Microbial consortia for soil improvement

Abstract

Provided herein are methods and systems for producing functional microbial consortia, particularly but not exclusively, microbial consortia having a desired functionality, as well as techniques relating to microbial consortia produced according to such methods. The microbial consortia can be used, for example, to improve soil for agricultural purposes.

Description

[Technical field]

[0001] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under P30 CA91842 and UL1TR002345 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0002] Sequence Listing The text of the computer readable sequence listing submitted herein, entitled "PLUTON-40093-601_SQL", created on March 20, 2023, and having a file size of 300,854 bytes, is incorporated herein by reference in its entirety.

[0003] Field Provided herein are methods and systems for producing functional microbial consortia, particularly, but not limited to, microbial consortia having a desired function, as well as technology relating to microbial consortia produced according to such methods. [Background technology]

[0004] Agricultural practices often include methods to improve soil to maintain or increase crop yields. In particular, plant nutrients (e.g., carbon (C), nitrogen (N), phosphorus (P), and other nutrients) are often provided to the soil in the form of chemical or biological soil amendments. Chemical amendments include fertilizers containing ammonia to provide fixed nitrogen and mined phosphates to provide phosphorus. In addition, farmers often plant cover crops to return nitrogen, carbon, and other nutrients to the soil. While growing, the symbiotic microorganisms of the cover crop fix the nitrogen released into the soil, and the death and tillage of the cover crop returns organic carbon and other nutrients to the soil. More carbon in the soil leads to better nutrient and water retention, generally resulting in healthier soils and increased crop production. Since the beginning of agriculture, much of the soil carbon has been released into the atmosphere worldwide as a result of increased tillage, which destroys the soil and promotes the breakdown of carbon-based materials by microorganisms. Returning carbon to the soil benefits soil health and removes greenhouse gases from the atmosphere.

[0005] With regard to nitrogen, about one-third of the nitrogen fixed for agricultural use is produced by biological nitrogen fixation (BNF) using cover crops or legume crop rotations, while the remaining two-thirds is applied in chemical form as ammonia produced by the Haber-Bosch process. In the Haber-Bosch process, N2 from the air reacts with H2, derived mainly from methane in natural gas, to produce NH3 (ammonia). Although the Haber-Bosch process has been successful in fixing nitrogen for agricultural use, the process consumes up to 5% of the natural gas produced annually worldwide as a source of hydrogen and energy. As a result, the Haber-Bosch process is a major source of the greenhouse gas CO2. Furthermore, inorganic N applied to soils as ammonia or ammonium nitrate is readily converted to nitric oxide (NOx) in a process called denitrification. Nitric oxide, specifically N2O, is hundreds of times more potent as a greenhouse gas than CO2. As a result, chemical fertilizers result in large amounts of nitrogen waste, the production of which is accompanied by large amounts of greenhouse gas production and climate impacts. Plants provide nitrogen to soils through biological nitrogen fixation, which is carried out by specific microorganisms associated with the particular plant. However, it may be impossible or impractical to grow a particular species of plant on every soil, soil type, and location. For example, soybean plants fix large amounts of nitrogen using microbial-infested root nodules, but maize has very limited interactions with rhizospheric diazotrophs (nitrogen-fixing microorganisms) and requires large amounts of fertilizer application to grow economically. Summary of the Invention [Means for solving the problem]

[0006] (Abstract) Thus, in some embodiments, the technology provided herein relates to the use of microbial consortia to fix and deliver carbon and nitrogen, along with phosphorus and other essential nutrients, to the soil (or other plant growth medium). In some embodiments, the technology provides nutrients to the soil with much less greenhouse gas impact than other current agricultural practices.

[0007] In some embodiments, the present technology provides microbial soil amendments (e.g., comprising a microbial consortium) that deliver carbon (C) and nitrogen (N) to a plant growth medium (e.g., soil or synthetic growth medium). In some embodiments, the microbial soil amendments further deliver other nutrients (e.g., phosphorus) to the plant growth medium (e.g., soil or synthetic growth medium).

[0008] In particular, the technology described herein provides biological fertilizers, including microbial consortia optimized for efficient delivery of one or more of N, C, P, and / or other required nutrients to soil, as well as methods for producing biological fertilizers and / or microbial consortia. In some embodiments, the technology provides microbial consortia that are grown in a bioreactor and applied in the field. In some embodiments, the microbial consortia provide one or more sources of, for example, immobilized nitrogen, organic carbon, phosphorus, and other nutrients. In some embodiments, the microbial consortia continue to grow after application to soil and provide carbon and nitrogen to the soil after being provided to the soil. That is, in some embodiments, the microbial consortia remove carbon and / or nitrogen from the air through fixation facilitated by photosynthesis. Thus, embodiments of the technology provide an efficient source of biological nitrogen fixation that can replace the Haber-Bosch process that produces CO2. Additionally, embodiments of the technology remove carbon from the air and deposit it in the soil, thereby improving soil quality while reducing greenhouse gases in the air. In some embodiments, the technology provides a bioavailable source of phosphorus and other nutrients in the soil for plant growth.

[0009] In some embodiments, the technology provides a microbial consortium that provides a specific function. In some embodiments, the technology described herein includes providing a sample that includes several microorganisms (e.g., from one or more environmental samples) and / or a microbial consortium (e.g., including one or more microorganisms from a natural consortium and / or one or more microorganisms from various environments, ecosystems, habitats, and / or ecological niches), and produces a novel consortium that includes a novel combination of microorganisms that act in concert. By testing for functional variables, the microorganisms and microbial consortium that provide the desired function are sequenced and subcultured until the desired microorganisms and microbial consortium are identified and / or isolated. In some embodiments, a machine learning model is developed using a statistically significant amount of data that assists in the selection of the microorganisms and microbial consortium. In some embodiments, the machine learning model is supplemented with phenotypic data for the component microorganisms.

[0010] As described herein, embodiments of the technology (e.g., methods of producing microbial consortia) include assessing or confirming the functionality of the cultures and / or consortia (e.g., by evaluating a test variable) rather than focusing on the phenotype of the microorganisms. In some embodiments, functionality may include measures of carbon sequestration, nitrogen fixation, survival time, and / or persistence of the microorganisms.

[0011] Thus, the present technology provides a method of accumulating carbon and nitrogen in an agricultural medium. For example, in some embodiments, the method includes applying a composition comprising photosynthetic and nitrogen fixing microorganisms to an agricultural medium and incubating the agricultural medium in the presence of nitrogen gas (N2) and carbon dioxide (CO2) with illumination by a light source. In some embodiments, the agricultural medium includes soil, greenhouse growth medium, or hydroponic growth medium. In some embodiments, the photosynthetic and nitrogen fixing microorganisms are bacteria. In some embodiments, the photosynthetic and nitrogen fixing microorganisms are members of the Cyanobacteriales, Cyanobacteriota, Nostocales, Pseudoanabaenales, Spirulinales, or Oscillatoriales orders. In some embodiments, the photosynthetic and nitrogen fixing microorganism is selected from the group consisting of Acaryochloris, Aliinostoc, Aliterella, Amazonocrinis, Anabaena, Anabaenopsis, Aphanizomenon, Atlanticothrix, Aulosira, Calenema, Calothrix, and the like. hrix, Camptylonemopsis, Chlorogloeopsis, Chroococcidiopsis, Chrysosporum, Compactonostoc, Constrictifilum, Crocosphaera, Cyanobacterium, Cyanocohniella,Cylindrospermopsis, Cylindrospermum, Dendronalium, Desikacharya, Desmonostoc, Dolichospermum, Erythrobacter, Fischerella, Fortiea, Fremyella, Geitlerinema ma), Gloeocapsa, Gloeocapsopsis, Gloeothece, Gloeotrichia, Goleter, Hapalosiphon, Hydrocoryne, Jaaginema, Johanseniella, Kamptonema, Komarekiella, Leptolyngbya ), Lyngbya, Macrochaete, Mastigocoleus, Microchaete, Nodosilinea, Nodularia, Nostoc, Nostocaceae, Nostochopsis, Oligotropha, Oscillatoria, Pantanalinema, Peratocladus, Pelatocladus, Planktothrichoides, Planktothrix, Polymorphum, Porphyrobacter, Prochlorococcus, Prochlorothrix, Pseudanabaena, Pseudoaliinostoc, Raphidiopsis,The photosynthetic and nitrogen fixing microorganism is a member of the genus Richelia, Rivularia, Roholtiella, Rubidibacter, Scytonema, Spirulina, Synechococcus, Synechocystis, Thermosynechococcus, Tolypothrix, Trichormus, Violetonostoc, Wollea, or Xenococcus. In some embodiments, the photosynthetic and nitrogen fixing microorganism is a Nostoc species. In some embodiments, the photosynthetic and nitrogen fixing microorganism has a ribosomal RNA gene nucleotide sequence that is at least 90%, 95%, 97%, or 99% identical to at least one of SEQ ID NOs: 1, 3, 7, 10, 13, 19, 22, 26, 30, 32, 34, 35, 39, 42, 45, 51, 54, 57, 61, 63, 66, 70, 74, 84, 89, 99, 103, 108, 116, 122, 123, 125, or 127.

[0012] In some embodiments, the agricultural medium and the composition together comprise a first microbial mass, and the incubating step is performed until a second microbial mass is produced that is at least two times larger than the first microbial mass. In some embodiments, the agricultural medium comprises a first microbial mass, and the incubating step is performed until a second microbial mass is produced that is at least two times larger than the first microbial mass.

[0013] In some embodiments, the composition further comprises a microorganism capable of metabolizing the carbon-containing and nitrogen-containing compounds produced by the photosynthetic and nitrogen-fixing microorganisms. In some embodiments, the composition comprises a consortium comprising the photosynthetic and nitrogen-fixing microorganisms. In some embodiments, the composition comprises a consortium comprising the photosynthetic and nitrogen-fixing microorganisms, wherein a first microorganism is capable of metabolizing the carbon-containing compounds produced by the photosynthetic and nitrogen-fixing microorganisms and / or is capable of metabolizing the nitrogen-containing compounds produced by the photosynthetic and nitrogen-fixing microorganisms. In some embodiments, the consortium comprises a second microorganism capable of metabolizing the carbon-containing compounds produced by the photosynthetic and nitrogen-fixing microorganisms and / or is capable of metabolizing the nitrogen-containing compounds produced by the photosynthetic and nitrogen-fixing microorganisms. In some embodiments, the consortium comprises a second microorganism capable of metabolizing the carbon-containing compounds produced by the first microorganisms and / or is capable of metabolizing the nitrogen-containing compounds produced by the first microorganisms. In some embodiments, the consortium includes a second microorganism, and the first microorganism is capable of metabolizing a carbon-containing compound produced by the second microorganism and / or is capable of metabolizing a nitrogen-containing compound produced by the second microorganism. In some embodiments, the consortium produces sustainable carbon compounds that provide a carbon sink in the soil. In some embodiments, the consortium produces melanin. In some embodiments, the consortium produces pheomelanin, eumelanin, and / or pyomelanin.

[0014] In some embodiments, the consortium includes a second photosynthetic microorganism. In some embodiments, the second photosynthetic microorganism is a bacterium. In some embodiments, the second photosynthetic microorganism is an algae. In some embodiments, the second photosynthetic microorganism is selected from the group consisting of Nodularia, Chrysosporum, Gloeocapsopsis, Lychelia, Mastigocoleus, Hapalosiphon, Gloeothece, Acaryochloris, Camptonema, Raphidiopsis, Crocosphera, Macrochaete, Thermosynthecoccus, Pseudoanabaena, Chroococcidiopsis, Prochlorothrix, Anabaena, Leptoryngbia, Callosthrix, Cylindrospermopsis, Dolichospermum, Cytonema, Lyngbia, Tolyposthrix, Fischerella, Fortiera, Alitherella, Hydrococcus, and the like. The bacteria is a member of the genus Lynae, Prochlorococcus, Planctotrichoides, Gaitrelinema, Xenococcus, Yaginema, Nostocopsis, Pantanalinema, Oscillatoria, Spirulina, Pellatocladus, Nodococcus, Aphanizomenon, Chlorogloepsis, Gloeocapsa, Karenema, Livularia, Trichorum, Synechococcus, Synechocystis, Cylindrospermum, Planktothrix, Bosea, Shinella, Novosphingobium, or Rubisibacter.

[0015] In some embodiments, the algae is selected from the group consisting of Tetraselmis, Chlorella, Coleochaete, Gonium, Paradoxia, Cymbomonas, Palmaria, Characiochloris, Marvania, Chlamydomonas, Parachlorella, Coccomyxa, Trebouxia, Cyanidium, Treubaria, Dinjarde, and the like. In some embodiments, the consortium is a member of the genus Dangeardinia, Galdieria, Nyholmiella, Porphyra, Chara, Ankistrodesmus, Picochlorum, Cyanophora, Chondrus, Pseudochloris, Interfilum, Gelidium, Symphyogyna, Chlorosarcina, or Cyanothece. In some embodiments, the consortium includes a second nitrogen-fixing microorganism.

[0016] In some embodiments, the consortium further includes organisms from the genus Variovorax, organisms from the phylum Proteobacteria, organisms from the genus Bosea, organisms from the genus Caulobacter, and / or organisms from the genus Pseudomonas.

[0017] In some embodiments, the light source provides light having a wavelength between about 380 nm and 750 nm, hi some embodiments, the light source is an artificial light source.

[0018] In some embodiments, the technology further includes a method of accumulating carbon and nitrogen in an agricultural medium, the method including applying a composition comprising a photosynthetic microorganism and a nitrogen fixing microorganism to the agricultural medium, and incubating the agricultural medium in the presence of nitrogen gas (N2) and carbon dioxide (CO2) with irradiation by a light source. In some embodiments, the agricultural medium includes soil, greenhouse growth medium, or hydroponic growth medium. In some embodiments, the photosynthetic microorganism is algae. In some embodiments, the photosynthetic microorganism is bacteria. In some embodiments, the nitrogen fixing microorganism is bacteria.

[0019] In some embodiments, the agricultural medium and the composition together comprise a first microbial consortium, and the incubating step is performed until a second microbial consortium is produced that is at least two times larger than the first microbial consortium. In some embodiments, the agricultural medium comprises a first microbial consortium, and the incubating step is performed until a second microbial consortium is produced that is at least two times larger than the first microbial consortium. In some embodiments, the algae is a member of the genus Tetraselmis, Chlorella, Coreochaeta, Gonium, Paradoxia, Symbomonas, Palmaria, Calasiochloris, Marvania, Chlamydomonas, Parachlorella, Coccomyxa, Trebuxia, Cyanidium, Troybaria, Danjardinia, Galdieria, Nuformia, Porphyra, Cala, Anchistrodesmus, Picochlorum, Cyanophora, Chondrus, Pseudochloris, Interphyllum, Gelidium, Symphyogina, Chlorosarquina, or Cyanothece.

[0020] In some embodiments, the bacteria is selected from the group consisting of Nodularia, Chrysosporum, Gloeocapsopsis, Lychearia, Mastigocoleus, Hapalosiphon, Gloeothece, Acaryochloris, Camptonema, Raphidiopsis, Crocosphera, Macrochaete, Thermosynthecoccus, Pseudoanabaena, Chroococcidiopsis, Prochlorothrix, Anabaena, Leptoryngbia, Callosthrix, Cylindrospermopsis, Dolichospermum, Cytonema, Lyngbia, Tolyposthrix, Fischella, Phor The member of the genera is Thiaea, Alitella, Hydrocoryne, Prochlorococcus, Planctotrichoides, Gaitrelinema, Xenococcus, Yaginema, Nostocopsis, Pantanalinema, Oscillatoria, Spirulina, Pellatocladus, Nodococcus, Aphanizomenon, Chlorogloeopsis, Gloeocapsa, Karenema, Livularia, Trichorum, Synechococcus, Synechocystis, Cylindrospermum, Planktothrix, Bosea, Synella, Novosphingobium, or Rubisibacter.

