System for producing products that promote nitrogen utilization efficiency in plants

Abstract

The present disclosure provides methods and systems for producing biostimulants that promote nitrogen utilization efficiency in plants. Described embodiments include methods of preparing a biostimulant composition in a bioreactor system comprising two or more vessels arranged in a series in series. The bioreactor system may comprise an established population of microbial strains that promote nitrogen utilization efficiency. Methods may include operating a 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 the product. The method may also include maintaining a concentration of microbial strains in the system that promote nitrogen utilization efficiency.

Description

Cross-reference to related applications

[0001] This application claims the benefits of U.S. Patent Application No. 18 / 541,671, filed December 15, 2023; U.S. Patent Application No. 18 / 541,917, filed December 15, 2023; U.S. Provisional Application No. 63 / 509,263, filed June 20, 2023; U.S. Provisional Application No. 63 / 510,615, filed June 27, 2023; and U.S. Provisional Application No. 63 / 610,535, filed December 15, 2023, each of which is incorporated herein by reference in its entirety. Background Technology

[0002] This disclosure generally relates to biostimulant compositions and methods of using such biostimulant compositions to promote plant growth.

[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 improve crop growth, enhance the effectiveness of agricultural inputs 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] On one hand, 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 containing a volume of working fluid, wherein a first container contains an established colony of a first microbial strain that promotes nitrogen use efficiency; (b) operating the bioreactor system for a duration by: (i) transferring an aqueous feedstock containing the microbial colony 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 a product effluent; (iii) maintaining, throughout the duration, at least 80% of the concentration of the first microbial strain that promotes nitrogen use efficiency in at least the first container at the beginning of the duration; and (iv) collecting at least a portion of the product effluent as a biostimulant composition; wherein the duration is at least 5 days; and wherein the first microbial strain that promotes nitrogen use efficiency is not present in the aqueous feedstock or any other input entering the bioreactor system during the duration at a concentration higher than 1% of the concentration of the first microbial strain that promotes nitrogen use efficiency in the first container.

[0005] In some embodiments, the primary microbial strain promoting nitrogen use efficiency is a microbial strain that performs nitrogen fixation, promotes nitrogen fixation in plant tissues, recruits nitrogen-fixing organisms to the plant rhizosphere or other tissues, or increases the organic nitrogen content and / or mineralization of organic nitrogen in the soil. In some embodiments, the primary microbial strain promoting nitrogen use efficiency is nifH gene positive. In some embodiments, the primary microbial strain promoting nitrogen use efficiency is a microbial strain that promotes plant growth in a nitrogen-poor growth medium. In some embodiments, the nitrogen-poor growth medium contains less than 10 ppm of nitrate.

[0006] In some implementations, the primary microbial strain that promotes nitrogen use efficiency belongs to the genus Cossackella (…). Kosakonia ), Klebsiella spp. Klebsiella ), Rahn's genus ( Rhynella ), Krüverella ( Kluyver ), Enterobacteriaceae ( Enterobacter ), Achromobacterium spp. ( Achromobacter Microbacteria ( Microbacterium Staphylococcus spp. Gluconobacter ), Methylobacterium ( Methylobacterium ), Pseudomonas spp. Pseudomonas Pantotheca ( ) Pantoea ), Azospirobacter spp. Azospirillum ), Sugarcane Species *Streptococcus* ( Herbaspirillum Burkholderia spp. Burkholderia ), cyanobacteria ( Cyanobacteria ), Bacillus spp. Bacillus ) and Bacillus spp. ( Paenibacillus In some implementations, the primary microbial strain promoting nitrogen use efficiency belongs to *Sacchariformis* (a member of the sugarcane family). Kosakonia sacchari ), Klebsiella variegata ( Klebsiella variicola ), Aquatic Raenella ( Rahnella aquatica ), Klüvora in the center ( Kluyvera intermedia ), pseudosugars Cossacella ( Kosakonia pseudosacchari ), Enterobacteriaceae species ( Enterobacter spp. ), Achromobacterium martensii ( Achromobacter marplatensis ), lipid production Azopyrillum ( Azopyrillum lipoferum ), Microbacterium tumefaciens ( Microbacterium murale ), Gluconobacter diazovorans ( Gluconobacter diazotrophicus ), symbiotic methylbacteria ( Methylobacterium symbiotic ), Bacillus subtilis ( Paenibacillus borealis ), Bacillus megaterium ( Bacillus megalith (Pristia giantiformis) Priestia megatherium )) or Bacillus subtilis ( Paenibacillus sonchi ) bacterial strains.

[0007] In some implementations, the first microbial strain that promotes nitrogen use efficiency is the strain deposited under ATCC accession number PTA-127654 (MS3907), the strain deposited under ATCC accession number PTA-127653 (MS3900), the strain deposited under ATCC accession number PTA-127655 (MS4921), or the strain deposited under ATCC accession number PTA-127652 (MS2748).

[0008] In some embodiments, the first microbial strain that promotes nitrogen use efficiency is not present in the aqueous feedstock or any other input entering the bioreactor system at a concentration exceeding 100 CFU / ml during the duration. In some embodiments, the first microbial strain that promotes nitrogen use efficiency is not present in the aqueous feedstock or any other input entering the bioreactor system during the duration. In some embodiments, maintaining step (b) (iii) includes maintaining the concentration of the first microbial strain that promotes nitrogen use efficiency at at least 1 x 10⁻⁶ CFU / ml. 3 CFU / ml.

[0009] In some embodiments, the first container further comprises an established population of a second microbial strain that promotes nitrogen use efficiency, and wherein step (b) further comprises: (v) maintaining the concentration of the second microbial strain that promotes nitrogen use efficiency at at least 80% of the concentration of the second microbial strain that promotes nitrogen use efficiency at the start of the duration; wherein the second microbial strain that promotes nitrogen use efficiency is not present in the aqueous feedstock or any other input entering the bioreactor system during the duration at a concentration higher than 1% of the concentration of the second microbial strain that promotes nitrogen use efficiency in the first container.

[0010] In some embodiments, the first container further comprises an established population of a third microbial strain that promotes nitrogen use efficiency, and step (b) further comprises: (v) maintaining the concentration of the third microbial strain that promotes nitrogen use efficiency at at least 80% of the concentration of the third microbial strain that promotes nitrogen use efficiency at the start of the duration; wherein the third microbial strain that promotes nitrogen use efficiency is not present in the aqueous feedstock or any other input entering the bioreactor system during the duration at a concentration higher than 1% of the concentration of the third microbial strain that promotes nitrogen use efficiency in the first container. In some embodiments, prior to step (b), the first container further comprises an established population of other microorganisms that promote nitrogen use efficiency, which are not the first microbial strain that promotes nitrogen use efficiency, the second microbial strain that promotes nitrogen use efficiency, or the third microbial strain that promotes nitrogen use efficiency, and step (b) (iii) further comprises maintaining the concentration of the other microorganism that promotes nitrogen use efficiency in at least the first container at at least 1 x 10⁻⁶ throughout the duration. 4 The concentration of other microorganisms that promote nitrogen use efficiency at the start of the duration is at least 80% of the concentration of CFU / ml or the concentration of other microorganisms that promote nitrogen use efficiency during the duration is not added to the bioreactor system at a concentration higher than 1% of the concentration of other microorganisms that promote nitrogen use efficiency in the first container.

[0011] In some implementations, the number of other microorganisms that promote nitrogen use efficiency does not exceed 10. 5 The concentration of CFU / ml is present in the aqueous feedstock or any other input entering the bioreactor system. In some embodiments, at the start of the duration, the population of other microorganisms promoting nitrogen use efficiency in the first vessel is at least 1 x 10⁻⁶. 4 CFU / ml. In some embodiments, other microorganisms that promote nitrogen use efficiency include those that perform nitrogen fixation, promote nitrogen fixation in plant tissues, recruit nitrogen-fixing organisms to the plant rhizosphere or other tissues, or increase the organic nitrogen content and / or mineralization of organic nitrogen in the soil. In some embodiments, other microorganisms that promote nitrogen use efficiency are those that exhibit... nifH Genetic positive.

[0012] In some embodiments, the method further includes adding an inoculum of a first microbial strain that promotes nitrogen use efficiency to the bioreactor system prior to step (a), wherein the inoculum of the first microbial strain that promotes nitrogen use efficiency produces at least 0.5 x 10⁻⁶ nitrogen-producing cells in at least one container. 4 An initial population of a first microbial strain that promotes nitrogen use efficiency at CFU / ml. In some embodiments, the concentration of the first microbial strain that promotes nitrogen use efficiency is less than 1 x 10⁻⁶ CFU / ml before adding the inoculum.2 CFU / ml.

[0013] In some embodiments, the aqueous feedstock also includes organic material that can be at least partially digested by microorganisms present in at least one of the containers.

[0014] In some embodiments, the organic material has been partially digested by its endogenous microorganisms prior to the transfer in step (b)(i). In some embodiments, the method further includes digesting the organic material in two or more containers connected in series prior to the transfer in step (b)(i).

[0015] In some embodiments, the organic material comprises manure and / or material produced from the microbial digestion of manure. In some embodiments, the aqueous feedstock also 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 includes partially digesting the phosphate rock particles in two or more containers connected in series prior to the transfer in step (b)(i).

[0016] In some implementations, the microbial aggregate contains at least 1 x 10 5 CFU / ml. In some embodiments, the microbial aggregate comprises microorganisms derived from manure and phosphate rock particles. In some embodiments, step (b) further includes the production of microbial metabolites that directly or indirectly promote nitrogen use efficiency in plants. In some embodiments, the transfer of step (b)(i), the transfer of step (b)(ii), and the collection of step (b)(iv) are performed continuously throughout the duration. In some embodiments, the transfer of step (b)(i), the transfer of step (b)(ii), and the collection of step (b)(iv) are performed periodically throughout the duration.

[0017] In some embodiments, the method further includes adding one or more carbon sources to at least one container of the bioreactor system. In some embodiments, the one or more carbon sources are contained in an aqueous feedstock. In some embodiments, the method further includes maintaining a malic acid concentration of at least 0.2% w / v relative to the volume of working fluid in at least one container of the bioreactor system. In some embodiments, the method further includes adding one or more nitrogen sources to at least one container of the bioreactor system.

[0018] In some embodiments, the one or more nitrogen sources include one or more of the following: ammonium sulfate, ammonium chloride, ammonium nitrate, sodium nitrate, yeast extract, yeast, or any combination thereof. In some embodiments, the method further includes adding one or more of the following to at least one container of the bioreactor system: soybean meal, lentil meal, chickpea meal, green pea meal, yellow pea meal, white soybean meal, corn meal, cereal meal, corn gluten, soy protein, or hydrolyzed soy protein, or any combination thereof. In some embodiments, soybean meal is added, and wherein the soybean meal is contained in an aqueous feedstock. In some embodiments, the method further includes maintaining a soybean meal concentration of at least 0.2% w / v relative to the volume of working fluid in at least one container of the bioreactor system.

[0019] In some embodiments, the bioreactor system includes a clarifier container containing a 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 container. 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, folding further includes releasing a population of a first microbial strain that promotes nitrogen use efficiency into the supernatant portion without introducing flocculent solids into the supernatant portion. In some embodiments, folding is performed via folding scrapers in a bottom portion of the clarifier container.

[0020] In some embodiments, the operation further includes transferring a portion of the flocculent material from the clarifier vessel to a forward vessel in the bioreactor system. In some embodiments, the product effluent includes a portion of the supernatant from the clarifier working fluid.

[0021] In some implementations, the method further includes generating at least 1 x 10⁻⁶ ppm in the product effluent stream. 2 A first microbial strain with a CFU / ml that promotes nitrogen use efficiency. In some embodiments, the bioreactor system includes a first container containing a first working fluid, a second container containing a second working fluid, and a third container containing 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 throughout the duration.

[0022] 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, operation includes maintaining a flow rate that produces a hydraulic retention time of at least 5 days. In some embodiments, operation includes maintaining a product effluent flow rate of at least 100 gallons / day. In some embodiments, the working fluid volume in each of two or more containers is at least 100 gallons.

[0023] 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.

[0024] In some embodiments, the method further includes maintaining at least one of two or more containers under aerobic conditions. In some embodiments, the method further includes maintaining at least one of two or more containers under microaerobic conditions. In some embodiments, the bioreactor system operates continuously for at least 90 days.

[0025] In some implementations, the five most abundant species in the microbial aggregate include one or more species belonging to one or more genera of the following: *Bacillus* ( Haliscomenobacter ), Levulina spp. Lewinella ), Thermophyton genus ( Hotline ), genus *Typhonium* Territorial ) and Acidobacteria ( Acidobacterium In some implementations, the five most abundant species in the microbial aggregate include one or more of the following species: Levonorgestrel (… Lewinella coherens ), Phenylacetylcholinesterol ( Thauera phenylacetica ), Herxolium mesenyi Thauera mechernichensis ), Canadian Solitaire and Nitrifying Spirochetes from Moscow ( Nitrospira Muscovite ).

[0026] In some embodiments, the microbial aggregate comprises endogenous microorganisms of the organic material. In some embodiments, at least a portion of the aqueous feedstock is produced by the method described herein. In some embodiments, at least one of the first, second, or third working fluids comprises a pH buffering system. 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 8 throughout the duration.

[0027] In some embodiments, the aqueous feedstock does not include a primary microbial strain that promotes nitrogen use efficiency at a concentration higher than 10 CFU / ml. In some embodiments, the primary microbial strain that promotes nitrogen use efficiency is not added to the bioreactor system at a concentration higher than 10 CFU / ml during the duration of the process.

[0028] In some embodiments, the bioreactor system includes at least one vessel positioned preceding a first vessel in a tandem series. In some embodiments, the working fluid volume of one of the two or more vessels contains microbial aggregates, wherein each microbial aggregate is distinct from all microbial aggregates in the other working fluids. 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 nitrogen-use-efficiency-enhancing first microbial strains in the product effluent. In some embodiments, the population of sporulated nitrogen-use-efficiency-enhancing first microbial strains contains at least 1 x 10⁻⁶ microorganisms. 2 CFU / ml. In some embodiments, the method further includes adding an additional population of a first microbial strain, a second microbial strain, or a third microbial strain that promotes nitrogen use efficiency to the biostimulant product.

[0029] 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 microbial aggregate, wherein the first working fluid contains a population of a first microbial strain that promotes nitrogen use efficiency, wherein the concentration of the first microbial strain promoting nitrogen use efficiency in the first working fluid is at least 100 times higher than the concentration of the first microbial strain promoting nitrogen use efficiency in the aqueous feed stream or any other input into the bioreactor system; (b) one or more additional containers arranged in a series including the first container, wherein each of the one or more additional containers contains a volume of working fluid and is in fluid communication with at least one other container in the series, and wherein at least one of the one or more additional containers includes a product effluent port; and (c) a product effluent stream in fluid communication with the product effluent port.

[0030] In some embodiments, the primary microbial strain promoting nitrogen use efficiency is a microbial strain that performs nitrogen fixation, promotes nitrogen fixation in plant tissues, recruits nitrogen-fixing organisms to the plant rhizosphere or other tissues, or increases the organic nitrogen content and / or mineralization of organic nitrogen in the soil. In some embodiments, the primary microbial strain promoting nitrogen use efficiency is nifH gene positive. In some embodiments, the primary microbial strain promoting nitrogen use efficiency is a microbial strain that promotes plant growth in a nitrogen-poor growth medium. In some embodiments, the nitrogen-poor growth medium contains less than 10 ppm of nitrate.

[0031] In some implementation schemes, the primary microbial strain that promotes nitrogen use efficiency belongs to the genera *Cochasacella*, *Klebsiella*, *Lahn's*, *Krugwall*, *Enterobacter*, *Achromobacter*, *Microbacterium*, *Staphylococcus*, *Methylobacterium*, *Pseudomonas*, *Pantospira*, and *Azotobacter*. Sugarcane Species include *Spp.*, *Burkholderia*, cyanobacteria, *Bacillus*, or *Bacillus-like* species. In some implementations, the primary microbial strain promoting nitrogen use efficiency belongs to *Sacchariformis sugarcane*, *Klebsiella variegata*, *Lahn's aquatic bacteria*, *Klüvora centralis*, etc. pseudosugars Cossacchariformis, Enterobacter species, Achromobacter madrapata, lipid-producing bacteria Azopyrillum Microbacterium tumefaciens, Glucosamine diazoxide, methyl symbiotic bacteria, Bacillus thuringiensis, Bacillus megaterium (Priestella megaterium), or Bacillus spp.

[0032] In some implementations, the first microbial strain that promotes nitrogen use efficiency is the strain deposited under ATCC accession number PTA-127654 (MS3907), the strain deposited under ATCC accession number PTA-127653 (MS3900), the strain deposited under ATCC accession number PTA-127655 (MS4921), or the strain deposited under ATCC accession number PTA-127652 (MS2748).

[0033] In some embodiments, the bioreactor system is a continuous flow bioreactor system, and the aqueous feedstock 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 is contained within a volume maintained at at least 1 x 10⁻⁶ during operation of the bioreactor system. 4The concentration of the first microbial strain that promotes nitrogen use efficiency is CFU / ml. In some embodiments, the aqueous feedstock and any other inputs entering the bioreactor system do not contain a population of the first microbial strain that promotes nitrogen use efficiency, or do not contain a concentration of the first microbial strain that promotes nitrogen use efficiency at a level higher than 100 CFU / ml. In some embodiments, the microbial aggregate contains at least 1 x 10⁻⁶ CFU / ml. 5 Microorganisms at CFU / ml.

[0034] In some embodiments, the aqueous feedstock further comprises organic material that can be digested by microorganisms present in the container. In some embodiments, the organic material comprises manure or manure-derived material. In some embodiments, the aqueous feedstock further comprises phosphate rock particles. In some embodiments, the microbial aggregate comprises microorganisms derived from manure and phosphate rock particles.

[0035] In some embodiments, the container including the product effluent port is a clarifier container configured to separate a portion of the working fluid within 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 settled flocculent within the clarifier container without resuspending solids in the flocculent portion back into the supernatant portion. In some embodiments, the system further includes a flocculent return stream flowing from the clarifier to a preceding 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 ppm. 4 A first microbial strain that promotes nitrogen use efficiency at CFU / ml. In some embodiments, the product effluent contains at least 1 x 10⁻⁶ CFU / ml. 2 A first microbial strain for promoting nitrogen use efficiency in sporulated form, CFU / ml. In some embodiments, the product effluent contains 0.2 to 2.5 mg / ml of total dry weight. In some embodiments, the product effluent has a chemical oxygen demand (COD) between 80 and 500 mg / L. In some embodiments, the product effluent has a conductivity between 1.3 and 3.0 mS / cm.

[0036] On the other hand, this disclosure provides a method comprising: (a) transferring water and phosphate rock into a first container containing a volume of a first working fluid, wherein products of microbial digestion of manure derived from manure are not transferred into the first container; (b) transferring a portion of the first working fluid into a second container containing a second working fluid; and (c) transferring the following into the second container: (i) a liquid comprising (A) a first microbial aggregate containing microorganisms derived from a first organic material, and (B) digestion products produced by the anaerobic digestion of the first organic material by the microorganisms; (ii) a second organic material; and (iii) yeast.

[0037] In some embodiments, the method further includes transferring a portion of the second working fluid to a third container containing a third working fluid, and transferring a portion of the third working fluid to a fourth container containing a fourth working fluid. In some embodiments, the method further includes separating a portion of the fourth working fluid into a flocculent portion and a supernatant portion. In some embodiments, the method further includes transferring the flocculent portion to a first container. In some embodiments, the method further includes maintaining the first, second, third, and / or fourth containers under aerobic conditions. In some embodiments, the first, second, third, and / or fourth containers are fluidized bed reactors, wherein the phosphate rock is continuously circulated within the first, second, third, and / or fourth containers. In some embodiments, the total volume of material added to the first container during a given time period is equal to the total volume of the first working fluid transferred to the second container during the same time period. In some embodiments, the total volume of material transferred to the second, third, and fourth containers during a given time period is equal to the total volume transferred out of the second, third, and fourth containers during the same time period. In some embodiments, the method further includes maintaining the volumes of the first, second, and third working fluids constant.

[0038] In some embodiments, the first organic material is manure. In some embodiments, the second organic material is manure. In some embodiments, the yeast is *Saccharomyces cerevisiae*. In some embodiments, the method further includes generating a product stream from a second, third, or fourth microbial aggregate, wherein the product stream comprises bacteria from one or more of the following species: *Levonorhynchus ligustus*, *Synthia phenylacetate*, *Synthia mescheniformis*, ... Solitary Canadian In some implementations, the five most abundant microorganisms in the second microbial aggregate include bacteria from one or more of the following species: Levulina ligandii, Solomonella phenylacetate, Solomonella mesennii, Canadian SolitaireAnd *Nitrospilurus moscoparia*. In some embodiments, the five most abundant microorganisms in the second microbial aggregate do not include bacteria from any of the following genera: *Bacillus*, *Thermophyton*, *Agromonella*, and *Acidobacter*. In some embodiments, the second microbial aggregate is contained in the fifth working fluid. In some embodiments, low-rank coal is not transferred to the first container.

[0039] On the one hand, this disclosure provides a biostimulant composition prepared by the methods or systems described herein.

[0040] On the one hand, this disclosure provides a method for promoting plant growth, which includes contacting a plant, seed or plant growth medium with the biostimulant composition described herein.

[0041] On the one hand, this disclosure provides a method for improving nitrogen use efficiency in plants, the method comprising contacting a plant, seed or plant growth medium with the biostimulant composition described herein.

[0042] On the one hand, this disclosure provides a method for improving phosphorus solubility in plant growth media, the method comprising contacting a plant, seed or plant growth media with the biostimulant composition described herein.

[0043] On one hand, 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 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 Gene sequence; and (b) carrier.

[0044] In some embodiments, the *Bacillus megaterium* strain is the MS3900 strain or an isolated clone thereof, deposited under ATCC accession number PTA-127653. In some embodiments, the composition further comprises the digestion product of the *Bacillus megaterium* strain on an organic substrate. In some embodiments, the carrier comprises a fertilizer. In some embodiments, the carrier is a solid coated with the *Bacillus megaterium* strain. In some embodiments, the carrier is a liquid. In some embodiments, the composition further comprises an excipient selected from wetting agents, spreading agents, dispersing agents, adhesives, dust suppressants, and binders. In some embodiments, the concentration of the *Bacillus megaterium* strain in the composition ranges from 1 x 10⁻⁶. 3 Up to 1 x 10 11 CFU / ml.

[0045] On one hand, this disclosure provides a composition comprising: (a) a strain of *Bacillus cerevisiae* 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 (iii) Gene sequence; at least 95% identical to SEQ ID NO: 8 rpoB Gene sequence; and (iv) at least 95% identical to SEQ ID NO: 13. nifH Gene sequence; and (b) carrier.

[0046] In some embodiments, the *Bacillus cerevisiae* strain is strain MS3907 or an isolated clone thereof, deposited under ATCC accession number PTA-127654. In some embodiments, the composition further comprises the digestion product of the *Bacillus cerevisiae* strain on an organic substrate. In some embodiments, the carrier comprises a fertilizer. In some embodiments, the carrier is a solid coated with *Bacillus cerevisiae* strain. In some embodiments, the carrier is a liquid. In some embodiments, the composition further comprises an excipient selected from wetting agents, spreading agents, dispersing agents, adhesives, dust suppressants, and binders. In some embodiments, the concentration of the *Bacillus cerevisiae* strain in the composition ranges from 1 x 10⁻⁶. 3 Up to 1 x 10 11 CFU / ml.

[0047] On one hand, this disclosure provides a composition comprising: (a) a *Bacillus solanaceus* 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 (iii) Gene sequence; at least 95% identical to SEQ ID NO: 9 rpoB Gene sequence; and (iv) at least 95% identical to SEQ ID NO: 14. nifH Gene sequence; and (b) carrier.

[0048] In some embodiments, the *Bacillus cuspidatum* strain is strain MS4921 or an isolated clone thereof, deposited under ATCC accession number PTA-127655. In some embodiments, the composition further comprises the digestion product of the *Bacillus cuspidatum* strain on an organic substrate. In some embodiments, the carrier comprises a fertilizer. In some embodiments, the carrier is a solid coated with *Bacillus cuspidatum* strain. In some embodiments, the carrier is a liquid. In some embodiments, the composition further comprises an excipient selected from wetting agents, spreading agents, dispersing agents, adhesives, dust suppressants, and binders. In some embodiments, the concentration of the *Bacillus cuspidatum* strain in the composition is in the range of 1 x 10⁻⁶. 3 Up to 1 x 10 11 CFU / ml.

[0049] On one hand, 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 SEQ ID NO: 10; (ii) a 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 11. gyrB Gene sequence; and (iii) at least 95% identical to SEQ ID NO: 12. rpoB Gene sequence; and (b) carrier.

[0050] In some embodiments, the *Bacillus megaterium* strain is *MS2748* strain or an isolated clone thereof, deposited under ATCC accession number PTA-127652. In some embodiments, the composition further comprises the digestion product of the *Bacillus megaterium* strain on 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 a liquid. In some embodiments, the composition further comprises an excipient selected from wetting agents, spreading agents, dispersing agents, adhesives, dust suppressants, and binders. In some embodiments, the concentration of the *Bacillus megaterium* strain in the composition ranges from 1 x 10⁻⁶. 3 Up to 1 x 10 11 CFU / ml. In some embodiments, the composition further comprises at least one or more of the following: (c) 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: 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. rpoBGene sequence; (d) a strain of *Bacillus niger* that has 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 (iii) Gene sequence; at least 95% identical to SEQ ID NO: 8 rpoB Gene sequence; and (iv) at least 95% identical to SEQ ID NO: 13. nifH Gene sequence; and (d) a *Bacillus solanaceus* 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 (iii) Gene sequence; at least 95% identical to SEQ ID NO: 9 rpoB Gene sequence; and (iv) at least 95% identical to SEQ ID NO: 14. nifH Gene sequence.

[0051] On the one hand, this disclosure provides an isolated strain of Bacillus megaterium that has 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.

[0052] In some implementations, the Bacillus megaterium strain is the MS3900 strain or its isolated clones deposited under ATCC accession number PTA-127653.

[0053] On the one hand, this disclosure provides an isolated strain of *Bacillus cerevisiae* species, which has 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; (c) at least 95% identical to SEQ ID NO: 8 rpoB Gene sequence; and (d) at least 95% identical to SEQ ID NO: 13. nifH Gene sequence.

[0054] In some implementations, the Bacillus niger strain is the MS3907 strain or its isolated clone preserved under ATCC accession number PTA-127654.

[0055] On the one hand, this disclosure provides an isolated strain of *Bacillus solanaceus* species, which has one or more of the following: (a) a 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 3; (b) a 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 6. gyrB Gene sequence; (c) at least 95% identical to SEQ ID NO: 9 rpoB Gene sequence; and (d) at least 95% identical to SEQ ID NO: 14. nifH Gene sequence.

[0056] In some implementations, the Bacillus subtilis strain is the MS4921 strain or its isolated clones, which is deposited under ATCC accession number PTA-127655.

[0057] On the one hand, this disclosure provides an isolated strain of Bacillus megaterium that has one or more of the following: (a) a 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 10; (b) a 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 11. gyrB Gene sequence; and (c) at least 95% identical to SEQ ID NO: 12. rpoB Gene sequence.

[0058] In some implementations, the Bacillus megaterium strain is the MS2748 strain or its isolated clones deposited under ATCC accession number PTA-127652.

[0059] On one hand, this disclosure provides a method for promoting the growth of plants grown in a culture medium, the method comprising contacting the plant or culture medium with the composition described herein or the isolated strain described herein.

[0060] In some embodiments, the contact increases the nitrogen content of the plant by at least 5%. In some embodiments, the contact increases nitrogen fixation activity and / or nitrogen use efficiency in plant tissues by at least 5%. In some embodiments, the contact increases the population of nitrogen-fixing bacteria in the plant roots and rhizosphere by at least 5%. In some embodiments, the contact causes nitrogen-fixing bacteria present in the culture medium to be recruited to the plant rhizosphere. In some embodiments, the contact results in an increase in plant growth of at least 10% compared to a control. In some embodiments, the culture medium includes soil, hydroponic medium, turface, or isolite.

[0061] On the one hand, this disclosure provides a method for enhancing the nitrogen-fixing activity of bacteria or the colonization ability of plant tissues, the method comprising incubating bacteria in the presence of strain MS3900.

[0062] In some implementations, the bacteria is strain MS3907.

[0063] On one hand, this disclosure provides a composition comprising: (a) a microbial aggregate comprising one or more bacterial strains selected from the group consisting of MS3900 (ATCC Accession No. PTA-127653), MS3907 (ATCC Accession No. PTA-127654), MS4921 (ATCC Accession No. PTA-127655), and MS2748 (ATCC Accession No. PTA-127652); and (b) metabolites produced by the digestion of an organic substrate by microorganisms within the microbial aggregate.

[0064] In some embodiments, the microbial aggregate further comprises an enriched community of nitrogen-fixing microorganisms. In some embodiments, the organic substrate is derived from cow dung, phosphate rock, or plant mills, or any combination thereof. In some embodiments, the microbial aggregate comprises microorganisms derived from cow dung, phosphate rock, or plant mills. In some embodiments, the plant mill is soybean flour, lentil flour, chickpea flour, green pea flour, yellow pea flour, white soybean flour, corn flour, cereal flour, corn gluten, soy protein, soy protein hydrolysate, or any combination thereof. In some embodiments, the microbial aggregate comprises 5 x 10 7 Up to 1.5 x 10 8 CFU / ml of bacteria. In some implementations, the microbial aggregate contains 5 x 10⁻⁶ CFU / ml of bacteria. 5 Up to 1.5 x 10 7 Nitrogen-fixing bacteria at CFU / ml. In some embodiments, the microbial aggregate contains 5 x 10⁻⁶ CFU / ml. 4 Up to 5 x 10 5 CFU / ml of sporulating bacteria. In some embodiments, the microbial aggregate contains 1 x 102 3 Up to 1 x 10 4 MS3900 spores, MS4921 spores, or any combination thereof, at CFU / ml. In some embodiments, the microbial aggregate comprises 1 x 10⁻⁶ spores. 2 Up to 1 x 10 4 MS3907 spores, CFU / ml. In some embodiments, the pH of the composition is 7 to 9. In some embodiments, the COD of the composition is 120 to 500 mg / L. In some embodiments, the conductivity of the composition is 0.5 to 2.0 mS / cm. In some embodiments, the total dry weight of the composition is 0.2 to 2.5 mg / ml.

[0065] On one hand, 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 containing a volume of working fluid, wherein at least one of the containers contains a population of a first microbial strain and a population of other microorganisms, the population of the first microbial strain being derived from an inoculum of the first microbial strain added to the bioreactor system; (b) operating the bioreactor system for a duration by: (i) transferring an aqueous feedstock containing microbial aggregates 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 a product effluent; (iii) collecting at least a portion of the product effluent as a biostimulant composition; and (iv) maintaining the population of the first microbial strain in at least the first container at at least 80% of the population of the first microbial strain at the start of the duration throughout the duration; wherein the duration is at least 5 days; and wherein the first microbial strain is not present in the aqueous feedstock or any other input entering the bioreactor system during the duration at a concentration higher than 1% of the concentration of the population of the first microbial strain in the first container.

[0066] In some embodiments, the first microbial strain possesses properties that promote plant growth. In some embodiments, the method further includes providing conditions in the bioreactor system that promote the formation of a rich colony of the first microbial strain relative to the population of the aqueous feedstock or any other input entering the bioreactor system. In some embodiments, the method further includes applying selective pressure to the bioreactor system that is favorable to the growth of the first microbial strain relative to other microorganisms. In some embodiments, the aqueous feedstock also contains organic material that can be digested by the first microbial strain and by at least some of the other microorganisms. In some embodiments, the method further includes generating metabolites with plant growth-promoting properties by digesting the organic material.

[0067] In some embodiments, the first working fluid contains at least 0.2% w / v malic acid. In some embodiments, the first working fluid contains at least 0.2% w / v soybean meal. In some embodiments, the first working fluid includes microaerophilic conditions. In some embodiments, the working fluid in at least one of one or more additional containers includes microaerophilic conditions. In some embodiments, the system has a hydraulic retention time of at least 5 days. In some embodiments, the first working fluid also contains an established population of a second microbial strain that promotes nitrogen use efficiency, wherein the concentration of the nitrogen-promoting second microbial strain in the first working fluid is at least 100 times higher than the concentration of the nitrogen-promoting second microbial strain in the aqueous feed stream entering the bioreactor system and any other input.

[0068] In some implementation schemes, the second microbial strain that promotes nitrogen use efficiency belongs to *Sacchariformis sugarcane*, *Klebsiella variegata*, *Lahn's bacterium*, *Klüvora cistern*, etc. pseudosugars Cossacchariformis, Enterobacter species, Achromobacter madrapata, lipid-producing bacteria Azopyrillum The strains include *Microbacterium tumefaciens*, *Gastrodinium diazogenes*, *Methylobacterium symbioticum*, *Bacillus auricula-judae*, *Bacillus megaterium* (*Priscilla megaterium*), or *Bacillus spp.* In some embodiments, the first working fluid further comprises an established community of a third microbial strain that promotes nitrogen use efficiency, wherein the concentration of the nitrogen-enhancing third microbial strain in the first working fluid is at least 100 times higher than the concentration of the nitrogen-enhancing third microbial strain in the aqueous feed stream entering the bioreactor system and any other input. In some embodiments, the nitrogen-enhancing third microbial strain belongs to *Sacchariformis sugarcane*, *Klebsiella variegata*, *Laenia aquaticis*, *Klüvora centralis*, etc. pseudosugars Cossacchariformis, Enterobacter species, Achromobacter madrapata, lipid-producing bacteria Azopyrillum Microbacterium tumefaciens, Glucosamine diazogenes, Methylobacterium symbioticum, Bacillus aurantium, Bacillus megaterium (Priestella megaterium), or Bacillus spp. In some embodiments, the first working fluid contains at least 1 x 10⁻⁶ bacteria. 5 CFU / ml nifH The total population of gene-positive microorganisms.

[0069] On one hand, this disclosure provides a method comprising: (a) transferring an aqueous raw material and an inoculum of isolated microorganisms capable of promoting nitrogen use efficiency in plants into a first container containing a first working fluid of a certain volume, wherein the aqueous raw material comprises: (i) a first microbial aggregate; and (ii) digestion products generated by the digestion of an organic substrate by microorganisms in the first microbial aggregate; and (b) incubating the inoculum under conditions that promote microbial growth.

