WO2025240938A2 - Production de consortiums microbiens - Google Patents

Production de consortiums microbiens

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Publication number
WO2025240938A2
WO2025240938A2 PCT/US2025/029874 US2025029874W WO2025240938A2 WO 2025240938 A2 WO2025240938 A2 WO 2025240938A2 US 2025029874 W US2025029874 W US 2025029874W WO 2025240938 A2 WO2025240938 A2 WO 2025240938A2
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WO
WIPO (PCT)
Prior art keywords
bacteria
different
grown
bacterial
bacterial production
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/029874
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English (en)
Other versions
WO2025240938A3 (fr
Inventor
James Gregory HOLLOWAY
Julia Christine BLATEZORE
Anna Elisabet EDLUND
Alex S. RAJANGAM
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yahoo Inc
Original Assignee
Oath Inc
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Filing date
Publication date
Application filed by Oath Inc filed Critical Oath Inc
Publication of WO2025240938A2 publication Critical patent/WO2025240938A2/fr
Publication of WO2025240938A3 publication Critical patent/WO2025240938A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms

Definitions

  • microbial consortia where the consortia comprise at least 1 bacterium grown on solid media.
  • methods for producing a microbial consortium comprising at least 2 different bacteria by growing the at least 2 different bacteria on separate solid media, harvesting the bacteria from the media, and combining the harvested bacteria, optionally with other microbes such as one or more fungi, to produce a microbial consortium.
  • a method comprises growing at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, or 35 and/or not more than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150, or 200, such as 2-200, preferably 3-200, more preferably 4-200, yet more preferably 2-150, still more preferably 2-100, yet still more preferably 5-100, such as 5-80, or 5-70, or 5-50 different bacteria on separate solid media under suitable conditions for growth of the respective bacteria.
  • at least 2 different bacteria are grown on a solid medium, then removed from the medium, however, for convenience, methods will be described in terms of growing different bacteria on separate solid media.
  • the term “different microbe,” e g., “different bacterium” includes a microbe, e.g., bacterium that is taxonomically different from another microbe, e.g., bacterium, such as different class, order, family, genus, species, or strain.
  • the term “different microbes,” e g., “different bacteria” includes two or more microbes, e.g two or more bacteria, each of which is taxonomically different from the other microbes, e.g., different class, order, family, genus, species, or strain.
  • viable units e g., viable bacterial cells and/or viable bacterial spores
  • methods and compositions disclosed herein are used to grow viable units, e g., viable bacterial cells and/or viable bacterial spores, in sufficient quantity and state to be used in dry mixtures, and not for obtaining one or more products from the bacteria, though one or more products may be produced during growth or other phases, they are not the object of the process, or for their effect on the substrate on which they are grown.
  • “Grown on solid media,” or similar terms, as used herein includes growth of bacteria on solid media that are then harvested for use in one or more bacterial consortia from the solid media; that is, growth on solid media is the final step in growing the bacteria.
  • a “bacterial consortium,” as that term is used herein includes a group of at least two different bacteria that are used together.
  • a “microbial consortium,” as that term is used herein includes a bacterial consortium, or a bacterial consortium that is combined with at least one other non-bacterial microbe,
  • any suitable bacteria may be used.
  • the bacteria comprise Streptomyces such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15 or 20 different Streptomyces.
  • any suitable Streptomyces bacteria may be used.
  • the bacteria may be of one or more Streptomyces species and/or one or more Streptomyces strains.
  • bacteria are bacteria of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30, 35, 40, 50, or 60 and/or not more than 3,
  • non-Slreplomyces bacteria may be used. Any suitable non-Slreplomyces bacteria may be used, depending on the intended use of the microbial consortium produced. In certain embodiments, at least one Streptomyces bacterium is used in combination with non-Slreplomyces bacteria comprising bacteria of at least 1, 2, 3, 4, 5, 6, 7, 8,
  • the non-Streptomyces bacteria comprises bacteria of one or more genera comprising Pseudomonas, Microvirga, Paenibacillus, Rhodococcus, Lysinibacillis, Actinoplanes, Agromyces, Allosphingosinicella, Aneurinibacilhis, Arthrobacter, Bacillus, Brevibacillus, Caldimonas, Cupriavidus, Erythrobacter, Kocuria, Microbacterium, Microbispora, Mycetocola A, Neorhizobium, Nocardia, Nocardioides, Nonomuraea, Peribacillus, Priestia, Pristimantibacillus, Stenotrophomonas, Stutzerimonas, Terrimonas, and
  • the in one or more non-Streptomyces bacteria comprise one or more Pseudomonas bacteria, e.g., one or more Pseudomonas bacteria of ATCC Deposit No. PTA-127849; additionally or alternatively, in certain embodiments the one or more non-Streptomyces bacteria comprise one or more Microvirga bacteria e.g., one or more Microvirga bacteria of ATCC Deposit No. PTA-127848; additionally or alternatively, in certain embodiments the one or more non-Streptomyces bacteria comprise one or more Paenibacillus bacteria, e.g., one or more Paenibacillus bacteria of ATCC Deposit No.
  • the one or more non-Streptomyces bacteria comprise one or more Rhodococcus bacteria, e.g., one or more Rhodococcus bacteria of ATCC Deposit No. PTA-127834; additionally or alternatively, in certain embodiments the one or more non-Streptomyces bacteria comprise one or more Lysinibacillus bacteria, e.g., one or more Lysinibacdlus bacteria of ATCC Deposit No. PTA- 127847; additionally or alternatively, in certain embodiments the one or more non-Streptomyces bacteria comprise one or more Actinoplanes bacteria, e g., one or more Actinoplanes bacteria of ATCC Deposit No.
  • the one or more non-Streptomyces bacteria comprise one or more Agromyces bacteria; additionally or alternatively, in certain embodiments the one or more non-Streptomyces bacteria comprise one or more Allosphingosinicella bacteria, e.g., one or more Allosphingosinicella bacteria of ATCC Deposit No. PTA-127850; additionally or alternatively, in certain embodiments the one or more non- Streptomyces bacteria comprise one or more Aneurinibacilhis bacteria, e.g., one or more Aneurinibacilhis bacteria of ATCC Deposit No.
  • the one or more non-Streptomyces bacteria comprise one or more Arthrobacter bacteria, e g., one or more Arthrobacter bacteria of ATCC Deposit No. PTA- 127834; additionally or alternatively, in certain embodiments the one or more non-Streptomyces bacteria comprise one or more Bacillus bacteria, e.g., one or more Bacillus bacteria of ATCC Deposit No. PTA-127847 or PTA-127848; additionally or alternatively, in certain embodiments the one or more non-Streptomyces bacteria comprise one or more Brevibacillus bacteria, e.g., one or more Brevibacillus bacteria of ATCC Deposit No.
  • the one or more non-Streptomyces bacteria comprise one or more Caldimonas bacteria, e.g., one or more Caldimonas bacteria of ATCC Deposit No. PTA-127850 ; additionally or alternatively, in certain embodiments the one or more non-Streptomyces bacteria comprise one or more Cupriavidus bacteria, e.g., one or more Cupriavidus bacteria of ATCC Deposit No. PTA-127850 ; additionally or alternatively, in certain embodiments the one or more non-Streptomyces bacteria comprise one or more Erythrobacter bacteria, e.g., one or more Erythrobacter bacteria of ATCC Deposit No.
  • the one or more non-Streptomyces bacteria comprise one or more Kocuria bacteria, e.g., one or more Kocuria bacteria of ATCC Deposit No. PTA-127849 ; additionally or alternatively, in certain embodiments the one or more non-Streptomyces bacteria comprise one or mor Microbacterium bacteria, e.g., one or more Microbacterium bacteria of ATCC Deposit No. PTA-127834; additionally or alternatively, in certain embodiments the one or more non- Streptomyces bacteria comprise one or more Microbispora bacteria, e.g., one or more Microbispora bacteria of ATCC Deposit No.
  • the one or more non-Streptomyces bacteria comprise one or more Mycetocola bacteria, e.g., one or more Mycetocola bacteria of ATCC Deposit No. PTA-127849; additionally or alternatively, in certain embodiments the one or more non-Streptomyces bacteria comprise one or more Neorhizobium bacteria, e.g., one or more Neorhizobium bacteria of ATCC Deposit No. PTA-127850; additionally or alternatively, in certain embodiments the one or more non-Streptomyces bacteria comprise one or more Nocardia bacteria, e.g., one or more Nocardia bacteria of ATCC Deposit No.
  • the one or more non-Streptomyces bacteria comprise one or more Nocardioides bacteria, e.g., one or more Nocardioides bacteria of ATCC Deposit No. PTA-127849; additionally or alternatively, in certain embodiments the one or more non-Streptomyces bacteria comprise one or more Nonomuraea bacteria, e.g., one or more Nonomuraea bacteria of ATCC Deposit No. PTA-127849; additionally or alternatively, in certain embodiments the one or more non-Streptomyces bacteria comprise one or more Peribacillus bacteria, e.g., one or more Peribacillus bacteria of ATCC Deposit No.
