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(12) Patent Application Publication (10) Pub. No.: US 2016/0073641 A1 Allen Et Al US 2016.007 3641A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0073641 A1 Allen et al. (43) Pub. Date: Mar. 17, 2016 (54) MICROBAL INOCULANT FORMULATIONS Publication Classification (71) Applicant: Newleaf Symbiotics, Inc., St. Louis, (51) Int. Cl. MO (US) AOIN 63/02 (2006.01) C05F II/08 (2006.01) (72) Inventors: Kimberly Allen, Ballwin, MO (US); CI2N I/04 (2006.01) Gregg Bogosian, Clarkson Valley, MO (52) U.S. Cl. (US) CPC A0IN 63/02 (2013.01); C12N I/04 (2013.01); C05F II/08 (2013.01) (21) Appl. No.: 14/856,020 (57) ABSTRACT Compositions comprising dried formulations of viable (22) Filed: Sep. 16, 2015 Methylobacterium as well as methods for making and using the formulations are provided. In particular the compositions Related U.S. Application Data provide for Methylobacterium formulations with improved (60) Provisional application No. 62/051,028, filed on Sep. viability, shelf-life, and plant or seed treatment characteris 16, 2014. tics. Patent Application Publication Mar. 17, 2016 Sheet 1 of 5 US 2016/0073641 A1 c: s was s & s's s --. 8. Patent Application Publication Mar. 17, 2016 Sheet 2 of 5 US 2016/0073641 A1 · Patent Application Publication Mar. 17, 2016 Sheet 3 of 5 US 2016/0073641 A1 Patent Application Publication Mar. 17, 2016 Sheet 4 of 5 US 2016/0073641 A1 & : : 3. Patent Application Publication Mar. 17, 2016 Sheet 5 of 5 US 2016/0073641 A1 O c C p CD s res O . O 2 US 2016/007 3641 A1 Mar. 17, 2016 MICROBAL INOCULANT FORMULATIONS growth, plant yield, seed germination, male fertility, and plant nutritional qualities has been disclosed in U.S. Pat. No. 5,512. CROSS-REFERENCE TO RELATED 069, U.S. Pat. No. 5,961,687, U.S. Pat. No. 6,174,837, U.S. APPLICATIONS Pat. No. 6,329,320, U.S. Pat. No. 7,435,878, and US Patent Application Pub. No. 2006/0228797. In addition, PPFM bac 0001. This U.S. Non-provisional patent application teria have been found to increase the yield of cultivated algae, claims the benefit of the U.S. Provisional Patent Application Suggesting their application to the production of algae-de No. 62/051,028, filed on Sep. 16, 2014 and incorporated rived biofuels (US Patent Application Pub. No. 2011/ herein by reference in its entirety. 0269219). BACKGROUND SUMMARY 0002 One-carbon organic compounds such as methane 0005 Provided herein are methods for efficient produc and methanol are found extensively in nature, and are utilized tion of large quantities of Methylobacterium. These methods as carbon Sources by bacteria classified as methanotrophs and can result in high titer Methylobacterium cultures where pro methylotrophs. Methanotrophic bacteria include species in duction time per batch is significantly reduced. The methods the genera Methylobacter, Methylomonas, Methylomicro of Methylobacterium production provided herein can also use bium, Methylococcus, Methylosinus, Methylocystis, Methy culture medium comprised of inexpensive and readily avail losphaera, Methylocaldum, and Methylocella (Lidstrom, able components. Also provide herein are useful fermentation 2006). Methanotrophs possess the enzyme methane broths, fermentation broth products, fermentation products, monooxygenase, that incorporates an atom of oxygen from and compositions comprising Methylobacterium. Methods of O into methane, forming methanol. All methanotrophs are using the fermentation broths, fermentation broth products, obligate one-carbon utilizers, unable to use compounds con fermentation products, and compositions comprising Methy taining carbon-carbon bonds. Methylotrophs, on the other lobacterium to treat plants or plant parts are also provided hand, can also utilize more complex organic compounds, herein. The methods and compositions provided herein can Such as organic acids, higher alcohols, Sugars, and the like. be used to produce large quantities of Methylobacterium for Thus, methylotrophic bacteria are facultative methylotrophs. application to plants or plant parts, for use as an inoculum in Methylotrophic bacteria include species in the genera Methy bioremediation, for production of useful products, and for lobacterium, Hyphomicrobium, Methylophilus, Methyloba production of recombinant proteins. Useful products obtain cillus, Methylophaga, Aminobacter, Methylorhabdus, Methy able by the methods and compositions provided herein lopilla, Methylosulfonomonas, Marinosulfonomonas, include, but are not limited to, poly-3-hydroxybutyric acid, Paracoccus, Xanthobacter, Ancylobacter (also known as 1,3-propanediol, and oxazopyrroloquinolines. Microcyclus). Thiobacillus, Rhodopseudomonas, Rhodo 0006 Provided herein are fermentation broths comprising bacter, Acetobacter, Bacillus, Mycobacterium, Arthobacter, a liquid phase and a solid phase that can be suspended