[0070] In some embodiments, promoting nitrogen use efficiency includes nitrogen fixation, promoting nitrogen fixation in plant tissues, recruiting nitrogen-fixing organisms to the plant rhizosphere and other tissues, or increasing the organic nitrogen content and / or mineralization of organic nitrogen in the soil for uptake. In some embodiments, conditions promote the growth of one or more microorganisms in a first microbial aggregate that can promote nitrogen fixation, promote nitrogen use efficiency, or promote the recruitment of nitrogen-fixing microorganisms to plant roots, or produce metabolites that promote plant growth, promote nitrogen fixation, promote nitrogen use efficiency, or promote the recruitment of nitrogen-fixing microorganisms to plant roots. In some embodiments, during incubation, the microorganisms or one or more microorganisms in the first microbial aggregate produce metabolites that promote nitrogen use efficiency. In some embodiments, incubation maintains or increases the population of one or more microorganisms in a microbial aggregate that promotes plant growth. In some embodiments, incubation results in at least maintaining the microbial population. In some embodiments, incubation results in an increase in the microbial population.

[0071] In some embodiments, incubation leads to the enrichment of a population of microorganisms that can promote nitrogen use efficiency. In some embodiments, conditions are maintained to support the growth of one or more microorganisms that can promote nitrogen fixation, promote nitrogen use efficiency, or promote the recruitment of nitrogen-fixing microorganisms to plant roots, or produce metabolites that can promote nitrogen fixation, promote nitrogen use efficiency, or promote the recruitment of nitrogen-fixing microorganisms to plant roots.

[0072] In some embodiments, the organic substrate comprises manure or lignocellulosic materials. In some embodiments, the aqueous feedstock also comprises an inorganic substrate. In some embodiments, the first microbial aggregate also comprises microorganisms derived from the inorganic substrate. In some embodiments, the inorganic substrate comprises phosphate rock.

[0073] In some implementations, the microorganism is a selection of bacteria from the following: Cossackella, Klebsiella, Laenella, Klüvora, Enterobacter, Achromobacter, Microbe, Staphylococcus, Methylobacterium, Pseudomonas, Pantotheca, and Azotobacter. Sugarcane *Syntrophus*, *Burkholderia*, cyanobacteria, *Bacillus*, and *Bacillus-like* species. In some embodiments, the microorganisms belong to *Sacchariformis*, *Klebsiella*, *Lahn's bacterium*, *Klüvora*, etc. pseudosugars Cossacchariformis, Enterobacter species, Achromobacter madrapata, lipid-producing bacteria AzopyrillumMicrobes, Staphylococcus aureus, Methylobacterium symbiosis, Bacillus thuringiensis, Bacillus megaterium (Priestella megaterium), and Bacillus solanaceus. In some embodiments, the microorganisms are strains deposited under ATCC accession number PTA-127654 (MS3907), under ATCC accession number PTA-127653 (MS3900), under ATCC accession number PTA-127655 (MS4921), or under ATCC accession number PTA-127652 (MS2748).

[0074] In some embodiments, the aqueous feedstock further comprises one or more carbon sources capable of being metabolized by microorganisms or microorganisms in a first microbial aggregate. In some embodiments, the one or more carbon sources comprise one or more simple sugars. In some embodiments, the one or more simple sugars include glucose, malic acid, lactose, sucrose, or pyruvate, or any combination thereof. In some embodiments, the aqueous feedstock further comprises a nitrogen source. In some embodiments, the nitrogen source comprises one or more of the following: ammonium sulfate, ammonium chloride, ammonium nitrate, sodium nitrate, yeast extract, or yeast, or any combination thereof. In some embodiments, the aqueous feedstock further comprises soy flour, corn flour, cereal flour, corn gluten, soy protein, or hydrolyzed soy protein, or any combination thereof.

[0075] In some embodiments, the aqueous feedstock and inoculum are transferred separately. In some embodiments, the aqueous feedstock and the inoculum containing isolated microorganisms are combined together before being transferred to a first container. In some embodiments, the first working fluid comprises (a) a second microbial aggregate derived from the aqueous feedstock, and / or (b) digestion products generated by the digestion of substances present in the aqueous feedstock by the first microbial aggregate and / or microorganisms. In some embodiments, the method further includes transferring 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) digestion products generated by the digestion of substances present in the first working fluid by the third microbial aggregate and microorganisms.

[0076] In some embodiments, the total amount of fluid transferred to the first container over a period of time is equal to the amount of the first working fluid transferred to the second container over the same period of time. In some embodiments, the volume of the first working fluid in the first container is maintained constant. In some embodiments, transferring an aqueous feedstock to the first container includes continuously flowing the aqueous feedstock into the first container at a first flow rate, and transferring a portion of the first working fluid to the second container includes continuously flowing a portion of the first working fluid into the second container at a second flow rate. In some embodiments, the first flow rate and the second flow rate are equal.

[0077] In some embodiments, the method further includes transferring a portion of the second working fluid to 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 a portion of the third working fluid to a fourth container containing a fourth working fluid, and incubating the fourth working fluid in the fourth container. In some embodiments, the volumes of the first, second, third, and fourth working fluids are maintained constant. In some embodiments, microorganisms are present in the second, third, and / or fourth working fluids. In some embodiments, the first working fluid and any subsequent working fluids are maintained under microaerophilic conditions.

[0078] In some embodiments, one or more of the five most abundant microbial species in the first microbial aggregate originate from the following genera: *Bacillus*, *Levonorhynchophylla*, *Thermophyton*, *Agromonella*, and *Acidobacter*. In some embodiments, one or more of the five most abundant microbial species in the first microbial aggregate include *Levonorhynchophylla*, *Synthia spp. phenylacetate*, *Synthia mescheniformis*, and *Synthia spp.* Solitalea canadensis Or *Nitrosporium musculosus* from Moscow.

[0079] In some embodiments, the method further includes transferring one or more additional isolated microorganisms capable of promoting nitrogen use efficiency in plants into a first container. In some embodiments, the one or more additional isolated microorganisms include one or more of MS3900, MS3907, MS4921, and MS2748, or any combination thereof. In some embodiments, two or more of MS3900, MS3907, MS4921, and MS2748 are transferred into the first container. In some embodiments, the method further includes transferring the microorganism capable of promoting nitrogen use efficiency in plants and / or one or more additional isolated microorganisms to a second, third, or fourth working fluid, or any subsequent working fluid (if more than four containers are fluidly connected in the method). In some embodiments, the one or more additional isolated microorganisms include one or more of MS3900, MS3907, MS4921, and MS2748, or any combination thereof.

[0080] In some embodiments, the method further includes filtering a first working fluid, a second working fluid, a third working fluid, a fourth working fluid, or any subsequent working fluid (if more than four containers are fluidly connected in the method). In some embodiments, filtration removes bacteria from the respective working fluids. In some embodiments, filtration removes at least 99% or at least 99.9% of all bacteria from the respective working fluids. In some embodiments, filtration produces a sterile fluid. In some embodiments, pH is monitored and adjusted during incubation. In some embodiments, the first working fluid is transferred directly to a clarifier, where flocculants from the first working fluid are separated from the first working fluid. In some embodiments, a portion of the first working fluid is not continuously transferred out of the first container. In some embodiments, incubation in the first container is operated in batch mode. In some embodiments, the dissolved oxygen content in the first working fluid is maintained between 0.1 and 0.8 mg / L during incubation in the first container. In some embodiments, the pH of the first working fluid is between 3.5 and 8 during incubation in the first container.

[0081] On one hand, 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 containing a volume of working fluid, wherein at least one of the containers contains a population of a first microbial strain and a population of other microorganisms, the population of the first microbial strain being derived from an inoculum of the first microbial strain added to the bioreactor system; (b) operating the bioreactor system for a duration by: (i) transferring an aqueous feedstock containing microbial aggregates 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 a product effluent; (iii) collecting at least a portion of the product effluent as a biostimulant composition; and (iv) maintaining the population of the first microbial strain in at least the first container at a level of at least 80% of the population of the first microbial strain at the start of the duration throughout the duration; wherein the duration is at least 5 days; and wherein the first microbial strain is not present in the aqueous feedstock or any other input entering the bioreactor system during the duration at a concentration higher than 1% of the concentration of the population of the first microbial strain in the first container.

[0082] In some implementations, the first microbial strain has properties that promote plant growth.

[0083] In some embodiments, the method further includes providing conditions in the bioreactor system that promote the formation of a rich colony of the first microbial strain relative to the population of the aqueous feedstock or any other input entering the bioreactor system. In some embodiments, the method further includes applying selective pressure to the bioreactor system that is favorable to the growth of the first microbial strain relative to other microorganisms. In some embodiments, the aqueous feedstock also contains organic material that can be digested by the first microbial strain and by at least some of the other microorganisms. In some embodiments, the method further includes generating metabolites with plant-promoting properties by digesting the organic material.

[0084] In some embodiments, the first working fluid contains at least 0.2% w / v malic acid. In some embodiments, the first working fluid contains at least 0.2% w / v soybean meal. In some embodiments, the first working fluid operates under microaerobic conditions. In some embodiments, the working fluid in at least one of one or more additional containers operates under microaerobic conditions. In some embodiments, the system has a hydraulic retention time of at least 5 days.

[0085] In some embodiments, the first working fluid further comprises an established community of a second microbial strain that promotes nitrogen use efficiency, wherein the concentration of the nitrogen-enhancing second microbial strain in the first working fluid is at least 100 times higher than the concentration of the nitrogen-enhancing second microbial strain in the aqueous feed stream entering the bioreactor system and any other input. In some embodiments, the nitrogen-enhancing second microbial strain belongs to the species *Sacchariformis canis*, *Klebsiella variegata*, *Laenia aquaticis*, *Klüberella centralis*, etc. pseusosacchari Cossacchariformis, Enterobacter species, Achromobacter madrapata, lipid-producing bacteria Azopirillum Microbacterium tumefaciens, Glucosamine diazoxide, methyl symbiotic bacteria, Bacillus thuringiensis, Bacillus megaterium (Priestella megaterium), or Bacillus spp.

[0086] In some embodiments, the first working fluid further comprises an established community of a third microbial strain that promotes nitrogen use efficiency, wherein the concentration of the nitrogen-enhancing third microbial strain in the first working fluid is at least 100 times higher than the concentration of the nitrogen-enhancing third microbial strain in the aqueous feed stream entering the bioreactor system and any other input. In some embodiments, the nitrogen-enhancing third microbial strain belongs to the family *Sacchariformis sugarcane*, *Klebsiella variegata*, *Laenia aquaticis*, *Klüberella centralis*, etc. pseusosacchari Cossacchariformis, Enterobacter species, Achromobacter madrapata, lipid-producing bacteria Azopirillum Microbacterium tumefaciens, Glucosamine diazogenes, Methylobacterium symbioticum, Bacillus aurantium, Bacillus megaterium (Priestella megaterium), or Bacillus spp. In some embodiments, the first working fluid contains at least 1 x 10⁻⁶ bacteria. 5 CFU / ml nifH The total population of gene-positive microorganisms.

[0087] On one hand, this disclosure provides a method for promoting plant growth, comprising: (a) contacting a plant and / or a culture medium in which the plant grows with a composition comprising one or more compounds, wherein the one or more compounds are selected from acinospesigenin-C, 3-cyclohexyl-6-[4-[3-(trifluoromethyl)phenyl]-1-piperazinyl]-1H-pyrimidin-2,4-dione, Arg-Thr-Ala-Arg, (2R)-2-[[4-(2,6-dipyrrolidone-1-ylpyrimidin-4-yl)piperazin-1-yl]methyl]-2,5,7,8-tetramethyl-3,4-dihydrobenzopyran-6-ol dihydrochloride, Gly-Leu-Arg-Val-Phe, wesiellamide, thrombin receptor activator for peptide 5. 5, TRAP-5), tungstic acid, fercomin, threonyl-isoleucine, BW-A868C, Lys-Ala-Leu-Glu, N-benzyloxycarbonylglycine, Glu-Asp-Asn, Glu-Asp-Asn, Ile-Glu-His-Lys, Chaps, didemethylcitalopram, Lys-Tyr-Thr-Ser-Ser, 3-amino-4,6-dimethyl-N-(1-phenylethyl)thiopheno[2,3-b]pyridine-2-carboxamide, Asn-Ala-Leu-Ala-His, Met-Asp-Arg, His-Arg-Lys-Glu, Asn-Cys-Phe, 7-hydroxylauric acid, Phe-Tyr-Lys-Arg, k -k-Strophanthoside, disopyramide, estradiol-4,9-diene-3,17-dione, HoPhe-Asp-OH, 5-methyl-6-oxo-5,6-dihydro-4h-imidazo[1,5-a]thieno[2,3-f][1,4]diaza-3-carboxylic acid tert-butyl ester, 2,6-naphthyldiol, zonisamide, Ser-Gln-Leu-Lys, Pro-Ala-Phe, Ala-Thr-Ile-Lys, mycophenolic acid, PC (15:0 / 18:3(6Z,9Z,12Z)), 6-[(2Z)-2-benzylheptaoxy]-3,4,5-trihydroxyoxacyclohexane-2-carboxylic acid, 7''-Deoxybonaspectin D 4''-methyl ether, Reciniferatoxin, trans-2-hepten-1-yl acetate, acetoxy-6-gingerol, Dubinidine, N-1-naphthylbenzamide, one or more of their derivatives, or combinations thereof.

[0088] In some embodiments, the plant growth characteristic is nitrogen use efficiency. In some embodiments, the concentration of one or more compounds in the composition is at least about 1 nanomolar (nM). In some embodiments, the concentration of one or more compounds in the composition is at least about 0.00001% of the dry weight of the composition. In some embodiments, contact includes contacting the plant with the composition. In some embodiments, contact includes contacting the plant seeds with the composition. In some embodiments, contact includes contacting the plant leaves with the composition. In some embodiments, the culture medium includes soil, hydroponic medium, turface, or isolite. In some embodiments, contact includes increasing the nitrogen content of the plant by at least 5%. In some embodiments, contact includes increasing the nitrogen fixation activity in plant tissues by at least 5%. In some embodiments, contact includes increasing the population of nitrogen-fixing bacteria in the plant roots and rhizosphere by at least 5%. In some embodiments, contact results in the recruitment of nitrogen-fixing bacteria present in the culture medium to the plant rhizosphere. In some embodiments, contact results in an increase in plant growth of at least 10% compared to plants and / or culture media that have not been exposed to one or more compounds.

[0089] On one hand, this disclosure provides a composition for promoting plant growth, comprising: (i) at least one microbial strain selected from *Bacillus megaterium* strains, *Bacillus cerevisiae* strains, or *Bacillus sophorae* strains; and (ii) one or more compounds selected from: phytolaccarin C, 3-cyclohexyl-6-[4-[3-(trifluoromethyl)phenyl]-1-piperazinyl]-1H-pyrimidin-2,4-dione, Arg-Thr-Ala-Arg, (2R)-2-[[4-(2,6-dipyrrolidine-1-ylpyrimidin-4-yl)piperazin-1- [Methyl]-2,5,7,8-tetramethyl-3,4-dihydrobenzopyran-6-ol dihydrochloride, Gly-Leu-Arg-Val-Phe, wesiellamide, thrombin receptor activating peptide 5 (TRAP-5), tungstate, fercomin, threonyl-isoleucine, BW-A868C, Lys-Ala-Leu-Glu, N-benzyloxycarbonylglycine, Glu-Asp-Asn, Glu-Asp-Asn, Ile-Glu-His-Lys, Chaps, desdimethylciphthaloyl Pluland, Lys-Tyr-Thr-Ser-Ser, 3-amino-4,6-dimethyl-N-(1-phenylethyl)thiopheno[2,3-b]pyridine-2-carboxamide, Asn-Ala-Leu-Ala-His, Met-Asp-Arg, His-Arg-Lys-Glu, Asn-Cys-Phe, 7-hydroxylauric acid, Phe-Tyr-Lys-Arg, k-toxigenic strophanthidin, disopyramide, estradiol-4,9-diene-3,17-dione, HoPhe-Asp-OH, 5-methyl- 6-Oxo-5,6-dihydro-4h-imidazo[1,5-a]thieno[2,3-f][1,4]diaza-3-carboxylic acid tert-butyl ester, 2,6-naphthyldiol, zonisamide, Ser-Gln-Leu-Lys, Pro-Ala-Phe, Ala-Thr-Ile-Lys, mycophenolic acid, PC (15:0 / 18:3(6Z,9Z,12Z)), 6-[(2Z)-2-benzylheptaoxy]-3,4,5-trihydroxyoxacyclohexane-2-carboxylic acid, 7''-Deoxybonaspectin D 4''-methyl ether, Reciniferatoxin, trans-2-hepten-1-yl acetate, acetoxy-6-gingerol, dupinidine, N-1-naphthylbenzamide, oxaloacetic acid, 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphocholine, undecano-2-ene-8,10-diyne isobutyramide, enantio-Corey PG-lactone diol, one or more of their derivatives, or combinations thereof.

[0090] In some embodiments, the *Bacillus soursopus* strain comprises 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; (iii) an rpoB gene sequence that is at least 95% identical to SEQ ID NO: 9; and (iv) a gyrB gene sequence that is at least 95% identical to SEQ ID NO: 14. nifH Gene sequences. In some embodiments, the *Bacillus niger* strain comprises 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; (iii) an rpoB gene sequence that is at least 95% identical to SEQ ID NO: 8; and (iv) a nifH gene sequence that is at least 95% identical to SEQ ID NO: 13. In some embodiments, the *Bacillus megaterium* strain 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; and (iii) an rpoB gene sequence that is at least 95% identical to SEQ ID NO: 7.

[0091] In some embodiments, among oxaloacetic acid, 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphate choline, undec-2-ene-8,10-diyne isobutyramide, and enantio-Corey PG-lactone diol, oxaloacetic acid has the highest abundance, and enantio-Corey PG-lactone diol has the lowest abundance. In some embodiments, the concentration of 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphate choline is 1%-2% of the concentration of oxaloacetic acid. In some embodiments, the concentration of undec-2-ene-8,10-diyne isobutyramide is 1%-2% of the concentration of oxaloacetic acid. In some embodiments, the concentration of enantio-Corey PG-lactone diol is 0.5%-1.5% of the concentration of oxaloacetic acid.

[0092] In some embodiments, the composition further comprises a carrier. In some embodiments, the carrier is formulated for application to plants and / or the culture medium in which the plants grow.

[0093] On one hand, this disclosure provides a composition for promoting plant growth, comprising: (i) two or more compounds selected from the group consisting of: phytolaccarin C, 3-cyclohexyl-6-[4-[3-(trifluoromethyl)phenyl]-1-piperazinyl]-1H-pyrimidin-2,4-dione, Arg-Thr-Ala-Arg, (2R)-2-[[4-(2,6-dipyrrolidone-1-ylpyrimidin-4-yl)piperazin-1-yl]methyl]-2,5,7,8-tetramethyl-3,4-dihydrobenzopyran-6-ol dihydrochloric acid Salt, Gly-Leu-Arg-Val-Phe, wesiellamide, thrombin receptor activating peptide 5 (TRAP-5), tungstate, fercomin, threonyl-isoleucine, BW-A868C, Lys-Ala-Leu-Glu, N-benzyloxycarbonylglycine, Glu-Asp-Asn, Glu-Asp-Asn, Ile-Glu-His-Lys, Chaps, desdimethylcitalopram, Lys-Tyr-Thr-Ser-Ser 3-Amino-4,6-dimethyl-N-(1-phenylethyl)thiopheno[2,3-b]pyridine-2-carboxamide, Asn-Ala-Leu-Ala-His, Met-Asp-Arg, His-Arg-Lys-Glu, Asn-Cys-Phe, 7-hydroxylauric acid, Phe-Tyr-Lys-Arg, k-strophanthidin, disopyramide, estradiol-4,9-diene-3,17-dione, HoPhe-Asp-OH, 5-methyl-6-oxo-5,6-dihydro -4h-Imidazolo[1,5-a]thieno[2,3-f][1,4]diaza-3-carboxylic acid tert-butyl ester, 2,6-naphthyldiol, zonisamide, Ser-Gln-Leu-Lys, Pro-Ala-Phe, Ala-Thr-Ile-Lys, mycophenolic acid, PC (15:0 / 18:3(6Z,9Z,12Z)), 6-[(2Z)-2-benzylheptaoxy]-3,4,5-trihydroxyoxacyclohexane-2-carboxylic acid, 7''-Deoxybonaspectin D 4''-methyl ether, Reciniferatoxin, trans-2-hepten-1-yl acetate, acetoxy-6-gingerol, dubinidine, N-1-naphthylbenzamide, oxaloacetic acid, 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphocholine, undecano-2-ene-8,10-diyne isobutyramide, enantio-CoreyPG-lactone diol, one or more of their derivatives, or combinations thereof; and (ii) a carrier.

[0094] In some embodiments, the carrier is formulated for application to plants and / or the culture medium in which the plants grow. In some embodiments, the carrier comprises a fertilizer. In some embodiments, the fertilizer is solid. In some embodiments, the carrier is liquid. In some embodiments, when applied to plants or plant growth media, the composition is configured to promote and / or be able to promote nitrogen use efficiency.

[0095] In some embodiments, among oxaloacetic acid, 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphate choline, undec-2-ene-8,10-diyne isobutyramide, and enantio-Corey PG-lactone diol, oxaloacetic acid has the highest abundance, and enantio-Corey PG-lactone diol has the lowest abundance. In some embodiments, the concentration of 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphate choline is 1%-2% of the concentration of oxaloacetic acid. In some embodiments, the concentration of undec-2-ene-8,10-diyne isobutyramide is 1%-2% of the concentration of oxaloacetic acid. In some embodiments, the concentration of enantio-Corey PG-lactone diol is 0.5%-1.5% of the concentration of oxaloacetic acid. In some embodiments, the concentration of one or more compounds in the composition is at least about 1 nM. In some embodiments, the concentration of one or more compounds in the composition is at least about 0.00001% of the total dry weight of the composition. In some embodiments, the composition further comprises an excipient selected from wetting agents, spreading agents, dispersants, adhesives, dust suppressants, and binders.

[0096] In some embodiments, the composition is configured to increase plant nitrogen content and / or be able to increase plant nitrogen content by at least 5%. In some embodiments, the composition is configured to increase nitrogen fixation activity in plant tissues and / or be able to increase nitrogen fixation activity in plant tissues by at least 5%. In some embodiments, the composition is configured to increase the population of nitrogen-fixing bacteria in the plant roots and rhizosphere and / or be able to increase the population of nitrogen-fixing bacteria in the plant roots and rhizosphere by at least 5%. In some embodiments, the composition is configured to cause and / or be able to cause nitrogen-fixing bacteria present in the plant culture medium to be recruited to the plant rhizosphere. In some embodiments, the composition is configured to cause and / or be able to cause an increase in plant growth of at least 10% compared to a control.

[0097] On one hand, this disclosure provides a method for promoting plant growth, comprising: (a) contacting a plant and / or a culture medium in which the plant grows with a composition comprising one or more compounds, wherein the one or more compounds are selected from 4-(2-pyridiniazo)-N,N-dimethylaniline, LPC (18:2 / 0:0), LPE (18:2 / 0:0), 7-[3-(dimethylamino)propoxy]-6-methoxy-2-(4-methyl-1,4-diazacycloheptane-1-yl)-N-(1-methylpiperidin-4-yl)quinazolin-4-amine, 7-chloro-2-(3,4-dimethoxyphenyl)-3,5,8-trihydroxy-6-methoxy-4H-benzopyran-4-one, 1 -(2,4,5-trimethoxyphenyl)-1,2-propanedione, 3-(4-hydroxy-2,3,5-trimethoxyphenyl)prop-2-enal, LPE (18:1 / 0:0), 9-((2-phosphonomethoxy)ethyl)guanine, PC (P-16:0 / 20:5(5Z,8Z,11Z,14Z,17Z)), Diellagilactone, 13-methylmyristic acid, D-limonene, one or more derivatives thereof, 3-phosphoadenosylselenic acid, 1,3-bis(4-bromophenyl)-5-phenyl-2,4-imidazolidinedione, F-amidine, LPC (0:0 / 18:3), undecanoic acid, Muzanzagenin, or combinations thereof.

[0098] In some embodiments, contact enhances nitrogen use efficiency in plants. In some embodiments, the concentration of one or more compounds in the composition is at least about 1 nM. In some embodiments, the concentration of one or more compounds in the composition is at least about 0.00001% of the total dry weight of the composition. In some embodiments, contact includes contacting the 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 increases the nitrogen content of the plant by at least 5%. In some embodiments, contact increases nitrogen fixation activity in plant tissues by at least 5%. In some embodiments, contact increases the population of nitrogen-fixing bacteria in the plant roots and rhizosphere by at least 5%. In some embodiments, contact causes nitrogen-fixing bacteria present in the culture medium to be recruited to the plant rhizosphere.

[0099] In some implementations, exposure results in at least a 10% increase in plant growth compared to plants and / or culture media that are not exposed to one or more compounds.

[0100] On one hand, this disclosure provides a composition for promoting plant growth, comprising: (i) at least one microbial strain selected from *Bacillus megaterium* strains, *Bacillus cerevisiae* strains, or *Bacillus sophorae* strains; and (ii) one or more compounds selected from the following: 4-(2-pyridiniazo)-N,N-dimethylaniline, LPC (18:2 / 0:0), LPE (18:2 / 0:0), 7-[3-(dimethylamino)propoxy]-6-methoxy-2-(4-methyl-1,4-diazacycloheptane-1-yl)-N-(1-methylpiperidin-4-yl)quinazolin-4-amine, 7-chloro-2-(3,4-dimethoxyphenyl)-3,5,8-trihydroxy-6-methoxy-4H-benzopyran-4-one, 1-(2,4,5-trimethoxyphenyl) 1,2-propanedione, 3-(4-hydroxy-2,3,5-trimethoxyphenyl)prop-2-enal, LPE (18:1 / 0:0), 9-((2-phosphonomethoxy)ethyl)guanine, PC (P-16:0 / 20:5(5Z,8Z,11Z,14Z,17Z)), Diellagilactone, 13-methylmyristic acid, D-limonene, 3-phosphoadenosylselenic acid, 1,3-bis(4-bromophenyl)-5-phenyl-2,4-imidazolidinedione, F-amidinium, LPC (0:0 / 18:3), undecanoic acid, Muzanzagenin, oxaloacetic acid, 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphocholine, undec-2-ene-8,10-diyne isobutyramide, enantio-Corey PG-lactone diol, one or more of its derivatives, or combinations thereof.

[0101] In some embodiments, the *Bacillus oryzae* strain comprises 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; (iii) an rpoB gene sequence that is at least 95% identical to SEQ ID NO: 9; and (iv) a nifH gene sequence that is at least 95% identical to SEQ ID NO: 14. In some embodiments, the *Bacillus oryzae* strain comprises 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; (iii) an rpoB gene sequence that is at least 95% identical to SEQ ID NO: 8; and (iv) a nifH gene sequence that is at least 95% identical to SEQ ID NO: 13. In some embodiments, the Bacillus megaterium strain 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; and (iii) an rpoB gene sequence that is at least 95% identical to SEQ ID NO: 7.

[0102] In some embodiments, among oxaloacetic acid, 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphate choline, undec-2-ene-8,10-diyne isobutyramide, and enantio-Corey PG-lactone diol, oxaloacetic acid has the highest abundance, and enantio-Corey PG-lactone diol has the lowest abundance. In some embodiments, the concentration of 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphate choline is 1%-2% of the concentration of oxaloacetic acid. In some embodiments, the concentration of undec-2-ene-8,10-diyne isobutyramide is 1%-2% of the concentration of oxaloacetic acid. In some embodiments, the concentration of enantio-Corey PG-lactone diol is 0.5%-1.5% of the concentration of oxaloacetic acid.

[0103] In some embodiments, the composition further comprises a carrier. In some embodiments, the carrier is formulated for application to plants and / or the culture medium in which the plants grow.

[0104] On one hand, this disclosure provides a composition for promoting plant growth, comprising: (i) two or more compounds selected from the group consisting of: 4-(2-pyridiniazo)-N,N-dimethylaniline, LPC (18:2 / 0:0), LPE (18:2 / 0:0), 7-[3-(dimethylamino)propoxy]-6-methoxy-2-(4-methyl-1,4-diazacycloheptane-1-yl)-N-(1-methylpiperidin-4-yl)quinazolin-4-amine, 7-chloro-2-(3,4-dimethoxyphenyl)-3,5,8-trihydroxy-6-methoxy-4H-benzopyran-4-one, 1-(2,4,5-trimethoxyphenyl)-1, 2-Propanedione, 3-(4-hydroxy-2,3,5-trimethoxyphenyl)prop-2-enal, LPE (18:1 / 0:0), 9-((2-phosphonomethoxy)ethyl)guanine, PC (P-16:0 / 20:5(5Z,8Z,11Z,14Z,17Z)), Diellagilactone, 13-methylmyristic acid, D-limonene, 3-phosphoadenosylselenic acid, 1,3-bis(4-bromophenyl)-5-phenyl-2,4-imidazolidinedione, F-amidine, LPC (0:0 / 18:3), undecanoic acid, Muzanzagenin, one or more of their derivatives, or combinations thereof; and (ii) a carrier.

[0105] In some embodiments, the carrier is formulated for application to plants and / or the culture medium in which the plants grow. In some embodiments, the carrier comprises fertilizer. In some embodiments, the fertilizer is solid. In some embodiments, the carrier is liquid. In some embodiments, when applied to plants, the composition is configured to improve and / or enhance nitrogen use efficiency.

[0106] In some embodiments, among oxaloacetic acid, 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphate choline, undec-2-ene-8,10-diyne isobutyramide, and enantio-Corey PG-lactone diol, oxaloacetic acid has the highest abundance, and enantio-Corey PG-lactone diol has the lowest abundance. In some embodiments, the concentration of 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphate choline is 1%-2% of the concentration of oxaloacetic acid. In some embodiments, the concentration of undec-2-ene-8,10-diyne isobutyramide is 1%-2% of the concentration of oxaloacetic acid. In some embodiments, the concentration of enantio-Corey PG-lactone diol is 0.5%-1.5% of the concentration of oxaloacetic acid. In some embodiments, the concentration of one or more compounds in the composition is at least about 1 nM. In some embodiments, the concentration of one or more compounds in the composition is at least about 0.00001%. In some embodiments, the composition further comprises an excipient selected from wetting agents, spreading agents, dispersants, adhesives, dust suppressants, and binders.

[0107] In some embodiments, the composition is configured to increase plant nitrogen content and / or be able to increase plant nitrogen content by at least 5%. In some embodiments, the composition is configured to increase nitrogen fixation activity in plant tissues and / or be able to increase nitrogen fixation activity in plant tissues by at least 5%. In some embodiments, the composition is configured to increase the population of nitrogen-fixing bacteria in the plant roots and rhizosphere and / or be able to increase the population of nitrogen-fixing bacteria in the plant roots and rhizosphere by at least 5%. In some embodiments, the composition is configured to cause and / or be able to cause nitrogen-fixing bacteria present in the plant culture medium to be recruited to the plant rhizosphere. In some embodiments, the composition is configured to cause and / or be able to cause an increase in plant growth of at least 10% compared to a control.

[0108] On one hand, this disclosure provides a method for promoting plant growth, comprising: (a) contacting a plant and / or a culture medium in which the plant grows with a composition comprising one or more compounds, wherein the one or more compounds are selected from zearalanone, dodecyl dimethyl acetal, 1-[5-ethyl-2-hydroxy-4-[[6-methyl-6-(1H-tetrazol-5-yl)heptyl]oxy]phenyl]ethyl ketone, N-[5-(1H-indol-3-ylmethyl)-1,3,4-thiadiazol-2-yl]-4-methoxybenzamide, trandolaprilat, lythidathion, chrysanthetriol, derivatives thereof, or combinations thereof.

[0109] In some embodiments, contact enhances phosphorus solubility in the plant growth medium. In some embodiments, the concentration of one or more compounds in the composition is at least about 1 nM. In some embodiments, the concentration of one or more compounds in the composition is at least about 0.00001% of the total dry weight of the composition. In some embodiments, contact includes contacting the plant with the composition. In some embodiments, contact includes contacting the plant seeds with the composition. In some embodiments, contact includes contacting the plant leaves with the composition. In some embodiments, the culture medium includes soil, hydroponic medium, turface, or isolite.

[0110] On one hand, this disclosure provides a composition for promoting plant growth, comprising: (a) at least one microbial strain selected from one or more of the following: Levonorgestrel ( Lewinella cohaerens ), Phenylacetylcholinesterol ( Thauera phenylacetica ), Herxolium mesenyi Thauera mechernichensis ), Solitalea canadensis and Moscow nitrifying bacteria ( Nitrospira moscoviensis (a) and (b) are compounds selected from one or more of the following: zearalenone, dodecyl dimethyl acetal, 1-[5-ethyl-2-hydroxy-4-[[6-methyl-6-(1H-tetrazol-5-yl)heptyl]oxy]phenyl]ethyl ketone, N-[5-(1H-indol-3-ylmethyl)-1,3,4-thiadiazol-2-yl]-4-methoxybenzamide, quintoprila, lythidathion, chrysanthemum triol, oxaloacetic acid, 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphate choline, undecano-2-ene-8,10-diyne isobutyramide, enantio-Corey PG-lactone diol or derivatives thereof, or combinations thereof.

[0111] In some embodiments, the concentration of one or more compounds in the composition is at least about 1 nM. In some embodiments, the concentration of one or more compounds in the composition is at least about 0.00001% of the total dry weight of the composition.

[0112] In some embodiments, among oxaloacetic acid, 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphate choline, undec-2-ene-8,10-diyne isobutyramide, and enantio-Corey PG-lactone diol, oxaloacetic acid has the highest abundance, and enantio-Corey PG-lactone diol has the lowest abundance. In some embodiments, the concentration of 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphate choline is 1%-2% of the concentration of oxaloacetic acid. In some embodiments, the concentration of undec-2-ene-8,10-diyne isobutyramide is 1%-2% of the concentration of oxaloacetic acid. In some embodiments, the concentration of enantio-Corey PG-lactone diol is 0.5%-1.5% of the concentration of oxaloacetic acid.

[0113] In some embodiments, the composition further comprises a carrier. In some embodiments, the carrier is formulated for application to plants and / or the culture medium in which the plants grow.

[0114] On one hand, this disclosure provides a composition for promoting plant growth, comprising: (a) two or more of the following: zearalenone, dodecyl dimethyl acetal, 1-[5-ethyl-2-hydroxy-4-[[6-methyl-6-(1H-tetrazol-5-yl)heptyl]oxy]phenyl]ethyl ketone, N-[5-(1H-indol-3-ylmethyl)-1,3,4-thiadiazol-2-yl]-4-methoxybenzamide, quintodextrin, lythidathion, chrysanthemum triol, oxaloacetic acid, 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphate choline, undecano-2-ene-8,10-diyne isobutyramide, enantio-Corey PG-lactone diol, derivatives thereof, or combinations thereof; and (b) a carrier.

[0115] In some embodiments, the carrier is formulated for application to plants and / or the culture medium in which the plants grow. In some embodiments, the carrier comprises a fertilizer. In some embodiments, the fertilizer is solid. In some embodiments, the carrier is liquid. In some embodiments, when applied to plants or a plant growth medium, the composition is configured to and / or to promote phosphorus solubility.