  • the one or more non-Streptomyces bacteria comprise one or more Priestia bacteria, e.g., one or more Priestia bacteria of ATCC Deposit No. PTA-127850; additionally or alternatively, in certain embodiments the one or more non-Streptomyces bacteria comprise one or more Pristimantibacillus bacteria, e.g., one or more Pristimantibacillus bacteria of ATCC Deposit No. PTA-127848; additionally or alternatively, in certain embodiments the one or more non-Streptomyces bacteria comprise one or more Stenotrophomonas bacteria, e.g., one or more Stenotrophomonas bacteria of ATCC Deposit No.
  • the one or more non-Streptomyces bacteria comprise one or more Stutzerimonas bacteria, e.g., one or more Stutzerimonas bacteria of ATCC Deposit No. PTA-127850; additionally or alternatively, in certain embodiments the one or more non- Streptomyces bacteria comprise one or more Terrimonas bacteria, e.g., one or more Terrimonas bacteria of ATCC Deposit No. PTA-127850; additionally or alternatively, in certain embodiments the one or more non-Streptomyces bacteria comprise one or more Zhihengliuella bacteria, e.g., one or more Zhihengliuella bacteria of ATCC Deposit No. PTA-127849.
  • bacteria used in a microbial composition wherein at least a portion of the bacteria are grown by solid state fermentation include some or all of the bacteria in the bacterial consortia deposited with the American Type Culture Collection (ATCC) on October 30, 2024 and assigned deposit numbers PTA127834-127850.
  • ATCC American Type Culture Collection
  • This deposit of the aforementioned bacteria will be maintained in the ATCC or other depository that conforms to the Budapest Treaty, which are public depositories, for a period of 30 years, or 5 years after the most recent request, or for the enforceable life of the patent, whichever is longer, and will be replaced if it ever becomes nonviable during that period.
  • Applicant will impose no restrictions on the availability of the deposited material; however, Applicant has no authority to waive any restrictions imposed by law on the transfer of biological material or its transportation in commerce.
  • a method comprises obtaining each different bacteria from a stock, e.g., a frozen stock, then, typically, treating the stock bacteria to increase bacterial numbers, e.g., by growing the bacteria in a liquid medium, then applying the increased bacteria to a solid medium.
  • a liquid medium comprises a carbohydrate, such as a monosaccharide, e.g., glucose, a source of amino acids, e.g. a protein hydrolysate or partial hydrolysate, such as peptone, and, in some cases, yeast extract, and in some cases a calcium salt, such as calcium chloride.
  • Stock bacteria may be inoculated into the liquid medium at any suitable amount, such as 1 CFU per 5 ml, then the liquid is incubated and agitated, e.g., by shaking in a fermentation flask, at an appropriate temperature for an amount of time suitable for growth to the desired level.
  • suitable temperatures include at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
  • a liquid inoculum is injected into a container comprises the solid medium under sterile conditions, without opening the container.
  • the solid medium may be any suitable medium.
  • all different bacteria are grown on the same type of solid medium; in certain embodiments, at least two of the different bacteria are grown on different types of solid media.
  • the solid medium comprises one or more starches, for example an oat starch, and a gelling agent, for example agar.
  • the solid medium comprises one or more starches, a gelling agent, and a plurality of vitamins.
  • the solid medium comprises ISP-3 or ISP-4 medium.
  • the gelling agent can be any suitable gelling agent, for example agar, pectin, gelatin, and the like, preferably at a concentration of at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, or 4 wt% and/or no more than 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, or 5 wt%, for example 0.1-5 wt%, preferably 0.2-2 wt%, more preferably 0.4-1 wt%.
  • the solid medium has been sufficiently dried prior to transfer of the at least portion of the stock such that there are not visible droplets on the surface of the solid medium.
  • the solid medium for each bacteria may be contained in any suitable container, such as a tray, a bag, e.g., paper bag, or any other container that can contain the solid medium and provide an environment that can be kept sterile.
  • the solid medium e.g., agar
  • the solid medium is contained in a tray. Any suitable tray may be used so long as it can contain sufficient agar for desired growth of bacteria. Sizes of trays may be determined by desired mass of bacteria to be harvested.
  • the tray can be contained in a sterile container, such as a plastic bag or the like. To prevent condensation, the tray/bag combination may be upside down for part or all of the growth of the bacteria, that is, positioned so that the surface of the solid medium faces down rather than up.
  • the solid medium comprises biomass. Any suitable biomass may be used.
  • biomass produced in an agricultural or other commercial enterprise is used, such as byproduct or waste of such enterprise.
  • Exemplary types of biomass include alfalfa, wood (e.g., biochar, forest residue), rice straw, switch grass, com stalks and/or leaves, and the like.
  • the biomass may be arranged in any suitable manner, such as in bales.
  • a pretreatment is used to free nutrients for growth of the bacteria; in certain embodiments, the biomass is treated with one or more fungi, which can be selected for their efficacy in providing the desired nutrients.
  • the biomass is sterilized, either as is or after pretreatment, then inoculated with a microbe; each different unit of biomass, e.g., different bale, is inoculated with a single microbe strain, so that a plurality of units of biomass are inoculated each with a different microbe, as described herein.
  • one or more units of biomass is inoculated with a plurality of different bacteria, e.g., 2, 3, 4, 5, 6, 7 8, 9, 10, or more than 10 different bacteria.
  • the inoculated biomass units are allowed to grow under suitable conditions, until a desired duration, and/or level of microbial growth is reached. Bacteria are then separated from the biomass units, e.g., bales, and treated as necessary to allow them to, e.g., be mixed into a microbial consortium.
  • a solid medium comprising grain is used as the solid substrate.
  • grain includes whole grain as well as one or more grain components or derivatives, such as wheat bran, or processed grain, such as rolled oats, ground corn, white rice, and the like.
  • Grains can provide all the necessary nutrients for bacterial growth and physical support matrix for bacterial colonization.
  • use of grains as opposed to a single monolithic substrate, such as an agar sheet, allows for far greater surface area for bacterial colonization, and thus in most cases grains are a preferable choice. Any suitable type of grain may be used.
  • the grain comprises a cereal grain.
  • the grain comprises amaranth, buckwheat, quinoa, teff, triticale, fonio, spelt, wild rice, wheat, rye, rice, oats, sorghum, millet, corn, barley, or a combination thereof.
  • the grain comprises wheat, rye, rice, oats, sorghum, millet, corn, barley, or a combination thereof.
  • the grain comprises wheat.
  • the grain comprises rye.
  • the grain comprises rice, such as brown rice or white rice.
  • the grain comprises oats.
  • the grain comprises sorghum.
  • the grain comprises millet.
  • the grain comprises corn.
  • the grain comprises barley.
  • two or more grains are combined.
  • a single grain is used.
  • the type of grain selected as a solid medium for bacterial growth can depend on one or more factors. For example, different bacteria can prefer different grains, certain grains may be more plentiful or available in the geographical location in which solid state growth is performed, and/or cost of the grain.
  • the solid substrate consists essentially of a grain.
  • biomass e.g., grains
  • the container is used both for sterilization of the biomass, e.g., grains, and for growth of the bacteria, increasing efficiency and minimizing the possibility of contamination.
  • the container is a bag, e.g., a vented bag, also referred to as a cultivation bag herein.
  • the use of autoclavable cultivation bags equipped with microporous filter patches is standard practice for many solid state fermentation (SSF) applications, including mushroom spawn production. These bags allow for sterilization of the solid substrate within the cultivation vessel itself.
  • SSF solid state fermentation
  • the filter patch permits essential gas exchange (influx of oxygen, efflux of carbon dioxide and metabolic heat) during the subsequent fermentation phase while acting as a barrier to prevent the entry of contaminating microorganisms after sterilization.
  • the grain can be prepared for use as the solid substrate, also referred to as solid media herein.
  • the grain is sterilized, typically in the container in which it will be used for growth of the bacteria.
  • Any suitable container can be used, but preferred containers are vented bags, described more fully herein, and the process will be described in terms of vented bags.
  • Any suitable method of sterilization may be used, so long as a sufficient reduction in the population of indigenous microorganisms naturally present on the grains, including bacteria, yeasts, fungal spores, and highly resistant bacterial endospores, is achieved to allow the selected bacteria to grow on the grain to the desired degree and purity. In some cases, complete sterilization is used. Heat is a common method of sterilization.
  • moist heat is preferable to dry heat methods, but in some cases, dry heat may be used.
  • the most common moist heat method of sterilization is autoclaving.
  • Autoclaving employs high-pressure steam to achieve temperatures significantly above the boiling point of water. Any suitable temperature, pressure, and time may be used for autoclaving, e.g., the standard temperature and pressure, 121 °C and 15 psi.
  • the duration of the autoclaving should be sufficient to achieve the desired degree of sterilization, and can depend on the grain type, size of grain particles, density of the grain, mass of grain in the container, container dimensions, and/or packing density of the containers within the autoclave. In particular, greater mass of grain will generally require longer autoclave duration.
  • the duration can be selected based on one or more of these parameters, but typically will be a minimum of 2-3 hours, e.g., for a container, such as a vented bag, containing 2-5 kg of grain.
