfer in step (b)(ii), and the collection in step (b)(iv) are driven by gravity.

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

53. The method of any one of claims 1-52, wherein the operation comprises maintaining the product effluent 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 the 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 micro-oxygen conditions.

58. The method of any one of claims 1-57, wherein the bioreactor system operates 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 the five most abundant species in the second microbial aggregate: *Bacillus*, *Lymans*, *Thermophyton*, *Geophysica*, and *Acidobacter*.

60. The method of any one of claims 1-59, wherein one or more of the following species are the five most abundant species in the said microbial aggregate: Lymannia glutinosa, Sophora phenylacetate, Sophora mesennii, Soritella canonis, and Nitrosporium mosqueensis.

61. The method of any one of claims 19-60, wherein the second microbial aggregate comprises microorganisms 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 circulates within its respective container.

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 during the duration.

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

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

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

67. The method of any one of claims 1-66, further comprising generating a colony of sporulated bacteria in the product effluent.

68. The method of any one of claims 1-67, further comprising generating a population of sporulated zinc-solubilizing bacterial strains in the product effluent.

69. The method of claim 68, wherein the population of the sporulated zinc-solubilizing bacteria strains comprises at least 1 x 10⁻⁶. 3 CFU / 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 preparing at least a portion of the aqueous raw material by a method including the following steps: (c) Transfer water, phosphate rock, and optionally the products of microbial digestion of the manure derived from the manure into a fourth container containing a volume of a fourth working fluid; (d) Transfer a portion of the fourth working fluid to a fifth container containing the fifth working fluid; (e) Transfer to the fifth container: (i) A liquid comprising (A) a third microbial aggregate containing microorganisms derived from manure and (B) digestion products produced by anaerobic digestion of the manure by said microorganisms; (ii) Manure; as well as (iii) Yeast.

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

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

74. The method of claim 73, further comprising transferring a portion of the raw material flocculent into 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 phosphate rock 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 the total volume of material added to the fourth container during a given time period is equal to the total volume of the fourth working fluid transferred to the fifth container during the same time period.

78. A bioreactor system comprising: (a) An aqueous feed stream in fluid communication with a first container containing a first working fluid of a certain volume, wherein the aqueous feed stream contains a first microbial aggregate, wherein the first working fluid contains a population of established zinc-solubilizing bacteria strains and a second microbial aggregate, wherein the concentration of zinc-solubilizing bacteria strains in the first working fluid is at least 100 times the concentration of zinc-solubilizing bacteria strains in the aqueous feed stream and any other input to the bioreactor system; (b) One or more additional containers arranged in series with the first container, each of the one or more additional containers containing a volume of working fluid and in fluid communication with at least one other container in the series, and at least one of the one or more additional containers including a product outflow port; as well as (c) A product outflow in fluid communication with the product outflow port.

79. The system of claim 78, wherein at least the first working fluid contains zinc molecules at a concentration of at least 5 mg / L.

80. The system of claim 78, wherein the second microbial aggregate comprises a first portion of microorganisms and a second portion of microorganisms, wherein at least the first working fluid contains zinc-containing molecules, the concentration of which inhibits the growth of the first portion of microorganisms relative to the zinc-solubilizing bacterial strain.

81. The system of claim 80, wherein the first portion of microorganisms comprises non-zinc solubilizers and / or non-zinc tolerant microorganisms.

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

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

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

85. The system according to any one of claims 78-84, wherein the zinc-solubilizing bacterial strain belongs to the species Bacillus safortus or Bacillus megaterium.

86. The system of any one of claims 78-85, wherein the zinc-solubilizing bacteria strain is one of the following strains: (a) A strain of Bacillus safranin with one or more of the following characteristics: (i) A 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 1; (ii) At least 95% identical to SEQ ID NO: 4 gyrB Gene sequence; as well as (iii) At least 95% identical to SEQ ID NO: 7 rpoB Gene sequence; (b) A *Bacillus megaterium* strain that has one or more of the following characteristics: (i) A 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 2; (ii) At least 95% identical to SEQ ID NO: 5 gyrB Gene sequence; as well as (iii) At least 95% identical to SEQ ID NO: 8 rpoB Gene sequence; or (b) A *Bacillus megaterium* strain that has one or more of the following characteristics: (i) A 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 3; (ii) At least 95% identical to SEQ ID NO: 6 gyrB Gene sequence; as well as (iii) At least 95% identical to SEQ ID NO: 9 rpoB Gene sequence.