[0021] In some embodiments, the bacterium is a member of the genus Alliinostoc, Amazonocrinis, Anabaena, Anabaenopsis, Atlanticothrix, Aurocilla, Caloshrix, Camptilonemopsis, Chrysosporum, Compactostoc, Constrictiphyllum, Cyanobacterium, Cyanocorniella, Cylindrospermum, Dendronarium, Desicacharia, Desmonostoc, Dolichospermum, Erythrobacter, Fortieria, Fremiella, Gaitrelinema, Gloeotrichia, Goleter, Johanseniella, Komarekiella, Microchaete, Nodularia, Nostoc, Nostocaseae, Oligotropha, Polymorpham, Porphyrobacter, Pseudoaliinostoc, Rohorchiella, Cytonema, Tolyposthrix, Trichorum, Violetnostoc, or Wallea.

[0022] In some embodiments, the bacteria is selected from the group consisting of Acaryochloris, Alliinostoc, Alitella, Amazonocrinis, Anabaena, Anabaenopsis, Aphanizomenon, Atlanticothrix, Aurocilla, Karenema, Calloshrix, Camptilonemopsis, Chlorogloeopsis, Chroococcidiopsis, Chrysosporum, Compactostoc, Constrictiphyllum, Crocosphera, Cyanobacterium, Cyanocorniella, Cylindrospermopsis, Cylindrospermum, Dendronarium, Desicacharia, Desmonostoc, Dolichospermum, Fischerella, Fortiera, Fremiella, Gaitrelinema, Gloeocapsa, Gloeocapsopsis, Gloeothece, Gloeotrichia, Goleter, Hapalosi The member of the genera is selected from the group consisting of Phon, Hydrocoryne, Yaginema, Johanseniella, Camptonema, Komarekiella, Leptolynbya, Lyngbya, Macrochaete, Mastigocoleus, Microchaete, Nodocilinea, Nodularia, Nostoc, Nostocaseae, Nostocoptis, Oscillatoria, Pantanalinema, Pelatocladus, Planctotrichoides, Planktothrix, Prochlorococcus, Prochlorothrix, Pseudoanabaena, Pseudoariinostoc, Raphidiopsis, Rickeria, Livularia, Rohorchiella, Rubisibacter, Cytonema, Spirulina, Synechococcus, Synechocystis, Thermosynechococcus, Tolyposthrix, Trichorum, Bioletnostoc, Wallea, and Xenococcus. In some embodiments, the bacterium has a ribosomal RNA gene nucleotide sequence that is at least 90%, 95%, 97%, or 99% identical to at least one of SEQ ID NOs: 1, 3, 7, 10, 13, 19, 22, 26, 30, 32, 34, 35, 39, 42, 45, 51, 54, 57, 61, 63, 66, 70, 74, 84, 89, 99, 103, 108, 116, 122, 123, 125, or 127.

[0023] In some embodiments, the composition further comprises a microorganism capable of metabolizing the carbon-containing and nitrogen-containing compounds produced by the photosynthetic and nitrogen-fixing microorganisms. In some embodiments, the composition comprises a consortium comprising the photosynthetic microorganisms and the nitrogen-fixing microorganisms. In some embodiments, the composition comprises a consortium comprising the photosynthetic microorganisms and the nitrogen-fixing microorganisms, wherein a first microorganism is capable of metabolizing the carbon-containing compounds produced by the photosynthetic microorganisms and the nitrogen-fixing microorganisms and / or is capable of metabolizing the nitrogen-containing compounds produced by the photosynthetic microorganisms and the nitrogen-fixing microorganisms. In some embodiments, the consortium comprises a second microorganism ... first microorganisms and / or is capable of metabolizing the nitrogen-containing compounds produced by the first microorganisms. In some embodiments, the consortium includes a second microorganism, and the first microorganism is capable of metabolizing a carbon-containing compound produced by the second microorganism and / or is capable of metabolizing a nitrogen-containing compound produced by the second microorganism. In some embodiments, the consortium produces sustainable carbon compounds that provide a carbon sink in the soil. In some embodiments, the consortium produces melanin. In some embodiments, the consortium produces pheomelanin, eumelanin, and / or pyomelanin.

[0024] In some embodiments, the consortium further includes organisms from the genus Variovorax, organisms from the phylum Proteobacteria, organisms from the genus Bosea, organisms from the genus Caulobacter, and / or organisms from the genus Pseudomonas.

[0025] In some embodiments, the light source provides light having a wavelength between about 380 nm and 750 nm. In some embodiments, the light source is an artificial light source. In some embodiments, the light source is the sun.

[0026] Further, in some embodiments, the present technology provides a method for producing a microbial consortium, the method comprising providing an aqueous medium free of fixed carbon and free of fixed nitrogen, inoculating the medium with a sample containing microorganisms to produce an inoculated medium, and exposing the inoculated medium to a gas containing N2 and CO2 and light. In some embodiments, the light comprises a wavelength of about 380 nm to 750 nm. In some embodiments, the gas containing N2 and CO2 is air. In some embodiments, the gas containing N2 and CO2 is provided by purified N2 and CO2. In some embodiments, the inoculated medium comprises a first microbial consortium, and the exposing step is performed until a second microbial consortium is produced that is at least two times larger than the first microbial consortium. In some embodiments, the consortium produces sustainable carbon compounds that provide a carbon sink in the soil. In some embodiments, the consortium produces melanin. In some embodiments, the consortium produces pheomelanin, eumelanin, and / or pyomelanin. In some embodiments, the community includes microorganisms having a ribosomal RNA gene nucleotide sequence that is at least 90%, 95%, 97%, or 99% identical to one or more of SEQ ID NOs: 1, 3, 7, 10, 13, 19, 22, 26, 30, 32, 34, 35, 39, 42, 45, 51, 54, 57, 61, 63, 66, 70, 74, 84, 89, 99, 103, 108, 116, 122, 123, 125, or 127.

[0027] In some embodiments, the present technology provides compositions comprising a microbial consortium including photosynthetic and nitrogen-fixing microorganisms or including photosynthetic and nitrogen-fixing microorganisms, and a microorganism capable of metabolizing carbon- and nitrogen-containing compounds produced by the photosynthetic and nitrogen-fixing microorganisms. In some embodiments, the photosynthetic and nitrogen-fixing microorganisms are bacteria. In some embodiments, the photosynthetic and nitrogen-fixing microorganisms are members of the Cyanobacteriales. In some embodiments, the photosynthetic and nitrogen-fixing microorganisms are members of the Cyanobacteriales, Cyanobacteriota, Nostocales, Pseudoanabaenales, Spirulinales, or Oscillatoryes orders. In some embodiments, the photosynthetic and nitrogen-fixing microorganisms are members of the Nostocales order.In some embodiments, the photosynthetic and nitrogen fixing microorganisms include Acaryochloris, Alliinostoc, Alitella, Amazonocrinis, Anabaena, Anabaenopsis, Aphanizomenon, Atlanticothrix, Aurocilla, Karenema, Calloshrix, Camptilonemopsis, Chlorogloeopsis, Chroococcidiopsis, Chrysosporum, Compactostoc, Constrictiphyllum, Crocosphera, Cyanobacterium, Cyanocorniella, Cylindrospermopsis, Cylindrospermum, Dendronarium, Desicacharia, Desmonostoc, Dolichospermum, Erythrobacter, Fischerella, Fortiera, Fremiella, Gaitrelinema, Gloeocapsa, Gloeocapsopsis, Gloeothece, Gloeotrichia, Goleter, Hapalosi, The bacteria may be a member of the genus Phon, Hydrocoryne, Yaginema, Johanseniella, Camptonema, Komarekiella, Leptolynbya, Lyngbya, Macrochaete, Mastigocoleus, Microchaete, Nodocilinea, Nodularia, Nostoc, Nostocaseae, Nostocoptis, Oligotropha, Oscillatoria, Pantanalinema, Pelatocladus, Planctotrichoides, Planktothrix, Polymorpham, Porphyrobacter, Prochlorococcus, Prochlorothrix, Pseudoanabaena, Pseudoariinostoc, Raphidiopsis, Richeria, Livularia, Rohorchiella, Rubisibacter, Cytonema, Spirulina, Synechococcus, Synechocystis, Thermosynechococcus, Tolyposthrix, Trichorum, Bioletnostoc, Wallea, or Xenococcus. In some embodiments, the photosynthetic and nitrogen fixing microorganism is a Nostoc species. In some embodiments, the photosynthetic and nitrogen fixing microorganism has a ribosomal RNA gene nucleotide sequence that is at least 90%, 95%, 97%, or 99% identical to one or more of SEQ ID NOs: 1, 3, 7, 10, 13, 19, 22, 26, 30, 32, 34, 35, 39, 42, 45, 51, 54, 57, 61, 63, 66, 70, 74, 84, 89, 99, 103, 108, 116, 122, 123, 125, or 127.

[0028] In some embodiments, the nitrogen fixing species is a microorganism from the genera Variovorax, Pseudomonas, Cupriavidus, Brevundimonas, Opitutus, Runella, Taylorella, Tahibacter, Polaromonas, Chitinophaga, Ferruginibacter, Enhydrobacter, Hymenobacter, or Ramlibacter. In some embodiments, the photosynthetic species is a microorganism from the genera Bosea, Cinella, or Novosphingobium.

[0029] In some embodiments, the consortium produces sustainable carbon compounds that provide a carbon sink in the soil. In some embodiments, the consortium produces melanin. In some embodiments, the consortium produces pheomelanin, eumelanin, and / or pyomelanin.

[0030] In some embodiments, the composition comprises a second microorganism capable of metabolizing the carbon-containing compounds produced by the photosynthetic and nitrogen-fixing microorganism and / or capable of metabolizing the nitrogen-containing compounds produced by the photosynthetic and nitrogen-fixing microorganism. In some embodiments, the consortium comprises a second microorganism capable of metabolizing the carbon-containing compounds produced by the first microorganism and / or capable of metabolizing the nitrogen-containing compounds produced by the first microorganism. In some embodiments, the consortium comprises a second microorganism, said first microorganism capable of metabolizing the carbon-containing compounds produced by the second microorganism and / or capable of metabolizing the nitrogen-containing compounds produced by the second microorganism.

[0031] In some embodiments, the consortium includes a second photosynthetic microorganism. In some embodiments, the second photosynthetic microorganism is a bacterium. In some embodiments, the second photosynthetic microorganism is an algae. In some embodiments, the photosynthetic bacteria include Acaryochloris, Aliinostoc, Alitella, Amazonocrinis, Anabaena, Anabaenopsis, Aphanizomenon, Atlanticothrix, Aulosira, Karenema, Calloshrix, Camptilonemopsis, Chlorogloeopsis, Chroococcidiopsis, Chrysosporum, Compactostoc, Constrictiphyllum, Crocosphera, Cyanobacterium, Cyanocorniella, Cylindrospermopsis, Cylindrospermum, Dendronarium, Desicacharia, Desmonostoc, Dolichospermum, Fischerella, Fortiera, Fremiella, Gaitrelinema, Gloeocapsa, Gloeocapsopsis, Gloeothece, Gloeotrichia, Goleter, Hapalosiphon, Hydro The member of the genera is Corynae, Yaginema, Johanseniella, Camptonema, Komarekiella, Leptoryngbia, Lyngbia, Macrochaete, Mastigocoleus, Microchaete, Nodocilinea, Nodularia, Nostoc, Nostococeae, Nostocoptis, Oscillatoria, Pantanalinema, Pelatocladus, Planctotrichoides, Planktothrix, Prochlorococcus, Prochlorothrix, Pseudoanabaena, Pseudoariinostoc, Raphidiopsis, Richeria, Livularia, Rohorchiella, Rubidibacter, Cytonema, Spirulina, Synechococcus, Synechocystis, Thermosynechococcus, Tolyposthrix, Trichorum, Bioletnostoc, Wallea, Bosea, Synella, Novosphingobium, or Xenococcus.In some embodiments, the algae is a member of the following genus: Tetraselmis, Chlorella, Coreochaeta, Gonium, Paradoxia, Symbomonas, Palmaria, Calasiochloris, Marvania, Chlamydomonas, Parachlorella, Coccomyxa, Trebuxia, Cyanidium, Troybaria, Danjardinia, Gardieria, Nuformia, Porphyra, Cara, Anchistrodesmus, Picochlorum, Cyanophora, Chondrus, Pseudochloris, Interphyllum, Gelidium, Symphyogina, Chlorosarquina, or Cyanothece.In some embodiments, the consortium is selected from the group consisting of Variovorax, Pseudomonas, Cupriavidus, Blevundimonas, Opitutus, Lunella, Teylorella, Tahibacter, Polaromonas, Chitinophaga, Ferruginibacter, Enhydrobacter, Hymenobacter, Ramulibacter, Acaryochloris, Aliinostoc, Aliterella, Amazonocrinis, Anabaena, Anabaenopsis, Aphanizomenon, Atlanticohthricus, Aurocilla, Calaminella ... Nema, Caloshrix, Camptilonemopsis, Chlorogloeopsis, Chroococcidiopsis, Chrysosporum, Compactostoc, Constrictiphyllum, Crocosphera, Cyanobacterium, Cyanocorniella, Cylindrospermopsis, Cylindrospermum, Dendronarium, Desicacharia, Desmonostoc, Dolichospermum, Erythrobacter, Fischerella, Fortiera, Fremiella, Gaitrelinema, Glomerulonema, Rhoeocapsa, Gloeocapsopsis, Gloeotese, Gloeotrichia, Goleter, Hapalosiphon, Hydrocoryne, Yergynema, Johanseniella, Camptonema, Komarekiella, Leptolyngbia, Lyngbia, Macrochaete, Mastigocoleus, Microchaete, Nodocilinea, Nodularia, Nostoc, Nostocaseae, Nostocoptis, Oligotropha, Oscillatoria, Pantanalinema, Pelatocladus, Planctotrichoides, Plan and a second nitrogen fixing microorganism from the genus K. spp., ...

[0032] In some embodiments, the consortium further includes organisms from the genus Variovorax, organisms from the phylum Proteobacteria, organisms from the genus Bosea, organisms from the genus Caulobacter, and / or organisms from the genus Pseudomonas.

[0033] A further embodiment provides a system that includes a fixed carbon-free, fixed nitrogen-free aqueous medium, a source of gas including N2 and CO2, a light source, and a sample including microorganisms.

[0034] Further embodiments of the system provide a system comprising a fixed carbon-free, fixed nitrogen-free aqueous medium, a source of gas including N2 and CO2, a light source, and a microbial consortium including photosynthetic and nitrogen-fixing microorganisms or including photosynthetic and nitrogen-fixing microorganisms, and a microorganism capable of metabolizing carbon-containing and nitrogen-containing compounds produced by the photosynthetic and nitrogen-fixing microorganisms. In some embodiments, the photosynthetic and nitrogen-fixing microorganisms are bacteria. In some embodiments, the photosynthetic and nitrogen-fixing microorganisms are members of the Cyanobacteriales. In some embodiments, the photosynthetic and nitrogen-fixing microorganisms are members of the orders Cyanobacteriales, Cyanobacteriota, Nostocales, Pseudoanabaenales, Spirulinales, or Oscillatoryes. In some embodiments, the photosynthetic and nitrogen-fixing microorganisms are members of the orders Nostocales.In some embodiments, the photosynthetic and nitrogen fixing microorganisms include Acaryochloris, Alliinostoc, Alitella, Amazonocrinis, Anabaena, Anabaenopsis, Aphanizomenon, Atlanticothrix, Aurocilla, Karenema, Calloshrix, Camptilonemopsis, Chlorogloeopsis, Chroococcidiopsis, Chrysosporum, Compactostoc, Constrictiphyllum, Crocosphera, Cyanobacterium, Cyanocorniella, Cylindrospermopsis, Cylindrospermum, Dendronarium, Desicacharia, Desmonostoc, Dolichospermum, Erythrobacter, Fischerella, Fortiera, Fremiella, Gaitrelinema, Gloeocapsa, Gloeocapsopsis, Gloeothece, Gloeotrichia, Goleter, Hapalosi, The bacteria may be a member of the genus Phon, Hydrocoryne, Yaginema, Johanseniella, Camptonema, Komarekiella, Leptolynbya, Lyngbya, Macrochaete, Mastigocoleus, Microchaete, Nodocilinea, Nodularia, Nostoc, Nostocaseae, Nostocoptis, Oligotropha, Oscillatoria, Pantanalinema, Pelatocladus, Planctotrichoides, Planktothrix, Polymorpham, Porphyrobacter, Prochlorococcus, Prochlorothrix, Pseudoanabaena, Pseudoariinostoc, Raphidiopsis, Richeria, Livularia, Rohorchiella, Rubisibacter, Cytonema, Spirulina, Synechococcus, Synechocystis, Thermosynechococcus, Tolyposthrix, Trichorum, Bioletnostoc, Wallea, or Xenococcus. In some embodiments, the photosynthetic and nitrogen fixing microorganism is a Nostoc species. In some embodiments, the photosynthetic and nitrogen fixing microorganism has a ribosomal RNA gene nucleotide sequence that is at least 90%, 95%, 97%, or 99% identical to one or more of SEQ ID NOs: 1, 3, 7, 10, 13, 19, 22, 26, 30, 32, 34, 35, 39, 42, 45, 51, 54, 57, 61, 63, 66, 70, 74, 84, 89, 99, 103, 108, 116, 122, 123, 125, or 127.