  • a container such as a vented bag
  • Preparation can include one or more of cleaning, e.g., removing foreign materials, dust, debris, and the like from the grain; reduction in grain size, e.g., by grinding or other suitable procedure, generally followed by sieving and, if necessary, additional grinding, to achieve a desired grain size; hydration; cooking or partial cooking, e.g., parboiling of rice; addition of one or more additives; and pre-germination. If grain size is reduced, any suitable method may be used, e.g., grinding and sieving.
  • the grain may be reduced in size to any suitable size, such as at least 10, 50, 100, 200, 500, 700, 1000, 1200, 1500, 1700, 2000, 2200, 2500, 3000, 4000, or 5000 urn and/or not more than 50, 100, 200, 500, 700, 1000, 1200, 1500, 1700, 2000, 2200, 2500, 3000, 4000, 5000, or 10,000 um, such as 10-10,000, preferably 200-5000, more preferably 1000-3000 um. It will be appreciated very small sizes, although they provide more surface area for growth, can also pack or stick together and inhibit movement of gases, e.g., oxygen and carbon dioxide, water, and heat.
  • gases e.g., oxygen and carbon dioxide, water, and heat.
  • Hydration of the grain can be important, as it is typically desirable to have the grain at a suitable initial moisture content for bacterial growth, as well as providing sufficient moisture for heat transfer during autoclaving. Hydration typically involves soaking the grain in water followed by draining excess water. Any suitable duration of soaking may be used, and it may vary depending on the grain (e g., 12 hours for millet, 8-24 hours suggested for general grain). Sometimes, a brief simmering or boiling step (e.g., 10-15 minutes) is included after soaking to ensure even hydration and potentially initiate gelatinization, followed by draining and surface drying until the grains are moist but not dripping wet.
  • the goal is to achieve the desired internal moisture content without excessive free water on the grain surface, which can lead to clumping and anaerobic conditions.
  • Any suitable moisture level may be used, such as at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, or 90% and/or 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 70, 80, 90, or 95%, such as 10-95%, preferably 15-80%, 20-70%, more preferably 25-60%, even more preferably 30-55%, yet more preferably 35-50%.
  • One or more additives can also be added.
  • Additives can include one or more additives to provide nutrients, e.g., one or more nutrients in which the grain may be deficient or sub-optimal for bacterial growth; to stimulate desired processes, e.g., an additive comprising calcium to induce sporulation; and/or to prevent grains from sticking together during sterilization and fermentation and/or to provide some pH buffering capacity, e.g., a combination of gypsum (CaSO4) and chalk (calcium carbonate, CaCCh).
  • Pre-germination allowing the fully hydrated grain to rest at room temperature, e.g, for 12-24 hours, before sterilization can encourage heat-resistant bacterial endospores to germinate into more heat-sensitive vegetative cells. This pre-germination step can potentially enhance the effectiveness of the subsequent autoclaving process by making these resistant forms more susceptible to inactivation by heat.
  • the grain optionally after pre-treatment, is loaded into the vented bag.
  • the quantity of the grain per bag depends on desired growth levels, adequacy of separation of grains to provide sufficient access to, e.g., air and water, and the like.
  • a vented bag contains at least 1, 2, 3, 4, 5, 6, 7, 9, 10, 12, 15, or 20 and/or not more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, or 25 kg of grain, such as 1-25, preferably 1-10, more preferably 1-8, still more preferably 1-7, yet more preferably 2-5 kg.
  • the grain is inoculated with the bacteria for growth in that bag.
  • a single strain of bacteria is inoculated in a given bag, as it is likely that different strains will compete with each other and/or grow at different rates, so that the final product is unpredictable; however, in certain embodiments, 2, 3, 4, 5, or 6 or more different strains are inoculated in the same bag; in such cases, one or more tests may be performed after growth of the bacteria to determine quantities of the different bacteria in the final growth product.
  • bacteria may be transferred from a storage container, in which they have been stored prior to solid state fermentation, directly to the grain on which they will grow.
  • bacteria typically, to prepare bacteria for inoculation, they will first be grown in a liquid medium to a sufficient density that adequate inoculation may be achieved.
  • the bacteria are grown in liquid media until a desired endpoint is reached, such as an optical density, or a duration of growth.
  • the bacteria are grown on the solid medium, e.g., grain, under suitable conditions until a desired time has elapsed and/or until a desired level of bacteria is reached.
  • the solid medium e.g., grain
  • each different bacteria will be grown separately under aseptic conditions, such as provided by vented bags, although in some cases two or more different bacteria may be grown on the same solid medium, e.g., grain.
  • Temperature is generally controlled to be at a suitable temperature or range of temperatures. Suitable temperatures include at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
  • Suitable duration of incubation can depend on one or more factors such as the bacteria and/or grain, amount of grain, and the like. Although the duration of fermentation can be determined by one or more observations or tests, in general, duration of incubation will be for a predetermined time, or range of times.
  • growth of bacteria to be used in a given consortium may be timed, e.g., inoculation times may be timed, so that all the bacteria to be used can be harvested at the same time, or within a day or two of each other, and processed together.
  • growth of bacteria to be used in a given consortium may be staggered, so that different bacteria or groups of bacteria are harvested at different times; such a system may be used when limited resources for harvesting and processing are available, so that all can’t be harvested at once.
  • bacteria that have already been harvested are stored under conditions that they retain desired viability until all bacteria for the consortium have been harvested.
  • humidity may be controlled to a desired degree, such as at least 50, 60, 70, 75, 77, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 and/or not more than 60, 70, 75, 77, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 92, 95, 96, 97, 98, 99, or 99.5%, preferably 50-99.5, more preferably 60-99.5, even more preferably 70-99.5, still more preferably 80-99.5, yet still more preferably 60-80-%, even yet still more preferably 85-98%. In certain embodiments, humidity is not controlled for part or all of the process.
  • bacteria are examined to determine if any contamination has occurred and, if so, appropriate measures are taken to ensure the contamination does not carry over into further stages of the process.
  • Conditions for growth for each bacteria such as inoculum size; amount of biomass, e.g., grain, needed for a given final amount of the bacteria; type of biomass, e.g., grain; pretreatment of biomass, e.g., grain, before bacterial growth; duration of growth; temperature for growth; and/or humidity for growth, and the like, may be determined empirically for each different bacteria.
  • the bacteria are harvested, generally after some processing of the grain/bacteria mixture, to be used in, e g., a bacterial consortium comprising a plurality of different bacteria.
  • the fermentation container e.g., bag
  • the fermentation container is opened; generally aseptic conditions are no longer required, although it can be desirable to prevent, e.g., bacteria from one fermentation contaminating the environment, e.g., other fermentations.
  • the grain/bacteria mixture is dried to a suitable degree, either in the same bag or in a container to which it is transferred, e.g., a bag or other container that has greater moisture exchange than the fermentation bag. Any suitable method of drying may be used.
  • Moisture content can be decreased to less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%, such as less than 10, preferably less than 8, more preferably less than 6, even more preferably less than 5%.
  • the bacteria/grain mixture is then removed from the container and bacteria harvested.
  • a technique is used that allows some or all of the grain to be carried forward with the bacteria to next steps. More typically, the bacteria are removed from the grain using any suitable technique, such as sieving and/or abrading, or, in the case of agar plates, scraping.
  • the bacteria may be used as is, or with only minor processing, after harvesting, greatly simplifying procedures when compared to growth in liquid medium, which requires removal of water.
  • the bacteria as dried to the desired level can provide various yields of gm viable units of bacteria (viable cells and/or viable spores)/kg dry weight of grain, depending on conditions and strain of bacteria.
  • the yield in some cases may be as low as not more than 1, 2, 3, 4, or 5 gm bacteria/kg dry grain and in others as high as not more than 150, 200, 250, or 300 gm bacteria/kg dry weight.
  • different bacteria may provide different yields, in terms of weight, viable bacterial units, or both.
  • a ratio of most containers to provide the needed amount of bacteria to least containers to provide the needed amount of bacteria for a given consortium may be, e.g., 2: 1, 3 : 1, 4: 1, 5:1, 6: 1, 7:1, 8: 1, 9:1, 10: 1 or more.
  • the harvested bacteria are further treated, e.g., to provide a bacterial harvest suitable for combining with other bacteria. This can include further drying the bacteria, homogenization, and the like.
  • the harvested bacteria can be assessed to determine a count for viable units that are viable cells and/or viable spores, such as spore count and/or CFU count.
  • a carrier and/or stabilizing substance for example a monosaccharide, di-saccharide, tri -saccharide, and/or a polysaccharide, e.g., glucose, lactose, maltose, and/or galactose.
  • the substance comprises glucose.
  • one or more bacteria are grown in liquid medium, then processed and, e.g., combined with bacteria grown by solid state fermentation.
  • the genera to be used in a final composition does not grow sufficiently on solid medium.
  • Such genera can include Bacillus, Paenibacillus, Lysinibacillus, Rhodococcus, Peribacillus, Priestia, Kocuria, and/or some strains of Streptomyces.
  • Techniques for liquid fermentation are well-known in the art.
  • Harvesting the bacteria grown in liquid medium generally requires more steps than for those grown on solid medium.
  • An exemplary process is filtration.
  • the filter can comprise any suitable filter such that at least a portion of the bacteria are thereby retained to produce a retentate comprising one or more bacteria and a filtrate comprising at least a portion of the growth medium.