87. The system according to any one of claims 78-86, wherein the zinc-solubilizing bacterial strain is a *Bacillus salsa* strain deposited with ATCC accession number PTA-127681, a *Bacillus megaterium* strain deposited with ATCC accession number PTA-127683, or a *Bacillus megaterium* strain deposited with ATCC accession number PTA-127682.

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

89. The system of any one of claims 78-88, wherein the volume of each 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 maintaining at least 1x10⁻¹⁰ during operation of the bioreactor system. 4 Zinc-solubilizing bacterial strains at a concentration of CFU / ml.

91. The system of any one of claims 78-90, wherein the aqueous feedstock and any other input into the bioreactor system do not contain the zinc-solubilizing bacterial strain, or do not contain zinc-solubilizing bacterial strains at concentrations higher than 100 CFU / ml.

92. The system of any one of claims 78-91, wherein the first microbial aggregate comprises at least 1 x 102 4 Microorganisms at CFU / ml.

93. The system of any one of claims 78-92, wherein the aqueous feedstock further comprises organic material that can be digested by microorganisms present in the container.

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

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

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

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

98. The system of claim 97, wherein the clarifier container includes one or more flocculant baffles configured to agitate settled flocculants in the clarifier container without resuspending solids in the supernatant portion.

99. The system of claim 97 or 98, further comprising a flocculent return stream flowing from the clarifier to a further container in the series.

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

101. The system of claim 100, wherein the product effluent comprises at least 1x10 4 Zinc-solubilizing bacterial strains at CFU / ml.

102. The system of claim 100 or 101, wherein the product effluent comprises at least 1 x 102 2 Zinc-solubilizing bacterial strains in sporulated form (CFU / ml).

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

104. The system according to any one of claims 100-103, wherein the chemical oxygen demand of the product effluent is 80 to 500 mg / L.

105. The system of any one of claims 100-104, wherein the conductivity of the product effluent is 0.1 to 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 working fluid in the first working fluid and / or at least one of the one or more additional containers comprises micro-oxygen conditions.

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

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

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

111. A composition comprising: (a) A strain of Bacillus safranin with one or more of the following characteristics: (i) A 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 1; (ii) At least 95% identical to SEQ ID NO: 4 gyrB Gene sequence; and (iii) At least 95% identical to SEQ ID NO: 7 rpoB Gene sequence; and (b) Carrier.

112. The composition of claim 111, wherein the *Bacillus safortiformis* strain is a strain deposited with ATCC accession number PTA-127681 or an isolated clone thereof.

113. The composition of claim 111 or 112, further comprising the product of the *Bacillus salsa* strain digesting an organic substrate.

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 with the *Bacillus salsa* strain.

116. The composition of claim 115, wherein the carrier is further coated with micronutrients.

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 additive selected from wetting agents, spreading agents, dispersants, adhesives, dust control agents or binders.

120. The composition of any one of claims 111-119, wherein the concentration of *Bacillus salsa* strain in the composition is in the range of 1 x 10⁻⁶. 3 Up to 1x10 11 cfu / ml.

121. The composition of any one of claims 111-120, wherein the concentration of *Bacillus salsa* strain in the composition is in the range of 1 x 10⁻⁶. 4 Up to 1x10 6 cfu / ml.

122. An isolated strain of *Bacillus safranin*, said isolated strain having one or more of the following characteristics: (a) A 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 1; (b) At least 95% identical to SEQ ID NO: 4 gyrB Gene sequence; and (c) At least 95% identical to SEQ ID NO: 7 rpoB Gene sequence.

123. The isolated strain of claim 122, wherein the *Bacillus safortifolius* strain is a strain deposited with ATCC accession number PTA-127681 or a clone thereof.

124. A composition comprising: (a) A Bacillus megater strain that has one or more of the following characteristics: (i) A 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 2; (ii) At least 95% identical to SEQ ID NO: 5 gyrB Gene sequence; and (iii) At least 95% identical to SEQ ID NO: 8 rpoB Gene sequence; and (b) Carrier.

125. The composition of claim 124, wherein the Bacillus megaterium strain is a strain deposited with ATCC accession number PTA-127683 or an isolated clone thereof.

126. The composition of claim 124 or 125, further comprising the product of the Bacillus megaterium strain digesting the organic substrate.

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

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

129. The composition of claim 128, wherein the carrier is further coated with micronutrients.

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 additive selected from wetting agents, spreading agents, dispersants, adhesives, dust control agents or binders.