therein, and Nocardia (Lidstrom, 2006). wherein the Solid phase comprises a Solid Substance wherein 0003 Most methylotrophic bacteria of the genus Methy a mono-culture or co-culture of Methylobacterium is adhered lobacterium are pink-pigmented. They are conventionally thereto and wherein the fermentation broth is essentially free referred to as PPFM bacteria, being pink-pigmented faculta of contaminating microorganisms. In certain embodiments, tive methylotrophs. Green (2005, 2006) identified twelve the broth can further comprise one or more microorganisms validated species in the genus Methylobacterium, specifically of pre-determined identity other than Methylobacterium. In M. aminovorans, M. chloromethanicum, M. dichlo certain embodiments, the Solid phase comprises at least about romethanicum, M. extorquens, M. filjisawaense, M. mesophi 0.02% to about 0.5% of the broth by mass. In certain embodi licum, M. Organophilum, M. radiotolerans, M. rhodesianum, ments, the Solid Substance is of animal, plant, microbial, M. rhodinum, M. thiocyanatum, and M. zatmanii. However, fungal, or mineral origin. In certain embodiments, the Solid M. nidulans is a nitrogen-fixing Methylobacterium that is not Substance is an agriculturally acceptable adjuvant or agricul a PPFM (Sy et al., 2001). Methylobacterium are ubiquitous in turally acceptable excipient. In certain embodiments, the nature, being found in soil, dust, fresh water, sediments, and Solid Substance is a polymer. In certain embodiments, the leaf Surfaces, as well as in industrial and clinical environ Solid Substance comprises a polysaccharide, diatomaceous ments (Green, 2006). earth, or a salt crystal. In certain embodiments, the polysac 0004. The existence of PPFM bacteria as colonizers of the charide is selected from the group consisting of a cellulosic leaf Surfaces of most (if not all) species of plants (ranging polysaccharide, a chitinous polysaccharide, and a galactan from algae, mosses and liverworts, and angiosperms and polysaccharide. In certain embodiments, the Methylobacte gymnosperms) suggests that PPFM bacteria may play an rium are at atiter of at least about 5x10 colony-forming units important role in plant physiology (Corpe and Rheem, 1989; per milliliter, at least about 1x10 colony-forming units per Holland and Polacco, 1994; Holland, 1997; Kutschera, milliliter, at least about 1x10" colony-forming units per mil 2007). The fact that plants produce and excrete methanol, liliter, or at least about 3x10" colony-forming units per mil probably as a waste product of pectin metabolism in growing liliter. In certain embodiments, the Methylobacterium are at a plant cell walls, Suggested to these researchers that a symbi titerofat least about 5x10 colony-forming units permilliliter otic relationship exists, with the PPFM bacteria feeding on to at least about 6x10' colony-forming units per milliliter. In the plant-produced methanol and in turn providing positive certain embodiments, at least one of the Methylobacterium is benefits to the plants. The suggested benefits of PPFM bac a Pink Pigmented Facultative Methylotroph (PPFM). In cer teria on plant physiology include positive effects on nitrogen tain embodiments, the Pink Pigmented Facultative Methy metabolism, seed germination, and stimulation of plant lotroph (PPFM) is selected from the group consisting of M. growth through the provision of PPFM-generated cytokinin aminovorans, M. chloromethanicum, M. dichloromethani plant hormones. The use of PPFM bacteria to improve plant cum, M. extorquens, M. fijisawaense, M. mesophilicum, M. US 2016/007 3641 A1 Mar. 17, 2016 Organophilum, M. radiotolerans, M. rhodesianum, M. rhodi growth of the Methylobacterium and wherein the media is num, M. thiocyancilium, M. cerastii, M. gossipicola, Methy essentially free of contaminating microorganisms. In certain lobacterium sp. strain LMG6378, M. phyllosphaerae, M. embodiments, the media further comprises one or more Olyzae, M. platani, M. populi, and M. zatmanii. In certain microorganisms of pre-determined identity other than Methy embodiments, at least one of the Methylobacterium is M. lobacterium. In certain embodiments, the Solid phase com nodulans. In certain embodiments of any of the aforemen prises at least about 0.02% to about 0.5% of the media by tioned broths, at least 10% of the Methylobacterium in the mass. In certain embodiments, the Solid Substance is an agri fermentation broth are Methylobacterium that are adhered to culturally acceptable adjuvant or agriculturally acceptable the solid substance. In certain embodiments of any of the aforementioned broths, the solid is not a photosynthetic excipient. In certain embodiments, the solid Substance pro microorganism. vides for adherent growth of the Methylobacterium. In certain embodiments, the Solid
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