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US-20260123633-A1 - METHANOTROPH STRAINS FOR MITIGATING METHANE AND METHODS RELATED THERETO

US20260123633A1US 20260123633 A1US20260123633 A1US 20260123633A1US-20260123633-A1

Abstract

Methanotroph strains that enhance early growth of plants, improve propagation/transplant vigor, increase nutrient uptake, improve stand establishment, improve stress tolerance and/or increase a plant's ability to utilize nutrients are provided herein. Uses of compostions comprising such strains and optionally methylotroph strains, for methane mitigation and crop improvement is provided. Also provided are methods to reduce green-house gas emission and convert methane to methanol with methanotroph strains.

Inventors

  • Natalie Breakfield
  • Desmond R. Jimenez
  • David Flack
  • Anthony Neumann

Assignees

  • NEWLEAF SYMBIOTICS, INC.

Dates

Publication Date
20260507
Application Date
20230712

Claims (20)

  1. 1 . A method for mitigating methane gas in an agricultural field that comprises: (a) applying a composition to a soil, field, plant, plant part or seed, wherein the composition comprises at least one methanotroph selected from the group consisting of a Type II methanotroph comprising a pMMO2 protein, and Type I methanotroph, (b) growing the methanotroph whereby the methanotroph uses methane as a carbon source; wherein use of the methane as the carbon source oxidizes methane or reduces methane emissions.
  2. 2 .- 3 . (canceled)
  3. 4 . The method of claim 1 , wherein the Type II methanotroph is Methylocystis hirsuta isolate comprising PmoA2, PmoB2 and PmoC2 proteins with sequences having at least 97% identity to SEQ ID NOS:76-78.
  4. 5 . The method of claim 1 , wherein the Type II methanotroph is Methylocystis hirsuta isolate selected from the group consisting of NLS1505 (NRRL B-68282), NLS1506 (NRRL B-68283), NLS1508 (NRRL B-68262), NLS1509 (NRRL B-68284), NLS1511 (NRRL B-68285), NLS1512 (NRRL B-68286), or a variant thereof.
  5. 6 . (canceled)
  6. 7 . The method of claim 1 , wherein said Type I methanotroph comprises NLS1501 (NRRL B-68261), NLS1504 (NRRL B-68281), or a variant thereof.
  7. 8 .- 11 . (canceled)
  8. 12 . A composition comprising a fermentation product comprising a methanotroph strain, wherein said fermentation product is essentially free of contaminating microorganisms, and wherein methanotroph strain comprises NLS1501 (NRRL B-68261), NLS1504 (NRRL B-68281), NLS1505 (NRRL B-68282), NLS1506 (NRRL B-68283), NLS1508 (NRRL B-68262), NLS1509 (NRRL B-68284), NLS1511 (NRRL B-68285), NLS1512 (NRRL B-68286), or a variant thereof.
  9. 13 . (canceled)
  10. 14 . The composition of claim 12 , wherein said composition further comprises a methylotroph.
  11. 15 . The composition of claim 14 , wherein said methylotroph is a Methylobacterium strain.