133. The composition of any one of claims 124-132, wherein the concentration of the Bacillus megaterium strain in the composition is in the range of 1 x 10⁻⁶. 3 Up to 1x10 11 cfu / ml.

134. The composition of any one of claims 124-133, wherein the concentration of the Bacillus megaterium strain in the composition is in the range of 1 x 10⁻⁶. 4 Up to 1x10 6 .

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

136. The isolated strain of claim 135, wherein the Bacillus megaterium strain is a strain deposited with ATCC accession number PTA-127683 or a clone thereof.

137. A composition comprising: (a) A Bacillus megater strain that has one or more of the following characteristics: (i) A 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 3; (ii) At least 95% identical to SEQ ID NO: 6 gyrB Gene sequence; and (iii) At least 95% identical to SEQ ID NO: 9 rpoB Gene sequence; and (b) Carrier.

138. The composition of claim 137, wherein the Bacillus megaterium strain is a strain deposited with ATCC accession number PTA-127682 or an isolated clone thereof.

139. The composition of claim 137 or 138, further comprising the product of the Bacillus megaterium strain digesting the organic substrate.

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

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

142. The composition of claim 141, wherein the carrier is further coated with micronutrients.

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 additive selected from wetting agents, spreading agents, dispersants, adhesives, dust control agents or binders.

146. The composition of any one of claims 137-145, wherein the concentration of the Bacillus megaterium strain in the composition is in the range of 1 x 10⁻⁶. 3 Up to 1x10 11 cfu / ml.

147. The composition of any one of claims 137-146, wherein the concentration of the Bacillus megaterium strain in the composition is in the range of 1 x 10⁻⁶. 4 Up to 1x10 6 .

148. An isolated strain of Bacillus megaterium, said isolated strain having one or more of the following: It has at least 95% identical 16S rRNA gene sequence to SEQ ID NO: 3; The gyrB gene sequence is at least 95% identical to that of SEQ ID NO: 6; and The rpoB gene sequence is at least 95% identical to that of SEQ ID NO:

9.

149. The isolated strain of claim 148, wherein the Bacillus megaterium strain is a strain deposited with ATCC accession number PTA-127682 or a clone thereof.

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

151. The method of claim 150, wherein the contact results in an increase of at least 5% in the amount of solubilized zinc available to the plant.

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

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

154. The method of claim 153, wherein the nutrient is zinc, and wherein the plant’s zinc uptake is increased by at least 5% 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 culture medium is soil or hydroponic culture medium.

158. A method for remedying zinc deficiency in a plant growth medium, the method comprising: (a) Measure the concentration of soluble zinc in the plant growth medium, the concentration being less than 0.5 ppm; as well as (b) After step (a), the plant growth medium is brought into contact with the biostimulant composition as claimed in claim 108, the composition as claimed in any one of claims 111-121, 124-134 or 137-147, or a composition containing the isolated strain as claimed in any one of claims 122, 123, 135, 136, 148 and 149.