[0116] In some embodiments, oxaloacetic acid has the highest abundance among oxaloacetic acid, 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphate choline, undec-2-ene-8,10-diyne isobutyramide, and enantio-Corey PG-lactone diol, and 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphate choline has the lowest abundance. In some embodiments, the concentration of 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphate choline is 1%-2% of the concentration of oxaloacetic acid. In some embodiments, the concentration of undec-2-ene-8,10-diyne isobutyramide is 8%-12% of the concentration of oxaloacetic acid. In some embodiments, the concentration of enantio-Corey PG-lactone diol is 8%-12% of the concentration of oxaloacetic acid. In some embodiments, the concentration of one or more compounds in the composition is at least about 1 nM. In some embodiments, the concentration of one or more compounds in the composition is at least about 0.00001% of the total dry weight of the composition. In some embodiments, the composition further comprises an excipient selected from wetting agents, spreading agents, dispersants, adhesives, dust suppressants, and binders. In some embodiments, the composition is configured to enhance and / or enhance phosphorus solubility in plant growth media. Incorporation

[0117] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent that each individual publication, patent or patent application is specifically and individually indicated to be incorporated by reference. Attached Figure Description

[0118] The novel features of this disclosure are expressly set forth in the appended claims. The features and advantages of this disclosure will be better understood by referring to the following detailed description of exemplary embodiments (which utilize the principles of this disclosure) and the accompanying drawings (also referred to herein as “Figures”), in which: Figures 1A-1B It is a series of graphs showing the nitrogen content and yield of maize compared to the control and the combination of MS3900 and MS3907 isolates. Figure 1A The percentage of nitrogen content in maize plants during the V9 growth period is shown. Figure 1B The corn yield is shown (in bushels / A).

[0119] Figures 2A-2B This is a series of graphs showing the nitrogen content and yield of maize plants among the control, the first prototype aggregate containing isolates (MS3900 and MS3907), the second prototype aggregate containing isolates (MS3900 and MS3907), and isolates only (MS3900 and MS3907). All conditions were tested under 80% standard growing conditions (GSP) and 100% GSP (where 80% GSP is 80% of 100% nitrogen content in the GSP). Figure 2A The results show the nitrogen content of maize plants during the V9 growing season. Figure 2B The results for corn yield are shown.

[0120] Figures 3A-3B This is a series of graphs showing the recruitment of beneficial nitrogen-fixing microorganisms in maize plants between control, first prototype aggregates containing isolates (MS3900 and MS3907), second prototype aggregates containing isolates (MS3900 and MS3907), and isolates only (MS3900 and MS3907). All conditions were tested at 80% GSP and 100% GSP. Figure 3A The copy number of the nifH gene in the rhizosphere is shown under different conditions. Figure 3B The acetylene reduction activity under different conditions is shown.

[0121] Figures 4A-4B It is a series of graphs showing the abundance levels of the nifH gene in the NTS system. Figure 4A The results showed that the basal product of NTS-4 (NTS 1.4) had a higher nifH enrichment compared to the basal inoculum. Figure 4B The results showed that, after the final process modification, the base product of NTS-4 (NTS 1.4) had a higher nifH content compared to the base products of other NTS systems.

[0122] Figures 5A-5C It is a series of graphs showing the plant growth promoting (PGP) characteristics of the NTS-4 (NTS 1.4) system and the control. Figure 5A The results showed that NTS-4 (NTS 1.4) had improved maize biomass compared to the untreated control (UTC) condition. Figure 5B The results show that NTS-4 (NTS 1.4) has improved nitrogen content compared to UTC. Figure 5C The results showed an increase in the number of associated nitrogen-fixing organisms in maize roots of NTS-4 (NTS 1.4) compared to UTC (measured by nifH copy number).

[0123] Figure 6 An exemplary schematic diagram of an NTS system with a packed bed reactor is shown.

[0124] Figure 7 An exemplary schematic diagram of an NTS system with a fluidized bed reactor is shown.

[0125] Figure 8 A- Figure 8 C is a series of graphs showing nitrogen use efficiency between control conditions, NTS-4 containing MS3900 and MS4921 isolates, NTS-4 containing MS4921, and high N conditions with an additional 10 lbs N / A. Figure 8 A shows the nitrogen-fixing capacity of the roots / basal stem. Figure 8 B shows the dry weight of the above-ground portion (shoot). Figure 8 C indicates the nitrogen content in the aboveground parts.

[0126] Figures 9A-9C This is a series of graphs showing nitrogen use efficiency between the control, isolates MS3900 and MS3907 (applied at 1 qt. / A), nitrogen-fixing isolates (applied at 2 qt. / A), and high-N conditions (including an additional 5 lbs N / A). The isolates were applied to maize in furrows. Figure 9A It shows the soil organic matter. Figure 9B The data shows soil organic nitrogen. Figure 9C The estimated nitrogen release is shown.

[0127] Figures 10A-10C The improved nitrogen fixation capacity of microorganisms on plants grown in a greenhouse under various test conditions was demonstrated. Figure 10A A sealed wide-mouth bottle containing roots, stems, and soil (with 10% acetylene) is shown. The ethylene content of the gas was analyzed. Figure 10B This demonstrates the concept of nitrogenase reducing acetylene. Figure 10C The results of acetylene reduction in maize roots / basal stems are shown in UTC, NTS-4 (NTS1.4) containing MS3900 and MS4921, NTS-4 (NTS 1.4) containing MS4921, and the control condition with 10 lbs N added.

[0128] Figures 11A-11B It is a series of graphs showing the chlorophyll content of leaves under various treatment conditions. Figure 11A The results of the maize V5 growth period are shown. Figure 11B The results of the growth period of maize V8 are shown.

[0129] Figure 12 This graph shows the photosynthetic quantum yield tested under various treatments during the growth period of maize V9. Both application ratios and the NTS treatment of the isolated bacteria showed improved light-harvesting efficiency of the photosystem.

[0130] Figure 13 This is a graph showing the photosynthetic electron transport rate under various treatment conditions during the growth period of maize V9.

[0131] Figure 14 This is a graph showing the number of maize tillers per maize plant measured under various treatment conditions during the V5 growth period.

[0132] Figure 15 This is a graph showing the maize plant height measured under various treatments during the V7 growth period.

[0133] Figure 16This is a graph showing the stem diameter under various treatment conditions.

[0134] Figure 17 This is a graph showing the stomatal conductance (CO2 and H2O gas exchange rate) under various treatment conditions during the growth period of maize V9.

[0135] Figure 18 This is a graph showing the transpiration rate (efficiency of water moving into and through the plant) measured under various treatments during the growth period of maize V9.

[0136] Figure 19 This is a graph showing the dry biomass of maize (measured by aboveground dry weight) under various treatment conditions.

[0137] Figure 20 This is a graph showing the acetylene reduction results of maize roots / basal stems measured under various treatment conditions.

[0138] Figures 21A-21B The increased plant nitrogen content was shown in plants treated with MS3907. Figure 21A The results of the corn plant are shown. Figure 21B The results of the sorghum plant were shown.

[0139] Figures 22A-22B The study showed plant growth-promoting traits after treatment with MS3900, MS3907, or both isolates. Figure 22A The results showed that under low nitrogen (20 mg N "NC-20") conditions, the addition of MS3900 and MS3907 synergistically improved plant growth. Figure 22B The addition of MS3900 and MS3907 results in a similar leaf area (measured in pixels) as when MS3907 is processed alone.

[0140] Figure 23 The results of a shake-flask study of sorghum grass isolated using different aeration conditions are shown. All treatments showed significantly larger leaf areas compared to the untreated control (UTC). Significant differences were found at the level of the ANOVA Student's t-test (p < 0.05) when no identical letters were connected.

[0141] Figure 24 The average pixel area of ​​the aboveground portion is displayed when the shaker performance is grouped according to aeration conditions.

[0142] Figure 25 The average performance of shake flasks using different proportions of PST WB is shown. The shake flask treatments were grouped according to the proportions of PST WB used under both aerobic and anaerobic conditions.

[0143] Figure 26Results of a rockwool test for promoting plant growth in Arabidopsis thaliana are presented, targeting NTS lines 1.1, 1.2, 1.3, and 1.4 with three reactors. All treatments showed mean leaf area measurements above UTC and all demonstrated significant plant growth promotion (p = 0.0008).

[0144] Figure 27 Results are shown for NTS 1.0 systems with three or four reactors. Overall, the performance of the output solution improved when the system residence time was increased by approximately 42.86%.

[0145] Figures 28A-28B The results of maize leaf area measurements are shown between NTS treatment solutions (with or without the added isolate). The NTS-4 system containing MS3900 or MS4921 shows an increase in maize leaf area compared to UTC.

[0146] Figure 29 The isolated bacteria (MS3900 and MS3907) treatments showed higher nitrogen-fixing organism recruitment compared to the control plants.

[0147] Figure 30 The results show significant differences in the nitrogen fixation community composition in the root zone among the different treatment conditions. Treatments 3300-3303 show a nitrogen fixation community of 80% GSP-N, while treatments 3304-3307 show a nitrogen fixation community of 100% GSP-N. 3000 is a nitrogen-fixing community under 80% GSP at UTC; 3301 is a nitrogen-fixing community under 80% GSP-N with PT1+Iso (isolated bacteria); 3302 is a nitrogen-fixing community under 80% GSP-N with PT2+Iso; 3303 is a nitrogen-fixing community under 80% GSP-N with only isolated bacteria; 3304 is a nitrogen-fixing community under 100% GSP-N at UTC; 3305 is a nitrogen-fixing community under 100% GSP-N with PT1+Iso; 3306 is a nitrogen-fixing community under 100% GSP-N with PT2+Iso; and 3307 is a nitrogen-fixing community under 100% GSP-N with only isolated bacteria.

[0148] Figure 31 The treatment with isolated bacteria (MS3900 and MS3907) showed the highest maize yield compared to other treatments.

[0149] Figure 32 Plants treated with only isolated bacteria (MS3900 and MS3907) showed greater nitrogen fixation activity in maize roots / basal stems.

[0150] Figure 33The isolated strains (MS3900 and MS3907) and the prototype polymer solution NTS-PT2 with the isolated strains added (e.g., NTS batch products) showed high plant nitrogen content.

[0151] Figures 34A-34B The results of titration determinations using MS3900 and MS3907 isolates are shown. Plants treated with low ratios (8 μl / plant and 16 μl / plant) of isolates showed higher aboveground dry weight ( Figure 34A ) and high root dry weight ( Figure 34B ).

[0152] Figures 35A-35B The results of the isolated bacteria treatment on soil nitrogen were shown. Figure 35A The ratios of 16 μl / plant and 32 μl / plant resulted in a higher percentage of nitrogen compared to other treatments. Figure 35B The ratio of all isolated bacteria MS3900 and MS3907 resulted in higher ppm nitrogen levels in the soil.

[0153] Figures 36A-36B The results of the treatment of isolated bacteria on dry weight are shown. Figure 36A The display shows that only MS4921 results in the highest aboveground dry weight. Figure 36B The results showed that only MS4921 and combinations of all three isolates (MS4921, MS3900 and MS3907) resulted in the highest root dry weight.

[0154] Figure 37 This diagram illustrates the determination of strong acetylene reduction in single isolate cultures in nitrogen-free nutrient medium (headspace nitrogen purging (N2) or no nitrogen purging). UTC represents the untreated control, i.e., uninoculated nitrogen-free medium; A or B = replicate sample A or replicate sample B; N2 represents headspace nitrogen purging to remove all oxygen before acetylene injection.

[0155] Figure 38 The addition of carbon and nutrients (Hoagland + C) to maize roots in a sterile system significantly improved the acetylene reduction of MS4921.

[0156] Figure 39 Treatment with NTS-1.4 solution containing MS4921 resulted in the highest root acetylene reduction (as measured by %UTC).

[0157] Figures 40A-40B Results show the nitrogen-fixing capacity of MS4921 or MS3907 as a seed irrigation or soaking solution for maize seedlings. Figure 40A Display 10 7 CFU / ml MS4921 irrigation resulted in optimal nitrogen fixation capacity. Figure 40BThe results show that the maize treatment leads to optimal nitrogen fixation capacity. In the MS3907 treatment, at 10 6 cfu / ml MS 3907 or 10 5 CFU / ml MS3907 soaking resulted in optimal nitrogen fixation capacity. The star above the column indicates that this column represents the average of the previous three columns.

[0158] Figure 41 This is a table summarizing the plant colonization characteristics of MS3900 and MS3907.

[0159] Figure 42 The results show that the NTS basal product is comparable to the starting inoculum. nifH The content was enriched.

[0160] Figure 43 The test NTS 1.0 system is shown. nifH The enrichment results. NTS-2 (e.g., NTS 1.2) compared to the starting inoculum, nifH Achieve maximum enrichment.

[0161] Figure 44 The results show the nitrogen fixation capacity of the NTS 1.0 system tested in an acetylene reduction assay. NTS-2 (e.g., NTS 1.2) showed the highest ethylene release level.

[0162] Figures 45A-45B Nitrogen-fixing bacteria of the NTS 1.0 system were shown in root extracts. Figure 45A Treatment with NTS-1.4 basal products suggests that nitrogen-fixing bacteria recruit nitrogen to the roots. Figure 45B The results showed that NTS-1.4 treatment led to improved rhizosphere compared to UTC plants. nifH content.

[0163] Figures 46A-46B The NTS 1.0 system base products were shown to affect the root extract ( Figure 46A ) and root zone ( Figure 46B The effect of the ratio of nifH gene content to 16S rRNA gene content in ( ).

[0164] Figure 47 The NTS 1.0 system was demonstrated in acetylene reduction assays (used as a surrogate indicator of nitrogenase activity). NTS-1.4 showed the highest level of ethylene production.

[0165] Figure 48 The NTS 1.0 system was demonstrated to be capable of acetylene reduction assays in maize roots with an additional high N (e.g., high nitrogen) control.

[0166] Figure 49The levels of the target isolate MS3907 in the base product of the NTS 1.0 system are shown. The isolate concentration was measured four months after inoculation in reactor 1.

[0167] Figures 50A-50B The results of MS3907 sporulation and retention in the NTS 1.0 system base product are shown. Figure 50A The results showed that adding malic acid had a positive effect on sporulation. Figure 50B The results show that in the NTS-1.2 and NTS-1.4 systems, the fluidized bed reactor has a positive effect on the retention of MS3907.

[0168] Figure 51 The average leaf area of ​​Arabidopsis thaliana in the 0.05% or 0.2% NTS 1.0 system is shown. The 0.05% NTS-1 system showed the highest plant growth promoting capacity compared to UTC and other NTS treatments.

[0169] Figure 52 The effect of NTS solutions on soil nitrogen levels was shown. The NTS-1.2 product showed the highest percentage of soil nitrogen compared to other NTS solutions.

[0170] Figure 53 The NTS-1.2 solution showed the highest estimated soil nitrogen release (ENR) compared to UTC and other NTS solutions. ENR is the estimated amount of nitrogen to be released seasonally (lbs / acre).

[0171] Figure 54 Plants treated with NTS-1.2 solution showed increased soil organic nitrogen compared to UTC and other NTS treatments.

[0172] Figures 55A-55B The abundance of total bacteria in plant root extracts treated with NTS 1.0 system solution is shown. Figure 55A The recruitment of total bacteria to plant roots treated with NTS-1.1 and NTS-1.4 solutions was shown. Figure 55B The NTS-1.4 treatment showed the highest total bacteria count in the rhizosphere compared to UTC and other NTS treatments.

[0173] Figures 56A-56B The plant growth promotion effect of plants treated with NTS 1.0 solution is shown. Figure 56A The NTS-1 treatment showed that plants had the highest aboveground dry weight compared to UTC plants and other NTS treatments. Figure 56B Plants treated with NTS-1.1, NTS-1.3, and NTS-1.4 showed significantly higher root dry weights compared to UTC plants.

[0174] Figure 57 The NTS-1.2 and 1.4 treatments showed increased plant height compared to the UTC treatment.

[0175] Figure 58 The results showed that all NTS-treated plants had significantly larger stem diameters compared to UTC-treated plants.

[0176] Figure 59 Plants treated with NTS-1.2 and NTS-1.3 showed significantly higher leaf chlorophyll content compared to UTC plants.

[0177] Figure 60 The effect of NTS solution on the aboveground dry weight of maize was shown. Plants treated with NTS-1.3 and NTS-1.4 had significantly higher aboveground dry weight compared to those treated with UTC.

[0178] Figure 61 This demonstrates the effect of NTS solution on the aboveground dry weight of maize, as shown in a separate study. Plants treated with NTS-4 had a significantly higher aboveground dry weight compared to those treated with UTC.

[0179] Figure 62 This is an exemplary schematic diagram of an NTS 2.0 system with a flocculant scraper in the clarifier.

[0180] Figure 63 This is an exemplary schematic diagram highlighting the flocculent scraper present in the clarifier of the NTS 2.0 system.

[0181] Figure 64 DNA markers for MS3907 and MS3900 in the roots of NTS 1.0-treated plants are shown. MS3907 was identified by MS3907-specific DNA markers in the root colonizing microbial communities of seedlings treated with NTS-1.2 and NTS-1.4.

[0182] Figure 65 The ARA activity in maize stalk tissue was demonstrated. Application of MS3900 and MS4921 together resulted in a stronger nitrogen fixation capacity compared to UTC.

[0183] Figure 66 This is an exemplary schematic diagram illustrating the components of the NTS digestive system.

[0184] Figures 67A-67B This is an exemplary schematic diagram of an NTS batch system. Figure 67A The prototype 1 (e.g., PT1) system is shown. Figure 67B The prototype 2 (e.g., PT2) system is shown.

[0185] Figure 68The effect of NTS batch system products (e.g., PT1 and PT2) on ethylene production is shown. The PT1 product shows the highest ethylene peak compared to UTC and PT2.

[0186] Figures 69A-69B Two graphs show the plant growth promotion test results for PT1 and PT2. The products of the NTs batch system were applied at 2 qt / acre or 4 qt / acre. On day 10 ( Figure 69A ) and the 14th day ( Figure 69B PT2 showed the largest blade area compared to PT1.

[0187] Figure 70 The application of MS3900 and MS3907 resulted in higher root and aboveground biomass compared to UTC.

[0188] Figure 71 The application of MS2748 resulted in higher root and aboveground biomass compared to UTC.

[0189] Figure 72 It shows P w An exemplary schematic diagram of the ST system. Water is the hydraulic power source.

[0190] Figure 73 This table shows the microbial characterization of the whole fermentation broth (WB) of water-based phosphorus solubilization technology (PwST) over four months. Zinc solubilization (Z-sol) and phosphorus solubilization (P-sol) were measured in media containing insoluble phosphates from different sources (National Institute of Botany Phosphate Growth Medium (NBRIP), Hydroxyapatite (HA) medium, and Phytate medium).

[0191] Figure 74 This is a table showing the top five bacterial species in PwST WB. WB is a blend of supernatant and flocculent in specific proportions for various applications.

[0192] Figure 75 This is a graph showing Arabidopsis plant growth yield (measured in average leaf area, cm2) from UTC and PwST samples over four months.

[0193] Figure 76 This is a graph showing the average ethylene production (in area / hour) of PwST samples over four months. The average ethylene production is compared with UTC.

[0194] Figure 77 This is a table showing the characteristics of the target functional enzyme in the (PwST)WB sample.

[0195] Figure 78This is a table showing the average abundance of PQQ, nitrogenase, gluconate 2-dehydrogenase, cellulase, and pectin lyase in PwST WB.

[0196] Figure 79 This is a graph showing the aboveground dry biomass (g) of maize treated with monoammonium phosphate (MAP) fertilizer applied with water (UTC) or PwST supernatant (SPN).

[0197] Figure 80 It indicates the amount of water used during planting (UTC) or P. w Figure 1. Aboveground dry biomass (g) of maize treated with ST supernatant ditch application.

[0198] Figure 81 This indicates the use of MAP fertilizer (using P) w A graph showing the nutrient uptake (in %UTC) of six macronutrients in the aboveground parts of maize treated with ST SPN coating (shown from left to right: nitrogen (N), sulfur (S), phosphorus (P), potassium (K), magnesium (Mg), and calcium (Ca)). An asterisk indicates a statistically significant difference from UTC at p = 0.1.

[0199] Figure 82 It is displayed using P w A graph showing the nutrient uptake (as a percentage of UTC) of six macronutrients in the aboveground parts of maize treated with ST SPN furrow application (shown from left to right: nitrogen (N), sulfur (S), phosphorus (P), potassium (K), magnesium (Mg), and calcium (Ca)). An asterisk indicates a statistically significant difference from UTC at p = 0.1 using ANOVA.

[0200] Figure 83 This is a graph showing the micronutrient uptake (as a percentage of UTC) of five micronutrients in the aboveground parts of maize treated with PwST SPN furrow application (shown from left to right: boron (B), zinc (Zn), manganese (Mg), iron (Fe), and copper (Cu)). An asterisk indicates a statistically significant difference from UTC at p = 0.1.

[0201] Figures 84A-84D It is a series of displays from P w ST cSPN coated MAP fertilizer contains micronutrients (e.g., magnesium). Figure 84A ),iron( Figure 84B ) and zinc ( Figure 84C A graph showing the release (in mg / L) of [a substance]. Figure 84D This is a table showing the measurements (in UTC percentages) of each micronutrient on day 6 after application.

[0202] Figures 85A-85BIt is a series showing untreated control (UTC) and P w A graph showing the phosphorus solubility (in mg / L) between ST and cSPN. Figure 85A The average dissolved phosphorus in water was measured for nutrients six days later. Figure 85B The data shows the dissolved phosphorus in water between the two conditions at each time point.

[0203] Figure 86 The plant growth-promoting capacity of complete NTS solutions and metabolites was demonstrated. Compared with UTC plants, complete NTS 1.0 system solutions and their metabolites showed an increase in average leaf area.

[0204] Figure 87 Showing in systems without nitrogen-fixing isolates, the nitrogen-fixing gene ( nifH ) is enriched.

[0205] Figure 88 The nitrogen fixation biocapacity of each reactor in the tandem NTS 2.3 system is shown (in terms of...). nifH (Enrichment measure).

[0206] Figures 89A-89B The growth of Arabidopsis plants was shown when only inorganic nitrogen was used as the nitrogen source in the NTS 1.4 and NTS 1.5 systems. Figure 89A The average leaf area under total nitrogen (30 mM N) is shown. Figure 89B The average leaf area is shown under reduced nitrogen (1 mMN).

[0207] Figures 90A-90B The growth of Arabidopsis plants was shown when only inorganic nitrogen was used as the nitrogen source in the NTS 2.2 and NTS 2.3 systems. Figure 90A The average leaf area under total nitrogen (30 mM N) is shown. Figure 90B The average leaf area is shown under reduced nitrogen (1 mMN).

[0208] Figures 91A-91B The growth of Arabidopsis plants was shown when inorganic and organic nitrogen were used as nitrogen sources in the NTS 2.2 and NTS 2.3 systems. Figure 91A The aboveground surface area is shown using inorganic nitrogen under total nitrogen (30 mM N) and reduced nitrogen conditions (10 mM, 1 mM N and 0.1 mM N). Figure 91B The aboveground surface area is shown under conditions of inorganic nitrogen (30 mM N) and reduced organic nitrogen (1 mM N).

[0209] Figure 92The growth of Arabidopsis plants using complete solutions or metabolites in the NTS 2.2 and NTS 2.3 systems is shown. All NTS 2.0 system treatments showed a larger mean leaf area than the untreated control.

[0210] Figures 93A-93B The growth of Arabidopsis plants using complete solutions or metabolites in NTS 1.4 and NTS 1.5 systems is shown. Figure 93A The results of complete solution treatment are shown. Figure 93B The results of metabolite treatments are shown. All NTS system treatments showed larger mean leaf area compared to the untreated control, with NTS 1.4 BP complete and metabolite treatments showing the greatest plant growth promotion.

[0211] Figure 94 The results show the chlorophyll content of maize leaves 10 days after the application of foliar treatment.

[0212] Figure 95 The image shows the corn plant height before and after foliar treatment. The black bars represent the plant height before treatment, and the gray bars represent the plant height 10 days after foliar treatment.

[0213] Figure 96 The figures show the diameter of maize stalks measured before harvest. Both NTS 1.4 treatments showed larger stalk diameters compared to the untreated control plants.

[0214] Figure 97 The image shows the maize leaf area measured before and after foliar treatment. The black bars represent the leaf area before treatment, and the gray bars represent the leaf area 10 days after foliar treatment.

[0215] Figure 98 The table shows the chlorophyll content of sorghum leaves during the vegetative growth stage V6 after applying NTS 1.4 containing MS3900 or MS3900 and MS4921 via furrow application or foliar treatment (gray bars represent foliar treatment).

[0216] Figure 99 The figures show sorghum plant height 10 days after application of NTS 1.4 containing MS3900 or MS3900 and MS4921 via furrow or foliar application (gray bars represent foliar application).

[0217] Figures 100A-100D The results of plant physiological trait tests after furrow or foliar application of NTS1.4 containing MS3900 or MS3900 and MS4921 are shown (gray bars represent foliar application). Figure 100A The results show the porosity conductance. Figure 100B The results show the transpiration rate. Figure 100C The results show the photosynthetic quantum yield. Figure 100DThe results show the photosynthetic electron transport rate.

[0218] Figure 101 The results show sorghum grain yield after furrow or foliar application of NTS 1.4 containing MS3900 or MS3900 and MS4921 (gray bars represent foliar application).

[0219] Figure 102 The results show the chlorophyll content of soybean leaves before and after foliar treatment for each NTS 2.0 system.

[0220] Figure 103 The acetylene reduction activity in soybeans was demonstrated after treatment with the NTS 2.0 system, either alone or with isolated bacteria.

[0221] Figures 104A-104C The results of plant physiological trait tests after soybean foliar treatment with NTS 2.0 solution containing MS4921 or MS3900 and MS4921 are shown. Figure 104A The results show the transpiration rate. Figure 104B The results of quantum yield are shown. Figure 104C The results show the photosynthetic electron transport rate.

[0222] Figure 105 The results of normalized vegetation index (NDVI) measurements are shown after soybean foliar treatment with NTS 2.0 solution containing MS4921 or MS3900 and MS4921.

[0223] Figure 106 The number of soybean pods per four plants is shown under various conditions of foliar treatment with NTS 2.0 solution containing MS4921 or MS3900 and MS4921.

[0224] Figure 107 The results show soybean grain yield (in grams) under various conditions after foliar treatment with NTS 2.0 solution containing MS4921 or MS3900 and MS4921.

[0225] Figure 108 The average chlorophyll content of maize leaves was measured before harvest after furrow application of NTS 1.4 solution containing MS3900 or MS3900 and MS4921.

[0226] Figure 109 The average maize plant height (in cm) was measured before harvest after furrow application of NTS 1.4 solution containing MS3900 or MS3900 and MS4921.

[0227] Figure 110The stem diameter (in millimeters) was measured before harvest after furrow application of NTS 1.4 solution containing MS3900 or MS3900 and MS4921.

[0228] Figure 111 The image shows maize leaf area measured 28 days after furrow application of NTS 1.4 solution containing MS3900 or MS3900 and MS4921.

[0229] Figure 112 The study shows the increased ethylene output from maize roots by acetylene reduction assay (ARA) by broadcast application of NTS 1.4 (36 uL / pot with MS3900 and MS4921).

[0230] Figure 113 The diagram shows a nonmetric dimensionality scaling (NMDS) plot of the whole bacterial community composition of the rhizosphere communities of plants treated with NTS 1.4, the isolated bacteria, and the control. Each symbol represents one DNA extraction (e.g., one rhizosphere soil community).

[0231] Figures 114A-114B The data shows the quantification of rhizosphere soil. Figure 114A The study showed the diversity of rhizosphere soil bacteria (* indicates a significant difference from the untreated control, p < 0.1). Figure 114B The abundance of nitrogen-fixing organisms in the rhizosphere soil is shown (* indicates a significant difference from the untreated control, p < 0.05).

[0232] Figure 115 The results show the chlorophyll content of maize leaves during the vegetative growth (V8) period after application of NTS 1.4 solution containing MS3900 or MS3900 and MS4921 to the untreated control or furrow treatment.

[0233] Figure 116 The figures show maize plant height during the vegetative growth phase (V7) after application of NTS 1.4 solution containing MS3900 or MS3900 and MS4921 to the untreated control or furrow treatment.

[0234] Figure 117 The figures show the diameter of maize stalks during the V7 growth period after application of NTS 1.4 solution containing MS3900 or MS3900 and MS4921 to the untreated control or furrow treatment.

[0235] Figure 118 The furrow application treatment showed increased ethylene output from the acetylene reduction assay (ARA) in maize root and neck regions compared to untreated control plants.

[0236] Figures 119A-119DThe measurements of plant physiological parameters in untreated control plants or plants treated with furrow application of NTS1.4 solution containing MS3900 or MS3900 and MS4921 are shown. Figure 119A The results show the stomatal conductance of maize. Figure 119B The results show the transpiration rate of maize. Figure 119C The results show the quantum yield of corn. Figure 119D The results show the photosynthetic electron transport rate of maize.

[0237] Figures 120A-120D The results show the grain yield and physical measurements of the corn ear. Figure 120A The results showed that furrow application of NTS 1.4 solution containing MS3900 or MS3900 and MS4921 increased corn ear length. Figure 120B The application of trench treatment with NTS 1.4 solution containing MS3900 or MS3900 and MS4921 is shown. Figure 120C The application of trench treatment with NTS 1.4 solution containing MS3900 or MS3900 and MS4921 is shown. Figure 120D The application of trench treatment with NTS 1.4 solution containing MS3900 or MS3900 and MS4921 is shown.

[0238] Figure 121 The results showed that NTS1.4, which was incorporating MS3900 and MS4921, significantly increased the abundance of nitrogen-fixing organisms in the rhizosphere soil at an application rate of 36 μL / pot (* indicates a significant difference compared with the untreated control of 80% GSP, p < 0.1).

[0239] Figure 122 The top blade ring height is shown after NTS 1.4 treatment with MS4921 and MS3900.

[0240] Figure 123 The stem diameters are shown after NTS 1.4 treatment with MS4921 and MS3900 or after NTS 1.4 treatment with MS3907 and MS3900.

[0241] Figure 124 The chlorophyll levels in maize leaves after treatment with NTS 1.4 containing MS3907 and MS3900 are shown.

[0242] Figure 125 The aboveground dry weight (e.g., aboveground biomass) of maize treated with NTS-1.4 containing MS4921+MS3900 is shown.

[0243] Figure 126The dry weight (e.g., root biomass) of maize roots is shown after treatment with NTS-1.4 containing MS4921+MS3900 or NTS-1.4 containing MS3907+MS3900.

[0244] Figure 127 The results show the chlorophyll content of maize leaves 17 days after furrow application of NTS-2.2 or NTS-2.3 in maize plants.

[0245] Figures 128A-128B The image shows maize plant height and stem diameter measured 17 days after furrow application. Both treatment groups receiving the NTS 2.0 system product showed increased plant height compared to the untreated control plants. Figure 128A ) and stem diameter ( Figure 128B ).

[0246] Figure 129 The image shows the leaf area of ​​maize 18 days after planting and furrow application of NTS 2.0 solution.

[0247] Figure 130 The results show the ethylene output from maize roots by acetylene reduction assay (ARA) in plants treated with NTS-2.2, untreated controls, and plants treated with NTS-2.3.

[0248] Figure 131 The study shows the nitrogen uptake of aboveground maize after furrow application of NTS-2.2 and NTS2.3 compared to untreated control plants.

[0249] Figures 132A-132B The maize biomass was shown across treatment conditions. Figure 132A The results showed that NTS2.2 and NTS2.3 significantly increased aboveground dry biomass when applied via furrow treatment. Figure 132B The results showed that NTS2.2 and NTS2.3 significantly increased root dry biomass when applied via furrow treatment.

[0250] Figure 133 The total dry weight of maize from the target isolate furrow application and foliar treatments is shown. MS4921 was applied alone as a furrow application treatment or in combination with MS3900, and MS4921 was also applied alone as a foliar treatment.

[0251] Figure 134 The results showed that foliar treatment with isolate MS4921 resulted in higher acetylene reduction activity in the roots and / or root collar compared to acetylene reduction activity quantified by untreated control (UTC) or furrow application treatment using MS4921 or MS4921+MS3900.

[0252] Figure 135 The chlorophyll content of maize leaves under various treatments was shown 7 days after application of urea ammonium nitrate (UAN) fertilizer. UAN with NTS containing MS3900 and MS4921 (18 μL / pot) showed higher chlorophyll content compared to UAN alone.

[0253] Figure 136 The maize plant height under each treatment condition was shown 7 days after application of urea ammonium nitrate (UAN32) fertilizer. Compared with UAN alone, UAN with NTS containing MS3900 and MS4921 (18 μL / pot) showed higher plant height.

[0254] Figure 137 The corn stalk diameters under various treatments were shown 7 days after application of urea ammonium nitrate (UAN32) fertilizer. UAN with NTS containing MS3900 and MS4921 (36 μL / pot) showed larger stalk diameters compared to UAN alone (18 μL / pot).

[0255] Figure 138 The table shows maize leaf area before (12 days post-planting, 12 dap) and after (20 days post-planting) application of the broadcast treatment. All UANs with the NTS treatment showed larger leaf area at 20 dap compared to UANs alone. Black bars represent 12 dap, and gray bars represent 20 dap.

[0256] Figure 139 The study showed the acetylene reduction activity (ARA) in maize roots / root collar after broadcast application (measured as ethylene output). Among the treatments, UAN (36 μL / pot) with NTS containing MS3900 showed the highest ARA activity.

[0257] Figure 140 The results show the stomatal conductance (the rate of CO2 and H2O gas exchange) in wheat after planting and treatment application, expressed as moles of H2O / m³. 2 / second (mol H2O / m 2 The value was measured in s / s. Pre-planting application at 1 qt / A significantly improved gas exchange even more than 2 qt / A. All treatments significantly increased stomatal conductance at heading stage, with 2 qt / A of UAN32+NTS-2.3+MS3900+MS4921 significantly increasing stomatal conductance. Furrow application, pre-planting application, and the UAN32 additive treatment significantly increased stomatal conductance in wheat.

[0258] Figure 141 The results show the transpiration rate (efficiency of water moving into and through the plant) in wheat, expressed in millimoles of H2O / m³. 2 / s (mmol H2O / m 2 The transpiration rate was measured in seconds (qt / s). All treatments significantly increased leaf transpiration rate at the heading stage, with 2 qt / A of UAN32+NTS-2.3+MS3900+MS4921 significantly increasing transpiration rate. Furrow application, pre-planting broadcast application, and UAN32 additive treatment significantly increased wheat transpiration rate.