  12. 16 . The composition of claim 15 , wherein the Methylobacterium comprises LGP2000 (NRRL B-50929), LGP2001 (NRRL B-50930), LGP2002 (NRRL B-50931), LGP2003 (NRRL B-50932), LGP2004 (NRRL B-50933), LGP2005 (NRRL B-50934), LGP2006 (NRRL B-50935), LGP2007 (NRRL B-50936), LGP2008 (NRRL B-50937), LGP2009 (NRRL B-50938), LGP2010 (NRRL B-50939), LGP2011 (NRRL B-50940), LGP2012 (NRRL B-50941), LGP2013 (NRRL B-50942), LGP2014 (NRRL B-67339), LGP2015 (NRRL B-67340), LGP2016 (NRRL B-67341), LGP2017 (NRRL B-67741), LGP2018 (NRRL B-67742), LGP2019 (NRRL B-67743), NLS0497 (NRRL B-67925), NLS0693 (NRRL B-67926), NLS1179 (NRRL B-67929), LGP2167 (NRRL B-67927), LGP2020 (NRRL-B-67892), LGP2021 (NRRL-B-68032), LGP2022 (NRRL-B-68033), LGP2023 (NRRL-B-68034), LGP2028 (NRRL B-68064), LGP2029 (NRRL B-68065), LGP2030 (NRRL B-68066), LGP2031 (NRRL B-68067), LGP2033 (NRRL B-68068), LGP2034 (NRRL B-68069), NLS0665 (NRRL B-68194), NLS0754 (NRRL B-68197), NLS0672 (NRRL B-68196), NLS0729 (NRRL B-68195), NLS0049 (NRRL-B-68236), NLS0591 (NRRL-B-68215), NLS0439 (NRRL-B-68216), NLS1310 (NRRL-B-68217), NLS1312 (NRRL-B-68218), NLS0612 (NRRL-B-68237), NLS0706 (NRRL B-68238), NLS0725 (NRRL-B-68239), NLS0770 (NRRL B-68075), NLS0737 (NRRL B-68074), NLS5278 (NRRL-B-68186), NLS5334 (NRRL-B-68187), NLS5480 (NRRL-B-68188), or NLS5549 (NRRL-B-68189), NLS7725 or a variant thereof, or a combination thereof.
  13. 17 . The composition of claim 15 , wherein the Methylobacterium comprises LGP2002 (NRRL B-50931), LGP2003 (NRRL B-50932), LGP2004 (NRRL B-50933), LGP2009 (NRRL B-50938), LGP2015 (NRRL B-67340), LGP2016 (NRRL B-67341), LGP2017 (NRRL B-67741), LGP2019 (NRRL B-67743), NLS0693 (NRRL B-67926), or LGP2020 (NRRL-B-67892), NLS7725 or a variant thereof, or a combination thereof.
  14. 18 . A plant, plant part or seed at least partially coated with the composition of claim 12 .
  15. 19 . A method for mitigating methanol that comprises: (a) treating a rice field with a composition comprising at least one methanotroph selected from the group consisting of a Type II methanotroph comprising a pMMO2 protein, and Type I methanotroph, wherein said Type I methanotroph is a Methylomicrobium sp. or a Methylosarcina sp.; and (b) growing the methanotroph in the field thereby mitigating methane.
  16. 20 . The method of claim 19 , wherein said composition comprises a methanotroph isolate comprising NLS1501 (NRRL B-68261), NLS1504 (NRRL B-68281), NLS1505 (NRRL B-68282), NLS1506 (NRRL B-68283), NLS1508 (NRRL B-68262), NLS1509 (NRRL B-68284), NLS1511 (NRRL B-68285), NLS1512 (NRRL B-68286), or a combination thereof.
  17. 21 . (canceled)
  18. 22 . An isolated methanotroph selected from NLS1501 (NRRL B-68261), NLS1504 (NRRL B-68281), NLS1505 (NRRL B-68282), NLS1506 (NRRL B-68283), NLS1508 (NRRL B-68262), NLS1509 (NRRL B-68284), NLS1511 (NRRL B-68285), NLS1512 (NRRL B-68286).
  19. 23 . A method for selecting a methanotroph isolate capable of utilizing methane as a food source, wherein said method comprises: a) detecting in the genome of a methanotroph isolate, one or more genetic elements, wherein said genetic element comprises a particulate methane monooxygenase; and b) treating a field, water, plant, plant part or seed with said methanotroph isolate, and measuring green-house gas emissions.
  20. 24 .- 49 . (canceled)