159. A method for promoting plant growth, the method comprising: (a) Contacting a plant or a culture medium in which the plant is grown with a composition comprising one or more compounds, said compounds including: 1-hexadecyl-2,3-di-o-acetylglycerol, diisodecyl phthalate, allothreonine, (S)-(-)-α-(1-naphthyl)ethylamine, indoleacetic acid (indole-3-acetic acid), salicylaniline, 25-hydroxycholesterol, 4-(2-aminophenyl)-2,4-dioxobutyric acid, 13,14-dihydro-15-one PGD2, 4-ethyloctanoic acid, phthalic acid, α-ketoisovaleric acid, acorn, Val-Ala, N-methylundec-10-enamide, Hi s-Ile-Lys-Arg, methylcarbamoyl PAF, hydantoin, Ala-Gly-Leu-Val-Ser, Arg-Val-Met, chapters, 2-phenylethanol, prostaglandin D3, methylphenidate, 5-hydroxyindoleacetaldehyde, azithromycin dihydrate, Pro-Ala-Phe, (4R,7S,7aR,12bS)-3-methyl-2,4,4a,7,7a,13-hexahydro-1H-4,12-bridged methylenebenzofurano[3,2-e]isoquinoline-7,9-diol, Glu-Pro-Thr, 2,6-naphthol, 3-aminoquinoline, acyl Phloroglucinol, L-pyrrolidone, tanikolide, Val-Arg-Glu, 3-methyl adipic acid, 10-propoxydecanoic acid, 7-hydroxy-3-(2-methoxyphenyl)-4H-chromene-4-one, N-methylundec-10-enamide, (5Z,9α,11α,13E,15S)-9,11-epidio-15-hydroperoxy-prostaglandin-5,13-diene-1-acid, Ile-Phe-Val-Lys, γ-glutamyl-Se-methylselenocysteine; 5-L-glutamyl-Se-methylselenocysteine, Trp-Ala-Lys, pantothenic acid Ester glucoside, daffodilone, LPC (O-16:0 / 2:0), sphingosine 1-phosphate, coenzyme Q6, dihydrocortisol, dibutyl ketone hydrochloride, LPE (18:0 / 0:0), nialamidine, androstenedione, 12-(2,3-dihydroxycyclopentyl)-2-dodecanoate, androstenedione-5-en-3,17-dione, hydroxytyrosol, 4-hydroxycinnamic acid, 2,4,5-trihydroxytoluene, isopropylamide, (1R,2R,5R,8R,9S,10R,12S)-12-hydroxy-11-methyl-6-methylene-16-oxo-15-oxapentane [9.3.2.15,8.01,10].[02,8] Heptadecano-9-carboxylic acid, allopurinol nucleoside, 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycerol-3-phosphate ethanolamine, uridine triacetate, JWH 018 7-hydroxyindole metabolite-d9, 1,6-dimethylphenazine, 2-(p-bromophenyl)-8-methyl-8H-thieno(2,3-b)indole, 3,4-dihydroxyphenyl glycol, dodecyl cis-9,10-epoxyoctadecanoate, heptadecanoyl alcohol, lauric acid, lignin, tetradecane, chromoyl alcohol, one or more derivatives thereof, or combinations thereof.

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

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

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

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

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

165. The method of any one of claims 159-164, wherein the composition further comprises an additive selected from wetting agents, spreading agents, dispersants, adhesives, dust control agents and binders.

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

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

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

169. A composition for promoting plant growth, said composition comprising: (i) at least one microbial strain, including *Bacillus safortiformis* or *Bacillus megaterium*; and (ii) One or more compounds, including 1-hexadecyl-2,3-di-o-acetylglycerol, diisodecyl phthalate, allothreonine, (S)-(-)-α-(1-naphthyl)ethylamine, indoleacetic acid (indole-3-acetic acid), salicylaniline, 25-hydroxycholesterol, 4-(2-aminophenyl)-2,4-dioxobutyric acid, 13,14-dihydro-15-one PGD2, 4-ethyloctanoic acid, phthalic acid, α-ketoisovaleric acid, acorn, Val-Ala, N-methylundec-10-enamide, His-Ile-Lys-Arg, methylcarbamoyl PAF Hypochondric acid, Ala-Gly-Leu-Val-Ser, Arg-Val-Met, chapters, 2-phenylethanol, prostaglandin D3, methylphenidate, 5-hydroxyindoleacetaldehyde, azithromycin dihydrate, Pro-Ala-Phe, (4R,7S,7aR,12bS)-3-methyl-2,4,4a,7,7a,13-hexahydro-1H-4,12-bridged methylenebenzofurano[3,2-e]isoquinoline-7,9-diol, Glu-Pro-Thr, 2,6-naphthol, 3-aminoquinoline, acylated phloroglucinol, L-pyrrolidone, t anikolide, Val-Arg-Glu, 3-methyl adipic acid, 10-propoxydecanoic acid, 7-hydroxy-3-(2-methoxyphenyl)-4H-chromen-4-one, N-methylundec-10-enamide, (5Z,9α,11α,13E,15S)-9,11-epidio-15-hydroperoxy-prostaglandin-5,13-diene-1-acid, Ile-Phe-Val-Lys, γ-glutamyl-Se-methylselenocysteine; 5-L-glutamyl-Se-methylselenocysteine, Trp-Ala-Lys, panthenolactone glucoside, duckbill Flower ketone, LPC(O-16:0 / 2:0), sphingosine 1-phosphate, coenzyme Q6, dihydrocortisol, dibutyl ketone hydrochloride, LPE(18:0 / 0:0), nialamidine, androstenedione, 12-(2,3-dihydroxycyclopentyl)-2-dodecanoate, androstenedione-5-en-3,17-dione, hydroxytyrosol, 4-hydroxycinnamic acid, 2,4,5-trihydroxytoluene, isopropylamide, (1R,2R,5R,8R,9S,10R,12S)-12-hydroxy-11-methyl-6-methylene-16-oxo-15-oxapentane [9.3.2.15,8.01,10].[02,8] Heptadecano-9-carboxylic acid, allopurinol nucleoside, 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycerol-3-phosphate ethanolamine, uridine triacetate, JWH 018 7-hydroxyindole metabolite-d9, 1,6-dimethylphenazine, 2-(p-bromophenyl)-8-methyl-8H-thieno(2,3-b)indole, 3,4-dihydroxyphenyl glycol, dodecyl cis-9,10-epoxyoctadecanoate, heptadecanoyl alcohol, lauric acid, lignin, tetradecane, chromoyl alcohol, one or more derivatives thereof, or combinations thereof.