[0259] Figure 142 The results show the quantum yield (% of light energy used in photosynthesis) in wheat, measured as the percentage of light used in photosynthesis. NTS treatments using two ratios and with two incorporated isolates significantly improved the light-harvesting efficiency of the photosynthetic system. NTS-2.3 with both isolates showed a significantly higher quantum yield at heading stage (furrow application at a ratio of 1 qt. / A), and UAN32+NTS-2.3+MS3900+MS4921 at 2 qt / A significantly increased quantum yield (pre-planting broadcast application). Furrow application, pre-planting broadcast application, and the UAN32 additive treatment significantly increased wheat quantum yield.

[0260] Figure 143 The results show the electron transport rate (photosynthetic capacity to assimilate carbon) in wheat, expressed in micromolar electrons / m². 2 / second (μmol electrons / m 2 The NTS treatment, measured in s / s, significantly increased photosystem energy transfer to ATP production, consistent with quantum yield. All treatments, using both ratios and incorporating both isolates, enhanced leaf electron transport rates at heading. Furrow application, pre-planting broadcast application, and UAN32 supplementation significantly improved wheat electron transport rates.

[0261] Figure 144 The results of the Normalized Differential Vegetation Index (NDVI) in wheat are shown. Treatment with the UAN32 additive significantly improved the NDVI of wheat leaves. NDVI values ​​range from -1 to 1, with higher values ​​indicating healthier plants.

[0262] Figure 145 The results show the chlorophyll index in wheat. Furrow application, pre-planting broadcast application, and UAN32 additive treatment significantly increased the chlorophyll index of wheat leaves.

[0263] Figure 146 The results of the Normalized Red Edge Index (NDRE) in wheat are shown. Furrow application, pre-planting broadcast application, and UAN32 additive treatment significantly increased the normalized red edge index (NDRE) of wheat leaves.

[0264] Figure 147The results show the acetylene reduction activity of winter wheat roots. Pre-planting application treatment increased the acetylene reduction activity of winter wheat roots.

[0265] Figure 148 The aboveground dry biomass results for winter wheat are shown (with standard error bars). Treatment with UAN32 additive increased the aboveground dry biomass of wheat.

[0266] Figures 149A-149C The results show the macronutrient results for the aboveground parts of winter wheat. Treatment with UAN32 additive significantly increased nitrogen uptake in the aboveground parts of wheat. Figure 149A Treatment with UAN32 additive increased phosphorus uptake in the aboveground parts of wheat. Figure 149B Treatment with UAN32 additive significantly increased potassium uptake in the aboveground parts of wheat. Figure 149C ).

[0267] Figures 150A-150E The micronutrient results for the aboveground parts of winter wheat are shown. Treatment with UAN32 additive significantly increased boron uptake in the aboveground parts of wheat. Figure 150A Treatment with UAN32 additives increased zinc uptake in the aboveground parts of wheat. Figure 150B Treatment with UAN32 additive increased manganese uptake in the aboveground parts of wheat. Figure 150C Treatment with UAN32 additive increased iron uptake in the aboveground parts of wheat. Figure 150D Treatment with UAN32 additive increased copper uptake in the aboveground parts of wheat. Figure 150E ).

[0268] Figure 151 The soil nutrient results are shown (with standard error bars). Treatment with UAN32 additive increased soil organic nitrogen.

[0269] Figure 152 The application of furrow application, pre-planting broadcast application, and UAN32 additive treatment significantly increased wheat grain yield.

[0270] Figure 153 This paper presents a non-metric dimensionality scaling (NMDS) plot showing the whole bacterial community composition of winter wheat root extracts treated with NTS 2.3 under different application methods, isolated bacteria, and control treatments. Each symbol represents a DNA extraction (e.g., a rhizosphere soil community). Connecting lines represent treatment groups. Treatment groups are distinguished by operational taxonomic units (OTUs).

[0271] Figure 154This diagram shows a non-metric dimensionality scaling (NMDS) plot of the total nitrogen-fixing bacterial community composition of winter wheat root extracts from NTS 2.3 treated with different application methods, isolated bacteria, and control treatments. Each symbol represents a DNA extraction (e.g., a rhizosphere soil community). Connecting lines represent treatment groups. Treatment groups are distinguished by operational taxonomic units (OTUs).

[0272] Figure 155 The results of total bacterial abundance in root extracts (with standard deviation bars) are shown by quantitative PCR of the 16S rRNA gene.

[0273] Figure 156 Nitrogen fixation biomass in root extracts is shown by quantitative PCR of the nifH gene (with standard deviation bars).

[0274] Figures 157A-157C The base peak chromatograms of UHPLC-Triple-TOF-MS / MS are shown, including T3 positive ion mode ( Figure 157A T3 negative ion mode ( Figure 157B ) and HILIC negative ion mode ( Figure 157C ).

[0275] Figure 158 The Orthogonal Partial Least Squares Discriminant Analysis (OPLSDA) score plots for NTS 1.4 and NTS 1.5 are displayed to show differences in metabolites between and within groups. The X-axis represents the predicted principal components, and between-group differences can be observed horizontally. The Y-axis represents the orthogonal principal components, and within-group differences are shown vertically. The percentage represents the component's explanatory power for the dataset. Each point in the plot represents a sample, samples within the same group are represented by the same ellipse, and the group represents the sample group.

[0276] Figure 159 The Orthogonal Partial Least Squares Discriminant Analysis (OPLSDA) score plots for NTS 1.4 and NTS 1.5 are displayed, showing the differences in the top metabolites between and within groups. The X-axis represents the predicted principal components, and between-group differences can be observed horizontally. The Y-axis represents the orthogonal principal components, and within-group differences are shown vertically. The percentage represents the explanatory power of the components in the dataset. Each point in the plot represents a sample, samples within the same group are represented by the same ellipse, and the group represents the sample group.

[0277] Figure 160 The figure shows the dynamic distribution of metabolite concentration differences. In the figure, the X-axis represents the ranking of metabolites based on their FC values. The Y-axis represents the log2 FC values. Each point represents one metabolite. The points marked in the lower left corner represent the 10 metabolites that were downregulated the most, and the points marked in the upper right corner represent the 10 metabolites that were upregulated the most.

[0278] Figure 161 Principal component analysis of the sample population PwST system output is shown compared to the sample NTS system output. PC1 represents the first principal component, and PC2 represents the second principal component. The percentage represents the percentage of the dataset explained by the principal components. Each shape in the figure represents a sample. FS = filtered sterile, IN = intact material (unfiltered sterile).

[0279] Figure 162 The results show the leaf area of ​​Arabidopsis thaliana plants treated with selected chemical compounds.

[0280] Figure 163 The results show the leaf area of ​​Arabidopsis thaliana plants treated with selected chemical compounds.

[0281] Figure 164 The results show the leaf area of ​​Arabidopsis thaliana plants treated with selected chemical compounds. Detailed Implementation

[0282] While various embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Many variations, modifications, and / or substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0283] This document describes systems and methods for the microbial digestion of multiple feedstocks. The systems disclosed herein may include continuous systems (e.g., sIP systems) capable of producing isolated microorganisms in a tandem serial manner. The production of isolated microorganisms in sIP systems can be carried out within mixed microbial aggregates. The target isolated microorganisms in the sIP digestion system can become enriched in the microbial environment and exhibit superior efficacy and function. The primary target function may be nitrogen use efficiency and / or nitrogen fixation from soil or fertilizer, as well as improved nutrient uptake by plants. The target isolated microorganisms may possess commercially valuable attributes and can be introduced into continuous (e.g., tandem serial) reactor systems consisting of complex microbial aggregates modified for specific functions (e.g., nitrogen use efficiency). Without wishing to be bound by theory, the target isolated microorganisms can contribute performance gains to the microbial aggregates of the digestion systems described herein, providing chemical and / or functional synergies as they grow within the system.

[0284] The products of the digestion methods and systems described herein may include microorganisms and metabolites produced by the microorganisms during digestion of a raw material 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 (e.g., soil) in which the plant grows, 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 that have the desired plant-growth-promoting properties, such that the biostimulant product possesses the desired plant-growth-promoting properties. The application of the digestion system products described herein may be applied to a dry fertilizer, in conjunction with fertilizer application, in a formulation comprising liquid fertilizer or micronutrient coating formulation with additional components, on a foliar surface, or any combination thereof. The application of the digestion system products 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., primary roots, 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.

[0285] I. Certain Definitions 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 the context otherwise expressly specifies, the singular forms “a,” “an,” and “the” used in this specification and the appended claims include plural references. It should also be noted that unless the context otherwise expressly specifies, 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.

[0286] The term "about" or "approximately" refers to a specific value within an acceptable range of error as determined by a person skilled in the art, which will depend in part on the method of measurement or determination of the value, 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 a specific value is described 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.

[0287] 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 tryptone soybean agar (TSA), zinc agar, nutrient medium, lysogenic broth (LB medium), and / or plate counting agar.

[0288] The term "digestive system" can refer to one or more reactors (e.g., containers) through which a certain volume of fluid can pass. The terms "digestive system" and "bioreactor system" are used interchangeably.

[0289] 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 proportion of the target isolated strain. An enriched culture may contain a higher amount of the target isolated strain and / or the target microbial population compared to the total microbial population of the culture. An enriched culture may contain a growth population of the target isolated strain and a microbial population enriched for a specific function (e.g., nitrogen use efficiency) over a period of time. An enriched culture may contain a certain proportion of the target isolated strain and a microbial population enriched for a specific function (e.g., nitrogen use efficiency). In some embodiments, an enriched culture may contain a certain proportion of the target isolated strain, a microbial population enriched for a specific function, and metabolites enriched for a specific function (e.g., nitrogen use efficiency). An enriched culture may contain a certain proportion of the total bacterial population in the container of the digestive system described herein. An enriched culture may contain a certain proportion of the total bacterial population in the output products (e.g., biostimulants) described herein. In some embodiments, the 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%.

[0290] 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.

[0291] 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 by one or more applications. “Effective microorganisms” can refer to a target strain that, at a statistically significant level, promotes plant health, growth, and / or yield compared to an untreated control. In some cases, the expression “effective amount” can be used herein to refer to the quantity of microbial treatments that can be used to obtain beneficial or desired results relative to the results occurring under suitable treatment conditions as described herein. For example, the expression “agricultural effective amount” can be used herein to refer to the quantity of microbial treatments that can be used to obtain agriculturally beneficial or desired results relative to the results occurring under suitable 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.

[0292] 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.

[0293] 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).

[0294] 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 alignment of sequences 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 algorithm (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 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 present in public or proprietary databases. Such searches can be performed using the National Center for Biotechnology Information Basic Local Alignment Search Tool (NCBI BLAST v 2.18).The NCBI BLAST program is available online from the National Center for Biotechnology Information (blast.ncbi.nlm.nih.gov / Blast.cgi). Typically, the following NCBI BLAST parameters can be used: filter options set to "default", comparison matrix set to "BLOSUM62", empty space cost set to "existence: 11, expansion: 1", word length set to 3, expected value (E threshold) set to 1e-3, and minimum local alignment length 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.

[0295] The term "plant growth promotion" (e.g., "PGP") can refer to processes that promote plant health, growth, yield, or any combination thereof. In some implementations, PGP can encompass improvements in a variety of plant characteristics, including but not limited to improved nitrogen fixation, improved phosphorus 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, improved tolerance to abiotic stresses, mitigation 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 stress tolerance (e.g., drought tolerance, waterlogging tolerance, salt tolerance, heat tolerance, insect resistance), improve nutrient uptake, plant health and growth, improve root development, increase leaf area, increase plant yield, increase macronutrient uptake, increase micronutrient uptake, increase seed germination, increase the abundance of functional enzymes, increase dry biomass, or increase the cumulative biomass of the plant. In some embodiments, the microbial strains, isolates, cultures, or compositions described herein increase the size or mass of the plant or its parts compared to control plants or untreated plants, or compared to predetermined standards. In some embodiments, the microbial strains, isolates, cultures, compositions, or synthetic aggregates described herein improve plant health, growth, and yield compared to control plants or untreated plants, and can also survive and proliferate in the microenvironment associated with the root surface.

[0296] 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.

[0297] For crops, "yield" can also refer to the amount of material harvested 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.

[0298] In some embodiments, the microbial strains, isolates, cultures, and compositions according to the embodiments of this application result in 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 result in 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 increase nitrogen fixation by the aforementioned percentage, increase nitrogen content by the aforementioned percentage, increase nitrogen acquisition by the aforementioned percentage, increase nitrogen uptake by the aforementioned percentage, or increase total root weight or leaf area or plant product yield by the aforementioned percentage (e.g., increase plant product weight by the aforementioned percentage).

[0299] 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) or (b) the target plant itself under conditions not exposed to a specific treatment (such as, for example, an inoculum or a combination of inoculum and / or other chemicals). A control plant can also refer to a plant that has not been treated. A control plant can also refer to a plant that has received standard fertilizer. A control plant can also refer to a plant that has only received water. 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 the microorganisms described herein. A treated plant can include a plant to which an inoculum or biostimulant composition of the microorganisms described herein has been applied using furrow application. A treated plant can include a plant to which an inoculum or biostimulant composition of the microorganisms described herein has been applied using lateral application. Treated plants may include plants to which an inoculum of the microorganisms described herein or a biostimulant composition has been applied to the soil. Untreated plants may include plants to which no inoculum of the microorganisms described herein or a biostimulant composition has been applied, directly or indirectly.

[0300] 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, yield, or any combination thereof. Many microorganisms suitable for use in inoculum form a symbiotic relationship with the target crop, in which 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, the isolated bacteria may colonize the plant, and root colonization may inhibit the entry of plant pathogens into the roots. The inoculum (e.g., a microbial inoculum) may be added at a point in time during digestive processes.

[0301] The term “tandem serial production of isolated microorganisms” (e.g., sIP) can refer to manipulated, specialized, continuous tandem serial reactors that can support the growth and enrichment of the microorganisms, isolated microorganisms, target isolated microorganisms, and / or microbial bodies described herein.

[0302] 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).

[0303] The term "whole fermentation broth" (e.g., WB) can refer to a mixture of supernatant and flocculants in a specific proportion used in the techniques described herein. Whole fermentation broth may contain microbial communities (e.g., microorganisms that promote nitrogen use efficiency), enzymes, fungi, biosolids, or any combination thereof. For example, the bacterial genera of whole fermentation broth may include *Bacillus*, *Levonorhynchus*, *Thermosynthesis*, *Agromonella*, *Acidobacter*, *Levonorhynchus ligus*, *Synthia spp.*, *Mesenni*, etc. Solitalea canadensis Nitrifying spirochetes from Moscow or any combination thereof. The complete fermentation broth can possess plant growth-promoting properties. For example, the complete fermentation broth can have nitrogen-fixing capabilities.

[0304] 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, molds, or any combination thereof.

[0305] The term "supernatant" (e.g., "basal product") can refer to the final product of a digestive system. The amount of isolated microorganisms, the number of members in microbial aggregates, or the types and amounts of microbial metabolites with plant growth-promoting capabilities can be measured in the supernatant.

[0306] 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 organic feedstock is introduced into the system. Hydraulic loading rate includes the rate at which hydraulic power is introduced into the system.

[0307] The term "internal recycling rate" can refer to the rate at which the working fluid is recycled within the phase space.

[0308] The term "hydraulic feed rate" can refer to the rate at which the working fluid transfers between phase spaces.

[0309] The term "hydraulic residence time" can refer to the duration of time that a working fluid exists in phase space.

[0310] 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 provides an enrichment environment for microorganisms within the digestive system.

[0311] 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.

[0312] 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.

[0313] II. Multiple Approaches to Nitrogen Use Efficiency Implementations of the systems and methods described herein produce biostimulant products that can promote efficient nitrogen utilization in plants in multiple modes. 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 media (e.g., soil). One mode of action of the products produced in some embodiments is the nitrogen-fixing activity of the nitrogen-fixing bacteria contained in the product. Another mode of action can be the recruitment of plant-associated nitrogen-fixing organisms already present in the soil to the plant roots, thereby enhancing nitrogen-fixing activity in the rhizosphere, and potentially enhancing nitrogen-fixing activity in other plant tissues if the recruited nitrogen-fixing microorganisms become endophytes and can move throughout the plant. This recruitment can be achieved through bacterial metabolites present in the biostimulant product and / or through bacterial isolates. Another mode of action can be the increase of soil organic nitrogen and the stimulation of organic nitrogen mineralization and uptake by microorganisms and / or microbial metabolites present in the products produced in the embodiments described herein.

[0314] The biostimulant products described herein can provide plants with microbial nitrogen fixation through: directly through the endophytic and symbiotic microorganisms present in the product, and indirectly through the mineralization or decomposition of organically bound nitrogen in the soil produced by soil nitrogen-fixing organisms. Plants can be provided with nitrogen through the decomposition of organically bound nitrogen in the soil. Implementations of the biostimulant products can also provide plants with the ability to acquire additional nitrogen from soil organic matter. Implementations of the biostimulant products can provide a combination of these strategies to better acquire nitrogen from biological / organic sources and improve nitrogen use efficiency (NUE).

[0315] The following genera of microorganisms can improve nitrogen use efficiency in plants: Cossackella, Klebsiella, Laenella, Klüvorella, Enterobacter, Achromobacter, Microbacterium, Staphylococcus, Methylobacterium, Pseudomonas, Pantotheca, and Azotobacter. Azocarus *Syntrophus*, *Burkholderia*, cyanobacteria, *Bacillus*, and *Bacillus-like* species. The following microbial species can promote nitrogen use efficiency in plants: *Sacchariformis* (Sugarcane Family), *Klebsiella* (Alternaria), *Lahn's bacterium* (Aquatic), *Kluwer's bacterium* (Centralized), pseusosacchari Cossacchariformis, Enterobacter species, Achromobacter madrapata, lipid-producing bacteria Azopirillum Microbes such as *Gastrobinacillus*, *Gastrobinacillus diazoxide*, *Methylobacterium symbioticum*, *Bacillus aurantium*, *Bacillus megaterium* (*Priestella megaterium*), and *Bacillus sophorae* are mentioned. Embodiments of the products described herein may include one or more of these microorganisms. These genera of microorganisms may include endophytic nitrogen-fixing organisms (nitrogenous organisms) of monocotyledonous plants.

[0316] Microbial inoculum, microbial aggregates, nitrogen-enhancing microorganisms, nitrogen-enhancing metabolites, or any combination thereof, can be nitrogen-enhancing microorganisms in the system's working fluid and / or in the digestive system's output products (such as basal products). These nitrogen-enhancing microorganisms may possess... nifH Gene. nifH Genes may include those having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.9%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO: 13-14. nifHThe gene may include a gene encoding a nitrogenase reductase polypeptide. In some embodiments, the nitrogenase reductase polypeptide has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.9%, or 100% sequence identity with the amino acid sequence encoded by SEQ ID NO: 13 or 14.

[0317] The biostimulant products described herein may include beneficial plant-associated nitrogen-fixing organisms that can associate with plant roots and other tissues and become endophytic nitrogen-fixing organisms, exhibit nitrogen-fixing activity when associated with roots and other plant tissues, and show a sustained increase in plant nitrogen use efficiency, or any combination thereof. In terms of nitrogen-fixing organism recruitment, biostimulant products may include the following capabilities: increasing the number of nitrogen-fixing organisms associated with plant roots and / or endophytic nitrogen-fixing organisms through nitrogen-fixing organism recruitment, nitrogen-fixing activity of treated plants, showing a sustained increase in nitrogen use efficiency, or any combination thereof. Microbial metabolites in biostimulant products may enhance the ability of plants to acquire organically bound nitrogen and other organic matter produced by soil nitrogen-fixing organisms, increase soil microbial respiration and biomass, and increase the recruitment of beneficial microorganisms.

[0318] In some embodiments, this document describes a series of tandem reaction chambers that can be used for methods of producing biostimulant products. In some embodiments, conditions within the reactor chamber can be established to selectively promote the production of one or more microorganisms that have specific desired effects on plant growth (e.g., nitrogen use efficiency).

[0319] On one hand, this disclosure provides a method comprising (a) transferring an aqueous organic feedstock and an inoculum of one or more microorganisms capable of promoting nitrogen use efficiency in plants into a first container containing a first working fluid of a certain volume, wherein the aqueous organic feedstock comprises: (i) a first microbial aggregate; and (ii) digestion products produced by microorganisms in the first microbial aggregate digesting the organic material; and (b) incubating the inoculum under conditions that promote the growth of nitrogen-fixing microorganisms supplied by the microbial aggregate and intentionally introduced microorganisms, thereby increasing the population of nitrogen-fixing microorganisms and enabling the intentionally introduced microorganisms to persist.

[0320] In some embodiments, the bioreactor system (e.g., a digestion system) contains a built-up population of one or more microbial strains that promote nitrogen use efficiency in one or more containers of the system. A “built-up 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, a built-up population is a population that has not decreased by more than 1%, 3%, 5%, 10%, 15%, 20%, or 25% during continuous operation of the bioreactor system for at least 5, 10, 15, 20, 25, 30, 60, or 90 days without being added to the bioreactor system at concentrations higher than 1, 10, 50, or 100 CFU / ml. In some embodiments, the built-up population of microbial strains has been built up by one or more inoculations of microbial strains into one or more containers of the bioreactor system. In some embodiments, the built-up population is a population derived from a population inoculated into the system at least 10, 30, 60, or 90 days prior.

[0321] In some embodiments, the bioreactor system comprises at least one microbial strain. In some embodiments, the bioreactor system comprises at least one microbial strain that promotes nitrogen use efficiency. In some embodiments, the bioreactor system comprises a built-up population of a first microbial strain that promotes nitrogen use efficiency and a built-up population of a second microbial strain that promotes nitrogen use efficiency. In some embodiments, the bioreactor system further comprises a built-up population of a third microbial strain that promotes nitrogen use efficiency. The built-up populations of the respective microbial strains can be established by inoculating the bioreactor system individually or in combination. Individual inoculation may comprise an inoculum containing one microbial strain (e.g., a microorganism). Combined inoculation may comprise an inoculum containing at least two microbial strains. Combined inoculation may comprise the same isolated microbial strain. Combined inoculation may comprise isolated microbial strains and non-isolated microbial strains. Combined inoculation may comprise two or more isolated microbial strains.

[0322] In some embodiments, the concentration of the second microbial strain that promotes nitrogen utilization efficiency in the first working fluid is at least 100 times higher than the concentration of the second microbial strain that promotes nitrogen utilization efficiency in the aqueous feed stream entering the bioreactor system and any other input. For example, the concentration of the second microbial strain that promotes nitrogen utilization efficiency in the first working fluid may be at least about 2 times higher, at least about 5 times higher, at least about 10 times higher, at least about 25 times higher, at least about 50 times higher, at least about 100 times higher, at least about 150 times higher, at least about 200 times higher, at least about 250 times higher, at least about 500 times higher, or greater than about 500 times higher. The concentration of the second microbial strain that promotes nitrogen utilization efficiency in the first working fluid can be up to about 500 times higher, up to about 250 times higher, up to about 200 times higher, up to about 150 times higher, up to about 100 times higher, up to about 50 times higher, up to about 25 times higher, up to about 10 times higher, up to about 5 times higher, up to about 2 times higher, or less than about 2 times higher than the concentration of the second microbial strain that promotes nitrogen utilization efficiency in the aqueous feed stream or any other input entering the bioreactor system.

[0323] In some embodiments, promoting nitrogen use efficiency may include increasing nitrogen fixation, promoting nitrogen fixation in plant roots and other tissues, recruiting nitrogen-fixing organisms to plant roots, increasing soil organic nitrogen content, and promoting the mineralization and uptake of soil organic nitrogen, or any combination thereof. In some embodiments, the microorganisms are capable of nitrogen fixation, promoting nitrogen fixation in plant roots and other tissues, recruiting nitrogen-fixing organisms to plant roots, increasing soil organic nitrogen content, and promoting the mineralization and uptake of soil organic nitrogen, or any combination thereof. In some embodiments, conditions promote the growth of one or more microorganisms in a first microbial aggregate that can promote plant growth, nitrogen fixation, nitrogen use efficiency, or the recruitment of nitrogen-fixing organisms to plant roots, or can produce metabolites that can promote plant growth, nitrogen fixation, nitrogen use efficiency, increase soil organic nitrogen, and the mineralization and uptake of organic nitrogen by plants, or the recruitment of nitrogen-fixing microorganisms to plant roots. In some embodiments, metabolites that promote plant growth and nitrogen use efficiency are produced during the incubation of microorganisms or one or more microorganisms in the first microbial aggregate. In some embodiments, incubation increases the population of one or more microorganisms in the microbial aggregate that can promote plant growth. In some embodiments, the aqueous organic feedstock further comprises an inorganic substrate. In some embodiments, the first microbial aggregate further comprises microorganisms derived from the inorganic substrate. In some embodiments, the inorganic substrate comprises phosphate rock. In some embodiments, one or more microorganisms belong to the species *Bacillus terrestris*, *Bacillus megaterium*, or *Bacillus solanaceus*. In some embodiments, the microorganisms are *Bacillus terrestris* strain (MS3907) deposited under ATCC accession number PTA-127654, *Bacillus megaterium* (or *Priestella megaterium*) strain (MS3900) deposited under ATCC accession number PTA-127653, *Bacillus solanaceus* strain (MS4921) deposited under ATCC accession number PTA-127655, or *Bacillus megaterium* strain (MS2748) deposited under ATCC accession number PTA-127652. In some embodiments, the first working fluid comprises (a) a second microbial aggregate derived from an aqueous organic feedstock and / or (b) digestion products produced by the first microbial aggregate and microorganisms digesting substances present in the organic feedstock. In some embodiments, the method further includes transferring a portion of the first working fluid to a second container. The second container may contain the second working fluid. The method may also include incubating the second working fluid in the second container. In some embodiments, the method further includes transferring a portion of the first working fluid to a second container containing the second working fluid and incubating the second working fluid in the second container. The second working fluid may contain a third microbial aggregate, and the third microbial aggregate may be derived from the first working fluid. The second working fluid may contain digestion products produced by the third microbial aggregate, microbial strains, or any combination thereof digesting substances present in the first working fluid.In some embodiments, the second working fluid comprises (a) a third microbial aggregate derived from the first working fluid and (b) digestion products produced by the third microbial aggregate and microbial digestion of substances present in the first working fluid. In some embodiments, the amount of aqueous organic feedstock transferred to the first container within a certain time period is equal to the amount of first working fluid transferred to the second container within the same time period. In some embodiments, the amount of aqueous organic feedstock transferred to the first container within a certain time period is not equal to the amount of first working fluid transferred to the second container within the same time period. In some embodiments, the volume of the first working fluid in the first container is maintained constant. In some embodiments, the volume of the first working fluid in the first container is not maintained constant. In some embodiments, transferring the aqueous organic feedstock to the first container includes continuously flowing the aqueous organic feedstock into the first container at a first flow rate, and transferring a portion of the first working fluid to the 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 is equal to the second flow rate. In some embodiments, the method further includes transferring a portion of the second working fluid to 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 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, second, third, and fourth working fluids are maintained at a constant volume. In some embodiments, the plant growth-promoting product prepared by the methods described herein can promote nitrogen use efficiency in plants. In some embodiments, methods for promoting plant nitrogen use efficiency include contacting the plant and / or the culture medium in which the plant grows with the product.

[0324] III. Microbial Digestion Methods and Systems A. System Overview Some embodiments disclosed herein include methods and systems for the digestion of substances provided in feedstocks by microorganisms contained in microbial aggregates. The digestion system may consist of a series of independent, fluidly connected containers (also referred to herein as “reactors”). In each reactor, different microbial aggregates can be built and maintained through 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 feedstock digestion can be carried out in different reactors, which can lead to (1) more complete digestion compared to other types of digestion systems—i.e., more complete degradation of macromolecules in the feedstock—and / or (2) the production of a variety of microbial digestion products with properties that promote plant growth (e.g., the ability to recruit nitrogen-fixing organisms to plant tissues or otherwise enhance nitrogen use efficiency).

[0325] In some embodiments, the reactor or series of reactors serves to facilitate the growth of one or more microorganisms with desired plant growth-promoting properties and / or to facilitate the production of digestible products with plant growth-promoting properties. The system may include two, three, four, five, six, 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 an inoculum. The one or more microorganisms may also be introduced into the system as part of a feed material containing a mixture of microorganisms. The one or more microorganisms may be endogenous microorganisms of organic materials (such as, for example, manure, plants, lignocellulosic materials, or algae). The one or more microorganisms may also be endogenous microorganisms of other types of feed materials (such as phosphate rock or coal). 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.

[0326] The inputs to a digestive system may include one or more of water, microbial inoculum, nutrients (e.g., carbon, nitrogen, phosphorus, or any combination thereof), and digestible substrate. The fluid within the reactors of the digestive system may be referred to herein as the "working fluid." Under continuous operation, each reactor may have a constant volume of working fluid, with the inflow rate matching the outflow rate. Since each reactor may contain different microbial aggregates and have conditions different from other reactors, the working fluid within each reactor can be considered distinct from the working fluids in 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.

[0327] The digestible substrates included in the input stream into the digestive system may include, for example, organic materials that can be digested by microorganisms in the digestive system. Such organic materials may include, for example, manure, lignocellulosic materials, wastewater biosolids, food waste, energy crops, yeast, agricultural waste, algae, or any combination thereof. Manure may be cow manure, chicken manure, horse manure, sheep manure, alpaca manure, rabbit manure, pig manure, bird manure, or any combination thereof. In some embodiments, the manure is a mixture of one, two, three, or more types of manure. The digestible substrate input into the digestive system may have been partially digested before being input into the system. Therefore, the system input may contain the products of endogenous microbial digestion of the original digestible substrate, as well as digestible material still present in the input. In some embodiments, the digestible substrates included in the input stream may include inorganic substrates. Inorganic substrates may include, for example, sand, vermiculite, perlite, pumice, or any combination thereof. In some embodiments, inorganic substrates include minerals. In some embodiments, inorganic substrates include phosphate rock.

[0328] In some embodiments, the microbial inoculum comprises a single isolated microorganism. In some embodiments, the microbial inoculum may comprise 1 to 5 isolated microorganisms. In some embodiments, the inoculum may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more isolated microorganisms. In some embodiments, the inoculum may comprise more than 5 isolated microorganisms. In some embodiments, the inoculum may comprise up to about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or fewer isolated microorganisms. In some embodiments, in addition to one or more isolated microorganisms, the microbial inoculum input into the digestive system may comprise a complex mixture of microorganisms, which may contain at least about 5, 10, 20, 25, 50, 100, 200, 225, 250, 275, 300, 350, 400, or more microbial species. In some implementations, in addition to one or more isolated microorganisms, the microbial inoculum input into the digestive system may contain a complex mixture of microorganisms, which may contain up to about 400, 350, 300, 275, 250, 225, 200, 100, 50, 25, 20, 10, 5 or fewer microbial species.

[0329] The inoculum of the microorganisms described herein may possess at least one plant growth-promoting characteristic (e.g., a plant growth property). Plant growth-promoting characteristics may include aboveground biomass, root biomass, nutrient uptake, crop yield, leaf area, chlorophyll content, increased photosynthesis, heat tolerance, cold tolerance, drought tolerance, or salt tolerance, or total biomass. The digestive system may be configured to increase the production of the microbial inoculum. The microorganisms may be bacterial strains, fungal strains, or algal species. The microbial inoculum may be a single-inoculation microbial strain.

[0330] In some embodiments, the inoculum of microorganisms may contain at least two isolated microorganisms. In some embodiments, the inoculum of microorganisms may contain at least one isolated microorganism and at least one non-isolated microorganism. The inoculum of microorganisms 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 of microorganisms may be transferred to a second, third, fourth, fifth, sixth, or any other container of the system described herein.

[0331] The concentration of the microbial inoculum can be at least about 1.0 x 10⁻⁶ before being transferred to the first container in the digestive system. 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 microbial inoculum before transfer to the first container in the digestive system can be up to approximately 1.0 x 10⁻⁶ cfu / ml. 12 cfu / ml, 1.0 x 10 11 cfu / ml, 1.0 x 10 10 cfu / ml, 1.0 x 10 9 cfu / ml, 1.0 x 10 8 cfu / ml, 1.0 x 10 7 cfu / ml, 1.0 x 10 6 cfu / ml, 1.0 x 10 5cfu / ml, 1.0 x 10 4 cfu / ml, 1.0 x 10 3 cfu / ml, 1.0 x 10 2 cfu / ml or less than approximately 1.0 x 10 2 cfu / ml.

[0332] The concentration of the microbial inoculum after incubation in the digestive system described herein can be at least 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 CFU / ml. The concentration of the microbial inoculum after incubation in the digestive system described herein can be up to about 1.0 x 10⁻⁶. 12 cfu / ml, 1.0 x 10 11 cfu / ml, 1.0 x 10 10 cfu / ml, 1.0 x 10 9 cfu / ml, 1.0 x 10 8 cfu / ml, 1.0 x 10 7 cfu / ml, 1.0 x 10 6 cfu / ml, 1.0 x 10 5 cfu / ml, 1.0 x 10 4 cfu / ml, 1.0 x 10 3 cfu / ml, 1.0 x 10 2 cfu / ml or less than approximately 1.0 x 10 2 cfu / ml.

[0333] In some embodiments, the aqueous organic feedstock and the microbial inoculum can be transferred to the first reactor separately. In some embodiments, the aqueous organic feedstock can be transferred to the first reactor before the microbial inoculum. In some embodiments, the microbial inoculum can be transferred to the first reactor before the aqueous organic feedstock. In some embodiments, the aqueous organic feedstock and the microbial inoculum can be transferred to the first reactor simultaneously.

[0334] In some embodiments, the aqueous feedstock may not contain the target isolated bacterial strain (e.g., an inoculum of the microorganism). For example, the aqueous feedstock may not contain the target isolated bacterial strain before being transferred to the first container. The concentration of the target isolated microbial strain (e.g., a strain that promotes nitrogen use efficiency) may be 0 cfu / ml. In some embodiments, the aqueous feedstock may contain the target isolated bacterial strain before being transferred to the first container. In some embodiments, the total composition of 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 isolated bacterial strain. In some embodiments, the aqueous feedstock may not contain microbial strains (e.g., microbial strains that promote nitrogen use efficiency) 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.

[0335] In some embodiments, the digestion system includes a clarifier chamber or clarifier tank (CLF). The terms "clarifier chamber," "clarifier tank," and "clarifier container" are used interchangeably. The clarifier may contain a working fluid (e.g., clarifier working fluid). The clarifier may include a single inlet port and a single outlet port. The clarifier may include a single inlet port and multiple outlet ports. In some embodiments, the clarifier includes one or more flocculant folding blades that can rotate and release microorganisms immobilized in the flocculant without introducing solids into the supernatant. The flocculant folding blades can move the working fluid in the clarifier to resuspend the microorganisms within the working fluid. In some embodiments, microorganisms and / or a certain amount of target isolated bacterial strains can be resuspended in the solution in the clarifier and transferred to the supernatant (e.g., base product). In some embodiments, the clarifier also includes a flow path for returning the flocculant to a first reactor. The flow path may include conduits from the clarifier to containers or combinations of containers 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). The clarifier may return flow 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 may 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, recirculating the flow from the clarifier to containers in the digestive system can help enrich the microbial community of the digestive system's microbial aggregates by providing the working fluid from the clarifier to different points in the system (e.g., containers). The recirculated working fluid may contain one or more organic materials, one or more microorganisms of the microbial aggregates, target isolated bacteria, one or more metabolites, or any combination thereof.