  • the filter is configured to retain at least 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 99, or 100% of the bacteria in the growth medium.
  • the filter can comprise a first filter layer.
  • the first filter layer can comprise a pore size of at least 0.2, 2, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, or 280 and/or not more than 2, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, or 300 um, for example, 0.2-300 um.
  • the first filter layer comprises a pore size of at least 0.2, 2, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, or 280 and/or not more than 2, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, or 300 um, for example, 0.2-300 um, preferably 120-240 um, more preferably 160-220 um.
  • the filter can further comprise a second filter layer under the first filter layer comprising a pore size smaller than the first filter layer.
  • the second filter layer can comprise a pore size of at least 0.2, 2, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, or 280 and/or not more than 2, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, or 300 um, for example, 0.2-300 um, preferably 20-100 um, more preferably 40-80 um.
  • the portion of bacteria retained comprises at least 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 99.5, 99.9, or 100% of the bacteria, for example at least 50, preferably at least 70, more preferably at least 90, even more preferably at least 95, yet more preferably at least 98, still more preferably at least 99%.
  • the bacteria after filtering, the bacteria are removed from the filter and placed in a collection vessel. Additionally or alternatively, the bacteria can be spray dried and/or lyophilized. In certain embodiments, after filtration, the bacteria retained on the filter are further dried, for example in a laminar flow hood.
  • the dried bacteria comprise less than 5, 2, 1, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, 0.005, or 0.001% water by weight, for example less than 1, preferably less than 0.5, more preferably less than 0.1, even more preferably less than 0.01, yet more preferably less than 0.005, still more preferably less than 0.001%.
  • the dried bacteria are then processed into a powder.
  • the powder comprises a median particle size of at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250, 370, 300, or 400 um and/or no more than 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250, 270, 300, 400, or 500 um, for example 20-500 um, preferably 90-270 um, more preferably 140-220 um, even more preferably 160-200 um, still more preferably 170-190 um. Any suitable technique can be used to process the dried first population of bacteria to the desired median particle size, for example blending.
  • bacteria produced by the methods described herein can be stored or used immediately, e.g., combined with other bacteria. In certain cases, they can be combined as is, that is, each different bacteria is added to the others essentially as harvested from the solid medium. In certain cases, one or more operations are performed to determine, e.g., total viable units, such as total number of spores and/or viable cells, and/or CFU, CFU per gram. In some cases, one or more additives are added to one or more of the harvested bacteria before combining. The bacteria can be combined to produce a consortium that is a dry product, or that can be dried to provide a dry product.
  • additional microbes e.g., fungi
  • bacteria produced by the methods provided herein to provide a microbial consortium.
  • Any suitable fungi may be used.
  • the fungi comprise mycorrhizal fungi.
  • one or more fungi are used of one or more of Aspergillus, Chaetomium, Coniothyrium, Paecilomyces, Penicillium, Phlebia, Rhizopogon, Scleroderma, Lecanicillium, Sebacina, A lie maria, Beauveria, Cladiosporium, Claroideoglomus, Funneliformis, Glomus, Laccaria, Lecanicillium, Metarhizium, Piriformospora, Pisolithus, Rhizophagus, Serendipita, Talaromyces, Trichoderma, Mucor, and/ or Phanerochaete genera.
  • one or more fungi are used of one or more of Aspergillus, Chaetomium, Coniothyrium, Paecilomyces, Penicillium, Phlebia, Rhizopogon, Scleroderma, Lecanicillium, Sebacina, Alternaria, Beauveria, Cladiosporium, Claroideoglomus, Funneliformis, Glomus, Laccaria, Lecanicillium, Metarhizium, Piriformospora, Pisolithus, Rhizophagus, Serendipita, Talaromyces, and/or Trichoderma genera.
  • one or more fungi are used of one or more of Glomus, Rhizopogon, and/or Scleroderma genera, such as Glomus aggregatum, Glomus etunicatum, Glomus intradices, Glomaus mosseae, Rhizopogon amylopogon, Rhizopogon fulvigleba, Rhizopogon luteolus, Rhizopogon villosullus, Scleroderma cepa, and/or Scleroderma citrinum.
  • Glomus aggregatum Glomus etunicatum
  • Glomus intradices Glomaus mosseae
  • Rhizopogon amylopogon Rhizopogon fulvigleba
  • Rhizopogon luteolus Rhizopogon villosullus
  • Scleroderma cepa and/or Scleroderma citrinum.
  • a microbial consortium comprising bacteria produced by the methods provided herein can be combined with one or more agriculturally acceptable adjuvants.
  • Any suitable agriculturally acceptable adjuvants can be used, such as an emulsifier, e.g., a surfactant, and/or a carrier.
  • the agriculturally acceptable adjuvant comprises an agent that helps the powder composition dissolve in water, e.g., a wetting agent.
  • the emulsifier comprises a surfactant and/or a lipid, for example soy lecithin, yucca powder, Tween 20, and/or Tween 80.
  • the emulsifier is at a concentration of at least 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 4, 5, 6, 7, 8, or 9 wt% and/or no more than 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10 wt%, for example 0.5- 10 wt%, preferably 1-3 wt%, more preferably 1.8-2.2 wt%.
  • the carrier comprises a saccharide, for example a monosaccharide, di-saccharide, tri-saccharide, and/or a polysaccharide, e.g., glucose, lactose, maltose, and/or galactose.
  • the carrier comprises glucose.
  • the carrier is at a concentration of at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 wt% and/or no more than 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 wt%, for example 50-100 wt%, preferably 70-100 wt%, more preferably 80-100 wt%, even more preferably 90-100 wt%, still more preferably 95-100 wt%.
  • the one or more carriers comprise a monosaccharide, a polysaccharide, a lipid, or a combination thereof. In certain embodiments, the one or more carriers comprises a monosaccharide. In certain embodiments, the monosaccharide comprises glucose.
  • the concentration of the monosaccharide is at least 20, 30, 40, 50, 60, 70, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 97, 98, or 99 wt% and/or not more than 30, 40, 50, 60, 70, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 97, 98, 99, 99.5, or 99.9 wt%, for example 20-99 wt%, preferably 70-95 wt%, more preferably 85-95 wt%.
  • the one or more agriculturally acceptable adjuvants comprise a polysaccharide.
  • the polysaccharide comprises a starch.
  • the starch is derived from yucca.
  • the starch is derived from oat.
  • the concentration of the polysaccharide is at least 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8. 2, 2.2, 2.4, 2.6, 2.8, 3, or 4 and/or not more than 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8.
  • the one or more agriculturally acceptable adjuvants comprises one or more surfactants.
  • the surfactant comprises a lipid.
  • the lipid comprises a lecithin.
  • the lecithin comprises a soy lecithin.
  • the concentration of surfactant is at least 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8.
  • the processed microbial consortium bacteria can be packaged in any suitable packaging.
  • the packaging may be a sealed bag, that optionally protects the bacteria from any environmental stressors, such as water and/or UV light.
  • the packaging comprises opaque, waterproof material into which the processed bacteria are vacuum-packed, e.g., under an inert atmosphere such as nitrogen.
  • the bacteria in the final composition comprises viable units, i.e., viable cells and viable spores, of at least 10 6 , 10 7 , 10 8 , 10 9 ,10 lo ,10 n , 10 12 , 10 13 , or 10 14 , and/or not more than 10 7 , 10 8 , 10 9 ,10 10 ,10 n , 10 12 , 10 13 , 10 14 , or 10 15 viable units/gm of composition, preferably 10 6 to 10 15 , more preferably 10 7 to 10 14 , even more preferably 10 7 to 10 13 , yet more preferably 10 7 to 10 11 , even yet more preferably 10 8 to 10 11 viable units/gm of composition.
  • viable units i.e., viable cells and viable spores
  • a composition that comprises a plurality of one type microbe, such as one of the bacteria described herein, and a solid medium on which the microbe are growing or have been growing.
  • the bacteria may be present at any suitable amount, such as at least 1, 2, 5, 10, 20, 30, 40, 50, 70, 100, 150, 200, 500, or 1000 gm or 1kg, 2kg, 5kg, 10kg, 50 kg, 100 kg, 500kg, 1000kg, or 5000 kg and/or not more than 2, 5, 10, 20, 30, 40, 50, 70, 100, 150, 200, 500, 1000 gm or 1kg, 2kg, 5kg, 10kg, 50 kg, 100 kg, 500kg, 1000kg, 5000, or 10,000 kg.
  • the solid medium such as a solid medium as described herein, can be present in a wt:wt ratio with the bacteria, such as at least 10:1, 50:1, 100: 1, 200: 1, 300: 1, 400: 1, 500: 1, 600: 1, 700:1, 800:1, 900:1, 1000:1, 2000: 1, or 5000: 1 and/or not more than 50:1, 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 2000: 1, 5000: 1, or 10:000: 1.
  • compositions comprising a plurality of containers, each of which comprises a one type bacteria, wherein the bacteria in each of the containers are different from each other, and a solid medium on which the microbe are growing or have been growing, such as at least 2, 3, 4, 5, 7, 10, 12, 15, 20, 50, 70, 100, 120, or 150 and/or not more than 3, 4, 5, 7, 10, 12, 15, 20, 50, 70, 100, 120, 150, or 200 different compositions.