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

7.

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

8.

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

9.

173. The composition according to 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 a culture medium in which the plant grows.

175. The composition of any one of claims 169-174, wherein the composition further comprises an additive selected from wetting agents, spreading agents, dispersants, adhesives, dust control agents and binders.

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

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

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

179. A composition for promoting plant growth, said composition comprising: (i) Two or more compounds comprising 1-hexadecyl-2,3-di-o-acetylglycerol, diisodecyl phthalate, allothreonine, (S)-(-)-α-(1-naphthyl)ethylamine, indoleacetic acid (indole-3-acetic acid), salicylaniline, 25-hydroxycholesterol, 4-(2-aminophenyl)-2,4-dioxobutyric acid, 13,14-dihydro-15-one PGD2, 4-ethyloctanoic acid, phthalic acid, α-ketoisovaleric acid, acorn, Val-Ala, N-methylundec-10-enamide, His-Ile-Lys-Arg, methylcarbamoyl PAF Hypochondric acid, Ala-Gly-Leu-Val-Ser, Arg-Val-Met, chapters, 2-phenylethanol, prostaglandin D3, methylphenidate, 5-hydroxyindoleacetaldehyde, azithromycin dihydrate, Pro-Ala-Phe, (4R,7S,7aR,12bS)-3-methyl-2,4,4a,7,7a,13-hexahydro-1H-4,12-bridged methylenebenzofurano[3,2-e]isoquinoline-7,9-diol, Glu-Pro-Thr, 2,6-naphthol, 3-aminoquinoline, acylated phloroglucinol, L-pyrrolidone, t anikolide, Val-Arg-Glu, 3-methyl adipic acid, 10-propoxydecanoic acid, 7-hydroxy-3-(2-methoxyphenyl)-4H-chromen-4-one, N-methylundec-10-enamide, (5Z,9α,11α,13E,15S)-9,11-epidio-15-hydroperoxy-prostaglandin-5,13-diene-1-acid, Ile-Phe-Val-Lys, γ-glutamyl-Se-methylselenocysteine; 5-L-glutamyl-Se-methylselenocysteine, Trp-Ala-Lys, panthenolactone glucoside, duckbill Flower ketone, LPC(O-16:0 / 2:0), sphingosine 1-phosphate, coenzyme Q6, dihydrocortisol, dibutyl ketone hydrochloride, LPE(18:0 / 0:0), nialamidine, androstenedione, 12-(2,3-dihydroxycyclopentyl)-2-dodecanoate, androstenedione-5-en-3,17-dione, hydroxytyrosol, 4-hydroxycinnamic acid, 2,4,5-trihydroxytoluene, isopropylamide, (1R,2R,5R,8R,9S,10R,12S)-12-hydroxy-11-methyl-6-methylene-16-oxo-15-oxapentane [9.3.2.15,8.01,10].[02,8] Heptadecano-9-carboxylic acid, allopurinol nucleoside, 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycerol-3-phosphate ethanolamine, triacetyluridine, JWH 018 7-hydroxyindole metabolite-d9, 1,6-dimethylphenazine, 2-(p-bromophenyl)-8-methyl-8H-thieno(2,3-b)indole, 3,4-dihydroxyphenyl glycol, dodecyl cis-9,10-epoxyoctadecanoate, heptadecanoylene, lauric acid, lignoceric acid, tetradecane, croterol, one or more derivatives thereof, or combinations thereof; and. (ii) Carrier.

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

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

182. The composition of claim 181, wherein the fertilizer is 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 additive selected from wetting agents, spreading agents, dispersants, adhesives, dust control agents and binders.

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

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

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

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