[0035] In some embodiments, the nitrogen fixing species is a microorganism from the genera Variovorax, Pseudomonas, Cupriavidus, Blevundimonas, Opitutus, Lunella, Teylorella, Tahibacter, Polaromonas, Chitinophaga, Ferruginibacter, Enhydrobacter, Hymenobacter, or Ramulibacter. In some embodiments, the photosynthetic species is a microorganism from the genera Bosea, Cinella, or Novosphingobium.

[0036] In some embodiments, the consortium produces sustainable carbon compounds that provide a carbon sink in the soil. In some embodiments, the consortium produces melanin. In some embodiments, the consortium produces pheomelanin, eumelanin, and / or pyomelanin.

[0037] In some embodiments, the composition comprises a second microorganism capable of metabolizing the carbon-containing compounds produced by the photosynthetic and nitrogen-fixing microorganism and / or capable of metabolizing the nitrogen-containing compounds produced by the photosynthetic and nitrogen-fixing microorganism. In some embodiments, the consortium comprises a second microorganism capable of metabolizing the carbon-containing compounds produced by the first microorganism and / or capable of metabolizing the nitrogen-containing compounds produced by the first microorganism. In some embodiments, the consortium comprises a second microorganism, said first microorganism capable of metabolizing the carbon-containing compounds produced by the second microorganism and / or capable of metabolizing the nitrogen-containing compounds produced by the second microorganism.

[0038] In some embodiments, the consortium includes a second photosynthetic microorganism. In some embodiments, the second photosynthetic microorganism is a bacterium. In some embodiments, the second photosynthetic microorganism is an algae. In some embodiments, the photosynthetic bacteria is selected from the group consisting of Acaryochloris, Alliinostoc, Alitella, Amazonocrinis, Anabaena, Anabaenopsis, Aphanizomenon, Atlanticothrix, Aulosira, Karenema, Calloshrix, Camptilonemopsis, Chlorogloeopsis, Chroococcidiopsis, Chrysosporum, Compactostoc, Constrictiphyllum, Crocosphera, Cyanobacterium, Cyanocorniella, Cylindrospermopsis, Cylindrospermum, Dendronarium, Desicacharia, Desmonostoc, Dolichospermum, Erythrobacter, Fischerella, Fortiera, Fremiella, Gaitrelinema, Gloeocapsa, Gloeocapsopsis, Gloeotese, Gloeotrichia, Goleter, Hapalosiphon, Hydrocoryne, Yaginema , Johanseniella, Camptonema, Komarekiella, Leptoryngbia, Lyngbia, Macrochaete, Mastigocoleus, Microchaete, Nodocilinea, Nodularia, Nostoc, Nostococeae, Nostocpsis, Oligotropha, Oscillatoria, Pantanalinema, Pelatocladus, Planctotrichoides, Planktothrix, Polymorpham, Porphyrobacter, Prochlorococcus, Prochlorothrix, Pseudoanabaena, Pseudoariinostoc, Raphidiopsis, Richeria, Livularia, Loforchiella, Rubidibacter, Cytonema, Spirulina, Synechococcus, Synechocystis, Thermosynechococcus, Tolyposthrix, Trichorum, Bioletnostoc, Wallea, Bosea, Synella, Novosphingobium, or Xenococcus.In some embodiments, the algae is a member of the following genus: Tetraselmis, Chlorella, Coreochaeta, Gonium, Paradoxia, Symbomonas, Palmaria, Calasiochloris, Marvania, Chlamydomonas, Parachlorella, Coccomyxa, Trebuxia, Cyanidium, Troybaria, Danjardinia, Gardieria, Nuformia, Porphyra, Cara, Anchistrodesmus, Picochlorum, Cyanophora, Chondrus, Pseudochloris, Interphyllum, Gelidium, Symphyogina, Chlorosarquina, or Cyanothece. In some embodiments, the consortium includes a second nitrogen-fixing microorganism from the genus: Variovorax, Pseudomonas, Cupriavidus, Blevundimonas, Opitutus, Lunella, Theylorella, Tahibacter, Polaromonas, Chitinophaga, Ferruginibacter, Enhydrobacter, Hymenobacter, or Ramulibacter.

[0039] In some embodiments, the consortium further includes organisms from the genus Variovorax, organisms from the phylum Proteobacteria, organisms from the genus Bosea, organisms from the genus Caulobacter, and / or organisms from the genus Pseudomonas.

[0040] In some embodiments, the present technology provides a composition comprising a synthetic growth medium and a microbial organism that collectively performs photosynthesis and nitrogen fixation. In some embodiments, a first microbial species performs photosynthesis. In some embodiments, the first microbial species that performs photosynthesis is a microorganism from the genus Bosea, Cinella, or Novosphingobium. In some embodiments, a second microbial species performs nitrogen fixation. In some embodiments, the second microbial species that performs nitrogen fixation is a microorganism from the genus Variovorax, Pseudomonas, Cupriavidus, Brevundimonas, Opitutus, Lunella, Teylorella, Tahibacter, Polaromonas, Chitinophaga, Ferruginibacter, Enhydrobacter, Hymenobacter, or Ramulibacter.

[0041] In some embodiments, the first microbial species performs photosynthesis and nitrogen fixation. In some embodiments, the first microbial species performs photosynthesis and nitrogen fixation is a bacterium. In some embodiments, the first microbial species performs photosynthesis and nitrogen fixation is a member of the Cyanobacteria order. In some embodiments, the first microbial species performs photosynthesis and nitrogen fixation is a member of the Cyanobacteriales, Cyanobacteriota, Nostocales, Pseudoanabaenales, Spirulinales, or Oscillatoryes order. In some embodiments, the first microbial species performs photosynthesis and nitrogen fixation is a member of the Nostocales order. In some embodiments, the first photosynthetic and nitrogen fixing microbial species is selected from the group consisting of Acaryochloris, Aliinostoc, Alitella, Amazonocrinis, Anabaena, Anabaenopsis, Aphanizomenon, Atlanticothrix, Aulosira, Karenema, Callothrix, Camptilonemopsis, Chlorogloeopsis, Chroococcidiopsis, Chrysosporum, Compactostoc, Constrictiphyllum, Crocosphera, Cyanobacterium, Cyanocorniella, Cylindrospermopsis, Cylindrospermum, Dendronarium, Desicacharia, Desmonostoc, Dolichospermum, Erythrobacter, Fischerella, Fortiera, Fremiella, Gaitrelinema, Gloeocapsa, Gloeocapsopsis, Gloeothece, Gloeotricia, Goleter, Hapa, The bacteria may be a member of the genus Rosiphon, Hydrocoryne, Yaginema, Johanseniella, Camptonema, Komarekiella, Leptolynbya, Lyngbya, Macrochaete, Mastigocoleus, Microchaete, Nodocilinea, Nodularia, Nostoc, Nostocaseae, Nostocoptis, Oligotropha, Oscillatoria, Pantanalinema, Pelatocladus, Planctotrichoides, Planktothrix, Polymorpham, Porphyrobacter, Prochlorococcus, Prochlorothrix, Pseudoanabaena, Pseudoariinostoc, Raphidiopsis, Richeria, Livularia, Rohorchiella, Rubidibacter, Cytonema, Spirulina, Synechococcus, Synechocystis, Thermosynechococcus, Tolyposthrix, Trichorum, Bioletnostoc, Wallea, or Xenococcus.In some embodiments, the first microbial species performing photosynthesis and nitrogen fixation is a Nostoc species. In some embodiments, the first microbial species performing photosynthesis and nitrogen fixation is a microorganism having a ribosomal RNA gene nucleotide sequence that is at least 90%, 95%, 97%, or 99% identical to SEQ ID NO: 1, 3, 7, 10, 13, 19, 22, 26, 30, 32, 34, 35, 39, 42, 45, 51, 54, 57, 61, 63, 66, 70, 74, 84, 89, 99, 103, 108, 116, 122, 123, 125, or 127. In some embodiments, the composition further comprises a second microbial species performing supplemental nitrogen fixation. In some embodiments, the second microbial species performing supplemental nitrogen fixation is a microorganism from the following genus: Variovorax, Pseudomonas, Cupriavidus, Blevundimonas, Opitutus, Lunella, Theylorella, Tahibacter, Polaromonas, Chitinophaga, Ferruginibacter, Enhydrobacter, Hymenobacter, or Ramulibacter.

[0042] In some embodiments, the composition further comprises a sustainable carbon compound that provides a carbon sink in the soil. In some embodiments, the microbial life form produces a sustainable carbon compound that provides a carbon sink in the soil. In some embodiments, the microbial life form produces melanin. In some embodiments, the microbial life form produces pheomelanin, eumelanin, and / or pyomelanin. In some embodiments, the composition further comprises melanin. In some embodiments, the composition further comprises pheomelanin, eumelanin, and / or pyomelanin.

[0043] In some embodiments, the microbial life forms comprise, consist of, or consist essentially of fewer than 10, fewer than 9, fewer than 8, fewer than 7, fewer than 6, fewer than 5, fewer than 4, or fewer than 3 species. In some embodiments, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the life forms of the microbial community are provided by the photosynthetic and nitrogen fixing microorganism and the second microorganism. In some embodiments, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 108%, 109%, 109%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109 ...9%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 1 %, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more are provided by microbial life forms that collectively perform photosynthesis and nitrogen fixation.

[0044] In some embodiments, the synthetic growth medium is a fixed carbon free medium. In some embodiments, the synthetic growth medium is a fixed nitrogen free medium. In some embodiments, the synthetic growth medium is a fixed carbon free, fixed nitrogen free medium. In some embodiments, the synthetic growth medium is a carbon free medium. In some embodiments, the synthetic growth medium is a nitrogen free medium. In some embodiments, the synthetic growth medium is a carbon free, nitrogen free medium. In some embodiments, a method for accumulating carbon and nitrogen in an agricultural medium is provided, the method comprising applying to the agricultural medium a composition comprising a synthetic growth medium and a microbial organism that collectively performs photosynthesis and nitrogen fixation. In some embodiments, the method further comprises exposing the composition to nitrogen gas (N2), carbon dioxide (CO2), and light. In some embodiments, the light is sunlight. In some embodiments, the agricultural medium is a crop field. In some embodiments, the agricultural medium comprises soil, greenhouse growth medium, or hydroponic growth medium. In some embodiments, the method comprises measuring fixed nitrogen and / or fixed carbon in the soil. In some embodiments, melanin provides fixed carbon in the soil. In some embodiments, pheomelanin, eumelanin, and / or pyomelanin provide fixed carbon in the soil.

[0045] Portions of this specification describe embodiments of the present technology in terms of algorithms and symbolic representations of operations on information. These algorithmic descriptions and representations are commonly used by those skilled in the art of data processing to effectively convey the substance of their work to others skilled in the art. These operations may be described functionally, computationally, or logically, and may be understood to be implemented by computer programs or equivalent electrical circuits, microcode, or the like. Furthermore, without loss of generality, it has also proven convenient at times to refer to arrangements of these operations as modules. The described operations and their associated modules may be embodied in software, firmware, hardware, or any combination thereof.

[0046] Certain steps, operations, or processes described herein may be implemented or performed by one or more hardware or software modules, alone or in combination with other devices. In some embodiments, a software module is implemented by a computer program product that includes a computer readable medium that includes computer program code, which can be executed by a computer processor to perform any or all of the steps, operations, or processes described.

[0047] In some embodiments, the system includes a computer and / or data storage that is provided virtually (e.g., as a cloud computing resource). In certain embodiments, the technology includes the use of cloud computing to provide a virtual computer system that includes components and / or performs the functions of the computers described herein. That is, in some embodiments, cloud computing provides the infrastructure, applications, and software described herein through a network and / or over the Internet. In some embodiments, computing resources (e.g., data analysis, calculations, data storage, application programs, file storage, etc.) are provided remotely over a network (e.g., the Internet and / or a cellular network).

[0048] An embodiment of the present technology may also relate to an apparatus for performing the operations herein. The apparatus may be specially constructed for the required purposes and / or may include a general-purpose computing device selectively activated or reconfigured by a computer program stored in the computer. Such a computer program may be stored in a non-transitory tangible computer-readable storage medium or any type of medium suitable for storing electronic instructions, which may be coupled to a computer system bus. Furthermore, any computing system referred to herein may include a single processor or may be an architecture employing a multi-processor design to increase computing power.

[0049] Further embodiments will be apparent to those skilled in the relevant art(s) based on the teachings contained herein.