  • the solid media is a grain, e.g., one of the grains described herein.
  • at least 2, 3, 4, 5, 6, or 7 and/or not more than 3, 4, 5, 6, 7, 8, 9, 10, 12, or 15 of the containers, or sets of the containers contains a different grain as solid media.
  • the bacteria are grown and, optionally, treated to provide a bacterial consortium, such as a bacterial consortium, or, if non-bacterial microbes are used in addition to the bacteria, a microbial consortium, combined with one or more agriculturally acceptable adjuvants, in one or more structures comprising, or produced from, one or more transportable units.
  • a bacterial consortium such as a bacterial consortium
  • a microbial consortium combined with one or more agriculturally acceptable adjuvants, in one or more structures comprising, or produced from, one or more transportable units.
  • a transportable unit can be any suitable unit that provides sufficient space and conditions for transporting various components, e.g., modular containers that can be shipped by ship, rail, and/or truck from an area where it is produced and/or packaged to an area where it is desirable to carry out one or more of the methods disclosed herein.
  • Components shipped in the container or containers to the desired location can include one or more of a tent or tents or other soft-sided structures for containing some or all of the materials for performing the methods; containers, e.g., vented bags, for containing solid media, e.g., grain, such as grain produced at or near the location to which the container or containers are shipped; materials used to produce a liquid inoculum for the solid medium; materials used to inoculate the solid medium in its container with the inoculum; materials to prepare the solid medium, e.g., grain, for growth of the bacteria, such as materials to reduce the size of the grain, clean it, sieve it, soak it in water, and the like, as well as one or more systems, e.g., autoclave, to sterilize the grains; materials to dry the grown bacteria on the media before separating bacteria from the media; materials to separate the grown bacteria from the solid media; one or more systems to provide temperature control in the stmcture for incubation; one or more systems to provide an as
  • kits comprising components of one or more structures, such as described herein, a system for providing and maintaining asceptic conditions in one or more of the structures, a system for providing and maintaining a desired temperature or range of temperatures in one or more of the structures, a system for providing and maintaining a desired humidity or range of humidities in one or more of the structures, a system to remove grown bacteria from the solid media, one or more systems to process the removed bacteria, one or more systems to combine the bacteria with one or more agriculturally acceptable adjuvants, and/or a system to package the processed bacteria, one or more of a plurality of microbial stocks, components of liquid media for growing the bacteria, components of solid media for growing the bacteria, and the like.
  • a method comprising growing at least 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, or 35 and/or not more than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150, or 200, such as 2-200, preferably 3-200, more preferably 4-200, yet more preferably 2-150, still more preferably 2-100, yet still more preferably 5-100, different bacteria on separate solid media under suitable conditions for growth of the respective bacteria; (ii) partially or completely removing the bacteria from their respective solid media after growth to provide harvested bacteria; and (iii) combining the harvested bacteria, whereby a bacterial consortium is produced.
  • the method can further include (iv) growing at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20 and/or not more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, or 50 additional bacteria, such as such as 1-50, or 1-40 or 1-30, or 1-20, or 1-10 additional bacteria, different from the other bacteria and from each other, in separate liquid media under conditions suitable for growth of the respective bacteria; (v) partially or completely removing the bacteria from their respective liquid media after growth to provide harvested bacteria; and (vi) combining the harvested bacteria from liquid media with the harvested bacteria from solid media.
  • Bacteria to be used in the method can include any suitable bacteria, such as those disclosed herein.
  • the method includes providing at least one fungus and combining it with the bacteria.
  • the solid media can comprise one or more grains, such as those described herein.
  • at least a portion of the plurality of different bacteria are grown in a plurality of geographically separate bacterial production facilities, such as described further herein.
  • the facility, or geographically separate facilities can transmit information regarding production of bacteria, conditions for production, supplies, storage, or a combination thereof, to a central processor, which processes the information.
  • Microbial consortia containing a plurality of different bacteria can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 150 and/or not more than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150, or 200 different bacteria, such as 2-200, or 2-150, or 2- 100, or 2-50, or 5-200, or 5-100, or 5-80, or 5-50 different bacteria.
  • a consortium also comprises fungi, as described herein.
  • a microbial consortium comprises a plurality of bacteria produced by liquid fermentation and a plurality of bacteria produced by solid state fermentation, such as at least 2, 3, 4, 5, 7, 10, 12, 15, 20, 30, 40, 50, 70, or 100 and/or not more than 3, 4, 5, 7, 10, 12, 15, 20, 30, 40, 50, 70, 100, or 150 bacteria produced by liquid fermentation and at least 2, 3, 4, 5, 7, 10, 12, 15, 20, 30, 40, 50, 70, or 100 and/or not more than 3, 4, 5, 7, 10, 12, 15, 20, 30, 40, 50, 70, 100, or 150 bacteria produced by solid fermentation.
  • kits comprising providing a plurality of different bacteria, wherein each of the different bacteria has been grown on solid media, and combining the plurality of different bacteria.
  • the plurality of different bacteria grown on solid media can comprises at least 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, or 35 and/or not more than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150, or 200, such as 2-200, preferably 3-200, more preferably 4-200, yet more preferably 2-150, still more preferably 2-100, yet still more preferably 5-100 different bacteria.
  • the method includes providing one or more additional bacteria, different from the bacteria grown on solid media and from each other, that has been grown in liquid media and combining the additional bacteria with the bacteria grown in solid media, such at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20 and/or not more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, or 50 additional bacteria, such as such as 1-50, or 1-40 or 1-30, or 1-20, or 1-10 additional bacteria grown in liquid media.
  • the bacteria can be any bacteria suitable for their intended purpose, such a bacteria described herein.
  • the bacteria are produced in a plurality of geographically separate bacterial production facilities. [0033]
  • a system comprising (i) one or more sources of a plurality of different bacteria grown on solid media; operably connected to (ii) a facility wherein the plurality of different bacteria are combined.
  • Choice of site for a facility to produce one or more different bacteria can be based on factors such as availability of grain or other biomass to be used in solid state fermentation, raw material costs, labor costs, transportation costs, and the like. Some or all of the different bacteria may be produced by methods described herein.
  • Each of the bacterial production facilities can produce at least one bacterium in sufficient quantity to be used in a particular microbial consortium, or a plurality of different microbial consortia, and in a form that can be shipped to a facility where different bacteria are combined to produce the consortium or consortia.
  • Bacteria produced by the methods described herein can be stored for up to 3, 6, 9, 12, 18, 24 months, or more than 24 months, and still retain sufficient viability to be used in a microbial consortium, so one or more bacterial production facilities may package bacteria produced at the facility in packaging and under conditions that allow a desired storage length.
  • a bacterial production facility can utilize a plurality of production runs for one or more of the different bacteria it produces, and the harvested bacteria stored until use.
  • Long-term storage conditions for bacteria are well-known; e.g., bacteria can be vacuum packed and sealed, e.g., under a non-oxygen atmosphere, in light- and water-proof containers and stored at an appropriate temperature until use.
  • sufficient quantities of the different bacteria produced at the different bacterial production facilities can be combined to produce the microbial consortium.
  • the bacteria may be combined at one or more dedicated locations, and/or at a location that comprises its own bacterial production facility, and stored and/or shipped to one or more locations. There will be at least one location where all different bacteria for a particular consortium are combined, although there may be separate facilities where a portion of the different bacteria are combined for later use in one or more overall consortia.
  • certain sets of different bacteria are used in a plurality of consortia and one or more facilities may be used to combine different bacteria from different facilities in the sets, which are then used to produce particular microbial consortia as needed.
  • a plurality of geographically different bacterial production facilities may each produce one or more different bacteria, in some cases where at least a portion of the bacterial production facilities, or all of the bacterial production facilities, produces at least one bacteria than is different from those produced at every other bacterial production facility.
  • the number of geographically separate bacterial production facilities can be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 50, 70, or 100 and/or not more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 50, 70, 100, 120, or 150 geographically separate bacterial production facilities, such as 2-150, preferably 2-120, more preferably 2-100, even more preferably 2-50, in some cases 5-150, or 5-120, or 5-100, or 5-80, or 5-60, or 5-40, or 5-30 geographically separate bacterial production facilities.
  • a portion of the facilities e.g., at least 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 98%, and/or, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 50, 70, or 100 and/or not more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 50, 70, 100, 120, or 150 of the different bacterial production facilities may produce at least one bacteria by solid state fermentation, such as at least 1, 2, 3, 4, 5, 7, 10, 15, 20, 30, 40 or 50 and/or not more than 2, 3, 4, 5, 7, 10, 15, 20, 30, 40, 50, or 100 different bacteria that are produced by solid state fermentation.
  • a portion of the facilities e.g., at least 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 98%, and/or, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 50, 70, or 100 and/or not more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 50, 70, 100, 120, or 150 of the geographically separate bacterial production facilities may produce at least one bacteria by liquid fermentation, such as at least 1, 2, 3, 4, 5, 7, 10, 15, 20, 30, 40 or 50 and/or not more than 2, 3, 4, 5, 7, 10, 15, 20, 30, 40, 50, or 100 different bacteria that are produced by liquid fermentation.
  • a portion or all of the geographically separate bacterial production facilities produce bacteria of only one genus, e.g., only Streptomyces, only Bacillus, and the like.