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of U.S. Provisional Patent Application No. 63/338,472, filed Jul. 12, 2022; U.S. Provisional Patent Application No. 63/383,405, filed Nov. 11, 2022; and U.S. Provisional Patent Application No. 63/489,104, filed Mar. 8, 2023; the entire disclosure of which are incorporated herein by reference. INCORPORATION OF SEQUENCE LISTING XML A computer readable form of the Sequence Listing XML containing the file named “NLSYM7007.WO Sequence Listing.xml,” which is 349,090 bytes in size (as measured in MICROSOFT WINDOWS® EXPLORER) and was created on Jul. 11, 2023, is provided herein and is herein incorporated by reference. This Sequence Listing consists of SEQ ID NOs: 1-128. BACKGROUND Concentrations of atmospheric methane are rapidly increasing, and sources of methane include agricultural sources, such as rice paddies and ruminants, wetlands, landfills, waste facilities, animal feed, and water. Biologic methods to reduce or mitigate methane from such sources are desired. In addition, methods of enhancing plant production by improving growth and/or increasing nutrient utilization by biologic methods are desired. One-carbon organic compounds such as methane and methanol are found extensively in nature, and may be utilized as carbon sources by a diverse group of bacteria. Methanotrophs possess the enzyme methane monooxygenase (MMO) which incorporates an atom of oxygen from O2 into methane, forming methanol. There are two forms of MMO, a soluble methane monooxygenase (sMMO), and a particulate methane monooxygenase (pMMO). Most known methanotrophs possess pMMO, and sMMO is also present in some methanotrophs. Methanotrophs are classified into three groups, Type I, Type II and Type X on the basis of various physiological and morphological differences. Type I and Type X methanotrophs are gammaproteobacteria, while Type II methanotrophs are alphaproteobacterial. Some methanotrophs have been reported to contain two distinct isozymes of particulate methane monooxygenase (pMMO). pMMO1 facilitates oxidation of methane under conditions where methane is at high concentrations, and pMMO2 facilitates oxidation of methane in environments where the methane concentration is low, including oxidation of atmospheric methane. pMMO consists of three protein subunits, PmoA, PmoB and PmoC, which are encoded on an operon present in the methanotroph genome. Methylotrophs, as defined herein, can utilize more complex organic compounds, such as organic acids, higher alcohols, sugars, and the like. Thus, methylotrophic bacteria can be facultative methylotrophs. Some methylotrophic bacteria in the Methylobacterium or Methylorubrum genera are pink-pigmented. They are conventionally referred to as PPFM bacteria, being pink-pigmented facultative methylotrophs. All nations have tried to frame a global regime to control green-house gas emissions and to assist with adaptation and yet emissions have continued to increase. Methane is a critical component of Earth's carbon cycle and contributes to global warming. Agriculture (e.g., enteric fermentation in livestock, manure management, and rice cultivation) is a contributor to global CH4 emission. Implementation of a biological methane oxidizing technology has the potential for mitigation of atmospheric methane levels and reduction of green-house gas emissions. SUMMARY Provided herein are strains of methanotrophic bacteria and compositions comprising one or more methanotroph strains, wherein the strains are capable of using methane as a carbon source for growth. Also provided are methods that utilize such strains for mitigating methane production, for example in agricultural applications, including plant production in flooded fields, for reducing methane produced in animal production, such as cattle or dairy industries, or for reducing natural methane sources that exist in wetlands or other natural water sources, (including but not limited to lakes, rivers, mangroves, marshes, bogs and streams), in geological sources, or in gases produced as the result of wildfires, wild animals, or insects. In some embodiments provided herein methanotrophs are applied to rice plants resulting in decreased levels of methane and enhanced plant growth. In some embodiments, methanotroph strains in the methods and compositions provided herein are species from a bacterial genus selected from the group consisting of Methyloacidimicrobium, Methyloacidiplilum, Methylobacter, Methylocaldum, Methylocapsa, Methylocella, Methylococcus, Methylocystis, Methyloferula, Methylogaea, Methyloglobus, Methylohalobius, Methylomagnum, Methylomarinum, Methylomicrobium, Methylomonas, Methyloparacoccus, Methyloperedens, Methyloprofundus, Methylosarcina, Methylosinus, Methylosoma, Methylosphaera, Methylothermus, and Methylovulum. In some embodiments, a methanotroph provided herein is a Methylocystis species selected from M. hirsuta, M. rosea and M. parvus. In some embodimen