[0050] The patent or application file contains at least one drawing executed in color. Copies of this invention or invention application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0051] These and other features, aspects, and advantages of the present technology will be better understood in connection with the following drawings. [Brief description of the drawings]

[0052] [Figure 1A] Light microscope images of BW01 cells are shown. The scale bar indicates a distance of 50 μm. Possible heterocyst structures are marked with "hc." [Figure 1B] Light microscope images of BW02 cells are shown. The scale bar indicates a distance of 50 μm. Possible heterocyst structures are marked with "hc." [Diagram 2] Heatmap showing the relative abundance of genera in the initial soil samples and the P1-P4 passages that produced the BW01, BW02, and BW05 consortia. Darker colors indicate higher read abundance as determined using Kraken2. [Figure 3A] Plot showing the relative abundance of Aminobacter in BW01, BW02, and BW05 as a function of passage from original soil sample through P1 to P4. Error bars indicate standard deviation (original soil sample n=1; P1 n=1; P2 n=2; P3 n=4; and P4 n=4). [Figure 3B] FIG. 13 is a plot showing the relative abundance of Bradyrhizobium in BW01, BW02, and BW05 as a function of passage from the original soil sample through P1 to P4. Error bars indicate standard deviation (original soil sample n=1; P1 n=1; P2 n=2; P3 n=4; and P4 n=4). [Figure 3C] Figure 1 shows a plot of the relative abundance of Mesorhizobium in BW01, BW02, and BW05 as a function of passage from original soil samples through P1 to P4. Error bars indicate standard deviation (original soil sample n=1; P1 n=1; P2 n=2; P3 n=4; and P4 n=4). [Figure 3D] Plot showing the relative abundance of Nostoc in BW01, BW02, and BW05 as a function of passage from the original soil sample through P1 to P4. Error bars indicate standard deviation (original soil sample n=1; P1 n=1; P2 n=2; P3 n=4; and P4 n=4). [Figure 3E] Plot showing the relative abundance of Pseudomonas in BW01, BW02, and BW05 as a function of passage from the original soil sample through P1 to P4. Error bars indicate standard deviation (original soil sample n=1; P1 n=1; P2 n=2; P3 n=4; and P4 n=4). [Figure 3F] Plot showing the relative abundance of Variovorax in BW01, BW02, and BW05 as a function of passage from the original soil sample through P1 to P4. Error bars indicate standard deviation (original soil sample n=1; P1 n=1; P2 n=2; P3 n=4; and P4 n=4). [Figure 3G]Figure 1 shows a plot of the relative abundance of Streptomyces in BW01, BW02, and BW05 as a function of passage from original soil samples through P1 to P4. Error bars indicate standard deviation (original soil sample n=1; P1 n=1; P2 n=2; P3 n=4; and P4 n=4). [Figure 3H] Plot showing the relative abundance of Azospirillum in BW01, BW02, and BW05 as a function of passage from original soil samples through P1 to P4. Error bars indicate standard deviation (original soil sample n=1; P1 n=1; P2 n=2; P3 n=4; and P4 n=4). [Figure 3I] Figure 1 shows a plot of the relative abundance of Rhizobium in BW01, BW02, and BW05 as a function of passage from original soil samples through P1 to P4. Error bars indicate standard deviation (original soil sample n=1; P1 n=1; P2 n=2; P3 n=4; and P4 n=4). [Figure 4] 1 is a plot showing the growth curves of all P4 samples for BW01, BW02, and BW05. The growth curves were determined by measuring the dry mass (grams) of cells in culture as a function of time (days). Dry mass is per 7.5 mL well in a standard 6-well plate. [Diagram 5] 1 is a series of plots showing the change in relative abundance of specific genes identified in nucleotide sequences from BW01, BW02, and BW05 consortia as a function of passage: nitrogenase subunit h (nifh), photosystem II subunits (psba and psbb), ribulose-1,5-bisphosphate carboxylase / oxygenase (rubiscoL and rubiscoS), and polysaccharide biosynthesis and export protein (wza) involved in EPS biosynthesis. Plots show gene presence by log10 gene count from original soil sample to P4. EggNOG orthologs with functional annotation are listed in Table 4. [Figure 6]9A-9C are a series of photographs showing the morphology of colony picks ("minimal consortia") when grown in liquid medium with illumination. Images were taken from bottom culture flasks illuminated from above. Culture morphology is indicated as "light," "dark," "filament," "restreaked," or "colonies." Picked colonies are identified as "P" numbers. See FIG. 9B. [Figure 7A] Figure 1 shows the taxonomy of the minimal community assessed by Phyloflash. Identified taxa are shown on the y-axis and sample ID and morphology on the x-axis. [Figure 7B] A series of plots showing the change in relative abundance of specific genes identified in nucleotide sequences obtained from colony picks ("minimal communities"). (B) Nitrogenase subunit h (nifh); (C and D) Photosystem II subunits (psba and psbb); (E) Ribulose-1,5-bisphosphate carboxylase / oxygenase (rubiscoL and rubiscoS); (F) Polysaccharide biosynthesis and export protein involved in EPS biosynthesis (wza). EggNOG orthologs with functional annotation are listed in Table 4. [Figure 7C] A series of plots showing the change in relative abundance of specific genes identified in nucleotide sequences obtained from colony picks ("minimal communities"). (B) Nitrogenase subunit h (nifh); (C and D) Photosystem II subunits (psba and psbb); (E) Ribulose-1,5-bisphosphate carboxylase / oxygenase (rubiscoL and rubiscoS); (F) Polysaccharide biosynthesis and export protein involved in EPS biosynthesis (wza). EggNOG orthologs with functional annotation are listed in Table 4. [Figure 7D]A series of plots showing the change in relative abundance of specific genes identified in nucleotide sequences obtained from colony picks ("minimal communities"). (B) Nitrogenase subunit h (nifh); (C and D) Photosystem II subunits (psba and psbb); (E) Ribulose-1,5-bisphosphate carboxylase / oxygenase (rubiscoL and rubiscoS); (F) Polysaccharide biosynthesis and export protein involved in EPS biosynthesis (wza). EggNOG orthologs with functional annotation are listed in Table 4. [Figure 7E] A series of plots showing the change in relative abundance of specific genes identified in nucleotide sequences obtained from colony picks ("minimal communities"). (B) Nitrogenase subunit h (nifh); (C and D) Photosystem II subunits (psba and psbb); (E) Ribulose-1,5-bisphosphate carboxylase / oxygenase (rubiscoL and rubiscoS); (F) Polysaccharide biosynthesis and export protein involved in EPS biosynthesis (wza). EggNOG orthologs with functional annotation are listed in Table 4. [Figure 7F] A series of plots showing the change in relative abundance of specific genes identified in nucleotide sequences obtained from colony picks ("minimal communities"). (B) Nitrogenase subunit h (nifh); (C and D) Photosystem II subunits (psba and psbb); (E) Ribulose-1,5-bisphosphate carboxylase / oxygenase (rubiscoL and rubiscoS); (F) Polysaccharide biosynthesis and export protein involved in EPS biosynthesis (wza). EggNOG orthologs with functional annotation are listed in Table 4. [Figure 8]A series of semi-logarithmic plots of growth curves for each of the minimal consortia grown in minimal medium (no nitrogen supplement) in 12-well plates. Dry biomass (y-axis, log2 scale) is scaled in kilograms per hectare. Wells have an area of ​​3.14 cm2. Three replicates were collected per culture, per time point. All wells were harvested at each time point and endpoint data was recorded. [Figure 9A] 1 is a bar plot showing saturation density for complete (blue bars) and minimal (red bars) consortia grown in 12-well plates in minimal medium under light. Error bars are standard error. [Figure 9B] Descriptions for the populations identified by P numbers in Figure 9A and the values ​​plotted on the plots are provided. [Figure 10] A series of plots showing the growth of three consortia in a 1 liter photobioreactor. Dry mass on the log2 y-axis is shown in grams per photobioreactor base. Data points are shown along with curve fits generated by the R package Growthcurver. Triplicate cultures were grown in nitrogen-free medium (M) or medium containing nitrogen in the form of nitrate (N) under light conditions with a photobioreactor sample size of n=2. [Figure 11] FIG. 1 is a series of bar plots showing the carbon and nitrogen content of cultures grown in photobioreactors. Fixed carbon and nitrogen are shown in grams per liter photobioreactor base. Error bars are standard deviations. Each set of bars represents the mean of two biological replicates. M indicates cultures grown in combined nitrogen-free minimal media. N indicates cultures grown in media supplemented with nitrate. C is carbon (red bars), N is nitrogen (green bars), and others (blue bars) are all other compounds in the dry cell pellet mass. [Figure 12A]A series of red fluorescence images of three consortia grown in sterile culture soil, with the day 0 blank subtracted and aligned by image analysis software. Excitation light is 450 nm blue light. A deep red filter was used to block blue wavelengths to the camera detector. A) BW01 day 1, B) BW01 day 10, C) BW02 day 1, D) BW02 day 10, E) BW05 day 1, F) BW05 day 10. [Figure 12B] The same series of fluorescence images as in FIG. 12B are shown, but color inverted and converted to grayscale to improve contrast and visibility of chlorophyll-containing regions and their abundance. [Figure 13] FIG. 1 is a series of plots showing the average Bray-Curtis dissimilarity index as a measure of passage for BW01 and BW02. Error bars represent standard deviation (n=4). [Figure 14] Plot of the first (PCo1) and second (PCo2) principal components obtained from principal coordinate analysis of the taxonomic composition determined from shotgun sequencing of the original soil microbiota and passages P1-P4 for BW01 and BW02. [Figure 15] FIG. 1 is a schematic diagram showing predicted growth in the field versus laboratory conditions. [Figure 16] Schematic diagram showing calculations of field growth potential assuming two different models. The calculations are based on thin-layer liquid cultures grown in 12-well plates and assume growth in two spatial dimensions, as cultures would grow in the field. [Figure 17] Schematic diagram showing metabolic functions in the consortium described herein, with red indicating electron sources, carriers, and sinks. [Figure 18A] Photographs of the cultures at the time of inoculation (top) and after growth (bottom) are shown. [Figure 18B] FIG. 1 is a schematic diagram showing the biosynthetic pathway for the production of melanins. [Figure 19]1 shows UV-visible absorption spectra of melanin reference material (0.02%, 0.01%, and 0.005% w / v) and aggregates isolated from cultures of BW02 (1×, 0.5×, 0.2×, 0.1×, and 0.05× dilutions). [Figure 20] 1 shows an array of photographs of solid medium culture plates containing minimal solid medium supplemented with 0.55 mM, 0.62 mM, 0.72 mM, 0.77 mM, 0.94 mM, 1.10 mM, 1.38 mM, 1.82 mM, 2.76 mM, and 5.52 mM L-tyrosine, on which BW02 cultures were grown under light for 14 days. [Figure 21] Images of culture supernatants from three independent cultures in multi-well plates are shown. Supernatants were obtained from cultures grown in minimal medium without added tyrosine ("Standard medium") or minimal medium with added tyrosine ("+L-tyrosine"). NaOH ("NaOH blank") and synthetic eumelanin ("Eumelanin") obtained from Sigma Aldrich were used as negative and positive controls, respectively. Uninoculated minimal medium ("-inoculum") was also used as a negative control. [Figure 22] Absorption spectra of supernatants inoculated with a microbial consortium supplemented with tyrosine and grown for two weeks are shown, as well as supernatants from cultures grown without and with added tyrosine ("+Tyr"), and melanin controls (0.0350% w / v and 0.0063% w / v). [Diagram 23] Photographs of minimal consortia MC1905, MC1909, and MC1918 produced from samples S1905, S1909, and S1918, respectively, in wells of a 96-well plate. Three replicates of each consortia were grown in minimal mineral medium (control, top row), and three replicates of each consortia were grown in the same medium supplemented with 2.77 mM homogentisic acid (HGA) (bottom row). [Figure 24] The absorption spectra of the samples shown in the photographs of Figure 23 are shown, with synthetic eumelanin shown as a control. [Diagram 25]The phyla, class, order, family, and genera identified for the minimal community organisms generated from BW01, BW02, and BW05, respectively, are shown. [Figure 26] The phyla, class, order, family, and genera identified for the minimal community organisms generated from BW01, BW02, and BW05, respectively, are shown. [Figure 27] The phyla, class, order, family, and genera identified for the minimal community organisms generated from BW01, BW02, and BW05, respectively, are shown. [Figure 28A] The phyla, class, order, family, and genus identified for the minimal community organisms generated from the FL community are shown. [Figure 28B] The phyla, class, order, family, and genus identified for the minimal community organisms generated from the FL community are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] It should be understood that the drawings are not necessarily drawn to scale, and that objects in the drawings are not necessarily drawn to scale in relation to each other. The drawings are representations intended to bring clarity and understanding to various embodiments of the devices, systems, and methods disclosed herein. Wherever possible, the same reference numbers are used throughout the drawings to refer to the same or similar parts. Furthermore, it should be appreciated that the drawings are not intended to limit the scope of the present teachings in any way.

[0054] Provided herein are methods and systems for producing functional microbial consortia, particularly, but not limited to, microbial consortia having a desired function, as well as technology relating to microbial consortia produced according to such methods.

[0055] In this detailed description of the various embodiments, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the disclosed embodiments. However, one of ordinary skill in the art will recognize that the various embodiments may be practiced with or without these specific details. In other instances, structures and devices are shown in block diagram form. Moreover, one of ordinary skill in the art will readily appreciate that the specific order in which the methods are presented and performed therein is illustrative, and that the order can be changed and is intended to be within the spirit and scope of the various embodiments disclosed herein.

[0056] All literature and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, papers, and Internet web pages, are expressly incorporated by reference in their entirety for all purposes. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the various embodiments described herein belong. If the definition of a term in an incorporated reference appears to differ from the definition provided in the present teachings, the definition provided in the present teachings shall prevail. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described in any way.

[0057] definition To facilitate the understanding of the present technology, several terms and phrases are defined below. Further definitions are set forth throughout the detailed description.

[0058] Unless otherwise defined herein, scientific and technical terms used in connection with the present technology shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In general, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. The methods and techniques of the present technology are generally performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification, unless otherwise indicated.See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989); Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2000); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992 and 2000 supplements); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons (1999); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990); Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998). each of which is incorporated herein by reference in its entirety.

[0059] As used herein, the phrase "in one embodiment" may, but does not necessarily, refer to the same embodiment. Further, as used herein, the phrase "in another embodiment" may, but does not necessarily refer to different embodiments. That is, as described below, various embodiments of the invention can be readily combined without departing from the scope or spirit of the invention.

[0060] Additionally, as used herein, the term "or" is an inclusive "or" operator and is equivalent to the term "and / or," unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for based on additional unlisted factors, unless the context clearly dictates otherwise. Additionally, throughout this specification, the meaning of "a, an, and the" includes plural references. The meaning of "in" includes "in" and "on."

[0061] As used herein, the terms "about," "approximately," "substantially," and "significantly" will be understood by those of ordinary skill in the art and will vary to some extent depending on the context in which they are used. When there are uses of these terms that are not clear to those of ordinary skill in the art given the context in which they are used, "about" and "approximately" will mean up to ±10% of the particular term, and "substantially" and "significantly" will mean more than ±10% of the particular term.

[0062] As used herein, the disclosure of a range includes the disclosure of all values ​​within the entire range and further divided ranges, including the endpoints and subranges given in the range.As used herein, the disclosure of a numerical range includes the endpoints and each intervening number to the same degree of precision.For example, for the range of 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range of 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0063] As used herein, the subscript "without" refers to an embodiment of a technology that excludes the underlying feature of the word to which it is appended. That is, the term "without X" as used herein means "absence of X," where X is the feature of the technology that is excluded in the "without X" technology. For example, a "no calcium" composition does not contain calcium, a "no mixing" method does not include a mixing step, etc.

[0064] The terms "first", "second", "third", etc. may be used herein to describe various steps, elements, compositions, components, regions, layers, and / or sections, but these steps, elements, compositions, components, regions, layers, and / or sections should not be limited by these terms unless otherwise indicated. These terms are used to distinguish one step, element, composition, component, region, layer, and / or section from another step, element, composition, component, region, layer, and / or section. Terms such as "first", "second", and other numerical terms, as used herein, do not imply a sequence or order unless clearly indicated by the context. That is, a first step, element, composition, component, region, layer, or section discussed herein may be named a second step, element, composition, component, region, layer, or section without departing from the technology.

[0065] As used herein, the words "presence" or "absence" (or alternatively "present" or "absent") are used in a relative sense to describe the amount or level of a particular entity (e.g., a component, an action, an element). For example, when an entity is said to be "present," this means that the level or amount of the entity is above a predetermined threshold, and conversely, when an entity is said to be "absent," this means that the level or amount of the entity is below a predetermined threshold. The predetermined threshold may be a detectability threshold or any other threshold associated with the particular test used to detect the entity. If an entity is "detected," it is "present." If an entity is "not detected," it is "absent."

[0066] As used herein, "increase" or "decrease" refers to a detectable (e.g., measured) positive or negative change in the value of a variable relative to a previously measured value of the variable, relative to an already established value, and / or relative to a standard control value, respectively. An increase is a positive change of preferably at least 10%, more preferably 50%, even more preferably 2-fold, even more preferably at least 5-fold, and most preferably at least 10-fold relative to a previously measured value of the variable, relative to an already established value, and / or relative to a standard control value. Similarly, a decrease is a negative change of preferably at least 10%, more preferably 50%, even more preferably at least 80%, and most preferably at least 90% relative to a previously measured value of the variable, relative to an already established value, and / or relative to a standard control value. Other terms indicating a quantitative change or difference, such as "more" or "less", are used herein in the same manner as above.