  • a bacterial production facility where a plurality of different bacteria are produced, at least some of which are grown on solid medium, or a plurality of geographically separate bacteria production facilities, e.g., as described herein, transmit information, e.g., that can influence the production of a microbial consortium or consortia, now or in the future, to a central processor, which processes the information. Any suitable information that can influence the production or other aspects of bacteria to be used for consortium or consortia, now or in the future, can be transmitted to the central processor.
  • Information can include any relevant information, for example information regarding supplies for producing each of the plurality of different bacteria, such as bacterial stocks, supplies of solid media, e.g., one or more grains, containers for growth, costs of labor or other components of production, and the like; availability of further supplies; timing of one or more production parameters; past production runs of each of the plurality of different bacteria, whether or not bacteria stored from previous runs at the first facility are available and if so, a quantity of the bacteria, or a combination thereof.
  • supplies for producing each of the plurality of different bacteria such as bacterial stocks, supplies of solid media, e.g., one or more grains, containers for growth, costs of labor or other components of production, and the like
  • availability of further supplies e.g., timing of one or more production parameters
  • past production runs of each of the plurality of different bacteria whether or not bacteria stored from previous runs at the first facility are available and if so, a quantity of the bacteria, or a combination thereof.
  • the central processor can further receive information regarding a microbial consortium to be produced, where the consortium includes one or more of the different bacteria produced at one or more of the bacterial production facilities, such as information regarding the composition of the consortium, date or date range when the consortium is need, transportation of the consortium, or a combination thereof.
  • the central processor can produce a protocol based, at least in part, on its processing of the information, for production of the consortium, which can include timing of inoculation of the solid or liquid media for each of the different bacteria, amount of inoculum for each inoculum, total amount of solid media, e.g., grain, for growing each different bacteria, nature and/or number of containers for growth of the bacteria, temperature for growth of each bacteria, humidity for growth of each bacteria, duration of growth of the different bacteria, and/or sourcing of solid or liquid media and can transmit or otherwise make available the protocol, or relevant portions thereof, to the one or more bacterial production facilities.
  • a protocol based, at least in part, on its processing of the information, for production of the consortium, which can include timing of inoculation of the solid or liquid media for each of the different bacteria, amount of inoculum for each inoculum, total amount of solid media, e.g., grain, for growing each different bacteria, nature and/or number of containers for growth of the bacteria, temperature for growth of each bacteria
  • Information can include information regarding past, current, and/or planned runs at one or more of the bacterial production facilities, stored materials such as grains or bacteria from previous runs, costs of one or more components of production, transportation, and any other relevant information.
  • the central processor can receive information regarding the outcome of production of the different bacteria using the protocol, such as yields, any adjustments necessary to the protocol, conditions during inoculation, growth, and/or harvesting of the bacteria, and any other relevant information.
  • the central processor can learn from the outcome and, in some cases, modify future protocols based, at least in part, on that learning.
  • a bacterial production facility can alter conditions for a production run for a particular bacteria to be different from conditions for previous runs with the same bacteria, or conditions may fortuitously or otherwise be different, and determine whether the altered conditions modulate, e.g., improve, yield, efficiency, or other parameters important to production, for future runs.
  • the processor can modify one or more algorithms used for production based, at least in part, on the information.
  • the central processor can transmit recommendations for altering conditions or other information for one or more runs at one or more bacterial production facilities, different from the one from which it received information, based at least in part on the processing. For example, information for one species or strain from a run at a first facility can be used to send information and/or recommendations to a second facility that produces the same or similar species or strain.
  • a network comprising a plurality of geographically separate bacterial production facilities, such as a plurality described herein, operably connected to at least one processing facility that receives bacteria produced at one or more of the geographically separate bacterial production facilities and combines them to produce a bacterial consortium from the bacteria. Also included may be one or more storage facilities for storing one or more bacteria produced at the one or more geographically separate bacterial production facilities; such storage facilities may be part of and/or separate from one or more of the geographically separate bacterial production facilities.
  • a portion or all of the geographically separate bacterial production facilities may be operably connected to a central processor, e.g, by one or more telemetry systems at the facilities, which receives information from the separate bacterial production facilities and processes the information.
  • the central processor may transmit one or more signals to the facilities, based, at least in part, on the information received, e.g., signals related to one or more production runs at one or more of the facilities.
  • the central processor generally will operate one or more algorithms that ensure that all geographically separate bacterial production facilities, and all relevant runs at the facilities, as well as any storage facilities, if used, are coordinated to produce a desired bacterial consortium at a desired time.
  • the central processor can also predict future conditions that can affect future runs, and modify algorithms or instructions to one or more entities based, at least in part, on the predicted future conditions.
  • the central processor can learn, e.g., from results of one or more production runs at one or more of the bacterial production facilities to modify one or more algorithms to modify production in a desired way, e.g., improve production efficiency, decrease costs, and the like.
  • the central processor may be configured to easily and automatically accommodate expansion or contraction in the number and type of bacterial production facilities in the network, and to accommodate expansion or contraction in inputs and outputs.
  • the central processor is typically remote from some or all of the geographically separate bacterial production facilities, that is, not located at the facilities, or at a single facility, or distributed among the facilities so that no one facility contains the entire processor.
  • Also provided are methods comprising (i) producing a plurality of different bacteria at a plurality of geographically separate different bacteria production facilities, such as a plurality of geographically separate different bacteria production facilities as described herein; transporting bacteria produced at least a portion of the bacterial production facilities to a facility where they are combined to produce a microbial consortium comprising the bacteria.
  • a method comprising (i) receiving at a central processor information from a first bacterial production facility regarding production of one or more different bacteria at the first bacterial production facility, wherein at least one of the different bacteria is grown on solid media, and (ii) processing the information at the central processor.
  • the central processor can receive information from a second, third, fourth, fifth, sixth, seventh, eighth, ninth, and/or tenth facility, or more than ten facilities, different from each other and from the first facility, e.g., geographically separate bacterial production facilities. In some cases at least one of the facilities grows bacteria in liquid media.
  • the information can comprise any suitable information, such as information regarding supplies for producing each of the plurality of different bacteria, such as bacterial stocks, supplies of solid media, and the like; availability of further supplies; timing of one or more production parameters; past production runs of each of the plurality of different bacteria, whether or not bacteria stored from previous runs at the first facility are available and if so, a quantity of the bacteria, or a combination thereof.
  • SSF solid-state fermentation
  • Seed cultures were prepared using a sterile glucose broth medium previously sterilized and stored. Select microbial strains from a proprietary 140-strain culture collection were retrieved from glycerol stocks stored at -80°C. See Table 2 for strains used, which included Streptomyces, Bacillus, Lysinibacillus, Brevibacillus, Microbacterium, and Paenebacillus . Using a sterile 10 pL inoculating loop, material from each working glycerol stock was aseptically transferred into a labeled sterile culture vessel containing 3 mL of glucose broth. These inoculated vessels were incubated at 28°C in a shaking incubator set at 240 rpm for two consecutive overnight periods to establish active seed cultures.
  • a total of 450 grams of the hydrated and homogenized grain substrate was transferred into autoclavable polypropylene bags (dimensions: 4” W x 13.5” H) equipped with a 0.2 pm filter patch.
  • the bags were folded and sealed before being sterilized via autoclaving at 121 °C and 1.5 atm pressure for 45 minutes. After sterilization, the substrate was allowed to cool to 28°C under aseptic conditions.
  • each cooled, sterile substrate bag was aseptically opened. A volume of 3 mL of liquid seed culture was pipetted directly onto the substrate, taking care to avoid contact with the inner surface of the bag. The bag was resealed approximately 1 inch from the top using a thermal bag sealer, then manually agitated to ensure uniform distribution of the inoculum throughout the substrate.
  • Inoculated substrate bags were incubated at 28°C for a duration of 7 to 15 days, depending on the microbial strain and the desired product. Visual assessments were performed at regular intervals to monitor microbial growth and colonization. Of the 21 strains tested, 15 demonstrated observable growth. Colonization was evaluated based on surface area coverage of the com substrate, with all but one strain achieving 90-100% colonization.
  • This example shows that use of a grain substrate, in this case, cracked corn, for microbial colonization can significantly increase biomass yield due to its substantially greater three-dimensional surface area.
  • cracked corn offers a complex, porous structure that far exceeds the flat, two-dimensional surface area of conventional agar plates.
  • the Example demonstrates that different bacteria may require different grain substrates, or liquid substrate, as some of the bacteria did not grow on the cracked com.
  • Embodiment 1 provide a method comprising (i) growing at least 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, or 35 and/or not more than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150, or 200, such as 2-200, preferably 3-200, more preferably 4-200, yet more preferably 2-150, still more preferably 2-100, yet still more preferably 5-100, different bacteria on separate solid media under suitable conditions for growth of the respective bacteria; (ii) partially or completely removing the bacteria from their respective solid media after growth to provide harvested bacteria; and (iii) combining the harvested bacteria, whereby a bacterial consortium is produced.
