IP Library › Granted Patent US 12,365,869
Granted Patent B2
US 12,365,869 · App. 18/541,917 · Granted Jul 22, 2025

Systems for production of products to promote nitrogen use efficiency in plants

Inventors: Luis Francisco Velazquez (Grand Prairie, TX); Allana Kay Welsh (Denton, TX); Neissa Maryann Pinzon (Frisco, TX); Maud Ann Wrightson Hinchee (Little Elm, TX); Robert D Chisholm (Mckinney, TX); Mohammad Kamrul Hassan (Aubrey, TX); Leslie Michelle Perry (Fort Worth, TX); Shashi S Rajbanshi (Frisco, TX); Katherine McElhany Williams (Corinth, TX)
Assignee: TENFOLD TECHNOLOGIES, LLC
C12N1/20A01N63/22A01N63/25A01P21/00C05F11/08C12M23/58C12M27/00C12M29/18C12M47/02
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Quick Facts
Patent No.
US 12,365,869
App. No.
18/541,917
Granted
Jul 22, 2025
Kind
B2
Abstract

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

Claims (47)

1. A bioreactor system comprising:

(a) a conduit comprising a stream of an aqueous feedstock in fluid communication with a first container comprising a volume of a first working fluid and a first container outflow port in fluid communication with the first working fluid and located below the top of the first working fluid, wherein the aqueous feedstock comprises a microbial consortium, wherein the first working fluid comprises an established population of a nitrogen use efficiency-promoting microbial strain;

(b) a second container in fluid communication with the first container, wherein the second container comprises a second working fluid and a second container inflow port in fluid communication with the first container outflow port and arranged to allow hydraulically balanced continuous flow of the first working fluid from the first container outflow port to the second container inflow port driven by gravity, and wherein the second container comprises a second container outflow port located below the top of the second working fluid, and wherein the second container is configured to maintain a population of the nitrogen use efficiency-promoting microbial strain;

(c) one or more additional containers arranged in series with the first container and the second container, wherein each of the one or more additional containers comprises a volume of a working fluid and is in fluid communication with at least one other container in the series, wherein either the second container or at least one of the one or more additional containers comprises a product outflow stream port, wherein at least two of the first container, the second container, or the one or more additional containers are fluidized bed reactors, and wherein the one or more additional containers are configured to maintain a population of the nitrogen use efficiency-promoting microbial strain; and

(d) a product outflow stream in fluid communication with the product outflow stream port, wherein the product outflow stream port is configured to allow hydraulically balanced continuous flow of the product outflow stream driven by gravity.

2. The bioreactor system of claim 1 , wherein the nitrogen use efficiency-promoting microbial strain is one that performs nitrogen fixation, promotes nitrogen fixation in the tissues of plants, recruits nitrogen fixers to the root zones or other tissues of plants, or increases organic nitrogen content and/or mineralization of organic nitrogen in soil.

3. The bioreactor system of claim 1 , wherein the nitrogen use efficiency-promoting microbial strain is positive for a nifH gene.

4. The bioreactor system of claim 1 , wherein the nitrogen use efficiency-promoting microbial strain is one that promotes plant growth in a nitrogen-poor growth medium.

5. The bioreactor system of claim 1 , wherein the nitrogen use efficiency-promoting microbial strain is of the genus Kosakonia, Klebsiella, Rahnella, Kluyvera, Enterobacter, Achromobacter, Microbacterium, Gluconobacter, Methylobacterium, Pseudomonas, Pantoea, Azospirillum, Azocarus, Herbaspirillum, Burkholderia, Cyanobacteria, Bacillus , or Paenibacillus.

6. The bioreactor system of claim 1 , wherein the nitrogen use efficiency-promoting microbial strain is of the species Kosakonia sacchari, Klebsiella variicola, Rahnella aquatilis, Kluyvera intermedia, Kosakonia pseusosacchari, Enterobacter spp., Achromobacter marplatensis, Azopirillum lipoferum, Microbacterium murale, Gluconobacter diazotrophicus, Methylobacterium symbioticum, Paenibacillus borealis, Bacillus megaterium ( Priestia megaterium ), or Paenibacillus sonchi.

7. The bioreactor system of claim 1 , wherein the bioreactor system is a continuous flow bioreactor system and the stream of the aqueous feedstock is a continuous stream.

8. The bioreactor system of claim 1 , wherein each of the first container and the one or more additional containers comprises a concentration of the nitrogen use efficiency-promoting microbial strain that remains at least 1×10 2 CFU/ml during operation of the bioreactor system.

9. The bioreactor system of claim 1 , wherein the microbial consortium comprises at least 1×10 5 CFU/ml of microbes.

10. The bioreactor system of claim 1 , wherein the aqueous feedstock further comprises an organic material digestible by microbes present in the first container, the second container, and the one or more additional containers.

11. The bioreactor system of claim 10 , wherein the organic material comprises manure or material derived from manure.

12. The bioreactor system of claim 1 , wherein the aqueous feedstock further comprises rock phosphate particles.

13. The bioreactor system of claim 1 , wherein the microbial consortium comprises microbes derived from manure and rock phosphate particles.

14. The bioreactor system of claim 1 , wherein the container comprising the product outflow stream port is a clarifier container configured to separate a portion of a working fluid in the clarifier container into a supernatant portion and a floc portion.

15. The bioreactor system of claim 14 , wherein the clarifier container comprises one or more floc folding flights configured to agitate settled floc in the clarifier container without resuspending solids in the floc portion into the supernatant portion.