[0067] As used herein, the term "improved" refers to improving an environmental characteristic compared to a control environment or compared to a known average amount associated with the characteristic in question. For example, an "improved" soil may refer to a soil that increases the production of plant biomass after application of beneficial microorganisms or microbial consortia to the soil relative to the plant biomass produced by soil that is not treated with beneficial microorganisms or microbial consortia, with other soil properties being substantially and / or essentially the same with respect to the effect on plant biomass production. Alternatively, the production of plant biomass after application of beneficial microorganisms or microbial consortia to the soil may be compared to the average biomass normally produced by the soil as shown in scientific or agricultural publications known to those skilled in the art. As used herein, "improved" does not necessarily require that the data be statistically significant (e.g., p<0.05), but rather, a quantifiable difference that demonstrates that one value (e.g., average treatment value) is different from another value (e.g., average control value) can reach the level of "improved."

[0068] As used herein, a "system" refers to a number of real and / or theoretical components that work together for a common purpose. In some embodiments, a "system" is an integrated collection of hardware and / or software components. In some embodiments, each component of a system interacts with and / or associates with one or more other components. In some embodiments, a system refers to a combination of components and software for controlling and directing a method. For example, a "system" or a "subsystem" may include one or more or any combination of the following: mechanical devices, hardware, hardware components, circuits, circuitry, logic designs, logic components, software, software modules, software or software module components, software procedures, software instructions, software routines, software objects, software functions, software classes, software programs, files containing software, and the like, for performing the functions of the system or subsystem. That is, the methods and apparatus of the embodiments, or certain aspects or portions thereof, may take the form of program code (e.g., instructions) embodied in a tangible medium, such as a floppy diskette, a CD-ROM, a hard drive, a flash memory, or any other machine-readable storage medium, such that when the program code is loaded into a machine, such as a computer, and executed, the machine becomes an apparatus for practicing the embodiments. In the case of program code execution on a programmable computer, the computing device generally includes a processor, a storage medium (e.g., volatile and non-volatile memory and / or storage elements) readable by the processor, at least one input device, and at least one output device. One or more programs may perform or utilize the processes described in connection with the embodiments, for example, through the use of application programming interfaces (APIs), reusable controls, or otherwise. Such programs are preferably implemented in a high-level procedural or object-oriented programming language that communicates with a computer system. However, if desired, the programs can be implemented in assembly or machine language.In any case, the language may be a compiled or interpreted language, and combined with hardware implementations.

[0069] As used herein, the term "biological system" refers to a collection of genes, enzymes, activities, or functions that work together to provide a metabolic pathway or network. A biological system may include genes, enzymes, activities, or functions provided by several individual organisms. That is, a biological system may be distributed across individual organisms in a group of microorganisms or a microbial community. Thus, a biological system may be described by a collection of genes, enzymes, activities, or functions without identifying the individual organisms that provide the genes, enzymes, activities, or functions. A biological system may also be described in terms of nutrient fluxes, energy fluxes, electrochemical gradients, metabolic inputs (biological reactants), and metabolic outputs (biological products) that provide, for example, the conversion of energy inputs to energy for biological processes, the anabolic synthesis of biomolecules, and the elimination of waste products.

[0070] As used herein, the term "metabolic pathway" refers to a set of linked metabolic, biochemical, and physical processes that convert metabolic inputs into metabolic outputs in a series of steps and intermediates.

[0071] As used herein, the term "metabolic network" refers to a set of linked metabolic pathways that can convert metabolic inputs into metabolic outputs in a series of steps and intermediates.

[0072] As used herein, the terms "microbial", "microbial organism", and "microorganism" refer to organisms that exist as microscopic cells contained within the Archaea, Bacteria, or Eukarya domains in the three-domain system (see Woese (1990) Proc Natl Acad Sci USA 87:4576-79, incorporated herein by reference), where eukaryotes include yeast and filamentous fungi, protozoa, algae, or higher protists. Thus, the terms are intended to encompass prokaryotic or eukaryotic cells or organisms that have microscopic dimensions, including all species of bacteria, archaea, and eubacteria, as well as eukaryotic microorganisms such as yeast and fungi. Also included are cell cultures of any species that can be cultured for the production of chemicals. The terms "microbial cells" and "microbes" are used interchangeably with the term "microorganism". The terms "bacteria" and "bacterium" and "archaea" and "archaeon" refer to prokaryotic organisms in the domains Bacteria and Archaea of ​​the three-domain system.

[0073] The term "archaebacteria" refers to a taxonomic domain of life forms that are typically found in unusual environments and are distinguished from the rest of the prokaryotes by several criteria, including the number of ribosomal proteins and the lack of muramic acid in the cell wall. Based on small subunit rRNA analysis, the archaea consist of two phylogenetically distinct groups, the crenarchaea and the euryarchaea. Based on their physiology, the archaea can be organized into three types: the methanogens (prokaryotes that produce methane), the extreme halophiles (prokaryotes that live at extremely high concentrations of salt (NaCl)), and the extreme (hyper)thermophiles (prokaryotes that live at extremely high temperatures). In addition to the unifying archaeal features that distinguish them from bacteria (e.g., the absence of murein in the cell wall, ester-linked membrane lipids, etc.), these prokaryotes exhibit unique structural or biochemical traits that adapt them to their specific habitats. The Crenarchaea consist mainly of hyperthermophilic sulfur-dependent prokaryotes, while the Euryarchaea include methanogens and extreme halophiles.

[0074] The term "bacteria" or "eubacteria" refers to the domain of prokaryotic life. Bacteria includes at least eleven distinct groups: (1) the Gram-positive (Gram+) bacteria, with two major divisions: (1) the high G+C group (Actinomycetes, Mycobacteria, Micrococcus, etc.) and (2) the low G+C group (Bacillus, Clostridia, Lactobacillus, Staphylococci, Streptococci, Mycoplasmas). (2) Proteobacteria, e.g., purple photosynthetic + non-photosynthetic Gram-negative bacteria (including most "general" Gram-negative bacteria), (3) Cyanobacteria, e.g., oxygenic phototrophs, (4) Spirochetes and related species, (5) Planctomyces, (6) Bacteroides, Flavobacteria, (7) Chlamydia, (8) Green sulfur bacteria, (9) Green non-sulfur bacteria (also anaerobic phototrophs), (10) Radiotolerant micrococci and related bacteria, (11) Thermotoga and Thermosipho thermophiles.

[0075] "Gram-negative bacteria" includes cocci, non-enteric bacilli, and enteric bacilli. Genera of Gram-negative bacteria include, for example, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, and the like. These include Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirilla, Serratia, Rhizobium, Chlamydia, Rickettsia, Treponema, and Fusobacterium.

[0076] "Gram-positive bacteria" includes cocci, non-spore-forming bacilli, and spore-forming bacilli. Genera of Gram-positive bacteria include, for example, Actinomyces, Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus, and Streptomyces.

[0077] As used herein, the term "genus" is defined as a taxonomic grouping of related species according to the Taxonomic Outline of Bacteria and Archaea (Garrity et al. (2007) The Taxonomic Outline of Bacteria and Archaea. TOBA Release 7.7, March 2007, Michigan State University Board of Trustees).

[0078] As used herein, the term "species" is defined as a collection of closely related organisms that have greater than 97% 16S ribosomal RNA sequence homology and greater than 70% genomic hybridization and that differs sufficiently from all other organisms to be recognized as a distinct unit.

[0079] As used herein, the term "strain" as used herein in reference to a microorganism describes an isolate of a microorganism that is considered to be of the same species but has a unique genome and, if the nucleotide changes are non-synonymous, a unique proteome that differs from other strains of the same organism. Strains may differ in their non-chromosomal genetic complement. Typically, strains are the result of isolation from different hosts or at different places and times, although multiple strains of the same organism may be isolated from the same host.

[0080] As used herein, the term "naturally-occurring" as applied to a nucleic acid, enzyme, cell, or organism refers to a nucleic acid, enzyme, cell, or organism that is found in nature. For example, a polypeptide or polynucleotide sequence that can be isolated from a natural source and is present in an organism that has not been intentionally modified by man in the laboratory is naturally-occurring.

[0081] As used herein, the term "non-naturally occurring" as applied to a nucleic acid, enzyme, cell, or organism refers to a nucleic acid, enzyme, cell, or organism that has at least one genetic change that is not normally found in a naturally occurring nucleic acid, enzyme, cell, or organism. Genetic changes include, for example, modifications that introduce expressible nucleic acids that code for metabolic polypeptides, addition of other nucleic acids, deletion of nucleic acids, and / or disruption of the function of other microbial genetic material. Such modifications include, for example, coding regions and functional fragments thereof for heterologous, homologous, or heterologous and homologous polypeptides for the referenced species. Further modifications include, for example, non-coding control regions in which the modifications alter the expression of a gene or operon.

[0082] As used herein, the term "in vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments include, but are not limited to, test tubes and cell cultures. The term "in vivo" refers to a natural environment (e.g., an animal or a cell) and to processes or reactions that occur within a natural environment.

[0083] As used herein, the term "cell culture" refers to any in vitro culture of cells, including, for example, prokaryotic and eukaryotic cells. The term includes continuous cell lines, primary cell cultures, transformed cell lines, finite cell lines (e.g., non-transformed cells), bacterial or archaeal cultures in or on solid or liquid media, and any other population of cells maintained in vitro.

[0084] As used herein, the term "culturable organism" refers to a living organism that can be maintained and grown in a laboratory. In some embodiments, a culturable organism cannot be maintained and grown in a laboratory in pure culture in the absence of other organisms, and thus may be referred to as a "non-culturable organism" with respect to growth in pure culture. However, in some embodiments, such an organism can be grown in a laboratory in a microbial consortium that includes at least one other organism, and thus may be both a "culturable organism" with respect to the consortium and a "non-culturable organism" with respect to growth in pure culture without other members of the consortium.

[0085] As used herein, the terms "isolate", "isolated", "isolated microorganism", and others are intended to mean that one or more microorganisms have been separated from at least one of the materials associated with the particular environment (e.g., soil, water, or higher multicellular life form) with which they are associated. That is, an "isolated microorganism" is not in its naturally occurring environment, but rather, the microorganism has been removed from its natural environment and placed in a non-naturally occurring state of existence by various techniques described herein. That is, an isolated strain may exist, for example, as a biologically pure culture, or as a spore (or other form of the strain) associated with a carrier composition. In certain embodiments of the present disclosure, an isolated microorganism exists as an isolated biologically pure culture. Those skilled in the art will recognize that an isolated biologically pure culture of a particular microorganism means that the culture is substantially free (within scientific reasonableness) of other living organisms and contains only the individual microorganism of interest. A culture may contain varying concentrations of the microorganism, and an isolated biologically pure microorganism is often necessarily different from a low-purity or impure material. Furthermore, in some aspects, the present disclosure provides certain quantitative measures of the concentration or purity limits found in isolated biologically pure microbial cultures. The presence of these purity values ​​is, in certain embodiments, an additional attribute that distinguishes the microorganisms disclosed herein from those present in nature.

[0086] As used herein, the term "selected environment" or "condition" refers to any external characteristic in which a particular organism or microbial community grows more efficiently (e.g., faster, in higher quantities or concentrations, with greater survival rates, etc.) than one or more other organisms or communities. Exemplary "conditions" or "environments" include, but are not limited to, a particular medium, volume, container, temperature, mixing, aeration, gravity, electromagnetic field, cell density, pH, nutrients, phosphate sources, nitrogen sources, symbiosis with one or more organisms, and / or interactions with a single organism or multiple organisms (e.g., a mixed population). "Conditions" or "environments" also include substances such as heavy metals, antibiotics, and chlorides that may be toxic to one or more organisms or communities of microorganisms. It should be understood that time can also be considered a "condition" since organisms are not static entities. That is, a culture grown over an extended period of time (e.g., days, weeks, months, years) may produce a culture that contains a particular organism or consortium in a proportion of the culture that is relatively higher than the relative amount of that particular organism or consortium in the culture prior to growth during that time.

[0087] As used herein, the term "selection" refers to increasing the frequency of individuals with different "types" in a population by removing or enriching for individuals of one type over others, whether deliberate or natural. The nature of a "type" can be defined by genetic characterization (e.g., gene or nucleotide sequence), functional characterization (e.g., enzymes, metabolic capabilities), taxonomic characterization (e.g., strain, subspecies, species, genus, family, or operational taxonomic units (OTUs) based on similarities or differences in nucleotide sequences), or physical characterization. Furthermore, a type can include one or many individuals. Prototypical examples of selection include, but are not limited to, growth rate selection, in which individuals that grow and reproduce faster become more prevalent in a population. An important consideration when performing selection is to determine "what to select for" or what is "selected for", i.e., determining the genetic, functional, and / or physical differences that are favorable or unfavorable in a particular environment. Growth rate selection is applied to select organisms that have a growth rate that is faster than other individuals in the population and can be passed on from parent cells to their progeny.

[0088] As used herein, the term "enrichment" refers to a process in which the abundance (e.g., expressed in absolute and / or relative terms) of one or more organisms, one or more functional capabilities, one or more genes or gene products, or one or more nucleotide sequences of interest is increased relative to the abundance of one or more other organisms, one or more other functional capabilities, one or more other genes or gene products, or one or more other nucleotide sequences. For example, in some embodiments, the term "enrichment" refers to a process of increasing the number (e.g., absolute and / or relative numbers) of one or more microorganisms present in a culture, for example, by culturing in a suitable medium under selective conditions.

[0089] As used herein, the term "medium" refers to the chemical environment to which an organism is exposed or provided access. The organism may be immersed in the medium or in physical proximity (e.g., in physical contact) with it. A medium typically includes water and other additional nutrients and / or chemicals that may contribute to the growth or maintenance of the organism. The ingredients may be purified chemicals (e.g., "defined" media) or uncharacterized mixtures of chemicals such as complexes, extracts made from milk or blood. Standardized media are widely used in laboratories. Examples of media for bacterial growth include, but are not limited to, LB and M9 minimal media. The term "minimal," when used with respect to a medium, refers to a medium that supports the growth of an organism but consists of only the simplest possible compounds. For example, M9 minimal medium may consist of the following components dissolved in water and sterilized: 48 mM Na2HPO4, 22 mM KH2PO4, 9 mM NaCl, 19 mM NH4Cl, 2 mM MgSO4, 0.1 mM CaCl2, 0.2% carbon and energy source (e.g., glucose).

[0090] A growth medium is also provided for plant growth. As used herein, the term "agricultural growth medium" refers to a medium suitable for supporting plant growth. Agricultural growth media can be natural or artificial, such as soil, culture mixture, bark, vermiculite, hydroponic solution, hydroponic solution applied to a solid plant support system, and tissue culture gel. Agricultural growth media can be used alone or in combination with one or more other media. It can be used with or without the addition of exogenous nutrients and a physical support system for the roots and leaves.

[0091] In some embodiments, the agricultural growth medium is a naturally occurring medium, such as soil, sand, mud, clay, humus, topsoil, rock, or water. In some embodiments, the agricultural growth medium is artificial. Such an artificial agricultural growth medium may be constructed to mimic the conditions of a naturally occurring medium. However, this is not necessary. An artificial agricultural growth medium can be made from one or more of any number of materials, including sand, minerals, glass, rock, water, metals, salts, nutrients, water, and combinations thereof. In some embodiments, the agricultural growth medium is sterile. In some embodiments, the agricultural growth medium is not sterile.

[0092] As used herein, the term "soil" refers to a growing medium of solid particles that contains organic matter and minerals, and typically further contains water and gas.

[0093] As used herein, the term "greenhouse growing medium" refers to a soil-free growing medium commonly used for greenhouse, raised bed, and container crop plant growth. Exemplary greenhouse growing media include peat moss, and may also include perlite, vermiculite, bark, and / or sand.

[0094] As used herein, the term "hydroponic medium" refers to a liquid growth medium that contains nutrients and / or minerals for plant growth and does not contain soil. The use of hydroponic medium may be used in combination with the use of an inert medium such as perlite, gravel, or other substrate that provides physical support for the plant.