[0336] 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. A flocculation scraper in the clarifier can aid in the separation of the clarifier's working fluid. In some embodiments, the separation may include gravity separation. Flocculation may settle to the bottom of the clarifier, and the supernatant may be collected. The scraping of the flocculation scraper may include the release of a microbial community. The microbial community may be released into the supernatant portion (e.g., the supernatant portion of the clarifier working fluid). The scraping can release the microbial community into the supernatant portion of the clarifier working fluid while preventing the introduction of solids (e.g., flocculent solids) from the flocculated portion into the supernatant portion of the clarifier working fluid.

[0337] The biostimulant compositions produced through the digestive process described herein can be used directly or after further processing. For example, the effluent from the digestive system (referred to herein as the “base product”) can be concentrated, sterilized, filtered, pasteurized, or dehydrated, or any combination thereof, before use. In some embodiments, the base product can be concentrated to at least about 2x, at least about 3x, at least about 4x, at least about 5x, at least about 6x, at least about 7x, at least about 8x, at least about 9x, at least about 10x, or greater than about 10x. In some embodiments, the base product can be concentrated to at most about 10x, at most about 9x, at most about 8x, at most about 7x, at most about 6x, at most about 5x, at most about 4x, at most about 3x, at most about 2x, or less than 2x. In some embodiments, the base product can be filtered to remove any bacteria or other microorganisms from the composition.

[0338] On one hand, this article provides a method comprising transferring an aqueous organic feedstock into a first container. An inoculum of microorganisms may be transferred into the first container. The aqueous organic feedstock may be transferred into the first container. The aqueous organic feedstock and the microbial inoculum may be transferred into the first container. The first container may contain a volume of a first working fluid. The aqueous organic feedstock may contain microbial aggregates. The aqueous organic feedstock may contain one or more digestion products generated from the digestion of one or more organic materials. The aqueous organic feedstock may contain microbial aggregates and digestion products generated from the digestion of organic materials. In some cases, the feedstock may contain at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than about 10 digestion products. In some cases, the feedstock may contain up to about 10, 9, 8, 7, 6, 5, 4, 3, 2, or fewer than 2 digestion products. The organic material may be digested by one or more microorganisms in the microbial aggregate. The organic material may be digested by a microbial community in the microbial inoculum. The digestion products described herein may comprise one or more sugars (e.g., monosaccharides, disaccharides, oligosaccharides, polysaccharides, or any combination thereof). The digestion products described herein may comprise sugars (e.g., xylose, mannose, glucose, or any combination thereof), one or more metabolites produced by microorganisms in the working fluid, one or more fatty acids, one or more dead microorganisms, one or more fragments of dead microorganisms, one or more microbial fermentation products, one or more enzymes, one or more plant growth regulators, one or more organic acids, one or more chelating agents, or any combination thereof. The method may also include incubating an inoculum of microorganisms under conditions that selectively promote microbial growth and increase the microbial population. The method may also include incubating an inoculum of microorganisms under one or more conditions described herein, said one or more conditions selectively promoting the growth of at least a portion of the microorganisms in the microbial aggregate. The terms "microbial digestion" and "digestion" are used interchangeably.

[0339] In some embodiments, digestion is anaerobic digestion. In some embodiments, digestion is aerobic digestion. In some embodiments, digestion is microaerophilic digestion. In some embodiments, digestion is aerobic digestion, microaerophilic digestion, anaerobic digestion, or a combination thereof. It is not intended to be theoretically construed as, during the digestion process, microorganisms digest biomolecules and other nutrients present in manure, yeast, or kelp, and / or produce digestion products containing compounds that promote plant growth and soil health. In some embodiments of the digestion process, the organic feedstock may be mixed with water to prepare an organic feedstock for use in an anaerobic digestion system. The anaerobic digestion system may include a mixing tank in which the organic feedstock is mixed to prepare a fluid feed mixture or working fluid. In some embodiments, the fluid feed mixture may contain manure, water, and Saccharomyces cerevisiae. In some embodiments, anaerobic digestion includes a process of anaerobic bacterial degradation of organic biomaterials. Biostimulants may also contain microorganisms that contribute to the plant-beneficial properties of the biostimulant product. The microorganisms in the biostimulant product may be derived from the microbial community present in the organic feedstock.

[0340] As an example system of this disclosure, a series of reactors serves to facilitate the growth of inoculum containing microorganisms (e.g., isolated bacteria) with desired plant-growth-promoting properties. A series of reactors (e.g., a tandem assembly of reactors) can also facilitate the production of microbial metabolites with desired plant-growth-promoting properties. The digestion system described herein can enrich inoculum containing target microorganisms, microbial communities within microbial aggregates possessing plant-growth-promoting properties (e.g., nitrogen use efficiency), communities of metabolites possessing plant-growth-promoting properties (e.g., nitrogen use efficiency), or any combination thereof. Compared to other digestion systems, this system offers the added advantage of targeting functional communities and / or metabolites of microorganisms with specific functions, and enriching and / or maintaining 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, a tandem reactor assembly enables the growth and enrichment of specific target microorganisms with optimal plant-growth-promoting properties. The system can guide the flow of a working fluid containing inoculum, a carbon source, and / or a nutrient source from the input organic feedstock to produce a base product (BP). A hydraulic source can flow into the reactor through an inlet port to form a first working fluid in the reactor tank. The hydraulic source can be input to the first reactor or any reactor in the system. 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 to the system described herein can flow into any reactor in the system, including but not limited to the first reactor, second reactor, third reactor, or any other reactor following the first reactor.

[0341] Microbial inoculum can be incubated in a reactor (e.g., a vessel) of the digestive system described herein. In some embodiments, the microbial inoculum can be incubated under conditions that selectively enrich and / or retain the microbial inoculum in the digestive system. In some cases, the microbial inoculum can survive in the digestive system in a trophic or sporulated state (e.g., a dormant state in the system). Not wishing to be bound by theory, the conditions of the digestive system reactor (e.g., hydraulic retention time of the system (e.g., flow rate), flocculant recirculation, system pH level, aerobic conditions, or any combination thereof) can alter the complex microbial aggregates of the digestive system to enrich at least a portion of the microorganisms within the microbial aggregates that possess plant growth-promoting properties (e.g., nitrogen use efficiency). The conditions of one or more reactors of the digestive system (e.g., hydraulic retention time of the system (e.g., flow rate), flocculant recirculation, system pH level, aerobic conditions, or any combination thereof) can alter the complex microbial aggregates of the digestive system to enrich microbial strain populations (e.g., provide an established population of microbial strains). Incubation of microbial inoculum and / or microbial aggregates containing certain microorganisms with plant growth-promoting properties can also produce metabolites with plant growth-promoting properties (e.g., nitrogen use efficiency).

[0342] The inoculum of microorganisms may comprise nitrogen-enhancing microbial strains that can be maintained as a population of microbial strains in the bioreactor system described herein. Maintenance (e.g., survival) of the microbial inoculum may include maintaining its initial quantity of microbial population under conditions induced by the conditions within the digester reactor. Maintenance (e.g., survival) of the microbial inoculum may include a condition where a certain amount of microbial inoculum is alive at the end of the residence period in the digester system (e.g., in a reactor or clarifier chamber). For example, after initial inoculation, a population of nitrogen-enhancing microbial strains may survive after incubation under the conditions of the digester system described herein (e.g., nutrients, flow rate, pH, aerobic parameters, or any combination thereof). In some embodiments, at least a portion of the microorganisms in the microbial aggregate may be enriched (e.g., grown or increased in number). These microorganisms in this portion of the microbial aggregate may possess nitrogen-enhancing capabilities. The proportion of nitrogen-enhancing microbial strains and nitrogen-enhancing microorganisms in the microbial aggregate relative to the total bacterial population count can be maintained in the first container of the digester system. In the second, third, fourth, fifth, sixth, seventh, or eighth container of the digestive system, the proportion of nitrogen-use-enhancing microbial strains and microbial aggregates relative to the total bacterial population can be maintained. In any combination of containers of the bioreactor system described herein, the proportion of nitrogen-use-enhancing microbial strains and microbial aggregates relative to the total bacterial population can be maintained. Over a period of time, the maintenance population of nitrogen-use-enhancing microbial strains can ensure that the change in their amount in the working fluid of the digestive system is 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%. The duration can include at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, 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, or more than 12 months. The duration can include up to about 10 years, 5 years, 4 years, 3 years, 24 months, 18 months, 12 months, 9 months, 6 months, 5 months, 4 months, 3 months, 8 weeks, 7 weeks, 6 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 24 hours, 18 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, or less than about 1 hour.

[0343] In some cases, parameters (including nutrients added to the system, system hydraulic retention time (e.g., flow rate), floc recirculation, system pH level, aerobic conditions, or any combination thereof) can promote the growth of microorganisms or at least a portion of the microorganisms in a microbial aggregate. These microorganisms may be those that promote nitrogen use efficiency. As the microorganisms or at least a portion of the microorganisms in the microbial aggregate are incubated under the conditions of the digester reactor (e.g., nutrients added to the system, system hydraulic retention time (e.g., flow rate), floc recirculation, system pH level, aerobic conditions, or any combination thereof), the amount of microorganisms or at least a portion of the microorganisms in the microbial aggregate can increase by at least about 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 30%). As at least a portion of the microorganisms or microbial aggregates are incubated under the conditions of a digester reactor (e.g., nutrients added to the system, system hydraulic retention time (e.g., flow rate), flocculant recirculation, system pH level, aerobic conditions, or any combination thereof), the quantity of microorganisms or at least a portion of the microorganisms in the microbial aggregates can increase to up to about 30%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01%, 0.001%, or 0.0001%). In some cases, without the addition of microbial inoculum, at least a portion of the microorganisms in the microbial aggregates that promote nitrogen use efficiency can accumulate and / or grow in the system.

[0344] Unwilling to be bound by theory, parameters (including nutrients added to the system, system hydraulic retention time (e.g., flow rate), floc recirculation, system pH level, aerobic conditions, or any combination thereof) may inhibit the growth of microorganisms and / or microbial communities. Unwilling to be bound by theory, parameters (including nutrients added to the system, system hydraulic retention time (e.g., flow rate), floc recirculation, system pH level, aerobic conditions, or any combination thereof) may inhibit the growth of microorganisms and / or microbial communities and increase the growth of target microorganisms and / or target microbial communities (e.g., microorganisms that promote nitrogen use efficiency). Unwilling to be bound by any theory, parameters of the bioreactor system may lead to selective alterations in the microbial community to favor microorganisms with a target function (e.g., nitrogen use efficiency). Nutrients (e.g., macronutrients, micronutrients, inorganic nutrients, or any combination thereof) may be present in the digestive system to provide an environment for bacterial growth. Microorganisms that promote nitrogen use efficiency may include microbial inoculum (e.g., a population of microbial strains that promote nitrogen use efficiency), microorganisms that promote nitrogen use efficiency in microbial aggregates, metabolites that promote nitrogen use efficiency produced by microbial inoculum and / or microorganisms that promote nitrogen use efficiency in microbial aggregates, or any combination thereof.

[0345] The microbial inoculum described herein can come into contact with plants (e.g., be applied to plants). In some embodiments, contact between the microbial 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 microbial inoculums can be transferred into the digestive system. One microbial inoculum and another microbial inoculum can be the same. One microbial inoculum and another microbial inoculum can be different. In some embodiments, the microbial inoculum and the aqueous organic feedstock are transferred simultaneously to the digestive system container. In some embodiments, the microbial inoculum and the aqueous organic feedstock are not transferred simultaneously to the digestive system container. In some embodiments, the microbial inoculum is transferred to the digestive system container before the aqueous organic feedstock. In some embodiments, the microbial inoculum is transferred to the digestive system container after the aqueous organic feedstock.

[0346] As the working fluid flows through the digestion system, the absolute number of nitrogen-enhancing microorganisms can increase. In some embodiments, the absolute number of nitrogen-enhancing microorganisms in the second container can be higher than that in the first container. In some embodiments, the absolute number of nitrogen-enhancing microorganisms in the third container can be higher than that in the first container. In some embodiments, the absolute number of nitrogen-enhancing microorganisms in the fourth container can be higher than that in the first container. In some embodiments, the absolute number of nitrogen-enhancing microorganisms in the fifth container can be higher than that in the first container. In some embodiments, the absolute number of nitrogen-enhancing microorganisms in the sixth container can be higher than that in the first container. In some embodiments, the absolute number of nitrogen-enhancing microorganisms in the seventh container can be higher than that in the first container. In some embodiments, the absolute number of nitrogen-enhancing microorganisms in the eighth container can be higher than that in the first container. In some embodiments, the absolute number of nitrogen-enhancing microorganisms in the ninth container may be higher than the absolute number of nitrogen-enhancing microorganisms in the first container. In some embodiments, the absolute number of nitrogen-enhancing microorganisms in the tenth container may be higher than the absolute number of nitrogen-enhancing microorganisms in the first container.

[0347] As the working fluid flows through the digestion system, the proportion of nitrogen-enhancing microorganisms relative to the total bacterial population can increase. In some embodiments, the proportion of nitrogen-enhancing microorganisms 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 nitrogen-enhancing microorganisms 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 nitrogen-enhancing microorganisms 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 nitrogen-enhancing microorganisms 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 nitrogen-enhancing microorganisms 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 nitrogen-enhancing microorganisms 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 nitrogen-enhancing microorganisms relative to the total bacterial population in the eighth container may be higher than that in the first container. In some embodiments, the proportion of nitrogen-enhancing microorganisms 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 nitrogen-enhancing microorganisms relative to the total bacterial population in the tenth container may be higher than that in the first container.

[0348] In some embodiments, the amount (e.g., concentration and / or number) of the microbial strain community (e.g., the established population of microbial strains) in one container of a bioreactor system may differ from that in another container by at least about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or more than about 20%. In some embodiments, the amount (e.g., concentration and / or number) of the microbial strain community (e.g., the established population of microbial strains) in one container of a bioreactor system may differ from that in another container by at most about 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or less than about 0.1%.

[0349] Microbial inoculants can produce one or more metabolites in the digestive system described herein. Microorganisms in a microbial aggregate and / or microbial inoculants can produce metabolites through catalytic enzyme metabolism. One or more metabolites can be produced through microbial metabolism. In the working fluid of the digestive system described herein, metabolites can be produced through enzymatic catalysis of the biochemical reaction of an organic substrate with an aqueous organic feedstock. Metabolites produced by microbial inoculants can possess plant growth-promoting properties. Metabolites produced by microbial inoculants can possess two or more plant growth-promoting properties. Plant growth-promoting properties may include aboveground biomass, root biomass, nutrient uptake, crop yield, photosynthesis, 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 directly applied to plants.

[0350] In some embodiments, the aqueous organic feedstock comprises one or more metabolites. In some embodiments, the aqueous organic feedstock comprises one or more metabolites produced by endogenous microorganisms of the organic feedstock. 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, cinnamides, or chromones), polyketides, eicosanoic acid-like substances, 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, xylose, or any combination thereof. 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, hydroxylated myristic acid, or any combination thereof.

[0351] One or more metabolites produced by an inoculum of microorganisms or by at least a portion of the 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 metabolite 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 metabolite weight 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 metabolite weight per 100 ml of basal product solution can be from about 10 mg to about 500 mg. In some embodiments, the metabolite weight per 100 ml of basal 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.

[0352] The term "organic feedstock" as used herein can refer to natural biological materials such as carbon compounds, proteins, and / or carbohydrates. In some embodiments, the organic feedstock may comprise organic substrates, including cottonseed, algae, neem, orange seed, linseed, jojoba, kusum, rubber seed, alfalfa, sugarcane, cactus, coffee beans, Deccan hemp, or any combination thereof. In some embodiments, the feedstock may comprise inorganic feedstocks. In some embodiments, the organic feedstock may include, but is not limited to, manure, giant kelp, lignocellulose, wastewater biosolids, food waste, energy crops, glucose solution, ammonium sulfate, oils, fats, and greases. In some embodiments, the feedstock is added at the beginning of the system (e.g., added to the first reactor and / or CMR). In some embodiments, the feedstock is added in an intermediate reactor of the system (e.g., not added to 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 organic raw material is a composition of natural biological materials. In some embodiments, the organic raw material is a blend of two, three, four, five, six, seven, eight, nine, ten, or more natural biological materials.

[0353] Organic feedstocks containing carbon and nitrogen sources can flow into reactor tanks. Organic feedstocks containing carbon and nitrogen sources can flow into reactor tanks via conduits (e.g., pipes). In some embodiments, reactor tanks circulate their internal working fluids for recycling, wherein reactor tanks may include outflow conduits to circulate and recycle 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 system terminal clarifier can be transferred from the reactor tank to the clarifier, producing a supernatant (e.g., base product). Working fluid flows through a series of reactor systems, which can facilitate the selective growth of added isolated bacteria and other microorganisms present with similar characteristics. The base product from the clarifier can be obtained and further analyzed for microbial composition. In the digestion system described herein, working fluid can exit from a mixing chamber, flow through at least one reaction, and flow into a clarifier chamber.

[0354] The organic feedstock may comprise one or more digestion products derived from the digestion of organic substrates present in the organic feedstock. The organic substrates can improve the stability of the fluid feed mixture. The organic substrates may comprise coconut fiber, peat moss, hemp, wood fiber, or any combination thereof. In some embodiments, the organic substrates comprise natural biological materials present in the aqueous organic feedstock. The digestion system may comprise a variety of microorganisms and / or microbial cells derived from the digestion of organic substrates from the aqueous organic feedstock.

[0355] The organic raw materials described herein may comprise one or more organic and / or biological materials. In some embodiments, the organic raw materials further comprise Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae yeast), tree yeast ( Saccharomyces arboricola yeast), tripartite yeast ( Saccharomyces mikatae yeast), Zhu's yeast ( Saccharomyces jurei yeast), Zhenbei yeast ( Saccharomyces eubayanus yeast), Kudriazwei yeast ( Saccharomyces kudriavzevii yeast), grape juice yeast ( Saccharomyces uvarum Yeast) or any combination thereof. In some embodiments, the organic raw material further comprises lignocellulosic material. In some embodiments, the organic raw material can be an aqueous mixture of at least one raw material and water. In some embodiments, the organic raw material can be an aqueous mixture of cow dung, brewer's yeast, water, or any combination thereof. The organic raw material can be an aqueous organic raw material (e.g., an organic raw material containing water).

[0356] Parameters of the digestive system (e.g., flow rate and solids content of the 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 organic feedstock of the digestive system to prepare an aqueous organic feedstock.

[0357] In some embodiments, the aqueous organic feedstock may further comprise an inorganic substrate. In some embodiments, the aqueous organic feedstock may comprise more than one inorganic substrate. Inorganic substrates can improve the stability of the fluid feed mixture. In some embodiments, the inorganic substrate comprises sand, vermiculite, perlite, diatomaceous earth, pumice, or any combination thereof. In some embodiments, the inorganic substrate comprises minerals. In some embodiments, the inorganic substrate comprises phosphate rock.

[0358] In some embodiments, the loading input into the reactor may comprise one or more carbon sources, one or more nitrogen sources, one or more powders, one or more isolated bacteria, or any combination thereof. In some embodiments, the powder is soybean flour. In some embodiments, the loading input comprises recycled flocculants from the system. In some embodiments, the loading input comprises whole fermentation broth (WB). The inoculum of the microorganisms described herein can metabolize carbon sources. Carbon metabolism of the microbial inoculum may include transferring the carbon-based portion of the carbon source to a substrate in the working fluid.

[0359] The inoculum of the microorganisms described herein can metabolize a nitrogen source. Nitrogen metabolism of the microbial inoculum may include transferring the nitrogen-based portion of the nitrogen source to a substrate in the working fluid. In some embodiments, the carbon source may be transferred to a first container of the digestive system. In some embodiments, the carbon source may be transferred to a second, third, fourth, fifth, or sixth container of the digestive system. In some embodiments, the nitrogen source may be transferred to a first container of the digestive system. In some embodiments, the nitrogen source may be transferred to a second, third, fourth, fifth, or sixth container of the digestive system.

[0360] In some embodiments, the organic feedstock is mixed inside the reactor. In some embodiments, the organic feedstock is mixed outside the reactor. In some embodiments, the organic feedstock is mixed between one, two, three, or more reactors. In some embodiments, the organic feedstock is a homogeneous mixture.

[0361] In some embodiments, the organic feedstock further comprises microbial aggregates. The terms “microorganism,” “microbial strain,” and “microbial body” can refer to microscopic organisms, including but not limited to bacteria, fungi, lichens, algae, protozoa, archaea, and / or molds. The terms “microorganism” and “microbial body” are used interchangeably herein. The organic feedstock may comprise microbial aggregates having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 10,000, or more types of microorganisms. Organic raw materials may contain microbial aggregates having up to about 10,000, 5,000, 4,000, 3,000, 2,000, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or fewer types of microorganisms. Organic feedstocks may contain microbial aggregates having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 10,000 or more groups of microorganisms. Organic feedstocks may contain microbial aggregates having up to about 10,000, 5,000, 4,000, 3,000, 2,000, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or fewer groups of microorganisms. Organic feedstocks may contain microbial aggregates having one group of microorganisms. Microbial aggregates may contain different microorganisms. Microbial aggregates may contain the same microorganisms. The microorganisms in the aggregate may originate from microorganisms originally present in the organic feedstock. Microorganisms can digest manure, yeast, other organic feedstocks, or any combination thereof to produce digestion products.

[0362] In some embodiments, the microorganisms may be added at the beginning of the system (e.g., to the first reactor). In some embodiments, the microorganisms may be added midway through the system (e.g., to a reactor that is neither the first nor the final reactor of the system), or the microorganisms may be added at the end of the system (e.g., to the final reactor). The microorganisms may be added simultaneously with the organic feedstock. The microorganisms may be added separately from the organic feedstock. In some embodiments, the microorganisms may be added to the system in the same reactor as the organic feedstock. In some embodiments, the microorganisms may be added to the system in different reactors than the organic feedstock. In some embodiments, the microorganisms may be added to the system before the organic feedstock. In some embodiments, the microorganisms may be added to the system after the organic feedstock. In some embodiments, the time interval between adding the microorganisms to the system and adding the 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 organic feedstock 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.

[0363] Microorganisms can possess nutrient-solubilizing properties and / or plant growth-promoting properties. For example, microorganisms can increase plant growth, increase aboveground and / or root biomass, increase crop yield, increase soil enzyme activity, increase photosynthetic efficiency, reduce heavy metal uptake, lower soil pH, or any combination thereof. Microorganisms can enhance plant growth in high-salinity soils, heavy metal-contaminated soils, or arid conditions. The microorganisms described herein (e.g., isolated bacteria) can possess nitrogen use efficiency properties.

[0364] In some implementations, the digestive system may include a residence time. Residence time may include the time spent by the microbial inoculum within the digestive system or the time spent by the microbial inoculum from transfer to the first container until collection from the digestive system. A longer residence time may favor the growth or enrichment of the microbial inoculum in the digestive system. A shorter residence time may favor the growth or enrichment of the microbial inoculum in the digestive system. The residence time of the digestive system may include at least 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, or more than about 9 months. The residence time of the digestive system may include at most about 9 months, 6 months, 3 months, 2 months, 1 month, 4 weeks, 3 weeks, 2 weeks, 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, or less than about 1 day. The terms "residence time" and "hydraulic residence time" may be used interchangeably.

[0365] 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 the 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 a submerged support that provides a surface for biofilm growth. This support can be referred to as a “fixed media substrate.” Reactor-to-reactor fluid flow can include a plug flow model, where the particles of the input fluid have the same velocity and direction of motion. In some embodiments, the fluid flow in the digestion system is gravity-driven. In some embodiments, the bioreactor system can be operated in a hydraulically balanced manner. A hydraulically balanced manner can involve transferring the working fluid at equal rates from one container in the system to another and to subsequent containers. In some embodiments, the transfer and / or collection of the methods described herein can be gravity-driven. The outlet port of one container can be higher than the inlet port of another container, allowing the working fluid to transfer between containers in the bioreactor system by gravity. In some embodiments, the opening of the outlet port of the first container is flush with or slightly below the level of the working fluid (i.e., the bottom of the outlet port opening may be 1, 2, 3, or 4 cm below the working fluid level), thereby allowing the rate of liquid flowing into the first container to be equal to the rate of liquid flowing out of the first container. The first container may be fluidly connected to the second container, for example, via a pipe or other conduit connected to the outlet port of the first container. This pipe or other conduit may be connected to the inlet port of the second container. The inlet port of the second container may be below the level of the outlet port of the first container, allowing the working fluid in the first container to flow into the second container solely by gravity. The second container may have an outlet port with an opening that is flush with or at least partially below the level of the working fluid in the second container (i.e., the bottom of the outlet port opening may be 1, 2, 3, or 4 cm below the working fluid level), thereby allowing the rate of liquid flowing into the first container to be equal to the rate of liquid flowing out of the first container solely by gravity. Additional containers (e.g., third, fourth, fifth, sixth, seventh, or more containers) may have inlet and outlet ports arranged in the same manner, with each subsequent container having inlet and outlet ports arranged such that flow between containers can be continuous and driven solely by gravity, i.e., the inlet and outlet ports of each subsequent container in the series are at a lower level than the outlet port of the preceding container. In some embodiments, all inlet and outlet ports of the containers in the system are kept open at all times to allow continuous flow driven solely by gravity (i.e., without any pumps to drive flow from any container to subsequent containers in the system). In some embodiments, fluid flow in the digestion system is driven by a pump. The effluent from the top of the last reactor can be used to produce a product. The product of the methods and systems described herein may be a biostimulant.Biostimulants can promote plant growth or improve soil quality.

[0366] B. Reactor The digestion process for producing biostimulants can take place in a digestion system comprising a series of tanks, containers, or vessels (e.g., reactors) through which the feedstock flows continuously. The reactor can be a fluidly connected container, system, vessel, or tank in which microbial aggregates containing the microorganisms, isolated strains, and / or microbial bodies described herein can be grown. The reactor can be discrete or continuous. The reactor can be a physically contained system 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) and packed bed reactors (PBRs).

[0367] In one aspect, this disclosure provides a bioreactor system. The bioreactor system may include an aqueous feed stream. This stream may be in fluid communication with a container (e.g., a first container). The container may contain a volume of working fluid. For example, the stream may be in fluid communication with a first container of the bioreactor system, which contains a volume of first working fluid. The aqueous feed may include microbial aggregates, wherein the microbial aggregates may include the microbial aggregates described herein. The working fluid (e.g., the first working fluid) may contain microbial strains. The microbial strains may be a population of microbial strains (e.g., nitrogen-use-efficient strains) and may contain properties that promote plant growth (e.g., desired plant growth-promoting properties). The first container of the bioreactor system may include a mixer. The mixer may be configured to aerate the working fluid (e.g., the first working fluid) in the container.

[0368] In some embodiments, the concentration of microbial strains (e.g., nitrogen-utilization-efficient strains) in the working fluid may be higher than that in the aqueous feedstock. In some embodiments, the concentration of microbial strains in the working fluid may be higher than that in any other input to the bioreactor. For example, the concentration of microbial strains (e.g., nitrogen-utilization-efficient strains) in the first working fluid may be at least about 2 times higher, at least about 5 times higher, at least about 10 times higher, at least about 25 times higher, at least about 50 times higher, at least about 100 times higher, at least about 150 times higher, at least about 200 times higher, at least about 250 times higher, at least about 500 times higher, or greater than about 500 times higher than the concentration of microbial strains in the aqueous feedstock stream or any other input entering the bioreactor system. The concentration of the microbial strain in the first working fluid can be up to about 500 times higher, up to about 250 times higher, up to about 200 times higher, up to about 150 times higher, up to about 100 times higher, up to about 50 times higher, up to about 25 times higher, up to about 10 times higher, up to about 5 times higher, up to about 2 times higher, or less than about 2 times higher than the concentration of the microbial strain (e.g., nitrogen-utilization-efficient strain) in the aqueous feed stream entering the bioreactor system or any other input.

[0369] The bioreactor system described herein may include one or more additional containers (e.g., one or more containers other than the first container). The containers may be arranged in a series. In some embodiments, the series of containers may include a first container, a second container, a third container, a fourth container, a fifth container, a sixth container, and any additional containers. One or more additional containers in the bioreactor system may contain a volume of working fluid. In some embodiments, each of the one or more additional containers contains a volume of working fluid. One or more additional containers may be in fluid communication with the containers in the series in the bioreactor system. In some embodiments, at least one of the containers in the bioreactor system may include a product effluent port. In some embodiments, each container in the series may include a product effluent port. A product effluent stream (e.g., a flow of a volume of working fluid from the container) may be in fluid communication with the product effluent port.

[0370] As an example, 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 contains microbial aggregates, wherein the first working fluid contains an established population of a first microbial strain that promotes nitrogen use efficiency, wherein the concentration of the first microbial strain that promotes nitrogen use efficiency in the first working fluid is at least 100 times higher than the concentration of the first microbial strain that promotes nitrogen use efficiency in the aqueous feed stream entering the bioreactor system and in any other input; (b) one or more additional containers arranged in a series including the first container, wherein each of the one or more additional containers contains a volume of working fluid and is in fluid communication with at least one other container in the series, and wherein at least one of the one or more additional containers includes a product effluent port; and (c) a product effluent stream in fluid communication with the product effluent port.

[0371] In some embodiments, the product effluent may contain a biostimulant composition. The biostimulant composition may contain a quantity of microbial strains (e.g., microbial strains that promote nitrogen use efficiency), wherein the quantity of the microbial strains (e.g., concentration and / or number of bacteria) may be the quantity described herein. The biostimulant composition may contain plant growth-promoting properties (e.g., desired plant growth-promoting properties). In some embodiments, the microbial strains may be configured to enhance plant growth-promoting properties (e.g., the ability to perform nitrogen fixation, promote nitrogen fixation in plant tissues, recruit nitrogen-fixing organisms to the plant rhizosphere or other tissues, increase organic nitrogen content in the soil and / or mineralize organic nitrogen, or any combination thereof).

[0372] A bioreactor system can be a continuous flow system. A continuous flow system can include a system where fluid flow is uninterrupted. The aqueous feed stream can be continuous. In some embodiments, the bioreactor system can include periodic flow, wherein the flow can be intermittent. In some embodiments, as a continuous flow bioreactor system, the volume of the working fluid can be constant.

[0373] In some embodiments, the bioreactor system may include the clarifier vessel described herein. The clarifier vessel may be configured to separate a portion of the working fluid within the clarifier vessel into a supernatant portion and a flocculent portion. In some embodiments, the system's product effluent may include the supernatant portion.

[0374] In some implementations, the product effluent contains at least about 100 CFU / ml and at least about 1x10 3 CFU / ml, at least approximately 1x10 4 CFU / ml, at least approximately 1x10 5CFU / ml, at least approximately 1x10 6 CFU / ml, at least approximately 1x10 7 CFU / ml, at least approximately 1x10 8 CFU / ml, at least approximately 1x10 9 CFU / ml, at least approximately 1x10 10 CFU / ml or greater than approximately 1x10 10 The microbial strain contains CFU / ml (e.g., a microbial strain that promotes nitrogen use efficiency). In some embodiments, the product effluent contains up to about 1 x 10⁻⁶ CFU / ml. 10 CFU / ml, up to approximately 1x10 9 CFU / ml, up to approximately 1x10 8 CFU / ml, up to approximately 1x10 7 CFU / ml, up to approximately 1x10 6 CFU / ml, up to approximately 1x10 5 CFU / ml, up to approximately 1x10 4 CFU / ml, up to approximately 1x10 3 Microbial strains with CFU / ml, up to about 100 CFU / ml, or less than about 100 CFU / ml (e.g., microbial strains that promote nitrogen use efficiency).

[0375] In some implementations, the product effluent contains at least about 100 CFU / ml and at least about 1x10 3 CFU / ml, at least approximately 1x10 4 CFU / ml, at least approximately 1x10 5 CFU / ml, at least approximately 1x10 6 CFU / ml, at least approximately 1x10 7 CFU / ml, at least approximately 1x10 8 CFU / ml, at least approximately 1x10 9 CFU / ml, at least approximately 1x10 10 CFU / ml or greater than approximately 1x10 10 CFU / ml of sporulated microbial strains (e.g., microbial strains that promote nitrogen use efficiency). In some embodiments, the product effluent contains up to about 1 x 10⁻⁶ CFU / ml. 10 CFU / ml, up to approximately 1x10 9 CFU / ml, up to approximately 1x10 8 CFU / ml, up to approximately 1x10 7 CFU / ml, up to approximately 1x10 6 CFU / ml, up to approximately 1x105 CFU / ml, up to approximately 1x10 4 CFU / ml, up to approximately 1x10 3 Microbial strains in sporulated form with CFU / ml, up to about 100 CFU / ml, or less than about 100 CFU / ml (e.g., microbial strains that promote nitrogen use efficiency).