16. The bioreactor system of claim 14 , further comprising a floc return stream that flows from the clarifier to an earlier container in the series.

17. The bioreactor system of claim 14 , wherein the product outflow stream comprises the supernatant portion.

18. The bioreactor system of claim 17 , wherein the product outflow stream comprises at least 1×10 4 CFU/ml of the nitrogen use efficiency-promoting microbial strain.

19. The bioreactor system of claim 17 , wherein the product outflow stream comprises at least 1×10 2 CFU/ml of a sporulated form of the nitrogen use efficiency-promoting microbial strain.

20. The bioreactor system of claim 17 , wherein the product outflow stream comprises a total dry weight of 0.2 to 2.5 mg/ml.

21. The bioreactor system of claim 17 , wherein the product outflow stream has a chemical oxygen demand of 80 to 500 mg/L.

22. The bioreactor system of claim 17 , wherein the product outflow stream has an electrical conductivity between 1.3 and 3.0 mS/cm.

23. The bioreactor system of claim 1 , wherein the first working fluid comprises malate at a concentration of at least 0.2% w/v.

24. The bioreactor system of claim 1 , wherein the first working fluid comprises soy flour at a concentration of at least 0.2% w/v.

25. The bioreactor system of claim 1 , wherein the first working fluid comprises microaerobic conditions.

26. The bioreactor system of claim 25 , wherein the working fluid in at least one of the one or more additional containers comprises microaerobic conditions.

27. The bioreactor system of claim 1 , wherein the system has a hydraulic retention time of at least 5 days.

28. The bioreactor system of claim 1 , wherein the first working fluid further comprises an established population of a second nitrogen use efficiency-promoting microbial strain.

29. The bioreactor system of claim 28 , wherein the second nitrogen use efficiency-promoting microbial strain is of the species Kosakonia sacchari, Klebsiella variicola, Rahnella aquatilis, Kluyvera intermedia, Kosakonia pseusosacchari, Enterobacter spp., Achromobacter marplatensis, Azopirillum lipoferum, Microbacterium murale, Gluconobacter diazotrophicus, Methylobacterium symbioticum, Paenibacillus borealis, Bacillus megaterium ( Priestia megaterium ), or Paenibacillus sonchi.

30. The bioreactor system of claim 1 , wherein the first working fluid comprises a total population of microbes positive for a nifH gene of at least 1×10 5 CFU/ml.

31. The bioreactor system of claim 1 , wherein the fluidized bed reactors are configured to maintain a higher concentration of the nitrogen use efficiency-promoting microbial strain in the bioreactor system as compared to packed bed reactors.

32. The bioreactor system of claim 1 , wherein the second container is configured to maintain a concentration of the nitrogen use efficiency-promoting microbial strain at at least the concentration of the nitrogen use efficiency-promoting microbial strain in the first working fluid in the first container.

33. The bioreactor system of claim 1 , wherein the fluidized bed reactors comprise particles of an inorganic substrate.

34. The bioreactor system of claim 33 , wherein the particles of the inorganic substrate are rock phosphate particles.

35. The bioreactor system of claim 28 , wherein the nitrogen use efficiency-promoting microbial strain is of the genus Paenibacillus , and the second nitrogen use efficiency-promoting microbial strain is of the genus Bacillus.

36. The bioreactor system of claim 35 , wherein the nitrogen use efficiency-promoting microbial strain is of the species Paenibacillus borealis and the second nitrogen use efficiency-promoting microbial strain is of the species Bacillus megaterium.

37. The bioreactor system of claim 36 , wherein the Paenibacillus borealis comprises the Paenibacillus borealis strain deposited under ATCC Accession No. PTA-127654 and the Bacillus megaterium comprises the Bacillus megaterium strain deposited under ATCC Accession No. PTA-127653.

38. The bioreactor system of claim 37 , wherein the first working fluid further comprises a Paenibacillus sonchi strain deposited under ATCC Accession No. PTA-127655.

39. The bioreactor system of claim 17 , wherein the product outflow stream comprises at least 1×10 2 CFU/ml of the nitrogen use efficiency-promoting microbial strain.

40. The bioreactor system of claim 1 , wherein the second container is configured to maintain the population of the nitrogen use efficiency-promoting microbial strain for at least 5 days.

41. The bioreactor system of claim 40 , wherein the one or more additional containers are configured to maintain the population of the nitrogen use efficiency-promoting microbial strain for at least 5 days.

42. The bioreactor system of claim 1 , wherein the second container is configured to maintain the population of the nitrogen use efficiency-promoting microbial strain for at least 2 months.

43. The bioreactor system of claim 42 , wherein the one or more additional containers are configured to maintain the population of the nitrogen use efficiency-promoting microbial strain for at least 2 months.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Apr 2, 2024
From: VELAZQUEZ, LUIS FRANCISCO; WELSH, ALLANA KAY; PINZON, NEISSA MARYANN; HINCHEE, MAUD ANN WRIGHTSON; CHISHOLM, ROBERT D.; HASSAN, MOHAMMAD KAMRUL; PERRY, LESLIE MICHELLE; RAJBANSHI, SHASHI S.; MCELHANY WILLIAMS, KATHERINE
To: TENFOLD TECHNOLOGIES, LLC
Reel/Frame 066978/0799 →
Continuity (4)
Provisional Application 63610535 · Dec 15, 2023
Provisional Application 63510615 · Jun 27, 2023
Provisional Application 63509263 · Jun 20, 2023
Related Publication 20240425789A1 · Dec 26, 2024
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