  • Embodiment 2 provides the method of embodiment 1 comprising (iv) growing at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20 and/or not more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 40, or 50 additional bacteria, such as 1-50, or 1-40 or 1-30, or 1-20, or 1-10 additional bacteria, different from the other bacteria and from each other, in separate liquid media under conditions suitable for growth of the respective bacteria (v) partially or completely removing the bacteria from their respective liquid media after growth to provide harvested bacteria; and (vi) combining the harvested bacteria from liquid media with the harvested bacteria from solid media.
  • additional bacteria such as 1-50, or 1-40 or 1-30, or 1-20, or 1-10 additional bacteria
  • Embodiment 4 provides the method of embodiment 3 wherein the bacteria grown in liquid media comprise bacteria of genus Bacillus.
  • Embodiment 5 provides the method of any previous embodiment wherein the different bacteria grown on separate solid media comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30, 35, 40, 50, or 60 and/or not more than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30, 35, 40, 50, 60, 70, 80, or 100, preferably 1-100, more preferably 2-100, even more preferably 3-80, still more preferably 4-40, yet more preferably 10-80, still yet more preferably 10-70, even more preferably 10-60, even still more preferably 10-40, further preferably 10-30, different strains of Streptomyces bacteria.
  • Embodiment 6 provides the method of embodiment 5 wherein at least a portion of the different strains of Streptomyces bacteria comprise bacteria from one or more ATCC deposit numbers PTA-127835- PTA- 127844, PTA- 127846, or PTA- 127849, as shown in Table 1.
  • Embodiment 7 provides the method of any previous embodiment wherein the different bacteria grown on separate solid or liquid media comprise one or more non- Streptomyces bacteria.
  • Embodiment 8 provides the method of embodiment 7 wherein the non-Streptomyces bacteria comprise one or more different bacteria of genus Actinoplanes.
  • Embodiment 9 provides the method of embodiment 7 or 8 wherein the non-Streptomyces bacteria comprise one or more different bacteria of genus Agromyces.
  • Embodiment 10 provides the method of any one of embodiments 7 through 9 wherein the non-Streptomyces bacteria comprise one or more different bacteria of genus Allosphingosinicella.
  • Embodiment 11 provides the method of any one of embodiments 7 through 10 wherein the non-Streptomyces bacteria comprise one or more different bacteria of genus Aneurinibacillus.
  • Embodiment 12 provides the method of any one of embodiments 7 through 11 wherein the non-Streptomyces bacteria comprise one or more different bacteria of genus Arthrobacter.
  • Embodiment 13 provides the method of any one of embodiments 7 through 12 wherein the non-Streptomyces bacteria comprise one or more different bacteria of genus Bacillus.
  • Embodiment 14 provides the method of any one of embodiments 7 through 13 wherein the non-Streptomyces bacteria comprise one or more different bacteria of genus Brevibacillus.
  • Embodiment 15 provides the method of any one of embodiments 7 through 14 wherein the non-Streptomyces bacteria comprise one or more different bacteria of genus Cal dimonas.
  • Embodiment 16 provides the method of any one of embodiments 7 through 15 wherein the non-Streptomyces bacteria comprise one or more different bacteria of genus Cupriavidus.
  • Embodiment 17 provides the method of any one of embodiments 7 through 16 wherein the non-Streptomyces bacteria comprise one or more different bacteria of genus Ery throb acter.
  • Embodiment 18 provides the method of any one of embodiments 7 through 17 wherein the non-Streptomyces bacteria comprise one or more different bacteria of genus Kocuria.
  • Embodiment 19 provides the method of any one of embodiments 7 through 18 wherein the non-Streptomyces bacteria comprise one or more different bacteria of genus Lysinibacillus.
  • Embodiment 20 provides the method of any one of embodiments 7 through 19 wherein the non-Streptomyces bacteria comprise one or more different bacteria of genus Microbacterium.
  • Embodiment 21 provides the method of any one of embodiments 7 through 20 wherein the non-Streptomyces bacteria comprise one or more different bacteria of genus Microbispora.
  • Embodiment 22 provides the method of any one of embodiments 7 through 21 wherein the non-Streptomyces bacteria comprise one or more different bacteria of genus Microvirga.
  • Embodiment 23 provides the method of any one of embodiments 7 through 22 wherein the non-Streptomyces bacteria comprise one or more different bacteria of genus Mycetocola_A.
  • Embodiment 24 provides the method of any one of embodiments 7 through 23 wherein the non-Streptomyces bacteria comprise one or more different bacteria of genus Neorhizobium.
  • Embodiment 25 provides the method of any one of embodiments 7 through 24 wherein the non-Streptomyces bacteria comprise one or more different bacteria of genus Nocardia.
  • Embodiment 26 provides the method of any one of embodiments 7 through 25 wherein the non-Streptomyces bacteria comprise one or more different bacteria of genus Nocardioides.
  • Embodiment 27 provides the method of any one of embodiments 7 through 26 wherein the non-Streptomyces bacteria comprise one or more different bacteria of genus Nonomuraea.
  • Embodiment 28 provides the method of any one of embodiments 7 through 27 wherein the non-Streptomyces bacteria comprise one or more different bacteria of genus Paenibacillus.
  • Embodiment 29 provides the method of any one of embodiments 7 through 28 wherein the non-Streptomyces bacteria comprise one or more different bacteria of genus Peribacillus.
  • Embodiment 30 provides the method of any one of embodiments 7 through 29 wherein the non-Streptomyces bacteria comprise one or more different bacteria of genus Priestia.
  • Embodiment 31 provides the method of any one of embodiments 7 through 30 wherein the non-Streptomyces bacteria comprise one or more different bacteria of genus Pristimantibacillus .
  • Embodiment 32 provides the method of any one of embodiments 7 through 31 wherein the non-Streptomyces bacteria comprise one or more different bacteria of genus Pseudomonas.
  • Embodiment 33 provides the method of any one of embodiments 7 through 32 wherein the non-Streptomyces bacteria comprise one or more different bacteria of genus Pseudomonas_K.
  • Embodiment 34 provides the method of any one of embodiments 7 through 33 wherein the non-Streptomyces bacteria comprise one or more different bacteria of genus Psuedomonas_E.
  • Embodiment 35 provides the method of any one of embodiments 7 through 34 wherein the non-Streptomyces bacteria comprise one or more different bacteria of genus Rhodococcus.
  • Embodiment 36 provides the method of any one of embodiments 7 through 35 wherein the non-Streptomyces bacteria comprise one or more different bacteria of genus Stenotrophomonas.
  • Embodiment 37 provides the method of any one of embodiments 7 through 36 wherein the non-Streptomyces bacteria comprise one or more different bacteria of genus Stutzerimonas.
  • Embodiment 38 provides the method of any one of embodiments 7 through 38 wherein the non-Streptomyces bacteria comprise one or more different bacteria of genus Terrimonas.
  • Embodiment 39 provides the method of any one of embodiments 7 through 39 wherein the non-Streptomyces bacteria comprise one or more different bacteria of genus Zhihengliuella.
  • Embodiment 40 provides the method of any previous embodiment comprising providing at least one fungus and combining it with the bacteria in the consortium to provide a microbial consortium.
  • Embodiment 41 provides the method of any previous embodiment wherein the separate solid media comprise one or more grains.
  • Embodiment 42 provides the method of embodiment 41 wherein the grain or grain derivative comprises amaranth, buckwheat, quinoa, teff, triticale, fonio, spelt, wild rice, wheat, rye, rice, oats, sorghum, millet, corn, barley, or a combination thereof.
  • Embodiment 43 provides the method of embodiment 41 wherein the grain or grain derivative comprises wheat, rye, rice, oats, sorghum, millet, com, barley, or a combination thereof.
  • Embodiment 44 provides the method of any one of embodiments 41 through 43 wherein one or more of the different bacteria are grown on different grains.
  • Embodiment 45 provides the method of any previous embodiment wherein at least a portion of the plurality of different bacteria are grown in a plurality of geographically separate bacterial production facilities.
  • Embodiment 46 provides the method of embodiment 45 wherein the plurality of geographically separate bacterial production facilities comprises at least
  • Embodiment 47 provides the method of embodiment 45 or 46 wherein the geographically separate bacterial production facilities transmit information regarding production of bacteria, conditions for production, supplies, storage, or a combination thereof, to a central processor, which processes the information.
  • Embodiment 48 provides the method of embodiment 47 wherein the central processor determines one or more production protocols for one or more microbial consortia.
  • Embodiment 49 provides the method of embodiment 48 wherein the central processor transmits at least a portion of the one or more production protocols to the geographically separate bacterial production facilities that will produce at least a portion of the bacteria to be used in a bacterial consortium.
  • Embodiment 50 provides the method of any previous embodiment wherein one or more of the harvested bacteria are stored prior to being combined to produce a microbial consortium.
  • Embodiment 51 provide a method comprising providing a plurality of different bacteria, wherein each of the different bacteria has been grown on solid media, and combining the plurality of different bacteria.
  • Embodiment 52 provides the method of embodiment 51 wherein the plurality of different bacteria grown on solid media comprises at least 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, or 35 and/or not more than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150, or 200, such as 2- 200, preferably 3-200, more preferably 4-200, yet more preferably 2-150, still more preferably 2- 100, yet still more preferably 5-100 different bacteria.
  • Embodiment 53 provides the method of embodiment 51 or 52 wherein the bacteria comprise Streptomyces bacteria.