[0376] In some embodiments, the product effluent may contain a total dry weight. The total dry weight of the product effluent may be at least about 0.05 mg / ml, at least about 0.1 mg / ml, at least about 0.2 mg / ml, at least about 0.3 mg / ml, at least about 0.4 mg / ml, at least about 0.5 mg / ml, at least about 1.0 mg / ml, at least about 1.5 mg / ml, at least about 2.0 mg / ml, at least about 2.5 mg / ml, at least about 3.0 mg / ml, at least about 3.5 mg / ml, at least about 4.0 mg / ml, at least about 5.0 mg / ml, or greater than about 5.0 mg / ml. The total dry weight of the product effluent can be up to about 5.0 mg / ml, up to about 4.0 mg / ml, up to about 3.5 mg / ml, up to about 3.0 mg / ml, up to about 2.5 mg / ml, up to about 2.0 mg / ml, up to about 1.5 mg / ml, up to about 1.0 mg / ml, up to about 0.5 mg / ml, up to about 0.4 mg / ml, up to about 0.3 mg / ml, up to about 0.2 mg / ml, up to about 0.1 mg / ml, up to about 0.05 mg / ml, or less than about 0.05 mg / ml. The total dry weight of the product effluent can be between about 0.05 mg / ml and about 4 mg / ml. The total dry weight of the product effluent can be from about 0.05 mg / ml to about 0.1 mg / ml, from about 0.05 mg / ml to about 0.2 mg / ml, from about 0.05 mg / ml to about 0.3 mg / ml, from about 0.05 mg / ml to about 0.4 mg / ml, from about 0.05 mg / ml to about 0.5 mg / ml, from about 0.05 mg / ml to about 1 mg / ml, from about 0.05 mg / ml to about 1.5 mg / ml, from about 0.05 mg / ml to about 2 mg / ml, from about 0.05 mg / ml to about 2.5 mg / ml, from about 0.05 mg / ml to about 3 mg / ml, from about 0.05 mg / ml to about 4 mg / ml, from about 0.1 mg / ml to about 0.2 mg / ml, from about 0.1 mg / ml to about 0.3 mg / ml, from about 0.1 mg / ml to about 0.4 mg / ml, from about 0.1 mg / ml to about 0.5 mg / ml, from about 0.1 mg / ml to about 1 mg / ml. mg / ml, about 0.1 mg / ml to about 1.5 mg / ml, about 0.1 mg / ml to about 2 mg / ml, about 0.1 mg / ml to about 2.5 mg / ml, about 0.1 mg / ml to about 3 mg / ml, about 0.1 mg / ml to about 4 mg / ml, about 0.2 mg / ml to about 0.3 mg / ml, about 0.2 mg / ml to about 0.4 mg / ml, about 0.2 mg / ml to about 0.5 mg / ml, about 0.2 mg / ml to about 1 mg / ml, about 0.2 mg / ml to about 1.5 mg / ml, about 0.2 mg / ml to about 2 mg / ml, about 0.2 mg / ml to about 2.5 mg / ml, about 0.2 mg / ml to about 3 mg / ml, about 0.2 mg / ml to about 4 mg / ml, about 0.3 mg / ml to about 0.4 mg / ml, about 0.3 mg / ml to about 0.5 mg / ml, about 0.3 mg / ml to about 1 mg / ml, about 0.3 mg / ml to about 1.5 mg / ml, about 0.3 mg / ml to about 2 mg / ml, about 0.3 mg / ml to about 2.5 mg / ml, about 0.3 mg / ml to about 3 mg / ml, about 0.3 mg / ml to about 4 mg / ml, about 0.4 mg / ml to about 0.5 mg / ml, about 0.4 mg / ml to about 1 mg / ml, about 0.4 mg / ml to about 1.5 mg / ml. mg / ml, about 0.4 mg / ml to about 2 mg / ml, about 0.4 mg / ml to about 2.5 mg / ml, about 0.4 mg / ml to about 3 mg / ml, about 0.4 mg / ml to about 4 mg / ml, about 0.5 mg / ml to about 1 mg / ml, about 0.5 mg / ml to about 1.5 mg / ml, about 0.5 mg / ml to about 2 mg / ml, about 0.5 mg / ml to about 2.5 mg / ml, about 0.5 mg / ml to about 3 mg / ml, about 0.5 mg / ml to about 4 mg / ml, about 1 mg / ml to about 1.5 mg / ml, about 1 mg / ml to about 2 mg / ml, about 1 mg / ml to about 2.5 mg / ml, about 1 mg / ml to about 3 mg / ml, about 1 mg / ml to about 4 mg / ml, about 1.5 mg / ml to about 2 mg / ml, about 1.5 mg / ml to about 2.5 mg / ml, about 1.5 mg / ml to about 3 mg / ml, about 1.5 mg / ml to about 3 mg / ml. The concentrations range from approximately 4 mg / ml to approximately 2.5 mg / ml, approximately 2 mg / ml to approximately 3 mg / ml, approximately 2 mg / ml to approximately 4 mg / ml, approximately 2.5 mg / ml to approximately 3 mg / ml, approximately 2.5 mg / ml to approximately 4 mg / ml, or approximately 3 mg / ml to approximately 4 mg / ml.

[0377] In some embodiments, the product effluent may contain a chemical oxygen demand (COD). The COD of the product effluent may be at least about 10 mg / L, at least about 20 mg / L, at least about 30 mg / L, at least about 40 mg / L, at least about 50 mg / L, at least about 60 mg / L, at least about 70 mg / L, at least about 80 mg / L, at least about 90 mg / L, at least about 100 mg / L, at least about 250 mg / L, at least about 500 mg / L, at least about 750 mg / L, at least about 1000 mg / L, or greater than about 1000 mg / L. The chemical oxygen demand (COD) of the product effluent can be up to about 1000 mg / L, up to about 750 mg / L, up to about 500 mg / L, up to about 250 mg / L, up to about 100 mg / L, up to about 90 mg / L, up to about 80 mg / L, up to about 70 mg / L, up to about 60 mg / L, up to about 50 mg / L, up to about 40 mg / L, up to about 30 mg / L, up to about 20 mg / L, up to about 10 mg / L, or less than about 10 mg / L. The COD of the product effluent can be between about 10 mg / L and about 1000 mg / L. The chemical oxygen demand (COD) of the product effluent can be approximately 10 mg / L to approximately 20 mg / L, approximately 10 mg / L to approximately 30 mg / L, approximately 10 mg / L to approximately 40 mg / L, approximately 10 mg / L to approximately 50 mg / L, approximately 10 mg / L to approximately 80 mg / L, approximately 10 mg / L to approximately 100 mg / L, approximately 10 mg / L to approximately 150 mg / L, approximately 10 mg / L to approximately 200 mg / L, approximately 10 mg / L to approximately 500 mg / L, approximately 10 mg / L to approximately 750 mg / L, approximately 10 mg / L to approximately 1000 mg / L, approximately 20 mg / L to approximately 30 mg / L, approximately 20 mg / L to approximately 40 mg / L, approximately 20 mg / L to approximately 50 mg / L, approximately 20 mg / L to approximately 80 mg / L, approximately 20 mg / L to approximately 100 mg / L, approximately 20 mg / L to approximately 15 ... mg / L to about 200 mg / L, about 20 mg / L to about 500 mg / L, about 20 mg / L to about 750 mg / L, about 20 mg / L to about 1,000 mg / L, about 30 mg / L to about 40 mg / L, about 30 mg / L to about 50 mg / L, about 30 mg / L to about 80 mg / L, about 30 mg / L to about 100 mg / L, about 30 mg / L to about 150 mg / L, about 30 mg / L to about 200 mg / L, about 30 mg / L to about 500 mg / L, about 30 mg / L to about 750 mg / L, about 30 mg / L to about 1,000 mg / L, about 40 mg / L to about 50 mg / L, about 40 mg / L to about 80 mg / L, about 40 mg / L to about 100 mg / L, about 40 mg / L to about 150 mg / L, about 40 mg / L to about 200 mg / L, about 40 mg / L to about 500 mg / L, about 40 mg / L to about 750 mg / L, about 40 mg / L to about 1,000 mg / L, about 50 mg / L to about 80 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 500 mg / L, about 50 mg / L to about 750 mg / L, about 50 mg / L to about 1,000 mg / L, about 80 mg / L to about 100 mg / L, about 80 mg / L to about 150 mg / L mg / L, about 80 mg / L to about 200 mg / L, about 80 mg / L to about 500 mg / L, about 80 mg / L to about 750 mg / L, about 80 mg / L to about 1,000 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 500 mg / L, about 100 mg / L to about 750 mg / L, about 100 mg / L to about 1,000 mg / L, about 150 mg / L to about 200 mg / L, about 150 mg / L to about 500 mg / L, about 150 mg / L to about 750 mg / L, about 150 mg / L to about 1,000 mg / L, about 200 mg / L to about 500 mg / L, about 200 mg / L to about 750 mg / L, about 200 mg / L to about 1,000 mg / L mg / L, approximately 500 mg / L to approximately 750 mg / L, approximately 500 mg / L to approximately 1,000 mg / L, or approximately 750 mg / L to approximately 1,000 mg / L.

[0378] In some embodiments, the product effluent may include conductivity. The conductivity of the product effluent may be at least about 0.01 mS / cm, at least about 0.05 mS / cm, at least about 0.1 mS / cm, at least about 0.5 mS / cm, at least about 1.0 mS / cm, at least about 1.5 mS / cm, at least about 2.0 mS / cm, at least about 2.5 mS / cm, at least about 3.0 mS / cm, at least about 4.0 mS / cm, at least about 5.0 mS / cm, at least about 10.0 mS / cm, or greater than about 10.0 mS / cm. The conductivity of the product effluent can be up to about 10.0 mS / cm, up to about 5.0 mS / cm, up to about 4.0 mS / cm, up to about 3.0 mS / cm, up to about 2.5 mS / cm, up to about 2.0 mS / cm, up to about 1.5 mS / cm, up to about 1.0 mS / cm, up to about 0.5 mS / cm, up to about 0.1 mS / cm, up to about 0.05 mS / cm, up to about 0.01 mS / cm, or less than about 0.01 mS / cm. The conductivity of the product effluent can be between about 0.01 mS / cm and about 10 mS / cm. The conductivity of the product effluent can range from about 0.01 mS / cm to about 0.05 mS / cm, from about 0.01 mS / cm to about 0.1 mS / cm, from about 0.01 mS / cm to about 0.5 mS / cm, from about 0.01 mS / cm to about 1 mS / cm, from about 0.01 mS / cm to about 1.5 mS / cm, from about 0.01 mS / cm to about 2 mS / cm, from about 0.01 mS / cm to about 2.5 mS / cm, from about 0.01 mS / cm to about 3 mS / cm, from about 0.01 mS / cm to about 4 mS / cm, from about 0.01 mS / cm to about 5 mS / cm, from about 0.01 mS / cm to about 10 mS / cm, from about 0.05 mS / cm to about 0.1 mS / cm, from about 0.05 mS / cm to about 0.5 mS / cm, and from about 0.05 mS / cm to about 1 mS / cm. mS / cm, about 0.05 mS / cm to about 1.5 mS / cm, about 0.05 mS / cm to about 2 mS / cm, about 0.05 mS / cm to about 2.5 mS / cm, about 0.05 mS / cm to about 3 mS / cm, about 0.05 mS / cm to about 4 mS / cm, about 0.05 mS / cm to about 5 mS / cm, about 0.05 mS / cm to about 10 mS / cm, about 0.1 mS / cm to about 0.5 mS / cm, about 0.1 mS / cm to about 1 mS / cm, about 0.1 mS / cm to about 1.5 mS / cm, about 0.1 mS / cm to about 2 mS / cm, about 0.1 mS / cm to about 2.5 mS / cm, about 0.1 mS / cm to about 3 mS / cm, about 0.1 mS / cm to about 4 mS / cm, about 0.1 mS / cm to about 5 mS / cm, about 0.1 mS / cm to about 10 mS / cm, about 0.5 mS / cm to about 1 mS / cm, about 0.5 mS / cm to about 1.5 mS / cm, about 0.5 mS / cm to about 2 mS / cm, about 0.5 mS / cm to about 2.5 mS / cm, about 0.5 mS / cm to about 3 mS / cm, about 0.5 mS / cm to about 4 mS / cm, about 0.5 mS / cm to about 5 mS / cm, about 0.5 mS / cm to about 10 mS / cm, about 1 mS / cm to about 1.5 mS / cm, about 1 mS / cm to about 2 mS / cm, about 1 mS / cm to about 2.5 mS / cm, about 1 mS / cm to about 3 mS / cm, about 1 mS / cm to about 4 mS / cm, about 1 mS / cm to about 5 mS / cm, about 1 mS / cm to about 10 mS / cm, about 1.5 mS / cm to about 2 mS / cm, about 1.5 mS / cm to about 2.5 mS / cm, about 1.5 mS / cm to about 3 mS / cm, about 1.5 mS / cm to about 4 mS / cm, about 1.5 mS / cm to about 5 mS / cm, about 1.5 mS / cm to about 10 mS / cm, about 2 mS / cm to about 2.5 mS / cm, about 2 mS / cm to about 3 mS / cm, about 2 mS / cm to about 4 mS / cm, about 2 mS / cm to about 5 mS / cm, about 2 mS / cm to about 10 mS / cm, about 2.5 mS / cm to about 3 mS / cm, about 2.5 mS / cm to about 4 mS / cm, about 2.5 mS / cm to about 5 mS / cm The values ​​are approximately 2.5 mS / cm to about 10 mS / cm, approximately 3 mS / cm to about 4 mS / cm, approximately 3 mS / cm to about 5 mS / cm, approximately 3 mS / cm to about 10 mS / cm, approximately 4 mS / cm to about 5 mS / cm, approximately 4 mS / cm to about 10 mS / cm, or between approximately 5 mS / cm and about 10 mS / cm.

[0379] 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 near the top of the working fluid in the reactor can continuously flow into the next reactor. Fluid in the middle of the reactor can continuously flow into the next reactor. Fluid at 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 between 0.1 inches and 35 inches below the top of the working fluid in the reactor. In some embodiments, the outlet port is at least about 1 inch, at least about 2 inches, at least about 5 inches, at least about 10 inches, at least about 20 inches, at least about 50 inches, at least about 100 inches, at least about 250 inches, at least about 500 inches, at least about 750 inches, at least about 1,000 inches, at least about 2,500 inches, at least about 5,000 inches, at least about 7,500 inches, or at least about 10,000 inches below the top of the working fluid in the reactor. In some embodiments, the effluent port is located at a maximum of 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 of the digestion system can be matched to the feed inflow rate to provide hydraulically balanced flow throughout the system. Reactors within the system can have unique, stable microbial aggregates with physiological characteristics and digestive capacities different from those in other tanks within the system. Reactors within the system can have microbial aggregates with similar physiological characteristics and digestive capacities to those in another reactor within the system. Each reactor within the system can have the same volumetric capacity. Each reactor within the system can have a different volumetric capacity. A digestion system may include at least two reactors. A digestion system may include at least about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more reactors. A digestion system may include at most about 10, 9, 8, 7, 6, 5, 4, 3, 2 or fewer reactors. The reactors of the digestion system may be arranged as a series-connected reactor assembly. The series-connected reactor assembly may have conduits (e.g., ports or outlets) connecting each reactor to adjacent reactors and / or vessels. The series-connected reactor assembly may have a continuous flow of working fluid flowing through each reactor to adjacent reactors.

[0380] In some embodiments, the reactor may have one inlet port and one outlet port. In some embodiments, the reactor may have multiple inlet ports and outlet ports. In some embodiments, the reactor may have one inlet port and multiple outlet ports. In some embodiments, the reactor may have multiple inlet ports and one outlet port. The reactor may have another inlet port to provide a carbon source and / or polymer inoculum. The inlet port may be located at any location 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, vents, or other conduits for transferring fluid from one vessel to another.

[0381] The reactor may have one fluid connection. The reactor may have multiple fluid connections (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more fluid connections). The 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 buildup at the bottom. 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 feedstock and prevent clogging within the reactor. The scraper can fold up flocs and facilitate floc return.

[0382] In some embodiments, the reactor may include one or more packed bed reactors. 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 interior of each packed bed reactor. The stationary medium contains a material that increases the surface area of ​​contact between the microbial community and the working fluid. Without being bound by theory, the increased surface area provided by the support can provide increased contact for the growth of biofilms (e.g., microbial aggregates, established microbial strains, or any combination thereof). The stationary medium also provides a stable platform for biofilm fixation. The packed bed reactor may be filled with a support to increase the surface area within the reactor. The support 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 various types, including durable plastics, polyvinyl chloride (PVC), metals, metal alloys, glass, glass composites, glass fibers, or any inert material of suitable strength. The design and configuration of the stationary medium can employ a variety of geometries that allow the working fluid to move freely through each packed bed reactor and prevent fouling. Free flow supports controlled hydraulic shear, which ultimately promotes uniform distribution of the working fluid. In this embodiment, the stationary medium is dispersed over the entire cross-sectional area of ​​each packed bed reactor.

[0383] The support may comprise one or more tubular components, one or more ring components, or other one or more packing materials. In some embodiments, the packed bed reactor provided herein may comprise a bundle of tubular components or columns. In some embodiments, the support may comprise hexagonal, mesh-like, perforated tubular structures, or any combination thereof. Without being bound by theory, hexagonal, mesh-like, and / or perforated supports may increase the surface area and increase flow through the columns within the vessel. In some embodiments, the tubular components or columns of the support may comprise diameters between 0.25 inches and 50 inches. In some embodiments, the support may comprise 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 include 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 employing packed-bed reactors can improve the production of bacterial isolates or other microorganisms. In some embodiments, reactors without supports (i.e., reactors that are not packed-bed reactors) improve the production of bacterial isolates or other microorganisms, or improve the digestion of digestible substrates.

[0384] In some embodiments, the reactor may include one or more fluidized bed reactors. In a fluidized bed reactor, solid particles can circulate in the working fluid of the reactor, which can provide a surface for microbial colonization. Such particles may include, for example, inorganic substrate particles, such as phosphate rock particles. In some embodiments, the fluidized bed reactors may be of the same volume. In some embodiments, the fluidized bed reactors may be of different volumes. In some embodiments, the fluidized bed reactor improves the uniformity of particle mixing within the digestive system. The solid material in the fluidized bed reactor may have inherent fluid-like properties and allow for more complete mixing. Reduction or elimination of radial and axial concentration gradients can provide better fluid-solid contact and achieve more uniform particle mixing. In some embodiments, the fluidized bed reactor improves the uniformity of temperature gradients within the digestive system. Without being bound by theory, the open vessel of the fluidized bed reactor can reduce isolated hot or cold spots within the vessel, thereby allowing for a more uniform distribution of fluid temperature.

[0385] 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 (a recycling rate of 25 to 35 gallons / min versus a hydraulic feed rate of 1 gallon / min). In some embodiments, the working fluid in each reactor is continuously recycled at a rate ratio of at least 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. In some implementations, the working fluid in each reactor is continuously recycled at a rate ratio of up to approximately 50:1, up to approximately 45:1, up to approximately 40:1, up to approximately 38:1, up to approximately 36:1, up to approximately 34:1, up to approximately 32:1, up to approximately 30:1, up to approximately 28:1, up to approximately 26:1, up to approximately 24:1, up to approximately 22:1, up to approximately 20:1, up to approximately 18:1, up to approximately 16:1, up to approximately 14:1, up to approximately 12:1, or up to approximately 10:1. The working fluid can be recycled by one or more pumps to prevent solids settling and to provide sufficient velocity with hydraulic shear to prevent excessive biofilm buildup and shearing. The digestion system provided 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.

[0386] 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. o C. At least about 20 o C. At least approximately 21 o C. At least approximately 22 o C. At least approximately 23 o C. At least approximately 24 o C. At least approximately 25 o C. At least approximately 26 o C. At least approximately 27 o C. At least approximately 28 o C. At least approximately 29 o C. At least about 30 o C. At least approximately 35 o C. At least about 40 o C. At least approximately 45 o C or at least about 50 oC. In some implementations, the reactor temperature is at most about 50°C. o C. At most about 45 o C. At most about 40 o C. At most about 35 o C. At most about 30 o C. At most about 29 o C. At most about 28 o C. At most about 27 o C. At most about 26 o C. At most about 25 o C. At most about 24 o C. At most about 23 o C. At most about 22 o C. At most about 21 o C. At most about 20 o C or at most about 15 o C. In some implementations, the temperature is at most about 45 degrees Celsius. o C. In some implementations, the temperature is maintained in a mid-range (e.g., less than 45°C). o C). In some implementations, the temperature may not be high (e.g., greater than 45°C). o C). In some embodiments, high-temperature conditions are avoided in one or more containers of the bioreactor system described herein.

[0387] In some implementations, the reactor temperature is approximately 15°C. o C to approximately 45 o C. In some implementations, the reactor temperature is approximately 15°C. o C to approximately 20 o C. Approximately 15 o C to approximately 22 o C. Approximately 15 o C to approximately 24 o C. Approximately 15 o C to approximately 26 o C. Approximately 15 o C to approximately 28 o C. Approximately 15 o C to approximately 30 o C. Approximately 15 o C to approximately 32 o C. Approximately 15 o C to approximately 34 o C. Approximately 15 o C to approximately 36 o C. Approximately 15 o C to approximately 40 o C. Approximately 15 o C to approximately 45 o C, approximately 20 o C to approximately 22o C, approximately 20 o C to approximately 24 o C, approximately 20 o C to approximately 26 o C, approximately 20 o C to approximately 28 o C, approximately 20 o C to approximately 30 o C, approximately 20 o C to approximately 32 o C, approximately 20 o C to approximately 34 o C, approximately 20 o C to approximately 36 o C, approximately 20 o C to approximately 40 o C, approximately 20 o C to approximately 45 o C, approximately 22 o C to approximately 24 o C, approximately 22 o C to approximately 26 o C, approximately 22 o C to approximately 28 o C, approximately 22 o C to approximately 30 o C, approximately 22 o C to approximately 32 o C, approximately 22 o C to approximately 34 o C, approximately 22 o C to approximately 36 o C, approximately 22 o C to approximately 40 o C, approximately 22 o C to approximately 45 o C, approximately 24 o C to approximately 26 o C, approximately 24 o C to approximately 28 o C, approximately 24 o C to approximately 30 o C, approximately 24 o C to approximately 32 o C, approximately 24 o C to approximately 34 o C, approximately 24 o C to approximately 36 o C, approximately 24 o C to approximately 40 o C, approximately 24 o C to approximately 45 o C, Approximately 26 o C to approximately 28 o C, Approximately 26 oC to approximately 30 o C, Approximately 26 o C to approximately 32 o C, Approximately 26 o C to approximately 34 o C, Approximately 26 o C to approximately 36 o C, Approximately 26 o C to approximately 40 o C, Approximately 26 o C to approximately 45 o C, Approximately 28 o C to approximately 30 o C, Approximately 28 o C to approximately 32 o C, Approximately 28 o C to approximately 34 o C, Approximately 28 o C to approximately 36 o C, Approximately 28 o C to approximately 40 o C, Approximately 28 o C to approximately 45 o C, approximately 30 o C to approximately 32 o C, approximately 30 o C to approximately 34 o C, approximately 30 o C to approximately 36 o C, approximately 30 o C to approximately 40 o C, approximately 30 o C to approximately 45 o C, approximately 32 o C to approximately 34 o C, approximately 32 o C to approximately 36 o C, approximately 32 o C to approximately 40 o C, approximately 32 o C to approximately 45 o C, approximately 34 o C to approximately 36 o C, approximately 34 o C to approximately 40 o C, approximately 34 o C to approximately 45 o C, approximately 36 o C to approximately 40 o C, approximately 36 o C to approximately 45 o C or approximately 40 o C to approximately 45 o C.

[0388] The reactor can be maintained under aerobic, microaerobic, or anaerobic conditions. A series of reactors in series within a digestion system can have different aerobic conditions. A series of reactors in series within a digestion system can have the same aerobic conditions. In some embodiments, the reactors can have the same aerobic conditions as adjacent reactors. In some embodiments, the reactors can have different aerobic conditions than adjacent reactors. In some embodiments, the digestion system can have aerobic, microaerobic, anaerobic conditions, or any combination thereof.

[0389] 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 dissolved oxygen measurements of 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 14 mg / L, about 3 mg / L to about 12 ... mg / L to about 15 mg / L, about 4 mg / L to about 5 mg / L, about 4 mg / L to about 6 mg / L, about 4 mg / L to about 7 mg / L, about 4 mg / L to about 8 mg / L, about 4 mg / L to about 9 mg / L, about 4 mg / L to about 10 mg / L, about 4 mg / L to about 12 mg / L, about 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 6 mg / L to about 8 mg / L, about 6 mg / L to about 9 mg / L, about 6 mg / L to about 10 mg / L 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 ranging from approximately mg / L to 15 mg / L. In some embodiments, aerobic conditions include dissolved oxygen measurements between 2 mg / L and 10 mg / L. In some embodiments, microaerobic conditions include dissolved oxygen measurements less than 2 mg / L. In some embodiments, microaerobic 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, microaerobic conditions include dissolved oxygen measurements of about 0.1 mg / L to about 1.99 mg / L. In some embodiments, microaerobic conditions include dissolved oxygen measurements of 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 1.4Conditions ranging from approximately 1.6 mg / L to approximately 1.8 mg / L, from approximately 1.6 mg / L to approximately 1.99 mg / L, or from approximately 1.8 mg / L to approximately 1.99 mg / L. In some embodiments, anaerobic conditions include conditions where the dissolved oxygen measurement is 0 mg / L.

[0390] The reactor of the digestion system described herein may include a working volume for containing a certain volume of working fluid. The working volume of the reactor of the digestion system described herein may be at least about 5 gallons, at least about 10 gallons, at least about 20 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 750 gallons, at least about 1,000 gallons, at least about 2,000 gallons, at least about 3,000 gallons, at least about 4,000 gallons, at least about 5,000 gallons, at least about 7,500 gallons, at least about 10,000 gallons, at least about 15,000 gallons, at least about 20,000 gallons, at least about 50,000 gallons, or greater than about 50,000 gallons. The reactor working volume of the digestion system described herein may be up to about 50,000 gallons, up to about 20,000 gallons, up to about 15,000 gallons, up to about 10,000 gallons, up to about 7,500 gallons, up to about 5,000 gallons, up to about 4,000 gallons, up to about 3,000 gallons, up to about 2,000 gallons, up to about 1,000 gallons, up to about 750 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 20 gallons, up to about 10 gallons, up to about 5 gallons, or less than about 5 gallons.

[0391] The reactor within the digestive system can be maintained at different pH levels. The reactor within the digestive system can be maintained at the same pH level. 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.

[0392] In some embodiments, the pH of the reactor within the digestive system can be from about 3 to about 9. In some embodiments, the pH of the reactor within 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 4 to about 7, from about 4 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.

[0393] In some embodiments, the reactor may include a distribution assembly (e.g., a distribution ring) located below the surface of the discharge volume. The distribution assembly can reduce the amount of surface disturbance and / or maintain an anaerobic environment within the reactor. The reactor environment can be anaerobic, microaerophilic, or aerobic. Without being bound by theory, the microaerophilic conditions of the digester reactor can enrich nitrogen-fixing microorganisms and / or microorganisms with nitrogen-use efficiency in the system's microbial aggregates.

[0394] C. Working fluids and microbial aggregates The working fluid may comprise a fluid-like substance that moves through the digestive system described herein. The working fluid may comprise solid components, liquid components, gaseous components, or any combination thereof. The working fluid may comprise microbial aggregates, inoculum of one or more isolated microorganisms or strains of microorganisms (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 microbial amplification or serve as a culture for the growth of microbial inoculum. The working fluid may include pH, viscosity, temperature, surface tension, adhesion, volume, or any combination thereof that enhances the growth and / or function of microorganisms or microbial cells. In some embodiments, the working fluid volume within each reactor is continuously replenished and discharged. In some embodiments, the working fluid volume within each reactor may be replenished and discharged in batches (e.g., discontinuously). In some embodiments, the working fluid within a first reactor may include a first working fluid. In some embodiments, the working fluid within a second reactor may include a second working fluid. In some embodiments, the working fluid within a third reactor may include a third working fluid. In some embodiments, the working fluid within 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 fluid may be mixed in a reactor (e.g., a chamber or container) preceding the first reactor. In some embodiments, the working fluid in each reactor may be different from the working fluid 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, algae, or any combination thereof). Different working fluids may contain different concentrations of target isolated bacteria. 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 isolated bacteria, different concentrations of carbon sources, different combinations of nitrogen sources, or any combination thereof. The working fluid in the reactors of the digestion system may be similar to the working fluid in the different reactors of the digestion system. The working fluid in each reactor may contain different microbial communities. Different microbial communities may include different bacteria, fungi, algae, or any combination thereof.

[0395] The working fluid within the vessel of a bioreactor system may contain one or more enzymes. The working fluid within one or more reactors (e.g., each reactor) may contain the same enzyme, which may be produced by microorganisms within the working fluid. The working fluid within one or more reactors (e.g., each reactor) may contain different enzymes, which may be produced by microorganisms within the working fluid. The enzymes within the working fluid may include dehydrogenases, hydrogenases, oxidases, catalases, peroxidases, phenol hydroxylases, glucanases, aminotransferases, thiocyanates, carboxylesterases, lipases, phosphatases, nucleases, phytases, arylsulfatases, amylases, cellulases, inulinases, xylanases, dextranases, 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), gluconate 2-dehydrogenase, cellulase, endo-1,3(4)-β-glucanase, pectin lyase, or any combination thereof. 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 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 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 greater than about 5%. In some embodiments, the enzyme may be present at an average abundance of up to about 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.001%, 0.0001%, or less than about 0.0001%. The working fluid within each reactor may contain enzymes with different activities. Enzyme activities may include, but are not limited to, nitrogen fixation, ammonia production, phosphorus solubility, nitrogen use efficiency, cell wall lysis, or any combination thereof.

[0396] The working fluid may contain digestion products that differ from those in the working fluids of other reactors in the system. In some embodiments, the working fluid may contain digestion products from aqueous organic feedstocks and microbial aggregates that are at least partially derived from previous working fluids.

[0397] The pH of the working fluid in each reactor can differ from that in other reactors. The pH of the working fluid in each reactor can be the same. The pH of the working fluid can be less than 6. The pH of the working fluid can be greater than 6. The pH range of the working fluid can be from 2 to 11. The pH of the working fluid can be at least 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, 11, or greater than 11. The pH of the working fluid can be at most about 11, 10.5, 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or less than 2.

[0398] The microbial colonies of the present invention can be stable. In stable microbial colonies, the identity and relative abundance of bacteria may not change significantly over time (e.g., at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or more than about 5 weeks). In stable microbial colonies, the identity and relative abundance of bacteria may not change significantly over time (e.g., at most about 5 weeks, 4 weeks, 3 weeks, 2 weeks, 1 week, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, or less than 1 day).

[0399] In some implementations, microbial aggregates can be characterized through population analysis. Population analysis may include community analysis, core community identification, and microbial community distance matrix calculation. In some implementations, microbial aggregates can be characterized in batches. Characterization of microbial aggregates can be measured in at least about 1, 2, 3, 4, 5, 6, 7, 8, or more batches. Characterization of microbial aggregates can be measured in at most about 7, 6, 5, 4, 3, 2, 1, or fewer batches. Based on population analysis, the most abundant species in the microbial aggregate can be identified. The most abundant species in the microbial aggregate can be referred to as the "top microbial species." In some implementations, the most abundant species in the microbial aggregate includes at least the top 2, top 3, top 4, top 5, top 10, top 15, or top 20 species. In some implementations, different reactors may have microbial aggregates with different top abundant species.

[0400] In some embodiments, a first microbial aggregate can be constructed in a mixing chamber where various inputs can be mixed into a homogeneous aqueous mixture for input 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 constructed. The first microbial aggregate may originate from microorganisms already present in one or more digestible substrates and / or from other inputs entering the mixing chamber. The first microbial aggregate may originate from inputs to the reactors in the digestion system described herein. In some embodiments, a second microbial aggregate is constructed in the first reactor. The second microbial aggregate may originate from microorganisms in the mixing chamber. In some embodiments, a third microbial aggregate is constructed in the second reactor. The third microbial aggregate may originate from a first working fluid present in the first reactor and transferred to the second reactor. In some embodiments, a fourth microbial aggregate is constructed in the third reactor. The fourth microbial aggregate may originate from a second working fluid present in the second reactor and transferred to the third reactor. In some embodiments, a fifth microbial aggregate is constructed in the fourth reactor. The fifth microbial aggregate may originate from a fourth working fluid present in the fourth reactor and transferred to the fifth reactor. Microbial aggregates can exist within any reactor of the digestive system described herein. Microbial aggregates can originate from the working fluid of the reactors in the digestive system described herein. A first microbial aggregate can originate from the input to the first reactor and can exist within the basic products of the digestive system. The first microbial aggregate can exist within the first reactor, second reactor, third reactor, or clarifier. Without being bound by theory, the first microbial aggregate within the working fluid can alter its microbial community and form a second microbial aggregate. The second microbial aggregate can exist within the first reactor, second reactor, third reactor, or clarifier. Without being bound by theory, the second microbial aggregate within the working fluid can alter its microbial community and form a third microbial aggregate. The third microbial aggregate can exist within the first reactor, second reactor, third reactor, or clarifier. Without being bound by theory, the third microbial aggregate within the working fluid can alter its microbial community and form a fourth microbial aggregate. The fourth microbial aggregate can exist within the first reactor, second reactor, third reactor, or clarifier. Without being bound by theory, the fourth microbial aggregate within the working fluid can alter its microbial community and form a fifth microbial aggregate. The fifth microbial aggregate can exist in the first reactor, second reactor, third reactor, or clarifier. The microbial aggregate of the digestive system described herein can be altered according to the conditions of the reactors and working fluids within the system (e.g., nutrients, residence time, flow rate, pH, oxygen content, digestion products).

[0401] A portion of the first working fluid may be transferred to a second container of the tandem serialized 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 may be transferred to a third container of the tandem serialized 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 may be transferred to a fourth container of the tandem serialized container assembly of the digestive system described herein. The working fluid in the fourth container may contain the fourth working fluid. A portion of the fourth working fluid may be transferred to a fifth container of the tandem serialized container assembly of the digestive system described herein. The working fluid in the fifth container may contain the fifth working fluid. A portion of the fifth working fluid may be transferred to a sixth container of the tandem serialized container assembly of the digestive system described herein. The working fluid in the sixth container may contain the sixth working fluid.

[0402] The working fluid in the containers of the digestive system can be incubated within the containers. The flow rate of the digestive system can increase or decrease the volume of the working fluid. The working fluid volume of the first, second, third, fourth, fifth, or sixth containers can increase over a period of time. The working fluid volume of the first, second, third, fourth, fifth, or sixth containers can decrease over a period of time. The working fluid volume of the first, second, third, fourth, fifth, or sixth containers may not increase or decrease over a period of time. The working fluid volume in each container of the digestive system can be the same. The working fluid volume in each container of the digestive system can be different. The volumes of the first, second, third, fourth, fifth, and / or sixth working fluids can be constant (e.g., unchanging over a period of time). Constant volumes can include volumes that do 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.

[0403] As the working fluid flows from each container of the digestion system, the working fluid may include flow rates. A first flow rate may include the flow rate of an aqueous feedstock entering the first container from a source outside the digestion system. 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. Flow rates (e.g., the first flow rate, the second flow rate, the third flow rate, the fourth flow rate, and the fifth flow rate) may be at least about 0.5 gallons / min, 1 gallon / min, 5 gallons / min, 10 gallons / min, 15 gallons / min, 20 gallons / min, 25 gallons / min, 30 gallons / min, 40 gallons / min, 50 gallons / min, 100 gallons / min, 200 gallons / min, 300 gallons / min, 400 gallons / min, 500 gallons / min, 1,000 gallons / min, or 10,000 gallons / min. The flow rate (e.g., first flow rate, second flow rate, third flow rate, fourth flow rate, fifth flow rate) can be up to about 10,000 gallons / min, 1,000 gallons / min, 500 gallons / min, 400 gallons / min, 300 gallons / min, 200 gallons / min, 100 gallons / min, 50 gallons / min, 40 gallons / min, 30 gallons / min, 25 gallons / min, 20 gallons / min, 15 gallons / min, 10 gallons / min, 5 gallons / min, 1 gallon / min, 0.5 gallons / min, or less than about 0.5 gallons / min.