[0095] As used herein, the term "culture" refers to a medium in a container or enclosure that contains at least one cell or individual viable organism, usually a medium in which the organism can grow. As used herein, the term "continuous culture" is intended to mean a liquid culture into which new medium is added at a rate equal to the rate at which medium is removed. Conversely, "batch culture" as used herein is intended to mean a culture of fixed size or volume in which new medium is not added or removed.

[0096] As used herein, the term "microbial community" refers to a set of microbial species or strains of a species that can be described as performing a common function or contributing to, resulting in, or correlating with a recognizable parameter or phenotypic characteristic. A community can include two or more taxonomic units of microorganisms (e.g., a family, a genus, a species, or a strain of a species). In some cases, microorganisms coexist symbiotically in a microbial community. A microbial community can be described by describing the taxonomic units present in the community (e.g., several strains, subspecies, species, genera, families, or operational taxonomic units (OTUs) based on nucleotide sequence similarities or differences), by describing the genes present in the community, by describing the nucleotide sequences present in the community, or by describing the functions present in and / or provided by the community. A microbial community can be a subset of organisms found in a microbial community. A microbial community can be described as a set of genes, enzymes, activities, or functions provided by several individual organisms. Additionally, a microbial community may be described in terms of a collection of genes, enzymes, activities, or functions without identifying the individual organisms that provide the genes, enzymes, activities, or functions. A microbial community may also be described in terms of nutrient fluxes, energy fluxes, electrochemical gradients, metabolic inputs (biological reactants), and metabolic outputs (biological products) that provide, for example, the conversion of energy inputs into energy for biological processes, the anabolic synthesis of biomolecules, and the elimination of waste products.

[0097] As used herein, the term "microbial community" refers to a group of microorganisms that includes two or more taxonomic units of microorganisms (e.g., a family, a genus, a species, or a strain of a species). Unlike a microbial community, a microbial community does not necessarily act in concert to perform a common function or contribute to, cause, or correlate with a recognizable parameter or phenotypic characteristic. See, for example, Doolittle (2010) "Metagenomics and the Units of Biological Organization" BioScience 60:102-112, which is incorporated herein by reference.

[0098] As used herein, the term "minimal consortium" refers to a microbial consortium that comprises a minimal set of members that are viable under given growth conditions. A minimal consortium can be produced from another more complex microbial consortium or group of microorganisms, for example, by streaking a culture of the microbial consortium or group of microorganisms on a solid minimal selective growth medium to separate individual colonies that make up the minimal consortium growing on the solid minimal selective growth medium, and, if appropriate to separate colonies with stable morphology, picking and restreaking the colonies on a solid minimal selective growth medium to separate individual colonies that make up the minimal consortium growing on the solid minimal selective growth medium. Thus, in this example, the minimal consortium comprises a minimal set of members that are viable on a minimal selective medium.

[0099] As used herein, the term "relative abundance" refers to the abundance of microorganisms of a particular taxonomic unit (e.g., OTU) in a first biological sample compared to the abundance of microorganisms of the corresponding taxonomic unit in one or other (e.g., second) samples. "Relative abundance" can be reflected, for example, in the number of isolated species corresponding to the taxonomic unit, or the degree to which biomarkers (e.g., nucleotide sequences) specific for the taxonomic unit are present or expressed in a given sample. The relative abundance of a particular taxonomic unit in a sample can be determined using culture-based or non-culture-based methods well known in the art. Non-culture-based methods include sequence analysis of amplified polynucleotides specific for the taxonomic unit, or comparison of proteomic-based profiles in the sample reflecting the number and extent of polypeptide-, lipid-, polysaccharide-, or carbohydrate-based biomarkers characteristic of one or more taxonomic units present in the sample. The relative abundance or abundance of a taxonomic unit or OTU can be calculated with reference to all detected taxonomic units / OTUs, or with reference to a certain set of invariant taxonomic units / OTUs. In some embodiments, taxonomic units are identified using sequence-based methods, for example as described in Wood (2014) "Kraken: ultrafast metagenomic sequence classification using exact alignments", Genome Biology 15:R46, and Wood (2019) "Improved metagenomic analysis with Kraken 2", Genome Biology 20:257, each of which is incorporated herein by reference.

[0100] As used herein, the term "significantly altered relative abundance" refers to a statistically significant increase or decrease in the relative abundance of the number of microorganisms of a particular taxonomic unit compared to the total microorganisms in a sample or the number of microorganisms of the corresponding taxonomic unit present in another sample. In some embodiments, a "significant increase" or "significant decrease" in relative abundance is defined as a statistically significant increase or a statistically significant decrease relative to a reference value. In some embodiments, a statistically significant increase or a statistically significant decrease is an increase or decrease that is two, three, or four times the standard deviation of the relative abundance. In some embodiments, a statistically significant increase or a statistically significant decrease is an increase or decrease with a P value of 0.1, 0.05, 0.01, or 0.005 or less.

[0101] In some embodiments, a "significant decrease" or "significant increase" in relative abundance refers to a statistically significant difference in one or more indicator species or taxonomic units compared to each other or to a reference species or taxonomic unit using a non-parametric statistical test such as a signed rank test. In some embodiments, a "significant decrease" or "significant increase" in relative abundance is determined using a model employing Bayesian inference and related approaches.

[0102] In certain embodiments, an increase in relative abundance reflects a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more increase over a reference value, hi some embodiments, an increase in relative abundance reflects a 2-fold, 3-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold increase over a reference value.

[0103] As used herein, "expression" refers to the process by which the information of a particular nucleic acid (e.g., a gene) is used to synthesize a product (e.g., a biomolecule (e.g., a nucleic acid, a polypeptide, a carbohydrate, a lipid, and combinations, derivatives, and / or metabolites thereof), a metabolite (e.g., a primary metabolite, a secondary metabolite), a fatty acid, a polyketide, a nucleotide, an amino acid, a cofactor, and combinations, derivatives, and / or metabolites thereof). The term "expression" includes one or more of the following, but is not limited to, transcription of a gene into a precursor mRNA, processing of the precursor mRNA to produce a mature mRNA, stability of the mRNA, translation of the mature mRNA into a protein (including codon usage and availability of tRNA), and / or glycosylation, and / or other modifications of the translation product. The term "expression" also includes transcription of non-coding RNA, such as transfer RNA, ribosomal RNA, microRNA, siRNA, piRNA, snoRNA, snRNA, exRNA, scaRNA, or long ncRNA. The term "expression" includes the production of functional products as well as the production of non-functional products that can be used to produce functional products by subsequent chemical or biochemical manipulation or synthesis.

[0104] As used herein, the term "phenotype" refers to the observable characteristics of an individual cell, cell culture, organism, or group of organisms (e.g., a microbial community) that result from the interaction between the genetic makeup of the individual cell, cell culture, organism, or group of organisms, and the environment (e.g., phenotype).

[0105] As used herein, the term "metagenome" is defined as "the collective genomes of all the microorganisms present in a given habitat" (Handelsman et al. (1998) Chem. Biol. 5:R245-R249). However, the term is also intended to include clones containing genomes or genes extracted from environmental samples.

[0106] As used herein, "metagenomic DNA" refers to the total genomic DNA associated with microorganisms isolated from a complex sample such as an open natural environment (e.g., soil, water) or from the microbiota of a multicellular organism (e.g., human).

[0107] As used herein, a "metagenomic library" refers to a clonal collection of the entire genomic DNA associated with microorganisms isolated from a complex sample such as an open natural environment (e.g., soil, water) or from the microbiota of a multicellular organism (e.g., human) in a recombinant vector.

[0108] As used herein, "genome" refers to the genetic material (eg, chromosomes) of an organism.

[0109] As used herein, the term "gene" refers to a nucleic acid molecule that includes a nucleic acid sequence that encodes a polypeptide or non-coding RNA and an expression control sequence operably linked to the nucleic acid sequence that encodes the polypeptide or non-coding RNA. For example, a gene can include a promoter, one or more enhancers, a nucleic acid sequence that encodes the polypeptide or non-coding RNA, downstream control sequences, and possibly other nucleic acid sequences involved in controlling the transcription of RNA from the gene.

[0110] As used herein, the term "gene-based" refers to the genetic or genomic factors that underlie a particular observation.

[0111] As used herein, the term "genetic" refers to heritable information encoded in a sequence of DNA nucleotides. Thus, the term "genetic characterization" is intended to mean sequencing, genotyping, comparison, mapping, or other assays of DNA-encoded information.

[0112] As used herein, the term "genetic material" refers to the DNA in an organism that is passed from one generation to the next. Usually, genetic material refers to the genome of the organism. Extrachromosomal elements, such as organelles or plasmid DNA, can also be part of the genetic material that determines the characteristics of the organism.

[0113] As used herein, the term "genetic change" or "genetic adaptation" refers to one or more mutations in the genome of an organism. As used herein, the term "mutation" refers to a difference in the sequence of DNA nucleotides in two related organisms, including, for example, substitutions, deletions, insertions, and rearrangements, or the movement of mobile genetic elements.

[0114] As used herein, the term "taxonomic unit" is a group of organisms that are deemed similar enough to be treated as a separate unit. Taxonomic units may include, but are not limited to, families, genus, species, or populations within species (e.g., strains).

[0115] As used herein, the term "operational taxonomic unit" (OTU) refers to a group of microorganisms that are deemed similar enough to be treated as an individual unit. OTUs can include, but are not limited to, taxonomic families, genuses, or species. OTUs are often defined by comparing nucleotide sequences between organisms. In certain cases, OTUs can include a group of microorganisms that are treated as a unit based on 97% or more, 95% or more, 90% or more, 80% or more, or 70% or more sequence identity, for example, among at least a portion of a discriminating biomarker, such as the 16S rRNA gene.

[0116] As used herein, the term "evaluation" is intended to mean the observation or measurement of an observable phenotype of an organism. Evaluation typically involves analysis, interpretation, and / or comparison with the phenotype of another organism. It is understood that phenotypes can be evaluated both at the gene level (e.g., with respect to nucleotide sequence) and at the level of gene products. Furthermore, phenotypes can be evaluated with respect to the behavior of the organism in the environment and / or the behavior of individual molecules or groups of molecules within the organism. Such comparisons are useful in determining the detailed function of mutated products resulting from genetic adaptations. Evaluation can be performed on a community or group by observing or measuring the observable phenotype of the community or group.

[0117] As used herein, the term "stepwise" is intended to mean a manner of a series of events where one follows the other over time. As used herein, the term "simultaneous" is intended to mean occurring at the same time.

[0118] As used herein, "kilobase" (kb) or "kilobase pair" (kbp) refers to 1000 nucleotides or 1000 base pairs of a nucleic acid (e.g., DNA or RNA).

[0119] As used herein, the term "stable" when used in reference to a group of microorganisms (e.g., a group of microorganisms, a microbial consortium, a microbial culture, or other group, set, or collection of microorganisms) refers to a group of microorganisms that does not change significantly (e.g., as measured by the similarity measures discussed above) from a first culture to a second culture when a portion of the first culture is used to inoculate a culture medium to produce a second culture, and the culture conditions, including external factors (light, nutrients, temperature, aeration, etc.), are the same for the first and second cultures. Thus, as used herein, the term "stability" when used in reference to a microbial population (e.g., "stability of a microbial population") refers to a qualitative or quantitative indication or measurement of the change (e.g., as measured by similarity measurements as discussed above) in a microbial population (e.g., a microbial population, microbial consortium, microbial culture, or other group, set, or consortium of microorganisms) from a first culture to a second culture when a portion of the first culture is used to inoculate a culture medium to produce a second culture, and culture conditions, including external factors (light, nutrients, temperature, aeration, etc.), are the same for the first and second cultures.

[0120] Additionally, as used herein, the term "stable," when used with respect to one or more functions provided and / or performed by a microbial population (e.g., a microbial population, microbial consortium, microbial culture, or other group, set, or collection of microorganisms), refers to one or more functions that do not change significantly from a first culture to a second culture (e.g., as measured by examination of metagenomic sequences and / or inferring function therefrom) when a portion of the first culture is used to inoculate a culture medium to produce a second culture, and the culture conditions, including external factors (light, nutrients, temperature, aeration, etc.), are the same for the first culture and the second culture. Thus, as used herein, the term "stability" when used in reference to one or more functions provided by a microbial population (e.g., "functional stability") refers to a qualitative or quantitative indication or measurement of the change (e.g., as measured by the similarity measurements discussed above) in a microbial population (e.g., a microbial population, microbial consortium, microbial culture, or other group, set, or consortium of microorganisms) from a first culture to a second culture when a portion of the first culture is used to inoculate a culture medium to produce a second culture, and the culture conditions, including external factors (light, nutrients, temperature, aeration, etc.), are the same for the first and second cultures. Thus, functional stability and microbial stability may be independent, such that a microbial population may be functionally stable, but the membership and / or abundance of members may change and the microbial population does not have stability as a microbial population. That is, a microbial community may have both functional stability and microbial community stability, a microbial community may have neither functional stability nor microbial community stability, a microbial community may have functional stability (e.g., regardless of the state of microbial community stability), and a microbial community may have microbial community stability (e.g., regardless of the state of functional stability).

[0121] In some embodiments, a microorganism may be "endogenous" to an environment. As used herein, a microorganism is considered "endogenous" to an environment if it is derived from the environment from which it is obtained. That is, a microorganism is endogenous to an environment if it is naturally found associated with said environment. In embodiments in which endogenous microorganisms are applied to an environment, the endogenous microorganisms are applied in amounts that differ from the levels found in the specified natural environment. That is, a microorganism that is endogenous to a given environment can still improve the environment if the microorganism is present in the environment at levels that do not occur naturally and / or if the microorganism is applied to the environment with other organisms that are exogenous to the environment and / or endogenous to the environment and present at levels that do not occur naturally.

[0122] In some embodiments, a microorganism may be "exogenous" (also referred to as "heterologous") to an environment. As used herein, a microorganism is considered "exogenous" if it is not derived from the environment from which it is obtained. That is, a microorganism is exogenous to an environment if it is not naturally found associated with the environment. For example, a microorganism that is normally associated with a first environment may be considered exogenous to a second environment that is naturally devoid of said microorganism.

[0123] As used herein, "environmental sample" means a sample taken or obtained from any part of the environment (e.g., ecosystem, ecological niche, habitat, etc.). Environmental samples can include liquid samples from rivers, lakes, ponds, oceans, glaciers, icebergs, rain, snow, sewage, reservoirs, tap water, drinking water, etc., solid samples from soil, compost, sand, rock, concrete, wood, brick, sewage, etc., and gas samples from air, aquatic hydrothermal sources, industrial exhaust, automobile exhaust, etc.

[0124] As used herein, the term "photosynthetic and nitrogen fixing microorganisms" refers to microorganisms that are capable of both photosynthesis and nitrogen fixation.

[0125] Detailed Description Provided herein is a technology for a microbial consortium comprising microorganisms capable of producing and / or delivering nutrients to soil, for example for agricultural applications. In some embodiments, the microbial consortium comprises several microorganisms. In some embodiments, the microbial consortium comprises several biological systems comprising components (e.g., enzymes, multienzyme complexes, metabolic pathways) provided by the microorganisms of the microbial consortium. In some embodiments, the biological systems provide several biological functions, such as photosynthesis (e.g., oxygenic and / or anoxygenic photosynthesis), nitrogen fixation, carbon fixation, and conversion (e.g., solubilization) of soil nutrients (e.g., phosphorus) into bioavailable forms for use by plants. In some embodiments, the microbial consortium comprises microorganisms and / or biological systems that fix N and / or C from the air and use photosynthetic energy to produce microbial biomass. In some embodiments, the biological system comprises multiple enzymes provided by multiple microorganisms. In some embodiments, the biological system comprises enzymes provided by one microorganism. In some embodiments, the mass (e.g., dry cell mass) of the microbial consortium is about 50% carbon and about 5% nitrogen.