  • Embodiment 54 provides the method of embodiment 53 wherein the Streptomyces bacteria comprise at least 1, 2,
  • Embodiment 55 provides the method of any one of embodiments embodiment 51 through 54 further comprising providing one or more additional bacteria, different from the bacteria grown on solid media and from each other, that has been grown in liquid media and combining the additional bacteria with the bacteria grown in solid media.
  • Embodiment 56 provides the method of embodiment 55 wherein the one or more bacteria grown in liquid media comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20 and/or not more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, or 50 additional bacteria, such as such as 1-50, or 1-40 or 1- 30, or 1-20, or 1-10 additional bacteria.
  • Embodiment 57 provides the method of any one of embodiments embodiment 51 through 56 wherein the different bacteria grown on separate solid or liquid media comprise one or more non-Streptomyces bacteria.
  • Embodiment 58 provides the method of embodiment 57 wherein the non-Streptomyces bacteria comprise bacteria of any one of embodiments 8 through 39.
  • Embodiment 59 provides the method of any one of embodiments 51 through 58 further comprising providing one or more fungi and combining them with the bacteria.
  • Embodiment 60 provides the method of any one of embodiments 51 through 59 wherein the different bacteria are produced at a plurality of geographically separate bacterial production facilities, each of which produces one or more bacteria to be combined.
  • Embodiment 61 provides the method of embodiment 60 wherein the plurality of geographically separate bacterial production facilities comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 50, 70, or 100 and/or not more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 50, 70, 100, 120, or 150 different bacterial production facilities, such as 2-150, preferably 2-120, more preferably 2-100, even more preferably 2-50, in some cases 5-150, or 5-120, or 5-100, or 5-80, or 5-60, or 5-40, or 5-30 geographically separate bacterial production facilities.
  • the plurality of geographically separate bacterial production facilities comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 50, 70, or 100 and/or not more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 50, 70, 100, 120, or 150 different bacterial production facilities, such as 2-150, preferably 2-120, more preferably 2-100, even more preferably 2-50, in some cases 5-150, or 5-120, or 5-100, or 5-80, or
  • Embodiment 62 provides a system comprising (i) one or more sources of a plurality of different bacteria grown on solid media; operably connected to (ii) a facility wherein the plurality of different bacteria are combined.
  • Embodiment 63 provides the system of embodiment 62 wherein the plurality of different bacteria grown on solid media comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 150 and/or not more than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150, or 200 different bacteria, such as 2-200, or 2-150, or 2-100, or 2-50, or 4-150, or 4-100, or 4-50, or 5-150, or 5-100, or 5-80, or 5-50, or 5-30 different bacteria.
  • Embodiment 64 provides the system of embodiment 62 or 63 wherein at least one of the one or more sources of bacteria grown on solid media is geographically separate from the facility where the bacteria are combined.
  • Embodiment 65 provides the system of any one of embodiments 62 through 64 wherein at least one of the one or more sources of bacteria grown on solid media is a source of a plurality of different bacteria.
  • Embodiment 66 provides the system of one of embodiments 62 through 65 comprising a plurality of sources of bacteria grown on solid media, each of which provides a different subset of the one or more different bacteria grown on solid media.
  • Embodiment 67 provides the system of embodiment 66 wherein the plurality of sources comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, 35, 40, or 50 and/or not more than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, 35, 40, 50, or 100 sources of bacteria grown on solid media.
  • Embodiment 68 provides the system of embodiment 66 or 67 wherein at least a portion of the plurality of sources are geographically separate from each other.
  • Embodiment 69 provides the system of any one of embodiments 66 through 68 wherein at least a portion of the plurality of sources are located together in a facility.
  • Embodiment 70 provides the system of any one of embodiments 62 through 69 further comprising one or more sources of one or more bacteria, different from the bacteria grown on solid media and different from each other, grown in liquid media, operably connected to the facility, wherein the one or more bacteria grown in liquid media are combined with those grown on solid media.
  • Embodiment 71 provides the system of any one of embodiments 62 through 70 further comprising one or more sources of one or more fungi operably connected to the facility, wherein the one or more fungi are combined with the bacteria.
  • Embodiment 72 provides the system of any one of embodiments 62 through 71 further comprising a central processor that receives information from the one or more sources of different bacteria.
  • Embodiment 73 provides the system of embodiment 72 wherein the central processor processes the information and provides one or more protocols for producing a consortium comprising the bacteria grown in solid media and/or bacteria grown on liquid media based, at least in part, on the processing, and transmits the one or more protocols, or relevant portions thereof, to the one or more sources.
  • Embodiment 74 provides the system of embodiment 72 or 73 wherein the central processor is remote from at least one of the one or more sources.
  • Embodiment 75 provides a method comprising (i) receiving at a central processor information from a first bacterial production facility regarding production of one or more different bacteria at the first bacterial production facility, wherein at least one of the different bacteria is grown on solid media, and (ii) processing the information at the central processor.
  • Embodiment 76 provides the method of embodiment 75 wherein the central processor receives information from a second bacterial production facility, geographically separate from the first bacterial production facility, regarding production of one or more different bacteria at the second bacterial production facility, wherein some or all of the different bacteria of the second bacterial production facility are different from the bacteria at the first facility.
  • Embodiment 77 provides the method of embodiment 76 wherein the central processor receives information from a third bacterial production facility, geographically separate from the first and second bacterial production facilities, regarding production of one or more different bacteria at the third bacterial production facility, wherein some or all of the different bacteria of the third bacterial production facility are different from the bacteria at the first and second bacterial production facilities.
  • Embodiment 78 provides the method of embodiment 77 wherein the central processor receives information from a fourth bacterial production facility, geographically separate from the first, second, and third bacterial production facilities, regarding production of one or more different bacteria at the fourth bacterial production facility, wherein some or all of the different bacteria of the fourth bacterial production facility are different from the bacteria at the first second, and third bacterial production facilities.
  • Embodiment 79 provides the method of embodiment 78 wherein the central processor receives information from a fifth bacterial production facility, geographically separate from the first, second, third, and fourth bacterial production facilities, regarding production of one or more different bacteria at the fifth bacterial production facility, wherein some or all of the different bacteria of the fifth bacterial production facility are different from the bacteria at the first second, third, and fourth bacterial production facilities.
  • Embodiment 80 provides the method of any one of embodiments 76 through 79 wherein at least one of the second, third, fourth, or fifth bacterial production facilities grows its bacteria in liquid media.
  • Embodiment 81 provides the method of any one of embodiments 76 through 79 wherein at least one of the second, third, fourth, or fifth bacterial production facilities grows its bacteria in solid media.
  • Embodiment 82 provides the method of any one of embodiments 75 through 81 wherein the processor receives information regarding production of a plurality of different bacteria at the first, second, third, fourth, and/or fifth facility, wherein each different bacteria is grown separately from the others.
  • Embodiment 83 provides the method of embodiment 82 wherein the plurality of different bacteria comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, or 50 different bacteria and/or not more than 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, 50 or 100 different bacteria.
  • Embodiment 84 provides the method of embodiment 83 wherein the number of different bacteria grown at a facility can be the same or different from the number grown at one or more of the other facilities.
  • Embodiment 85 provides the method of any one of embodiments 75 through 84 wherein the information comprises information regarding supplies for producing each of the plurality of different bacteria, such as bacterial stocks, supplies of solid media, and the like; availability of further supplies; timing of one or more production parameters; past production runs of each of the plurality of different bacteria, whether or not bacteria stored from previous runs at the first facility are available and if so, a quantity of the bacteria, or a combination thereof.
  • Embodiment 86 provides the method of any one of embodiments 75 through 85 wherein the central processor further receives information regarding a microbial consortium to be produced that includes one or more of the different bacteria.
  • Embodiment 87 provides the method of embodiment 86 wherein the information comprises composition of the microbial consortium, date or date range that the microbial consortium is needed, transportation of the microbial consortium, or a combination thereof.
  • Embodiment 88 provides the method of embodiment 86 or 87 wherein the central processor produces a protocol based, at least in part, on the processing of step (ii), for production of the microbial consortium.
  • Embodiment 89 provides the method of embodiment 88 wherein the protocol comprises timing of inoculation of the solid or liquid media for each of the different bacteria, amount of inoculum for each inoculum, duration of growth of the different bacteria, sourcing of solid or liquid media, or a combination thereof.
  • Embodiment 90 provides the method of embodiment 88 or embodiment 89 wherein the central processor transmits or otherwise makes available the protocol, or a portion of the protocol, to the one or more bacterial production facilities.
  • Embodiment 91 provides the method of embodiment 90 wherein the central processor receives information regarding the outcome of production of the different bacteria using the protocol.
  • Embodiment 92 provides the method of embodiment 91 wherein the central processor learns from the outcome and modifies future protocols based, at least in part, on the learning.
  • Embodiment 93 provides the method of any one of embodiments 75 through 90 wherein the central processor is remote from the one or more facilities.

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Abstract

L'invention concerne des procédés et des compositions pour faire croître une pluralité de bactéries différentes, chacune des bactéries étant cultivée sur un milieu séparé des autres, retirer les bactéries des milieux, et les associer pour produire un consortium microbien.
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