[0404] Microbial aggregates may contain one or more microbial communities. These microbial communities can be generated by input into a digestive system. The aqueous organic feedstock input into the digestive system may contain microbial aggregates. Incubation 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 in the microbial aggregate, may possess desired plant growth-promoting characteristics. Plant growth-promoting characteristics may include aboveground biomass, root biomass, nutrient uptake, photosynthetic activity, crop yield, deaminase activity, acid production, leaf area, chlorophyll content, or total biomass.

[0405] The reactors of the digestive 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 the containers of the digestive system described herein can be continuous. Continuous flow of the working fluid can include uninterrupted flow of the working fluid or flow 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 digestive system described herein (e.g., between the containers of the digestive system) can have a first flow rate. Continuous flow of the working fluid within the digestive system described herein (e.g., between the containers of the digestive system) can have a second flow rate. In some embodiments, the first flow rate is equal to the second flow rate. The first flow rate can include the flow rate of fluid transferred from a source outside the digestive system into the first container. The second flow rate may include the fluid flow rate from the first container to the second container. In some embodiments, the amount of working fluid and / or aqueous organic feedstock transferred to the first container over a period of time is equal to the amount of working fluid and / or aqueous organic feedstock transferred to the second fluidly connected container over the same period of time. In some embodiments, the first flow rate and the second flow rate are different.

[0406] In some embodiments, the first container of the digestive system contains 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 fluid flow through the digestive system described herein.

[0407] In some embodiments, the digestive system can be inoculated with an inoculum of microorganisms (e.g., an inoculum of a microbial strain). The inoculum of a microbial strain can be an isolated microorganism. The isolated microorganism can 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 can comprise a mixture of multiple isolated microorganisms. The inoculum of microorganisms can comprise a mixture of at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, or more isolated microorganisms. The inoculum of microorganisms can comprise a mixture of up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2, or fewer isolated microorganisms.

[0408] In some cases, after the initial inoculation of the digestive system, it may not be necessary to inoculate again with isolated bacteria (e.g., microbial strains) or combinations of isolated bacteria. 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 isolated bacteria (e.g., microbial strains) into the container of the digestive system at a point in time during digestive system operations. In some cases, the digestive system may be re-inoculated with isolated bacteria or combinations of isolated bacteria 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 for longer periods. In some cases, the digestive system may be inoculated with isolated bacteria or combinations of isolated bacteria on day 1 of the digestive process, and then inoculated at least once, twice, three times, four times, five times, or more after day 1 of the digestive process. In some cases, the digestive system may be inoculated with isolated bacteria or combinations of isolated bacteria on day 1 of the digestive process, and then inoculated at most once, twice, three times, four times, five times, or more after day 1 of the digestive process. In some cases, the digestive system can be re-inoculated with the microbial strains described herein after a period of time following digestive system manipulation. For example, the digestive system can be re-inoculated with the microbial strains described herein after a duration of at least approximately 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, or 10 years of digestive system manipulation. For example, after a digestive system operation lasting up to approximately 10, 5, 4, 3, 24, 18, 12, 9, 6, 5, 4, 3, 8, 7, 6, 5, 4, 3, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, the digestive system can be re-inoculated with the microbial strains described herein.

[0409] In some embodiments, the concentration of microbial strains (e.g., the concentration of microbial strains) is maintained (e.g., retained) from the concentration of microbial strains added to the container (e.g., a first container) of the digestive system at 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 about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about ...

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 containing a volume of working fluid, wherein the first container contains an established population of a first microbial strain that promotes nitrogen use efficiency; (b) The bioreactor system is operated for a period of time in the following manner: (i) Transferring the aqueous raw material containing microbial aggregates 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) Throughout the duration, the concentration of the first microbial strain promoting nitrogen use efficiency is maintained in at least the first container at at least 80% of the concentration of the first microbial strain promoting nitrogen use efficiency 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 the first microbial strain that promotes nitrogen use efficiency is not present in the aqueous feedstock or any other input entering the bioreactor system during the duration at a concentration higher than 1% of the concentration of the first microbial strain that promotes nitrogen use efficiency in the first container.

2. The method according to claim 1, wherein the first microbial strain that promotes nitrogen use efficiency is a microbial strain that performs nitrogen fixation, promotes nitrogen fixation in plant tissues, recruits nitrogen-fixing organisms to the plant rhizosphere or other tissues, or increases the organic nitrogen content and / or mineralizes organic nitrogen in the soil.

3. The method according to claim 1, wherein the first microbial strain that promotes nitrogen use efficiency is positive for the nifH gene.

4. The method according to claim 1, wherein the first microbial strain that promotes nitrogen use efficiency is a microbial strain that promotes plant growth in a nitrogen-poor growth medium.

5. The method of claim 4, wherein the nitrogen-poor growth medium contains less than 10 ppm of nitrate.

6. The method according to claim 1, wherein the first microbial strain that promotes nitrogen use efficiency belongs to the genera *Coccus*, *Klebsiella*, *Lahn's*, *Krugwall*, *Enterobacter*, *Achromobacter*, *Microbacterium*, *Staphylococcus*, *Methylobacterium*, *Pseudomonas*, *Pantospira*, *Azotobacter*, etc. Azocarus , spirilla, Burkholderia, cyanobacteria, Bacillus and Bacillus-like bacteria.

7. The method according to claim 1, wherein the first microbial strain that promotes nitrogen use efficiency belongs to *Sacchariformis sugarcane*, *Klebsiella variegata*, *Laenia aquaticis*, *Kluwera cirrhosa*, or *Central Kluwera*. pseusosacchari Cossacchariformis, Enterobacter species, Achromobacter madrapata, lipid-producing bacteria Azopirillum Microbacterium tumefaciens, Glucosamine diazoxide, methyl symbiotic bacteria, Bacillus thuringiensis, Bacillus megaterium (Priestella megaterium), or Bacillus spp.

8. The method according to claim 1, wherein the first microbial strain that promotes nitrogen use efficiency is a strain (MS3907) preserved under ATCC accession number PTA-127654, a strain (MS3900) preserved under ATCC accession number PTA-127653, a strain (MS4921) preserved under ATCC accession number PTA-127655, or a strain (MS2748) preserved under ATCC accession number PTA-127652.

9. The method according to any one of claims 1 to 8, wherein the first microbial strain that promotes nitrogen use efficiency is not present in the aqueous feedstock or any other input entering the bioreactor system during the duration at a concentration greater than 100 CFU / ml.

10. The method according to any one of claims 1 to 9, wherein the first microbial strain that promotes nitrogen use efficiency is not present in the aqueous feedstock or any other input entering the bioreactor system during the duration.

11. The method of any one of claims 1 to 10, wherein the maintaining of step (b)(iii) comprises maintaining the concentration of the first microbial strain that facilitates nitrogen use efficiency at at least 1 x 10 3 CFU / ml.

12. The method according to any one of claims 1 to 11, wherein the first container further comprises an established population of a second microbial strain that promotes nitrogen use efficiency, and wherein the operation of step (b) further comprises (v) maintaining the concentration of the second microbial strain that promotes nitrogen use efficiency at at least 80% of the concentration of the second microbial strain that promotes nitrogen use efficiency at the start of the duration; wherein the second microbial strain that promotes nitrogen use efficiency is not present in the aqueous feedstock or any other input entering the bioreactor system during the duration at a concentration higher than 1% of the concentration of the second microbial strain that promotes nitrogen use efficiency in the first container.

13. The method of claim 12, wherein the first container further comprises an established population of a third microbial strain that promotes nitrogen use efficiency, and wherein the operation of step (b) further comprises (v) maintaining the concentration of the third microbial strain that promotes nitrogen use efficiency at at least 80% of the concentration of the third microbial strain that promotes nitrogen use efficiency at the start of the duration; wherein the third microbial strain that promotes nitrogen use efficiency is not present in the aqueous feedstock or any other input entering the bioreactor system during the duration at a concentration higher than 1% of the concentration of the third microbial strain that promotes nitrogen use efficiency in the first container.

14. The method according to any one of claims 1 to 13, wherein prior to step (b), the first container further comprises an established community of other microorganisms that promote nitrogen use efficiency, said other microorganisms not being the first microbial strain that promotes nitrogen use efficiency, the second microbial strain that promotes nitrogen use efficiency, or the third microbial strain that promotes nitrogen use efficiency, and wherein step (b) (iii) further comprises maintaining the concentration of said other microorganisms that promote nitrogen use efficiency at least 1 x 10⁻⁶ in at least the first container throughout the entire duration. 4 CFU / ml or maintained at at least 80% of the concentration of the other microorganisms that promote nitrogen use efficiency at the start of the duration, wherein the other microorganisms that promote nitrogen use efficiency are not added to the bioreactor system at a concentration higher than 1% of the concentration of the other microorganisms that promote nitrogen use efficiency in the first container during the duration.

15. The method of claim 14, wherein the other microorganisms promoting nitrogen use efficiency are not greater than 10 5 The concentration of CFU / ml is present in the aqueous feedstock or any other input entering the bioreactor system.

16. The method of claim 14 or 15, wherein at the start of the duration, the population of the other microorganisms promoting nitrogen use efficiency in the first container is at least 1 x 10⁻⁶. 4 CFU / ml.

17. The method according to any one of claims 14 to 16, wherein the other microorganisms that promote nitrogen use efficiency include microorganisms that perform nitrogen fixation, promote nitrogen fixation in plant tissues, recruit nitrogen-fixing organisms to plant rhizospheres or other tissues, or increase the organic nitrogen content and / or organic nitrogen mineralization in the soil.

18. The method according to any one of claims 14 to 16, wherein the other microorganism promoting nitrogen use efficiency is positive for the nifH gene.

19. The method according to any one of claims 1 to 18, further comprising, prior to step (a), adding an inoculum of the first microbial strain for promoting nitrogen use efficiency to the bioreactor system, wherein the inoculum of the first microbial strain for promoting nitrogen use efficiency produces at least 0.5 x 10⁻⁶ nitrogen-producing microorganisms in at least one container. 4 The initial population of the first microbial strain that promotes nitrogen use efficiency, with a concentration of CFU / ml.

20. The method of claim 19, wherein the concentration of the first microbial strain for promoting nitrogen use efficiency is less than 1 x 10⁻⁶ before adding the inoculum. 2 CFU / ml.

21. The method according to any one of claims 1 to 20, wherein the aqueous raw material further comprises organic material that can be at least partially digested by microorganisms present in at least one of the containers.

22. The method of claim 21, wherein prior to the transfer in step (b)(i), the organic material has been partially digested by microorganisms endogenous to the organic material.

23. The method of claim 21, further comprising digesting the organic material in two or more containers connected in series prior to the transfer in step (b)(i).

24. The method according to any one of claims 21 to 23, wherein the organic material comprises manure and / or material produced by microbial digestion of manure.

25. The method according to any one of claims 1 to 24, wherein the aqueous raw material further comprises inorganic materials.

26. The method of claim 25, wherein the inorganic material comprises phosphate rock particles.

27. The method of claim 26, wherein prior to the transfer in step (b)(i), the phosphate rock particles have been partially digested by microorganisms present in the aqueous feedstock.

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

29. The method according to any one of claims 1 to 28, wherein the microbial aggregate comprises at least 1 x 10 5 CFU / ml.

30. The method of claim 26, wherein the microbial aggregate comprises microorganisms derived from manure and microorganisms derived from phosphate rock particles.

31. The method according to any one of claims 1 to 30, wherein the operation in step (b) further comprises generating microbial metabolites that directly or indirectly promote nitrogen use efficiency in plants.

32. The method according to any one of claims 1 to 31, wherein the transfer in step (b)(i), the transfer in step (b)(ii), and the collection in step (b)(iv) are performed continuously throughout the entire duration.

33. The method according to any one of claims 1 to 31, wherein the transfer in step (b)(i), the transfer in step (b)(ii), and the collection in step (b)(iv) are performed periodically throughout the entire duration.

34. The method according to any one of claims 1 to 33, further comprising adding one or more carbon sources to at least one container of the bioreactor system.

35. The method of claim 34, wherein the one or more carbon sources are contained in the aqueous feedstock.

36. The method of claim 34 or 35, further comprising maintaining the malic acid concentration and / or glucose concentration at a concentration of at least 0.2% w / v relative to the volume of the working fluid in at least one vessel of the bioreactor system.

37. The method according to any one of claims 1 to 36, further comprising adding one or more nitrogen sources to at least one container of the bioreactor system.

38. The method of claim 37, wherein the one or more nitrogen sources comprise one or more of the following: ammonium sulfate, ammonium chloride, ammonium nitrate, sodium nitrate, yeast extract, yeast, or any combination thereof.

39. The method according to any one of claims 1 to 38, further comprising adding one or more of the following to at least one container of the bioreactor system: soybean flour, lentil flour, chickpea flour, green pea flour, yellow pea flour, white soybean flour, corn flour, cereal flour, corn gluten, soy protein, or soy protein hydrolysate, or any combination thereof.

40. The method of claim 39, wherein the soybean flour is added, and wherein the soybean flour is contained in the aqueous raw material.

41. The method of claim 39 or 40, further comprising maintaining a soybean flour concentration at a concentration of at least 0.2% w / v relative to the volume of the working fluid in at least one container of the bioreactor system.

42. The method according to any one of claims 1 to 41, wherein the bioreactor system includes a clarifier container containing a clarifier working fluid.

43. The method of claim 42, further comprising separating the supernatant portion of the clarifier working fluid from the flocculent portion of the clarifier working fluid within the clarifier container.

44. The method of claim 43, wherein the separation comprises gravity separation.

45. The method according to any one of claims 42 to 44, further comprising folding the flocculent portion of the clarifier working fluid.

46. ​​The method of claim 45, wherein the folding further comprises releasing a population of the first microbial strain that promotes nitrogen use efficiency into the supernatant portion without introducing flocculent solids into the supernatant portion.

47. The method of claim 45 or 46, wherein the folding is performed by a folding scraper in the bottom portion of the clarifier container.

48. The method according to any one of claims 43 to 47, wherein the operation further comprises transferring a portion of the flocculent from the clarifier container to a forward container in the bioreactor system.

49. The method according to any one of claims 43 to 47, wherein the product effluent comprises the supernatant portion of the clarifier working fluid.

50. The method according to any one of claims 1 to 49, wherein the method further comprises generating at least 1 x 10⁻⁶ ppm in the product effluent. 2 The first microbial strain that promotes nitrogen use efficiency (CFU / ml).

51. The method according to any one of claims 1 to 50, wherein the bioreactor system comprises a first container containing a first working fluid of a certain volume, a second container containing a second working fluid of a certain volume, and a third container containing a third working fluid of a certain volume.

52. The method of claim 51, 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.

53. The method of claim 52, wherein the third container includes an outlet port in fluid connection with the clarifier container.

54. The method according to any one of claims 51 to 53, further comprising maintaining the volume of each of the first working fluid, the second working fluid, and the third working fluid constant throughout the entire duration.

55. The method according to any one of claims 1 to 54, wherein step (b) comprises operating the bioreactor system in a hydraulically balanced manner.

56. The method according to any one of claims 1 to 55, wherein the transfer in step (b)(i), the transfer in step (b)(ii), and the collection in step (b)(iv) are driven by gravity.

57. The method according to any one of claims 1 to 56, wherein the operation comprises maintaining a flow rate that produces a hydraulic residence time of at least 5 days.

58. The method of any one of claims 1 to 57, wherein the operation comprises maintaining the product effluent at a flow rate of at least 100 gallons per day.

59. The method according to any one of claims 1 to 58, wherein the volume of the working fluid in each of the two or more containers is at least 100 gallons.

60. The method according to any one of claims 1 to 59, wherein at least one of the two or more containers is a fluidized bed reactor.

61. The method according to any one of claims 1 to 60, wherein at least one of the two or more containers is a packed bed reactor.

62. The method according to any one of claims 1 to 61, further comprising maintaining at least one of the two or more containers under aerobic conditions.

63. The method according to any one of claims 1 to 62, further comprising maintaining at least one of the two or more containers under microaerobic conditions.

64. The method according to any one of claims 1 to 63, wherein the bioreactor system operates continuously for at least 90 days.

65. The method according to any one of claims 1 to 64, wherein the five most abundant species in the microbial aggregate include one or more species belonging to one or more of the following genera: *Bacillus*, *Levonorhynchus*, *Thermocybe*, *Typhonium*, and *Acidobacter*.

66. The method according to any one of claims 1 to 65, wherein the five most abundant species in the microbial aggregate include one or more of the following species: Levulina ligata, Solanum phenylacetate, Solanum mesennii, Solitalia canadensis, and Nitrifying spirochetes.

67. The method according to any one of claims 1 to 66, wherein the microbial aggregate comprises endogenous microorganisms of the organic material.

68. The method according to any one of claims 1 to 67, wherein at least a portion of the aqueous raw material is produced by the method according to any one of claims 105 to 121.

69. The method according to any one of claims 51 to 68, wherein at least one of the first working fluid, the second working fluid, or the third working fluid comprises a pH buffer system.

70. The method according to any one of claims 51 to 69, 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 8 throughout the entire duration.

71. The method according to any one of claims 1 to 70, wherein the aqueous raw material does not contain the first microbial strain that promotes nitrogen use efficiency at a concentration higher than 10 CFU / ml.

72. The method according to any one of claims 1 to 71, wherein the first microbial strain that promotes nitrogen use efficiency is not added to the bioreactor system at a concentration higher than 10 CFU / ml during the said duration.

73. The method according to any one of claims 1 to 72, wherein the bioreactor system comprises at least one container positioned preceding the first container in the tandem series.

74. The method according to any one of claims 1 to 73, wherein the volume of the working fluid in one of the two or more containers contains the microbial aggregate, wherein each microbial aggregate is different from all said microbial aggregates in the other working fluids.

75. The method according to any one of claims 1 to 74, further comprising generating a colony of sporulated bacteria in the product effluent.

76. The method according to any one of claims 1 to 75, further comprising generating a population of the first microbial strain that promotes nitrogen use efficiency and sporulation in the product effluent.

77. The method of claim 76, wherein the population of the sporulated first microbial strain that promotes nitrogen use efficiency comprises at least 1 x 10⁻⁶ micrograms per cubic meter. 2 CFU / ml.

78. The method according to any one of claims 1 to 77, further comprising adding an additional population of the first microbial strain that promotes nitrogen use efficiency, the second microbial strain that promotes nitrogen use efficiency, or the third microbial strain that promotes nitrogen use efficiency to the biostimulant product.

79. A bioreactor system, comprising: (a) A stream of aqueous feedstock in fluid communication with a first container containing a first working fluid of a certain volume, wherein the aqueous feedstock contains a microbial aggregate, wherein the first working fluid contains a population of a first strain of microorganisms that promotes nitrogen use efficiency, wherein the concentration of the first microbial strain that promotes nitrogen use efficiency in the first working fluid is at least 100 times higher than the concentration of the first microbial strain that promotes nitrogen use efficiency in the aqueous feedstock stream entering the bioreactor system or in any other input; (b) One or more additional containers arranged in a series including the first container, wherein each of the one or more additional containers contains a volume of working fluid and is in fluid communication with at least one other container in the series, and wherein at least one of the one or more additional containers includes a product outflow port. as well as (c) Product outflow, which is in fluid communication with the product outflow port.

80. The system of claim 79, wherein the first microbial strain that promotes nitrogen use efficiency is a microbial strain that performs nitrogen fixation, promotes nitrogen fixation in plant tissues, recruits nitrogen-fixing organisms to the plant rhizosphere or other tissues, or increases the organic nitrogen content and / or mineralizes organic nitrogen in the soil.

81. The system of claim 79, wherein the first microbial strain that promotes nitrogen use efficiency is positive for the nifH gene.

82. The system of claim 79, wherein the first microbial strain that promotes nitrogen use efficiency is a microbial strain that promotes plant growth in a nitrogen-poor growth medium.

83. The system of claim 79, wherein the nitrogen-poor growth medium contains less than 10 ppm of nitrate.

84. The system according to claim 79, wherein the first microbial strain that promotes nitrogen use efficiency belongs to the genera *Coccus*, *Klebsiella*, *Lahn's*, *Krugwall*, *Enterobacter*, *Achromobacter*, *Microbacterium*, *Staphylococcus*, *Methylobacterium*, *Pseudomonas*, *Pantospira*, *Azotobacter*, etc. Azocarus , spirilla, Burkholderia, cyanobacteria, Bacillus or Bacillus-like bacteria.

85. The system according to claim 79, wherein the first microbial strain that promotes nitrogen use efficiency belongs to *Sacchariformis sugarcane*, *Klebsiella variegata*, *Laenia aquaticis*, *Kluweria centralis*, etc. pseusosacchari Cossacchariformis, Enterobacter species, Achromobacter madrapata, lipid-producing bacteria Azopirillum Microbacterium tumefaciens, Glucosamine diazoxide, methyl symbiotic bacteria, Bacillus thuringiensis, Bacillus megaterium (Priestella megaterium), or Bacillus spp.

86. The system according to claim 79, wherein the first microbial strain that promotes nitrogen use efficiency is a strain (MS3907) deposited under ATCC accession number PTA-127654, a strain (MS3900) deposited under ATCC accession number PTA-127653, a strain (MS4921) deposited under ATCC accession number PTA-127655, or a strain (MS2748) deposited under ATCC accession number PTA-127652.

87. The system of claim 79, wherein the bioreactor system is a continuous flow bioreactor system, and the flow of the aqueous feedstock is continuous.

88. The system according to any one of claims 79 to 87, wherein each of the volumes of the working fluid is constant.

89. The system according to any one of claims 79 to 88, wherein each of the first container and the one or more additional containers is contained in a configuration that remains at least 1 x 10⁻⁶ during operation of the bioreactor system. 4 The concentration of the first microbial strain that promotes nitrogen use efficiency is CFU / ml.

90. The system according to any one of claims 79 to 89, wherein the aqueous feedstock and any other input entering the bioreactor system do not contain the population of the first microbial strain that promotes nitrogen use efficiency, or do not contain a concentration of the first microbial strain that promotes nitrogen use efficiency at a level higher than 100 CFU / ml.

91. The system according to any one of claims 79 to 90, wherein the microbial aggregate comprises at least 1 x 10 5 Microorganisms at CFU / ml.

92. The system according to any one of claims 79 to 91, wherein the aqueous feedstock further comprises organic material that can be digested by microorganisms present in the container.

93. The system of claim 92, wherein the organic material comprises manure or a material derived from manure.

94. The system according to any one of claims 79 to 93, wherein the aqueous feedstock further comprises phosphate rock particles.

95. The system of claim 94, wherein the microbial aggregate comprises microorganisms derived from manure and phosphate rock particles.

96. The system according to any one of claims 79 to 95, wherein the container at 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.

97. The system of claim 96, wherein the clarifier container includes one or more flocculant baffles configured to agitate settled flocculants in the clarifier container without resuspending solids in the flocculant portion into the supernatant portion.

98. The system of claim 96 or 97 further includes a flocculant return stream flowing from the clarifier to the preceding container in the series.

99. The system according to any one of claims 96 to 98, wherein the product effluent comprises the supernatant portion.

100. The system of claim 99, wherein the product effluent comprises at least 1 x 10 4 The first microbial strain that promotes nitrogen use efficiency (CFU / ml).

101. The system of claim 99 or 100, wherein the product effluent comprises at least 1 x 10 2 The first microbial strain that promotes nitrogen use efficiency, in sporulated form at CFU / ml.

102. The system according to any one of claims 99 to 101, wherein the product effluent comprises a total dry weight of 0.2 to 2.5 mg / ml.

103. The system according to any one of claims 99 to 102, wherein the product effluent has a chemical oxygen demand between 80 and 500 mg / L.

104. The system according to any one of claims 99 to 103, wherein the product effluent has a conductivity between 1.3 and 3.0 mS / cm.

105. A method comprising: (a) Transferring water and phosphate rock into a first container containing a certain volume of a first working fluid, wherein the products of microbial digestion of said manure derived from manure are not transferred into the first container; (b) Transfer a portion of the first working fluid to a second container containing the second working fluid; (c) Transfer to the second container: (i) A liquid comprising (A) a first microbial aggregate containing microorganisms derived from a first organic material, and (B) digestion products produced by the anaerobic digestion of the first organic material by the microorganisms; (ii) Second organic material; and (iii) Yeast.

106. The method of claim 105, further comprising transferring a portion of the second working fluid to a third container containing a third working fluid, and transferring a portion of the third working fluid to a fourth container containing a fourth working fluid.

107. The method of claim 106 further comprises separating a portion of the fourth working fluid into a flocculent portion and a supernatant portion.

108. The method of claim 107, further comprising transferring a portion of the flocculent to the first container.

109. The method according to any one of claims 106 to 108, further comprising maintaining the first container, the second container, the third container and / or the fourth container under aerobic conditions.

110. The method according to any one of claims 106 to 108, wherein the first container, the second container, the third container and / or the fourth container are fluidized bed reactors, wherein the phosphate rock is continuously circulated within the first container, the second container, the third container and / or the fourth container.

111. The method according to any one of claims 105 to 110, wherein the total volume of material added to the first container during a given time period is equal to the total volume of the first working fluid transferred to the second container during the same time period.

112. The method according to any one of claims 106 to 111, wherein the total volume of material transferred to the second container, the third container and the fourth container within a given time period is equal to the total volume transferred out of the second container, the third container and the fourth container within the same time period.

113. The method according to any one of claims 106 to 112, further comprising maintaining the volume of the first working fluid, the volume of the second working fluid, and the volume of the third working fluid constant.

114. The method according to any one of claims 105 to 113, wherein the first organic material is manure.

115. The method according to any one of claims 105 to 114, wherein the second organic material is manure.

116. The method according to any one of claims 105 to 115, wherein the yeast is Saccharomyces cerevisiae.

117. The method according to any one of claims 106 to 116, further comprising generating a product stream from a second, third, or fourth microbial aggregate, wherein the product stream comprises bacteria from one or more of the following species: Levulina ligandii, Sylvius phenylacetate, Sylvius mesennii, Solitalea canadensis .

118. The method of claim 117, wherein the five most abundant microorganisms in the second microbial aggregate comprise bacteria from one or more of the following species: Levulina ligandii, Solomonella phenylacetate, Solomonella mesennii, Solitalea canadensis And Nitrifying Spirulina musculosus.

119. The method according to claim 117 or 118, wherein the five most abundant microorganisms in the second microbial aggregate do not include bacteria from any of the following genera: *Bacillus*, *Thermophyton*, *Germospora*, and *Acidobacter*.

120. The method according to any one of claims 117 to 119, wherein the second microbial aggregate is contained in the fifth working fluid.

121. The method according to any one of claims 105 to 120, wherein low-rank coal is not transferred into the first container.

122. A biostimulant composition prepared by the method of any one of claims 1 to 78, the system of any one of claims 79 to 104, or the method of any one of claims 105 to 121.

123. A method for promoting plant growth, comprising contacting a plant, seed, or plant growth medium with the biostimulant composition of claim 122.

124. A method for improving nitrogen use efficiency in plants, the method comprising contacting a plant, seed, or plant growth medium with the biostimulant composition of claim 122.

125. A method for increasing phosphorus solubility in a plant growth medium, the method comprising contacting a plant, seed, or the plant growth medium with the biostimulant composition of claim 122.

126. A composition comprising: (a) A strain of Bacillus megaterium 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: 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.

127. The composition according to claim 126, wherein the Bacillus megaterium strain is MS3900 strain or its isolated clone preserved under ATCC accession number PTA-127653.

128. The composition according to claim 126 or 127 further comprises the product of the digestion of the organic substrate by the Bacillus megaterium strain.

129. The composition according to any one of claims 126 to 128, wherein the carrier comprises a fertilizer.

130. The composition according to any one of claims 126 to 129, wherein the carrier is a solid coated with the Bacillus megaterium strain.

131. The composition according to any one of claims 126 to 130, wherein the carrier is a liquid.

132. The composition according to any one of claims 126 to 131, wherein the composition further comprises an excipient selected from wetting agents, spreading agents, dispersants, adhesives, dust-proofing agents, and binders.

133. The composition according to any one of claims 126 to 132, wherein the concentration of the *Bacillus megaterium* strain in the composition is in the range of 1 x 10⁻⁶. 3 Up to 1 x 10 11 CFU / ml.

134. A composition comprising: (a) A strain of *Bacillus niger* 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; (iii) At least 95% identical to SEQ ID NO: 8 rpoB Gene sequence; and (iv) At least 95% identical to SEQ ID NO: 13 nifH Gene sequence; and (b) Carrier.

135. The composition according to claim 134, wherein the Bacillus niger strain is MS3907 strain or its isolated clone preserved under ATCC accession number PTA-127654.

136. The composition according to claim 134 or 135 further comprises the product of digestion of the organic substrate by the *Bacillus niger* strain.

137. The composition according to any one of claims 134 to 136, wherein the carrier comprises a fertilizer.

138. The composition according to any one of claims 134 to 137, wherein the carrier is a solid coated with the *Bacillus cerevisiae* strain.

139. The composition according to any one of claims 134 to 138, wherein the carrier is a liquid.

140. The composition according to any one of claims 134 to 139, wherein the composition further comprises an excipient selected from wetting agents, spreading agents, dispersants, adhesives, dust-proofing agents, and binders.

141. The composition according to any one of claims 134 to 140, wherein the concentration of the *Bacillus cerevisiae* strain in the composition is in the range of 1 x 10⁻⁶. 3 Up to 1 x 10 11 CFU / ml.

142. A composition comprising: (a) A *Bacillus* strain of *Sonchus* 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; (iii) At least 95% identical to SEQ ID NO: 9 rpoB Gene sequence; and (iv) At least 95% identical to SEQ ID NO: 14 nifH Gene sequence; and (b) Carrier.

143. The composition according to claim 142, wherein the Bacillus oryzae strain is MS4921 strain or its isolated clone preserved under ATCC accession number PTA-127655.

144. The composition according to claim 142 or 143 further comprises the product of digestion of the organic substrate by the sow thistle bacillus strain.

145. The composition according to any one of claims 142 to 144, wherein the carrier comprises a fertilizer.

146. The composition according to any one of claims 142 to 145, wherein the carrier is a solid coated with the Bacillus trichomoniasis strain.

147. The composition according to any one of claims 142 to 145, wherein the carrier is a liquid.

148. The composition according to any one of claims 142 to 147, wherein the composition further comprises an excipient selected from wetting agents, spreading agents, dispersants, adhesives, dust-proofing agents, and binders.

149. The composition according to any one of claims 142 to 148, wherein the concentration of the *Bacillus solanacearum* strain in the composition is in the range of 1 x 10⁻⁶. 3 Up to 1 x 10 11 CFU / ml.

150. A composition comprising: (a) A strain of Bacillus megaterium 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: 10; (ii) At least 95% identical to SEQ ID NO: 11 gyrB Gene sequence; and (iii) At least 95% identical to SEQ ID NO: 12 rpoB Gene sequence; and (b) Carrier.

151. The composition according to claim 150, wherein the Bacillus megaterium strain is MS2748 strain or its isolated clone preserved under ATCC accession number PTA-127652.

152. The composition according to claim 150 or 151 further comprises the product of the digestion of the organic substrate by the Bacillus megaterium strain.

153. The composition according to any one of claims 150 to 152, wherein the carrier comprises a fertilizer.

154. The composition according to any one of claims 150 to 153, wherein the carrier is a solid coated with the Bacillus megaterium strain.

155. The composition according to any one of claims 150 to 154, wherein the carrier is a liquid.

156. The composition according to any one of claims 150 to 155, wherein the composition further comprises an excipient selected from wetting agents, spreading agents, dispersants, adhesives, dust-proofing agents, and binders.

157. The composition according to any one of claims 150 to 156, wherein the concentration of the *Bacillus megaterium* strain in the composition is in the range of 1 x 10⁻⁶. 3 Up to 1 x 10 11 CFU / ml.

158. The composition according to any one of claims 150 to 157, further comprising at least one or more of the following: (b) A strain of Bacillus megaterium 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: 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; (c) A strain of *Bacillus niger* 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; (iii) At least 95% identical to SEQ ID NO: 8 rpoB Gene sequence; and (iv) At least 95% identical to SEQ ID NO: 13 nifH Gene sequence; and (d) A *Bacillus* strain of *Sonchus* 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; (iii) At least 95% identical to SEQ ID NO: 9 rpoB Gene sequence; and (iv) At least 95% identical to SEQ ID NO: 14 nifH Gene sequence.

159. An isolated strain of Bacillus megaterium, 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.

160. The isolated strain according to claim 159, wherein the Bacillus megaterium strain is the MS3900 strain or its isolated clone preserved under ATCC accession number PTA-127653.

161. An isolated strain of *Bacillus cerevisiae*, having one or more of the following characteristics: (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; (c) At least 95% identical to SEQ ID NO: 8 rpoB Gene sequence; and (d) At least 95% identical to SEQ ID NO: 13 nifH Gene sequence.

162. The isolated strain according to claim 161, wherein the Bacillus thuringiensis strain is the MS3907 strain or its isolated clone preserved under ATCC accession number PTA-127654.

163. An isolated strain of a *Bacillus solanacearum* species, having one or more of the following characteristics: (a) A 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 3; (b) At least 95% identical to SEQ ID NO: 6 gyrB Gene sequence; (c) At least 95% identical to SEQ ID NO: 9 rpoB Gene sequence; and (d) At least 95% identical to SEQ ID NO: 14 nifH Gene sequence.

164. The isolated strain according to claim 163, wherein the Bacillus soursoprothiolane strain is the MS4921 strain or its isolated clone preserved under ATCC accession number PTA-127655.

165. An isolated strain of Bacillus megaterium, having one or more of the following characteristics: (a) A 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO: 10; (b) At least 95% identical to SEQ ID NO: 11 gyrB Gene sequence; and (c) At least 95% identical to SEQ ID NO: 12 rpoB Gene sequence.

166. The isolated strain according to claim 165, wherein the Bacillus megaterium strain is MS2748 strain or its isolated clone preserved under ATCC accession number PTA-127652.

167. 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 composition of any one of claims 126 to 158 or an isolated strain of any one of claims 159 to 166.

168. The method of claim 167, wherein the contact increases the nitrogen content of the plant by at least 5%.

169. The method according to claim 167 or 168, wherein the contact increases nitrogen fixation activity and / or nitrogen use efficiency in plant tissue by at least 5%.

170. The method according to any one of claims 167 to 169, wherein the contact increases the population of nitrogen-fixing bacteria in the roots and rhizosphere of the plant by at least 5%.

171. The method according to any one of claims 167 to 170, wherein the contact causes nitrogen-fixing bacteria present in the culture medium to be recruited to the root zone of the plant.

172. The method according to any one of claims 167 to 171, wherein the contact results in an increase in plant growth of at least 10% compared to the control.

173. The method according to any one of claims 167 to 172, wherein the culture medium comprises soil, hydroponic medium, turface, or isolite.

174. A method for enhancing the nitrogen-fixing activity of bacteria or the colonization capacity of plant tissues, the method comprising incubating the bacteria in the presence of strain MS3900.