[0126] In some embodiments, the technology includes methods for producing and / or optimizing microbial consortia to be applied directly to crop fields (e.g., by spraying) and for growing the microbial consortia with or instead of traditional plant-based cover crops. In some embodiments, the method includes growing the microbial consortia in a bioreactor. In some embodiments, the bioreactor includes a growth pond, and the microbial consortia are grown in the growth pond under controlled conditions (e.g., controlled composition of the growth medium in the growth pond, controlled temperature, controlled light conditions (e.g., incident wavelength and light flux)). In some embodiments, the microbial consortia are grown without providing immobilized nitrogen in the growth medium. In some embodiments, the microbial consortia are grown without providing immobilized carbon in the growth medium. In some embodiments, the microbial consortia are grown without providing immobilized nitrogen in the growth medium and without providing immobilized carbon in the growth medium. In some embodiments, the nitrogen and / or carbon are provided by atmospheric nitrogen (e.g., atmospheric N2 gas) and / or atmospheric carbon (e.g., atmospheric CO2 gas). That is, in some embodiments, the techniques include growing a microbial consortium under conditions in which nitrogen and / or carbon is provided by atmospheric nitrogen (e.g., atmospheric N2 gas) and / or atmospheric carbon (e.g., atmospheric CO2 gas).

[0127] In some embodiments, the controlled conditions include providing light of a controlled wavelength, a controlled spectrum of light, and / or a controlled photon flux of light. In some embodiments, broad spectrum (e.g., "white") light is provided, e.g., light having a spectrum of wavelengths from about 380 to about 750 nm. In some embodiments, "far red" light is provided, e.g., light having a peak wavelength of about 740 nm (e.g., light having a spectrum of about 730 to 750 nm).

[0128] In some embodiments, the technology provides a method for formulating a composition comprising a microbial consortium and applying the composition directly to a crop field (e.g., by spraying). In some embodiments, the microbial consortium continues to grow after being applied to the crop field. In some embodiments, the technology provides a method for formulating a composition comprising a microbial consortium and applying the composition directly to a crop field (e.g., by spraying) and growing the microbial consortium together with or instead of a conventional plant-based cover crop. Thus, applying a composition comprising a microbial consortium to a field provides N to the crop growing in the field, while removing CO2 from the air and depositing fixed carbon in the soil. High carbon content in the soil is accompanied by high soil performance, including nutrient retention capacity and stability against runoff. Thus, in some embodiments, the technology has application to provide a technology for fixing carbon and nitrogen at low cost to growers, for example, during intermediate seasons.

[0129] As described herein, embodiments of the present technology deliver fixed N and C to agricultural fields and reduce greenhouse gases. However, crops require additional nutrients in addition to N and C, such as phosphorus (P), potassium (K), manganese, magnesium, calcium, iron, boron, sodium, etc.

[0130] Regarding phosphorus, phosphorus cannot be captured from the air in the way that N can; that is, phosphorus is supplied by mining phosphate minerals from the earth and adding them to fertilizers in this form. Availability of free P in soils can be problematic, as P tends to bind tightly to soil components and is therefore difficult for plants to obtain and utilize. Soil P availability to plants depends on pH, with P being most available to plants at a pH of about 6-7. At soil pHs below 6, phosphate tends to bind to metal ions such as iron or aluminum, and at pHs above 7, P tends to form complexes with calcium. Additionally, overapplication of phosphorus to fields has negatively impacted water supplies and fisheries through anoxic eutrophication. Thus, there is a need for technologies that deliver phosphorus more efficiently, so that it is accessible to crops and does not enter waterways.

[0131] That is, certain embodiments of the present technology provide P to the biomass (e.g., in a biologically available form present in the biomass of the microbial consortium). In some embodiments, the microbial consortium includes microorganisms that readily accumulate phosphorus and incorporate it into the biomass. In some embodiments, the microbial consortium includes microorganisms that accumulate polyphosphate (see, e.g., Kornberg (1999) "Inorganic polyphosphate: a molecule of many functions," Annu Rev Biochem 68:89-125, incorporated herein by reference). In some embodiments, the microbial consortium includes microorganisms that accumulate P to meet the nutritional needs of the field-applied microbial consortium and associated crops. In some embodiments, the microbial consortium includes microorganisms that mobilize bound phosphate from the soil and provide it to the crop or other microorganisms in a bioavailable form.

[0132] As described herein, the technology provides a biological approach to producing nitrogen fertilizer that can partially or completely replace chemical production of fertilizer. Additionally, the technology described herein removes carbon from the atmosphere and, in some embodiments, provides nutrients (e.g., P) to the soil in a bioavailable form. The technology is advantageous because, for example, it reduces greenhouse gases, reduces fossil fuel use (e.g., by reducing or minimizing the use of the Haber-Bosch process), increases the efficiency of phosphorus use, and reduces denitrification in agricultural lands.

[0133] During the development of embodiments of the present technology, experiments were performed to show that a method for producing microbial consortia using selective conditions reduces the complexity of the microbial population while selecting for the function of carbon and nitrogen fixation. Although most environmental samples contain phototrophic organisms that can survive without supplementing the medium with nitrogen or carbon compounds, three cultures (BW01, BW02, and BW05) performed particularly strongly in early passages. Data showed that these cultures were capable of high productivity levels in liter-scale liquid culture and could form colonies in sterile soil. Furthermore, experiments showed that reduced-complexity versions of these consortia grew well in liquid culture.

[0134] Function of microbial communities As described herein, the technology relates to producing, providing and / or using a microbial consortium that includes several microorganisms and provides several functions. In some embodiments, the microbial consortium includes 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more microorganisms. In some embodiments, the microbial consortium includes about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more microorganisms.

[0135] In some embodiments, the microbial consortium comprises several enzymes, multienzyme complexes, biological systems, biological pathways, or biological functions. In some embodiments, the enzymes, multienzyme complexes, biological systems, biological pathways, or biological functions provide photosynthesis (e.g., oxygenic and / or nonoxygenic photosynthesis), nitrogen fixation, carbon fixation, and conversion of nutrients into bioavailable forms for use by the plant.

[0136] For example, embodiments of the present technology provide a microbial consortium that performs photosynthesis (e.g., oxygenic and / or nonoxygenic photosynthesis) and nitrogen fixation, and transfers carbon and electrons between metabolic functions and / or microbial cells of the consortium (Figure 17). In particular, in some embodiments, the microbial consortium performs photosynthesis (e.g., oxygenic photosynthesis) by capturing light energy (e.g., from the sun when grown in the field or from lighting when grown in a photobioreactor), and uses this energy to remove electrons from water to drive the Calvin-Benson cycle and fix carbon from atmospheric carbon dioxide. The fixed carbon is recycled to produce molecules including simple and complex carbohydrates. The microbial consortium uses the energy, electrons, and protons produced from photosynthesis to perform nitrogen fixation (e.g., biological nitrogen fixation). Nitrogen fixation can be performed in phototrophic organisms (as in Nostoc spp.) or in separate life forms that consume sugars or other carbon compounds secreted by the phototrophic organisms. The microbial community includes metabolic functions and / or nitrogen flux and / or transfer (e.g., in the form of fixed nitrogen compounds) between cells in the community. The microbial community includes metabolic functions and / or carbon flux and / or transfer (e.g., in the form of fixed carbon compounds (e.g., carbon compounds containing four or more carbons) between cells in the community. The microbial community includes metabolic functions and / or electron flux and / or transfer (e.g., transported by electron shuttle molecules) between cells in the community. Oxygenic phototrophs form the primary electron source for the community, for example, by removing electrons from water in the reaction center of photosystem II using solar energy. These electrons are transported by various carbon-containing compounds (e.g., sugars and polysaccharides) secreted by the phototrophs. Nitrogen fixation requires reducing equivalents (electrons) and energy transported by ATP, both of which can be supplied by the oxidation of these carbon-containing compounds. Additionally, electrons can be recovered from hydrogen gas released during nitrogen fixation. This hydrogen can also serve as a source of electrons for non-oxygenic photosynthesis in the anaerobic regions of the biofilm.

[0137] That is, without being bound by theory, it is assumed that the microorganisms of the microbial consortium capture light energy (e.g., in the field or in a photobioreactor) by photosynthesis (e.g., oxygenic photosynthesis), which is then used to transfer electrons from water to atmospheric carbon dioxide (e.g., in the Calvin-Benson cycle) to fix carbon, e.g., in the form of simple and complex carbohydrates. It is further assumed that the energy, electrons, and protons produced by photosynthesis are used for biological nitrogen fixation (BNF) by the photosynthetic organisms of the consortium (e.g., Nostoc species) or another organism that consumes the fixed carbon compounds (e.g., simple carbohydrates, complex carbohydrates, other carbon compounds) secreted by the phototrophic organisms. The fixed carbon compounds secreted by the consortium but not consumed by the members of the consortium provide soil organic carbon (SOC) or humus, which improves the soil and sequesters carbon from the atmosphere. That is, in some embodiments, the present technology relates to a consortium that produces carbon compounds that sequester carbon from the atmosphere. In some embodiments, the technology provides a consortium including members of the consortium that produce carbon compounds that sequester carbon from the atmosphere. In some embodiments, the embodiments provide a consortium that produces melanin (e.g., pheomelanin, eumelanin, and / or pyomelanin). That is, the embodiments provide a consortium that produces sustainable carbon compounds that provide a stable carbon sink that removes carbon (e.g., CO2) from the atmosphere and stores the carbon in sustainable carbon compounds (e.g., melanin) in the soil. The embodiments provide microbial organisms that produce sustainable carbon compounds that provide a stable carbon sink that removes carbon (e.g., CO2) from the atmosphere and stores the carbon in sustainable carbon compounds (e.g., melanin) in the soil.

[0138] In some embodiments, it is contemplated that the consortium includes phosphate solubilizing and / or mobilizing microorganisms or that the consortium includes an enzyme, multienzyme complex, biological system, biological pathway, or biological function for solubilizing and / or mobilizing phosphate. It is contemplated that the phosphate solubilizing and / or mobilizing microorganisms consume fixed carbon compounds (e.g., simple carbohydrates, complex carbohydrates, and other carbon compounds) secreted by the phototrophic organism.

[0139] In some embodiments, the technology provided herein relates to providing, producing, and / or using a microbial consortium including photosynthetic, nitrogen-fixing, and / or carbon-fixing microorganisms. In some embodiments, the photosynthetic life forms of the consortium also fix nitrogen and / or also fix carbon. For example, in some embodiments, the microbial consortium includes a cyanobacterium (e.g., Nostoc spp.). Furthermore, during the development of embodiments of the technology, data was collected indicating that certain life forms (e.g., Nostoc spp.) grow faster in a microbial consortium (in association with other life forms) than these life forms (e.g., Nostoc spp.) grow alone (in the absence of association with other life forms in the microbial consortium). It is assumed that some life forms (non-photosynthetic heterotrophs) in the consortium process organic molecules and hydrogen, which increases the growth rate of other life forms (e.g., photosynthetic life forms such as Nostoc spp.). Furthermore, in some embodiments, the microbial consortium includes nitrogen-fixing microorganisms from genera such as Rhizobium, Bradyrhizobium, or Azospirillum. Rhizobium and Bradyrhizobium are known to fix nitrogen when associated with legumes, whereas Azospirillum is a free-living nitrogen-fixing microorganism.

[0140] During the development of embodiments of the technology described herein, experiments were performed to select for strong carbon fixation and nitrogen fixation activity while applying controlled selection conditions to environmental samples to reduce the complexity of the microbial community. Most environmental samples appeared to contain phototrophic organisms that could survive without nitrogen or carbon combinations, but three cultures (referred to herein as "BW01", "BW02" and "BW05") performed particularly strongly. See below and in the Examples, e.g., Example 20. Additionally, during the development of embodiments of the technology described herein, experiments were performed to produce minimal consortia from additional environmental samples using the process for producing minimal consortia described herein. See, e.g., Example 21.

[0141] method In some embodiments, the microbial consortium is produced using a method of selection. In some embodiments, the method includes the method of producing a functional microbial consortium described in U.S. Patent Application No. 17 / 544,879, which is expressly incorporated herein by reference in its entirety. For example, in some embodiments, the microbial consortium is produced using a method that includes growing a culture under specific conditions (e.g., selective conditions) and screening the culture by evaluating nucleotide sequences (e.g., metagenomic sequences) present in the culture after growth and / or by testing the functional properties of the culture. In some embodiments, one or more environmental samples (e.g., organic-rich environmental samples) may be collected. If a single environmental sample is collected, the method includes homogenizing the environmental sample to provide an input sample. If multiple environmental samples are collected, the method includes mixing the multiple environmental samples to provide a mixed environmental sample and homogenizing the mixed environmental samples to provide an input sample.

[0142] In embodiments involving the step of using multiple environmental samples to generate an input sample, collecting and mixing multiple environmental samples can serve not only to maximize the statistical sample space of microorganisms to be screened from, but also to maximize the combinations of microorganisms present in the microbial consortium identified and / or generated using the techniques described herein applied to the input sample. Furthermore, collecting and mixing multiple environmental samples to generate an input sample to which the techniques described herein are applied can generate novel microbial consortia that do not exist in nature by combining microorganisms that do not normally live in the same environment in nature. In some embodiments, various environmental samples from geographically disparate areas can be mixed to further increase the statistical sample space of microbial consortium combinations. For example, embodiments provide that multiple environmental samples are obtained, each environmental sample taken from a different ecosystem, habitat, and / or ecological niche. Embodiments also provide that the multiple environmental samples are obtained from locations that are 1 m, 10 m, 100 m, 1000 m, 10,000 m, or more than 10,000 m away from each other. In some embodiments, samples are obtained from two or more locations anywhere on Earth, including above and below the surface of the Earth's land and water bodies.

[0143] In some cases, multiple input samples may be created during collection. Each input sample of the multiple input samples may include a different combination of individual environmental samples mixed together. For example, environmental samples A, B, and C (from one or more different ecosystems, habitats, and / or ecological niches) may be mixed to provide an input sample that includes A and B, B and C, or A and C. As a further example, environmental samples A, B, C, and D (from one or more different ecosystems, habitats, and / or ecological niches) may be mixed to provide an input sample that includes A, B, and C; A, B, and D; A, C, and D; or B, C, and D. As another example, environmental samples A, B, C, D, and E (from one or more different ecosystems, habitats, and / or ecological niches) may be mixed to provide an input sample including A and B; A and C; A and D; A and E; B and C; B and D; B and E; C and D; C and E; D and E; A, B, and C; A, B, and D; A, B, and E; A, C, and D; A, C, and E; A, D, and E; B, C, and D; B, C, and E; B, D, and E; C, D, and E; A, B, C, and D; A, B, C, and E; A, B, D, and E; A, C, D, and E; B, C, D, and E; or A, B, C, D, and E. Each input sample of the multiple input samples can include a range of fraction compositions of any two individual environmental samples of the multiple individual samples that are mixed together to provide the input sample. For example, any two individual environmental samples may be mixed together to provide an input sample that comprises a fractional composition of the first environmental sample in the range of 0.01 to 0.99 (e.g., 0.01, 0.05, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 0.95, or 0.99 of the first environmental sample) and a fractional composition of the second environmental sample in the range of 0.99 to 0.01 (e.g., 0.99, 0.95, 0.90, 0.80, 0.70, 0.60, 0.50, 0.40, 0.30, 0.20, 0.10, 0.05, or 0.01 of the second environmental sample).

[0144] The input sample can be separated and developed with varying amounts and types of environmental samples mixed together. This is because it is recognized that combinations of microorganisms can be beneficial, but also that individual microorganisms may become ineffective or that microorganisms from foreign environmental samples may become dominant. Furthermore, embodiments of the present technology include the use of a single environmental sample that is homogenized to provide an input sample. Those skilled in the art will appreciate that a single environmental sample may contain multiple individual ecosystems or ecological niches that are not naturally mixed, but are mixed when the single sample is homogenized. For example, an environmental sample may contain multiple individual sub-samples that exist as strata, layers, or subgroups, such as a strata of cylindrical soil core samples, a strata of microbial mat samples, a strata of water column samples, a subgroup of microbial communities including biofilms, etc.