175. The method of claim 174, wherein the bacteria is strain MS3907.

176. A composition comprising: (a) A microbial aggregate comprising one or more bacterial strains selected from the following: MS3900 (ATCC accession number PTA-127653), MS3907 (ATCC accession number PTA-127654), MS4921 (ATCC accession number PTA-127655) and MS2748 (ATCC accession number PTA-127652); and (b) Metabolites produced by the digestion of organic substrates by microorganisms within the said microbial aggregate.

177. The composition of claim 176, wherein the microbial aggregate further comprises an enriched community of nitrogen-fixing microorganisms.

178. The composition according to claim 176 or 177, wherein the organic substrate is derived from cow dung, phosphate rock, or plant abrasive, or any combination thereof.

179. The composition according to any one of claims 176 to 178, wherein the microbial aggregate comprises microorganisms derived from cow dung, phosphate rock, or plant mills.

180. The composition according to claim 178 or 179, wherein the plant-based mill is soybean flour, lentil flour, chickpea flour, green pea flour, yellow pea flour, white soybean flour, corn flour, cereal flour, corn gluten, soy protein, soy protein hydrolysate, or any combination thereof.

181. The composition according to any one of claims 176 to 180, wherein the microbial aggregate comprises 5 x 10 7 Up to 1.5x10 8 CFU / ml of bacteria.

182. The composition according to any one of claims 176 to 181, wherein the microbial aggregate comprises 1 x 10 5 Up to 1.5x10 7 Nitrogen-fixing bacteria at CFU / ml.

183. The composition according to any one of claims 176 to 181, wherein the microbial aggregate comprises 5 x 10 4 Up to 5x10 5 Sporulating bacteria at CFU / ml.

184. The composition according to any one of claims 176 to 183, wherein the microbial aggregate comprises 1 x 10 3 Up to 1x10 4 MS3900 spores, MS4921 spores, or any combination thereof, at CFU / ml.

185. The composition according to any one of claims 176 to 184, wherein the microbial aggregate comprises 1 x 10 2 Up to 1x10 4 MS3907 spores at CFU / ml.

186. The composition according to any one of claims 176 to 185, wherein the pH of the composition is 7 to 9.

187. The composition according to any one of claims 176 to 186, wherein the COD of the composition is 120 to 500 mg / L.

188. The composition according to any one of claims 176 to 187, wherein the conductivity of the composition is 0.5 to 2.0 mS / cm.

189. The composition according to any one of claims 176 to 188, wherein the total dry weight of the composition is 0.2 to 2.5 mg / ml.

190. 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 containing a volume of working fluid, wherein at least one of the containers contains a population of a first microbial strain and a population of other microorganisms, the population of the first microbial strain being derived from an inoculum of the first microbial strain added to the bioreactor system. (b) The bioreactor system is operated for a period of time in the following manner: (i) Transferring the aqueous feedstock containing microbial aggregates 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) Collect at least a portion of the product effluent as the biostimulant composition; as well as (iv) Throughout the duration, the population of the first microbial strain is maintained in at least the first container at a level of at least 80% of the population of the first microbial strain at the start of the duration; The duration is at least 5 days; and the first microbial strain is not present in the aqueous feedstock or any other input entering the bioreactor system during the duration at a concentration higher than 1% of the concentration of the population of the first microbial strain in the first container.

191. The method of claim 190, wherein the first microbial strain has the property of promoting plant growth.

192. The method of claim 190 or 191 further comprises providing conditions in the bioreactor system that promote the formation of a rich cluster of the first microbial strain relative to the population of the aqueous feedstock or any other input entering the bioreactor system.

193. The method of claim 190 or 191 further comprises applying a selective pressure to the bioreactor system, the selective pressure being favorable to the growth of the first microbial strain relative to the other microorganisms.

194. The method according to any one of claims 191 to 193, wherein the aqueous raw material further comprises organic material that can be digested by the first microbial strain and by at least some of the other microorganisms.

195. The method of claim 194, further comprising generating a metabolite having the plant growth-promoting properties by digesting the organic material.

196. The system according to any one of claims 79 to 104, wherein the first working fluid comprises malic acid at a concentration of at least 0.2% w / v.

197. The system according to any one of claims 79 to 104 and 196, wherein the first working fluid comprises soybean flour at a concentration of at least 0.2% w / v.

198. The system according to any one of claims 79 to 104, 196 and 197, wherein the first working fluid comprises microaerobic conditions.

199. The system of claim 198, wherein the working fluid in at least one of the one or more additional containers comprises microaerobic conditions.

200. The system according to any one of claims 79 to 104 and 196 to 199, wherein the system has a hydraulic residence time of at least 5 days.

201. The system according to any one of claims 79 to 104 and 196 to 200, wherein the first working fluid further comprises an established population of a second microbial strain that promotes nitrogen utilization efficiency, wherein the concentration of the second microbial strain that promotes nitrogen utilization efficiency in the first working fluid is at least 100 times higher than the concentration of the second microbial strain that promotes nitrogen utilization efficiency in the aqueous feed stream and any other input entering the bioreactor system.

202. The system according to claim 201, wherein the second microbial strain that promotes nitrogen use efficiency belongs to *Sacchariformis sugarcane*, *Klebsiella variegata*, *Laenia aquaticis*, *Kluweria centralis*, etc. pseusosacchari Cossacchariformis, Enterobacter species, Achromobacter madrapata, lipid-producing bacteria Azopirillum Microbacterium tumefaciens, Glucosamine diazoxide, methyl symbiotic bacteria, Bacillus thuringiensis, Bacillus megaterium (Priestella megaterium), or Bacillus spp.

203. The system according to any one of claims 79 to 104 and 196 to 202, wherein the first working fluid further comprises an established population of a third microbial strain that promotes nitrogen utilization efficiency, wherein the concentration of the third microbial strain that promotes nitrogen utilization efficiency in the first working fluid is at least 100 times higher than the concentration of the third microbial strain that promotes nitrogen utilization efficiency in the aqueous feed stream and any other input entering the bioreactor system.

204. The system according to claim 203, wherein the third microbial strain that promotes nitrogen use efficiency belongs to *Sacchariformis sugarcane*, *Klebsiella variegata*, *Laenia aquaticis*, *Kluweria centralis*, etc. pseusosacchari Cossacchariformis, Enterobacter species, Achromobacter madrapata, lipid-producing bacteria Azopirillum Microbacterium tumefaciens, Glucosamine diazoxide, methyl symbiotic bacteria, Bacillus thuringiensis, Bacillus megaterium (Priestella megaterium), or Bacillus spp.

205. The system according to any one of claims 79 to 104 and 196 to 204, wherein the first working fluid comprises at least 1 x 10 5 CFU / ml nifH The total population of gene-positive microorganisms.

206. A method for promoting plant growth, comprising: (a) Contacting a plant and / or a culture medium in which the plant grows with a composition comprising one or more compounds, wherein the one or more compounds are selected from phytolaccoside C, 3-cyclohexyl-6-[4-[3-(trifluoromethyl)phenyl]-1-piperazinyl]-1H-pyrimidin-2,4-dione, Arg-Thr-Ala-Arg, (2R)-2-[[4-(2,6-dipyrrolidone-1-ylpyrimidin-4-yl)piperazin-1-yl]methyl]-2,5,7,8-tetramethyl-3,4-dihydrobenzopyran-6- Dihydrochloride, Gly-Leu-Arg-Val-Phe, wesiellamide, thrombin receptor activating peptide 5 (TRAP-5), tungstate, fercomin, threonyl-isoleucine, BW-A868C, Lys-Ala-Leu-Glu, N-benzyloxycarbonylglycine, Glu-Asp-Asn, Glu-Asp-Asn, Ile-Glu-His-Lys, Chaps, desdimethylcitalopram, Lys-Tyr-Thr-Ser-Se r, 3-amino-4,6-dimethyl-N-(1-phenylethyl)thiopheno[2,3-b]pyridine-2-carboxamide, Asn-Ala-Leu-Ala-His, Met-Asp-Arg, His-Arg-Lys-Glu, Asn-Cys-Phe, 7-hydroxylauric acid, Phe-Tyr-Lys-Arg, k-strophanthidin, disopyramide, estradiol-4,9-diene-3,17-dione, HoPhe-Asp-OH, 5-methyl-6-oxo-5,6-di Hydrogen-4h-imidazo[1,5-a]thieno[2,3-f][1,4]diaza-3-carboxylic acid tert-butyl ester, 2,6-naphthyldiol, zonisamide, Ser-Gln-Leu-Lys, Pro-Ala-Phe, Ala-Thr-Ile-Lys, mycophenolic acid, PC(15:0 / 18:3(6Z,9Z,12Z)), 6-[(2Z)-2-benzylheptaoxy]-3,4,5-trihydroxyoxacyclohexane-2-carboxylic acid, 7''-Deoxybonaspectin D 4''-methyl ether, Reciniferatoxin, trans-2-hepten-1-yl acetate, acetoxy-6-gingerol, dupinidine, N-1-naphthylbenzamide, one or more derivatives thereof, or combinations thereof.

207. The method according to claim 206, wherein the plant growth characteristic is nitrogen use efficiency.

208. The method according to claim 206 or 207, wherein the concentration of the one or more compounds in the composition is at least about 1 nanomolar (nM).

209. The method according to any one of claims 206 to 208, wherein the concentration of the one or more compounds in the composition is at least about 0.00001% of the dry weight of the composition.

210. The method according to any one of claims 206 to 209, wherein the contact comprises contacting the plant with the composition.

211. The method according to any one of claims 206 to 210, wherein the contact comprises contacting the plant seed with the composition.

212. The method according to any one of claims 206 to 211, wherein the contact comprises contacting the leaves of the plant with the composition.

213. The method according to any one of claims 206 to 212, wherein the culture medium comprises soil, hydroponic medium, turface, or isolite.

214. The method according to any one of claims 206 to 213, wherein the contact comprises increasing the nitrogen content of the plant by at least 5%.

215. The method according to any one of claims 206 to 214, wherein the contact comprises increasing nitrogen fixation activity in plant tissue by at least 5%.

216. The method according to any one of claims 206 to 215, wherein the contact comprises increasing the population of nitrogen-fixing bacteria in the roots and rhizosphere of the plant by at least 5%.

217. The method according to any one of claims 206 to 216, wherein the contact causes nitrogen-fixing bacteria present in the culture medium to be recruited to the root zone of the plant.

218. The method according to any one of claims 206 to 217, wherein the contact results in an increase in plant growth of at least 10% compared to the plant and / or the culture medium not exposed to the one or more of the compounds.

219. A composition for promoting plant growth, comprising: (i) at least one microbial strain selected from Bacillus megaterium, Bacillus nereis, or Bacillus sophorae; and (ii) One or more compounds selected from the following: Phytolaccarin C, 3-cyclohexyl-6-[4-[3-(trifluoromethyl)phenyl]-1-piperazinyl]-1H-pyrimidin-2,4-dione, Arg-Thr-Ala-Arg, (2R)-2-[[4-(2,6-dipyrrolidone-1-ylpyrimidin-4-yl)piperazin-1-yl]methyl]-2,5,7,8-tetramethyl-3,4-dihydrobenzopyran-6-ol dihydrochloride, Gly-Leu-Arg-Val-Phe westiellamide, thrombin receptor activating peptide 5 (TRAP-5), tungstate, fercomin, threonyl-isoleucine, BW-A868C, Lys-Ala-Leu-Glu, N-benzyloxycarbonylglycine, Glu-Asp-Asn, Glu-Asp-Asn, Ile-Glu-His-Lys, Chaps, desdimethylcitalopram, Lys-Tyr-Thr-Ser-Ser, 3-amino-4,6-dimethyl-N- (1-Phenylacetyl)thiopheno[2,3-b]pyridine-2-carboxamide, Asn-Ala-Leu-Ala-His, Met-Asp-Arg, His-Arg-Lys-Glu, Asn-Cys-Phe, 7-hydroxylauric acid, Phe-Tyr-Lys-Arg, k-strophanthin, disopyramide, estradiol-4,9-diene-3,17-dione, HoPhe-Asp-OH, 5-methyl-6-oxo-5,6-dihydro-4h-imidazo[ 1,5-a]thieno[2,3-f][1,4]diaza-3-carboxylic acid tert-butyl ester, 2,6-naphthyldiol, zonisamide, Ser-Gln-Leu-Lys, Pro-Ala-Phe, Ala-Thr-Ile-Lys, mycophenolic acid, PC (15:0 / 18:3(6Z,9Z,12Z)), 6-[(2Z)-2-benzylheptaoxy]-3,4,5-trihydroxyoxacyclohexane-2-carboxylic acid, 7''-Deoxybonaspectin D 4''-methyl ether, Reciniferatoxin, trans-2-hepten-1-yl acetate, acetoxy-6-gingerol, dupinidine, N-1-naphthylbenzamide, oxaloacetic acid, 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphocholine, undecano-2-ene-8,10-diyne isobutyramide, enantio-Corey PG-lactone diol, one or more of their derivatives, or combinations thereof.

220. The composition of claim 219, wherein the *Bacillus soursoprothiolane* strain comprises 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; (iii) an rpoB gene sequence that is at least 95% identical to SEQ ID NO: 9; and (iv) The nifH gene sequence is at least 95% identical to SEQ ID NO:

14.

221. The composition according to claim 219 or 220, wherein the *Bacillus nereis* strain comprises 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; (iii) an rpoB gene sequence that is at least 95% identical to SEQ ID NO: 8; and (iv) The nifH gene sequence is at least 95% identical to SEQ ID NO:

13.

222. The composition according to any one of claims 219 to 221, wherein the Bacillus megaterium strain 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; and (iii) The rpoB gene sequence is at least 95% identical to SEQ ID NO:

7.

223. The composition according to any one of claims 219 to 222, wherein oxaloacetic acid has the highest abundance and enantio-Corey PG-lactone diol has the lowest abundance among oxaloacetic acid, 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphocholine, undecano-2-ene-8,10-diyne isobutyramide, and enantio-Corey PG-lactone diol.

224. The composition according to any one of claims 219 to 223, wherein the concentration of 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphocholine is 1%-2% of the concentration of oxaloacetic acid.

225. The composition according to any one of claims 219 to 224, wherein the concentration of undecano-2-ene-8,10-diyne isobutyramide is 1%-2% of the concentration of oxaloacetic acid.

226. The composition according to any one of claims 219 to 225, wherein the concentration of enantio-Corey PG-lactone diol is 0.5% to 1.5% of the concentration of oxaloacetic acid.

227. The composition according to any one of claims 219 to 226, further comprising a carrier.

228. The composition of claim 228, wherein the carrier is formulated for application to a plant and / or a culture medium in which the plant grows.

229. A composition for promoting plant growth, comprising: (i) Two or more compounds selected from the following: Phytolaccarin C, 3-cyclohexyl-6-[4-[3-(trifluoromethyl)phenyl]-1-piperazinyl]-1H-pyrimidin-2,4-dione, Arg-Thr-Ala-Arg, (2R)-2-[[4-(2,6-dipyrrolidone-1-ylpyrimidin-4-yl)piperazin-1-yl]methyl]-2,5,7,8-tetramethyl-3,4-dihydrobenzopyran-6-ol dihydrochloride, Gly-Leu-Arg-Val-Phe westiellamide, thrombin receptor activating peptide 5 (TRAP-5), tungstate, fercomin, threonyl-isoleucine, BW-A868C, Lys-Ala-Leu-Glu, N-benzyloxycarbonylglycine, Glu-Asp-Asn, Glu-Asp-Asn, Ile-Glu-His-Lys, Chaps, desdimethylcitalopram, Lys-Tyr-Thr-Ser-Ser, 3-amino-4,6-dimethyl-N- (1-Phenylacetyl)thiopheno[2,3-b]pyridine-2-carboxamide, Asn-Ala-Leu-Ala-His, Met-Asp-Arg, His-Arg-Lys-Glu, Asn-Cys-Phe, 7-hydroxylauric acid, Phe-Tyr-Lys-Arg, k-strophanthin, disopyramide, estradiol-4,9-diene-3,17-dione, HoPhe-Asp-OH, 5-methyl-6-oxo-5,6-dihydro-4h-imidazo[ 1,5-a]thieno[2,3-f][1,4]diaza-3-carboxylic acid tert-butyl ester, 2,6-naphthyldiol, zonisamide, Ser-Gln-Leu-Lys, Pro-Ala-Phe, Ala-Thr-Ile-Lys, mycophenolic acid, PC (15:0 / 18:3(6Z,9Z,12Z)), 6-[(2Z)-2-benzylheptaoxy]-3,4,5-trihydroxyoxacyclohexane-2-carboxylic acid, 7''-Deoxybonaspectin D 4''-methyl ether, Reciniferatoxin, trans-2-hepten-1-yl acetate, acetoxy-6-gingerol, dupinidine, N-1-naphthylbenzamide, oxaloacetic acid, 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphocholine, undecano-2-ene-8,10-diyne isobutyramide, enantio-Corey PG-lactone diol, one or more of their derivatives, or combinations thereof; and (ii) Carrier.

230. The composition of claim 229, wherein the carrier is formulated for application to a plant and / or a culture medium in which the plant grows.

231. The composition according to claim 229 or 230, wherein the carrier comprises a fertilizer.

232. The composition according to claim 231, wherein the fertilizer is solid.

233. The composition according to any one of claims 229 to 232, wherein the carrier is a liquid.

234. The composition according to any one of claims 219 to 233, wherein when applied to a plant or a plant growth medium, the composition is configured to promote and / or be able to promote nitrogen use efficiency.

235. The composition according to any one of claims 229 to 234, wherein oxaloacetic acid has the highest abundance and enantio-Corey PG-lactone diol has the lowest abundance among oxaloacetic acid, 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphocholine, undecano-2-ene-8,10-diyne isobutyramide, and enantio-Corey PG-lactone diol.

236. The composition according to claim 235, wherein the concentration of 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphocholine is 1%-2% of the concentration of oxaloacetic acid.

237. The composition according to claim 235, wherein the concentration of undecano-2-ene-8,10-diyne isobutyramide is 1%-2% of the concentration of oxaloacetic acid.

238. The composition according to claim 235, wherein the concentration of enantio-Corey PG-lactone diol is 0.5%-1.5% of the concentration of oxaloacetic acid.

239. The composition according to any one of claims 219 to 238, wherein the concentration of one or more compounds in the composition is at least about 1 nM.

240. The composition according to any one of claims 219 to 239, wherein the concentration of one or more compounds in the composition is at least about 0.00001% of the total dry weight of the composition.

241. The composition according to any one of claims 219 to 240, wherein the composition further comprises an excipient selected from wetting agents, spreading agents, dispersants, adhesives, dust-proofing agents, and binders.

242. The composition according to any one of claims 219 to 241, wherein the composition is configured to increase the nitrogen content of plants and / or be able to increase the nitrogen content of plants by at least 5%.

243. The composition according to any one of claims 219 to 242, wherein the composition is configured to increase nitrogen fixation activity in plant tissues and / or be able to increase nitrogen fixation activity in plant tissues by at least 5%.

244. The composition according to any one of claims 219 to 243, wherein the composition is configured to increase the population of nitrogen-fixing bacteria in the roots and rhizosphere of the plant and / or to increase the population of nitrogen-fixing bacteria in the roots and rhizosphere of the plant by at least 5%.

245. The composition according to any one of claims 219 to 244, wherein the composition is configured to cause and / or be able to cause nitrogen-fixing bacteria present in the culture medium of the plant to be recruited to the root zone of the plant.

246. The composition according to any one of claims 219 to 245, wherein the composition is configured to cause and / or be able to cause an increase in plant growth of at least 10% compared to a control.

247. A method for promoting plant growth, comprising: (a) Contacting a plant and / or a culture medium in which said plant grows with a composition comprising one or more compounds, said one or more compounds being selected from 4-(2-pyridiniazo)-N,N-dimethylaniline, LPC (18:2 / 0:0), LPE (18:2 / 0:0), 7-[3-(dimethylamino)propoxy]-6-methoxy-2-(4-methyl-1,4-diazacycloheptane-1-yl)-N-(1-methylpiperidin-4-yl)quinazolin-4-amine, 7-chloro-2-(3,4-dimethoxyphenyl)-3,5,8-trihydroxy-6-methoxy-4H-benzopyran-4-one, 1-(2,4,5-trimethoxy) 1,2-propanedione, 3-(4-hydroxy-2,3,5-trimethoxyphenyl)prop-2-enal, LPE (18:1 / 0:0), 9-((2-phosphonomethoxy)ethyl)guanine, PC (P-16:0 / 20:5(5Z,8Z,11Z,14Z,17Z)), Diellagilactone, 13-methylmyristic acid, D-limonene, one or more derivatives thereof, 3-phosphoadenosylselenic acid, 1,3-bis(4-bromophenyl)-5-phenyl-2,4-imidazolidinedione, F-amidine, LPC (0:0 / 18:3), undecanoic acid, Muzanzagenin, or combinations thereof.

248. The method of claim 247, wherein the contact improves the nitrogen use efficiency of the plant.

249. The method according to claim 247 or 248, wherein the concentration of one or more compounds in the composition is at least about 1 nM.

250. The method according to any one of claims 248 to 249, wherein the concentration of one or more compounds in the composition is at least about 0.00001% of the total dry weight of the composition.

251. The method according to any one of claims 248 to 250, wherein the contact comprises contacting the plant with the composition.

252. The method according to any one of claims 248 to 251, wherein the contact comprises contacting the plant seed with the composition.

253. The method according to any one of claims 248 to 252, wherein the contact comprises contacting the leaves of the plant with the composition.

254. The method according to any one of claims 248 to 253, wherein the culture medium comprises soil, hydroponic medium, turface, or isolite.

255. The method according to any one of claims 248 to 254, wherein the contact increases the nitrogen content of the plant by at least 5%.

256. The method according to any one of claims 248 to 255, wherein the contact increases nitrogen fixation activity in plant tissue by at least 5%.

257. The method according to any one of claims 248 to 256, wherein the contact increases the population of nitrogen-fixing bacteria in the roots and rhizosphere of the plant by at least 5%.

258. The method according to any one of claims 248 to 257, wherein the contact causes nitrogen-fixing bacteria present in the culture medium to be recruited to the root zone of the plant.

259. The method according to any one of claims 248 to 258, wherein the contact results in an increase in plant growth of at least 10% compared to the plant and / or the culture medium not exposed to the one or more of the compounds.

260. A composition for promoting plant growth, comprising: (i) at least one microbial strain selected from Bacillus megaterium, Bacillus nereis, or Bacillus sophorae; and (ii) One or more compounds selected from the following: 4-(2-pyridiniazo)-N,N-dimethylaniline, LPC (18:2 / 0:0), LPE (18:2 / 0:0), 7-[3-(dimethylamino)propoxy]-6-methoxy-2-(4-methyl-1,4-diazacycloheptane-1-yl)-N-(1-methylpiperidin-4-yl)quinazolin-4-amine, 7-chloro-2-(3,4-dimethoxyphenyl)-3,5,8-trihydroxy-6-methoxy-4H-benzopyran-4-one, 1-(2,4,5-trimethoxyphenyl)-1,2-propanedione, 3-(4-hydroxy-2,3,5-trimethoxyphenyl)prop-2-enal LPE (18:1 / 0:0), 9-((2-phosphonomethoxy)ethyl)guanine, PC (P-16:0 / 20:5(5Z,8Z,11Z,14Z,17Z)), Diellagilactone, 13-methylmyristic acid, D-limonene, 3-phosphoadenosylselenic acid, 1,3-bis(4-bromophenyl)-5-phenyl-2,4-imidazolidinedione, F-amidine, LPC (0:0 / 18:3), undecanoic acid, Muzanzagenin, oxaloacetic acid, 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphocholine, undecano-2-ene-8,10-diyne isobutyramide, enantio-Corey PG-lactone diol, one or more of their derivatives, or combinations thereof.

261. The composition according to claim 260, wherein the *Bacillus soursoprothiolane* strain comprises 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; (iii) an rpoB gene sequence that is at least 95% identical to SEQ ID NO: 9; and (iv) The nifH gene sequence is at least 95% identical to SEQ ID NO:

14.

262. The composition according to claim 260 or 261, wherein the *Bacillus nereis* strain comprises 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; (iii) an rpoB gene sequence that is at least 95% identical to SEQ ID NO: 8; and (iv) The nifH gene sequence is at least 95% identical to SEQ ID NO:

13.

263. The composition according to any one of claims 260 to 262, wherein the Bacillus megaterium strain 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; and (iii) The rpoB gene sequence is at least 95% identical to SEQ ID NO:

7.

264. The composition according to any one of claims 260 to 263, wherein oxaloacetic acid has the highest abundance and enantio-Corey PG-lactone diol has the lowest abundance among oxaloacetic acid, 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphocholine, undecano-2-ene-8,10-diyne isobutyramide, and enantio-Corey PG-lactone diol.

265. The composition according to claim 264, wherein the concentration of 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphocholine is 1%-2% of the concentration of oxaloacetic acid.

266. The composition according to claim 264, wherein the concentration of undecano-2-ene-8,10-diyne isobutyramide is 1%-2% of the concentration of oxaloacetic acid.

267. The composition according to claim 264, wherein the concentration of enantio-Corey PG-lactone diol is 0.5%-1.5% of the concentration of oxaloacetic acid.

268. The composition according to any one of claims 260 to 267, further comprising a carrier.

269. The composition of claim 268, wherein the carrier is formulated for application to a plant and / or a culture medium in which the plant grows.

270. A composition for promoting plant growth, comprising: (i) Two or more compounds selected from the following: 4-(2-pyridiniazo)-N,N-dimethylaniline, LPC (18:2 / 0:0), LPE (18:2 / 0:0), 7-[3-(dimethylamino)propoxy]-6-methoxy-2-(4-methyl-1,4-diazacycloheptane-1-yl)-N-(1-methylpiperidin-4-yl)quinazolin-4-amine, 7-chloro-2-(3,4-dimethoxyphenyl)-3,5,8-trihydroxy-6-methoxy-4H-benzopyran-4-one, 1-(2,4,5-trimethoxyphenyl)-1,2-propanedione, 3-(4- Hydroxy-2,3,5-trimethoxyphenyl)prop-2-enal, LPE (18:1 / 0:0), 9-((2-phosphonomethoxy)ethyl)guanine, PC (P-16:0 / 20:5(5Z,8Z,11Z,14Z,17Z)), Diellagilactone, 13-methylmyristic acid, D-limonene, 3-phosphoadenosylselenic acid, 1,3-bis(4-bromophenyl)-5-phenyl-2,4-imidazolidinedione, F-amidinium, LPC (0:0 / 18:3), undecanoic acid, Muzanzagenin, one or more derivatives thereof, or combinations thereof; and (ii) Carrier.

271. The composition of claim 270, wherein the carrier is formulated for application to a plant and / or a culture medium in which the plant grows.

272. The composition according to claim 270 or 271, wherein the carrier comprises a fertilizer.

273. The composition according to claim 272, wherein the fertilizer is solid.

274. The composition according to any one of claims 270 to 273, wherein the carrier is a liquid.

275. The composition according to any one of claims 260 to 274, wherein when applied to plants, the composition is configured to improve and / or be able to improve nitrogen use efficiency.

276. The composition according to any one of claims 270 to 274, wherein oxaloacetic acid has the highest abundance and enantio-Corey PG-lactone diol has the lowest abundance among oxaloacetic acid, 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphocholine, undecano-2-ene-8,10-diyne isobutyramide, and enantio-Corey PG-lactone diol.

277. The composition according to claim 276, wherein the concentration of 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphocholine is 1%-2% of the concentration of oxaloacetic acid.

278. The composition according to claim 276, wherein the concentration of undecano-2-ene-8,10-diyne isobutyramide is 1%-2% of the concentration of oxaloacetic acid.

279. The composition according to claim 276, wherein the concentration of enantio-Corey PG-lactone diol is 0.5%-1.5% of the concentration of oxaloacetic acid.

280. The composition according to any one of claims 260 to 279, wherein the concentration of one or more compounds in the composition is at least about 1 nM.

281. The composition according to any one of claims 260 to 280, wherein the concentration of the one or more compounds in the composition is at least about 0.00001%.

282. The composition according to any one of claims 260 to 281, wherein the composition further comprises an excipient selected from wetting agents, spreading agents, dispersants, adhesives, dust-proofing agents, and binders.

283. The composition according to any one of claims 260 to 282, wherein the composition is configured to increase the nitrogen content of plants and / or is capable of increasing the nitrogen content of plants by at least 5%.

284. The composition according to any one of claims 260 to 283, wherein the composition is configured to increase nitrogen fixation activity in plant tissues and / or be able to increase nitrogen fixation activity in plant tissues by at least 5%.

285. The composition according to any one of claims 260 to 284, wherein the composition is configured to increase the population of nitrogen-fixing bacteria in the roots and rhizosphere of the plant and / or to increase the population of nitrogen-fixing bacteria in the roots and rhizosphere of the plant by at least 5%.

286. The composition according to any one of claims 260 to 285, wherein the composition is configured to cause and / or enable nitrogen-fixing bacteria present in the culture medium of the plant to be recruited to the rhizosphere of the plant.

287. The composition according to any one of claims 260 to 286, wherein the composition is configured to cause and / or be able to cause an increase in plant growth of at least 10% compared to a control.

288. A method for promoting plant growth, comprising: (a) Contacting a plant and / or a culture medium in which the plant grows with a composition comprising one or more compounds, wherein the one or more compounds are selected from zearalenone, dodecyl dimethyl acetal, 1-[5-ethyl-2-hydroxy-4-[[6-methyl-6-(1H-tetrazol-5-yl)heptyl]oxy]phenyl]ethyl ketone, N-[5-(1H-indol-3-ylmethyl)-1,3,4-thiadiazol-2-yl]-4-methoxybenzamide, quintodextrin, lythidathion, chrysanthemum triol, their derivatives, or combinations thereof.

289. The method of claim 288, wherein the contact enhances phosphorus solubility in the plant growth medium.

290. The method according to claim 288 or 289, wherein the concentration of the one or more compounds in the composition is at least about 1 nM.

291. The method according to any one of claims 288 to 290, wherein the concentration of the one or more compounds in the composition is at least about 0.00001% of the total dry weight of the composition.

292. The method according to any one of claims 288 to 291, wherein the contact comprises contacting the plant with the composition.

293. The method according to any one of claims 288 to 292, wherein the contact comprises contacting the plant seed with the composition.

294. The method according to any one of claims 288 to 293, wherein the contact comprises contacting the leaves of the plant with the composition.

295. The method according to any one of claims 288 to 294, wherein the culture medium comprises soil, hydroponic medium, turface, or isolite.

296. A composition for promoting plant growth, comprising: (a) At least one microbial strain selected from one or more of the following: Levulva linkerella, Sorbus phenylacetate, Sorbus mesennii, Solitalea canadensis and Moscow nitrifying spirochete; and (b) One or more compounds selected from the following: zearalenone, dodecyl dimethyl acetal, 1-[5-ethyl-2-hydroxy-4-[[6-methyl-6-(1H-tetrazol-5-yl)heptyl]oxy]phenyl]ethyl ketone, N-[5-(1H-indol-3-ylmethyl)-1,3,4-thiadiazol-2-yl]-4-methoxybenzamide, quintopril, lythidathion, chrysanthemum triol, oxaloacetic acid, 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphate choline, undecano-2-ene-8,10-diyne isobutyramide, enantio-Corey PG-lactone diol or derivatives thereof, or combinations thereof.

297. The composition according to claim 296, wherein the concentration of one or more compounds in the composition is at least about 1 nM.

298. The composition according to claim 296 or 297, wherein the concentration of the one or more compounds in the composition is at least about 0.00001% of the total dry weight of the composition.

299. The composition according to any one of claims 296 to 298, wherein oxaloacetic acid has the highest abundance and enantio-Corey PG-lactone diol has the lowest abundance among oxaloacetic acid, 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphocholine, undecano-2-ene-8,10-diyne isobutyramide, and enantio-Corey PG-lactone diol.

300. The composition according to claim 299, wherein the concentration of 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphocholine is 1%-2% of the concentration of oxaloacetic acid.

301. The composition according to claim 299, wherein the concentration of undecano-2-ene-8,10-diyne isobutyramide is 1%-2% of the concentration of oxaloacetic acid.

302. The composition according to claim 299, wherein the concentration of enantio-Corey PG-lactone diol is 0.5%-1.5% of the concentration of oxaloacetic acid.

303. The composition according to any one of claims 296 to 302, further comprising a carrier.

304. The composition of claim 303, wherein the carrier is formulated for application to a plant and / or a culture medium in which the plant grows.

305. A composition for promoting plant growth, comprising: (a) Two or more of the following: zearalenone, dodecyl dimethyl acetal, 1-[5-ethyl-2-hydroxy-4-[[6-methyl-6-(1H-tetrazol-5-yl)heptyl]oxy]phenyl]ethyl ketone, N-[5-(1H-indol-3-ylmethyl)-1,3,4-thiadiazol-2-yl]-4-methoxybenzamide, quintodextrin, lythidathion, chrysanthemum triol, oxaloacetic acid, 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphate choline, undecyl-2-ene-8,10-diyne isobutyramide, enantio-CoreyPG-lactone diol, derivatives thereof, or combinations thereof; and (b) Carrier.

306. The composition of claim 305, wherein the carrier is formulated for application to a plant and / or a culture medium in which the plant grows.

307. The composition according to claim 305 or 306, wherein the carrier comprises a fertilizer.

308. The composition according to claim 307, wherein the fertilizer is solid.

309. The composition according to any one of claims 305 to 308, wherein the carrier is a liquid.

310. The composition according to any one of claims 296 to 309, wherein when applied to a plant or a plant growth medium, the composition is configured to and / or to promote phosphorus solubility.

311. The composition according to any one of claims 305 to 309, wherein oxaloacetic acid has the highest abundance among oxaloacetic acid, 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphate choline, undecano-2-ene-8,10-diyne isobutyramide, and enantio-Corey PG-lactone diol, and 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphate choline has the lowest abundance.

312. The composition according to claim 311, wherein the concentration of 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphocholine is 1%-2% of the concentration of oxaloacetic acid.

313. The composition according to claim 311, wherein the concentration of undecano-2-ene-8,10-diyne isobutyramide is 8%-12% of the concentration of oxaloacetic acid.

314. The composition according to claim 311, wherein the concentration of enantio-Corey PG-lactone diol is 8%-12% of the concentration of oxaloacetic acid.

315. The composition according to any one of claims 296 to 314, wherein the concentration of one or more compounds in the composition is at least about 1 nM.

316. The composition according to any one of claims 296 to 315, wherein the concentration of the one or more compounds in the composition is at least about 0.00001% of the total dry weight of the composition.

317. The composition according to any one of claims 296 to 316, wherein the composition further comprises an excipient selected from wetting agents, spreading agents, dispersants, adhesives, dust-proofing agents, and binders.

318. The composition according to any one of claims 296 to 317, wherein the composition is configured to enhance and / or enhance phosphorus solubility in plant growth media.

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