[0145] That is, embodiments of the methods provided herein include the use of a single environmental sample that is homogenized to provide an input sample, and / or include the use of multiple environmental samples that are mixed and homogenized to provide an input sample.

[0146] In some embodiments, a selection of input samples (e.g., environmental samples or mixed environmental samples among mixed environmental samples) based on one or more criteria may be performed. The culture of the input sample may be grown under one or more environmental conditions. In some embodiments, the culture medium is provided without nitrogen compounds (e.g., without a source of immobilized nitrogen) or without carbon compounds (e.g., without a source of immobilized carbon). That is, in some embodiments, the culture medium is described as a nitrogen-free or carbon-free medium, or a "C / N-free medium". The input sample may be subjected to nitrogen for fixation by providing nitrogen from ambient air enrichment, or by bubbling anoxic N2 and providing salts and other nutrients known to be necessary for microorganisms to perform nitrogen fixation. The input sample may also be subjected to CO2, for example, by ambient air enrichment, or by bubbling in CO2.

[0147] In some embodiments, the culture medium is a fresh water replacement medium (e.g., BG-11, see Examples). In some embodiments, the method includes the use of a salt water or seawater medium (or a salt water or seawater replacement medium), and nitrogen fixing and carbon sequestering microorganisms adapted to seawater or other salt water sources are selected according to the methods described herein. In some embodiments, the salt water or seawater medium (or the salt water or seawater replacement medium) provides micronutrients. In some embodiments, the salt water or seawater medium (or the salt water or seawater replacement medium) is supplemented with micronutrients. In some embodiments, the culture medium is produced by adding water and other nutrients (e.g., iron) from evaporated seawater. In some embodiments, trace elements (e.g., including compounds containing one or more of boron, manganese, zinc, molybdenum, copper, cobalt, and / or iron) are added to the culture medium.

[0148] After incubation and time, the cultures are tested based on one or more variables, for example, for increased carbon and nitrogen or increased activity to fix CO2 and / or nitrogen. Measurements may be by quantity. In some embodiments, metagenomic and / or genomic DNA of the microorganisms comprising the candidate microbial consortium is isolated and sequenced for identification. In some embodiments, biomarkers are used to identify one or more microorganisms. In some embodiments, cultures are tested on nitrogen-free and carbon-free media (on liquid or solid media) to measure survival time and / or persistence. In some embodiments, selection of one or more microbial cultures and / or specific portions of one or more microbial cultures is performed to provide cultures for testing. In some embodiments, testing includes evaluating cultures for carbon capture, nitrogen fixation, and / or persistence. In some cases, additives are provided to the cultures to facilitate uptake of the microbial consortium by the environment (e.g., soil) or culture medium. For example, the microbial consortium may require carbon, energy, nitrogen, micronutrients, and reducing equivalents. The above process may be repeated several times through multiple iterations, each iteration further isolating and generating specific information about microorganisms and specific microbial consortia that have achieved the desired results with respect to selected variables, such as climate variables, carbon sequestration, nitrogen fixation, and survival time / persistence. In some embodiments, the selection of microorganisms and microbial consortia for further testing is aided by computational methods, including statistical models and machine learning. See, for example, U.S. Patent Application No. 17 / 544,879, which is expressly incorporated herein by reference in its entirety. For example, embodiments provide that the data is developed into a machine learning model that correlates microorganisms and biomarkers, as well as combinations of microorganisms with the variables being tested. The selection of microbial consortia for testing and / or the selection of properties of the environmental sample are suggested by the machine learning model as a result based on the variables being tested. In some embodiments, the desired phenotype is input along with the desired results for the variables being tested, and the associated microorganisms can be suggested for further testing by the machine learning model.

[0149] In some embodiments, the technology provides machine learning techniques for identifying microbial species and other information associated with one or more variables. In some embodiments, the process includes a series of operations that can be performed in hardware, software, or a combination thereof. With respect to software, the method includes computer-executable instructions that perform the recited operations when executed by one or more processors. In general, computer-executable instructions can include routines, code segments, programs, objects, components, data structures, etc. that perform a particular function or implement a particular abstract data type. The order in which the operations are described is not intended to be construed as a limitation, and any number of the described blocks can be combined in any order and / or in parallel to perform the process.

[0150] In some embodiments, the technology provides further methods for selecting microbial consortia that provide a specified function. In some embodiments, the technology provides methods for screening microbial populations, microbial consortia, and / or multiple microorganisms to generate and / or identify microbial consortia that provide a specified function. In some embodiments, the technology generates microbial consortia that are not found in nature by combining microorganisms from different environments, ecological niches, and / or habitats (e.g., microorganisms that are not found together in nature).

[0151] In some embodiments, the method includes providing a sample containing a plurality of microorganisms, inoculating an Nth volume of growth medium with a portion of the sample to provide an Nth culture, growing the Nth culture under a set of selective conditions, producing an Nth taxonomic classification of microorganisms in the Nth culture, inoculating an N+1th volume of growth medium with a portion of the Nth culture, growing the N+1th culture under a set of selective conditions, producing an N+1th taxonomic classification of microorganisms in the N+1th culture, and deriving a measure of microbial community stability for the N+1th culture using the N+1th taxonomic classification and the Nth taxonomic classification. The measure of microbial community stability is monitored to identify when the measure of microbial community stability has reached a plateau value. If the measure of microbial community stability has not reached a plateau value, the inoculation, growth, taxonomic classification, and deriving the measure of microbial community stability steps of the method are repeated by providing the N+1th sample as the Nth sample. If the measure of microbial population stability reaches a plateau value, the method includes providing the stable N+1 culture as a culture comprising a microbial consortium that performs the identified function. In some embodiments, the steps of inoculating, growing, producing a taxonomic classification, and deriving the measure of microbial population stability are repeated 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times.

[0152] In some embodiments, the method further comprises isolating each of the microorganisms in the stable microbial consortium in pure culture. In some embodiments, the method further comprises obtaining a genomic sequence of each of the microorganisms in the stable microbial consortium in pure culture. In some embodiments, the method further comprises preserving the stable microbial consortium and / or each of the microorganisms in the stable microbial consortium (e.g., by freezing (e.g., at -80°C)). In some embodiments, the method further comprises measuring an identified function of the stable microbial consortium using a test substrate and measuring an output of the function.

[0153] In some embodiments, the technology provides an iterative method in which a portion of a first culture is used to inoculate a second volume of fresh medium. Thus, in some embodiments, a portion of a first culture (e.g., a culture produced by inoculating a selective growth medium with an environmental sample) is used to inoculate a second culture (e.g., comprising the same or different growth medium as the first sample). In some embodiments, a portion of the second culture is used to inoculate a third culture. In some embodiments, a portion of the third culture is used to inoculate a fourth culture. In some embodiments, a portion of the fourth culture is used to inoculate a fifth culture. In some embodiments, a portion of the fifth culture is used to inoculate a sixth culture. In some embodiments, a portion of the sixth culture is used to inoculate a seventh culture. In some embodiments, a portion of the seventh culture is used to inoculate an eighth culture. In some embodiments, a portion of the Nth culture is used to inoculate an N+1th culture. In some embodiments, the Nth culture is a first culture inoculated with at least a portion of the environmental sample. In some embodiments, the Nth culture is a second, third, fourth, fifth, sixth, seventh, eighth, etc. culture inoculated with at least a portion of a culture inoculated with a preceding culture (e.g., the first, second, third, fourth, fifth, sixth, or seventh culture, respectively). As used herein, the process of repeated culturing by using a portion of the Nth culture to inoculate the N+1th culture is referred to as "passaging" the culture.

[0154] Additionally, cultures inoculated directly from environmental samples may be referred to herein as P0 (zero) cultures. The first passage comprises inoculating a fresh culture medium with a portion of the P0 culture to produce a P1 culture, the second passage comprises inoculating a fresh culture medium with a portion of the P1 culture to produce a P2 culture, the third passage comprises inoculating a fresh culture medium with a portion of the P2 culture to produce a P3 culture, the fourth passage comprises inoculating a fresh culture medium with a portion of the P3 culture to produce a P4 culture, the fifth passage comprises inoculating a fresh culture medium with a portion of the P4 culture to produce a P5 culture, the sixth passage comprises inoculating a fresh culture medium with a portion of the P5 culture to produce a P6 culture, the seventh passage comprises inoculating a fresh culture medium with a portion of the P6 culture to produce a P7 culture, the eighth passage comprises inoculating a fresh culture medium with a portion of the P7 culture to produce a P8 culture, and the Nth passage comprises using a portion of the P(N-1) culture to produce a PN culture. As used herein, the term "passage number" refers to a particular passage designated by a number, e.g., passage number 1 refers to the first passage, passage number 2 refers to the second passage, and so on.

[0155] In some embodiments, the volume of the portion of the Nth (e.g., first) culture used to inoculate the N+1th (e.g., second) culture ranges from 100 μl to 100 L, or more, depending on the scale of the culture process (e.g., from research scale to pilot scale to commercial production scale). Thus, embodiments provide for removing a volume of 100 μl to 100 L (e.g., 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 μl; 1, 2, 5, 10, 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 mL; or 1, 2, 5, 10, 20, 50, or 100 L) from one culture and adding that volume to fresh culture medium. In some embodiments, the ratio of inoculation volume to fresh culture medium volume is about 1:10 to 1:1000. Thus, in some embodiments, the volume of fresh culture medium is between 1 ml and 100,000 L (e.g., 1, 2, 5, 10, 20, 50, 100, 200, 500, or 1000 mL, 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, or 100,000 L).

[0156] Growth of cultures In some embodiments, the technology provides methods for reducing the complexity of a population of microorganisms (e.g., present in an environmental sample) while selecting a microbial consortium that performs a specified function and / or identifying a microbial consortium that performs a specified function. Exemplary functions for which a microbial consortium may be selected and / or identified include, for example, photosynthesis (e.g., oxygenic and / or anoxygenic photosynthesis), phosphorus solubilization or mobilization, biodegradation, fermentation, production of chemical precursors, biosensing, nitrogen fixation, carbon fixation, and / or production of sustainable carbon compounds that provide a carbon sink to remove atmospheric carbon (e.g., production of melanin).

[0157] In some embodiments, the environmental sample is used to inoculate a culture medium, and the inoculated culture medium grows under selective conditions provided by the culture medium (e.g., presence, absence, or type of carbon source; presence, absence, or type of nitrogen source; presence, absence, or type of cofactors, minerals, vitamins, or other nutrients; presence, absence, or type of cations and / or anions; presence, absence, or type of trace minerals, cations, and / or anions; presence, absence, or type of solid growth substrate, such as sand or other solid substrate) or with selective conditions provided externally to the growth medium (e.g., temperature; humidity; presence, absence, wavelength, and / or intensity of light; light / dark cycle; pressure; culture medium; culture volume material; size or shape; presence, absence, type, or intensity of culture agitation; presence, absence, and / or type of gas provided).

[0158] In some embodiments, the culture is inoculated and incubated for a length of time, e.g., 30-60 minutes (e.g., 30.0, 30.5, 31.0, 31.5, 32.0, 32.5, 33.0, 33.5, 34.0, 34.5, 35.0, 35.5, 36.0, 36.5, 37.0, 37.5, 38.0, 38.5, 39.0, 39.5, 40.0, 40.5, 41.0, 41.5, 42.0, 42.5, 43.0, 43.5, 44.0, 44.5, 45.0, 45.5, 46.0, 46.5, 47.0, 47.5, 48.0, 48.5, 49.0, 49.5, 50 0.0, 50.5, 51.0, 51.5, 52.0, 52.5, 53.0, 53.5, 54.0, 54.5, 55.0, 55.5, 56.0, 56.5, 57.0, 57.5, 58.0, 58.5, 59.0, 59.5, or 60.0 minutes; 1 to 24 hours (e.g., 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0 , 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 20.5, 21.0, 21.5, 22.0, 22.5, 23.0, 23.5, or 24.0 hours; 1 to 30 days (e.g., 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 1 6.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 20.5, 21.0, 21.5, 22.0, 22.5, 23.0, 23.5, 24.0, 24.5, 25.0, 25.5, 26.0, 26.5, 27.0, 27.5, 28.0, 28.5, 29.0, 29.5, or 30.0 days; and / or 1 to 10 weeks (e.g., 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 weeks).

[0159] In some embodiments, experimental measurements of growth rate, time to exponential growth phase, time to culture saturation, or other culture growth characteristics are measured to identify the length of time for culture growth. In some embodiments, a growth time is selected that provides a culture at or near the end of the exponential growth phase to provide a culture with a robust type and number of microorganisms for further characterization and / or selection. In some embodiments, growth is measured quantitatively and / or qualitatively using measurements of absolute or relative numbers of microorganisms in a defined culture volume. In some embodiments, absolute or relative numbers of microorganisms in a defined culture volume are measured using light scattering, measuring dry or wet mass of solids (e.g., cells) separated from the culture, counting colonies grown on solid media using a portion of the culture, or measuring some other characteristic of the culture or a portion thereof that correlates or causally relates to the number of microorganisms in the culture. In some embodiments, growth is characterized by determining a growth curve. In some embodiments, growth is characterized by determining doubling time and / or time to half saturation. In some embodiments, the growth rate is modeled using experimental data (eg, using a logarithmic model of growth).

[0160] Genetic characterization In some embodiments, the microorganisms in the culture are characterized by shotgun metagenomic sequencing. Techniques and systems for obtaining gene sequences from multiple organisms in samples, such as environmental or clinical samples, are well known to those skilled in the art. For example, Zhou et al. (Appl. Environ. Microbiol. (1996) 62:316-322) provide a robust extraction and purification of nucleic acids. This protocol can also be modified depending on the experimental objectives and the type of environmental sample, such as soil, sediment, and groundwater. Many commercially available kits for DNA extraction and purification can also be used. Samples containing less than 2 pg of purified DNA may require amplification, which can be performed using conventional techniques known in the art, such as the whole-community genome amplification (WCGA) method (Wu et al., Appl. Environ. Microbiol. (2006) 72, 4931-4941). Techniques and systems for obtaining purified RNA from environmental samples are also well known to those skilled in the art. For example, the approach described by Hurt et al. (Appl. Environ. Microbiol. (2001) 67:4495-4503) can be used. This method can simultaneously separate DNA and RNA in the same sample. Gel electrophoresis can also be used to separate RNA populations (McGrath et al., J. Microbiol. Methods (2008) 75:172-176). Samples containing less than 5 pg of purified RNA may require amplification, which can be performed using conventional techniques known in the art, such as the Whole Population RNA Amplification Approach (WCRA) to obtain cDNA (Gao et al., Appl. Environ. Microbiol. (2007) 73:563-571). In some embodiments, environmental sample collection and DNA extraction are performed as previously described (DeSantis et al., Microbial Ecology, 53(3):371-383, 2007).

[0161] The isolated nucleic acid (e.g., metagenomic DNA) can be subjected to a sequencing method to obtain metagenomic sequence data. Sequencing methods can be broadly divided into those that typically use template amplification and those that do not. Methods that require amplification include pyrosequencing (e.g., GS 20 and GS FLX), commercialized as the 454 technology platform by Roche, the Solexa platform commercialized by Life Technologies / Ion Torrent, Illumina, and GnuBio, and the Supported Oligonucleotide Ligation and Detection (SOLiD) platform commercialized by Applied Biosystems. Non-amplification approaches, also known as single molecule sequencing, are exemplified by the HeliScope platform commercialized by Helicos BioSciences, and emerging platforms commercialized by VisiGen, Oxford Nanopore Technologies Ltd., and Pacific Biosciences, respectively. Thus, metagenomic shotgun sequencing, in some embodiments, includes pyrosequencing, sequencing by ligation, single molecule sequencing, sequencing by synthesis (SBS), semiconductor sequencing, nanopore sequencing, massive parallel clonal, massive parallel single molecule SBS, massive parallel single molecule real-time, massive parallel single molecule real-time nanopore technology, etc. Morozova and Marra provide a review of some such techniques in Genomics, 92:255 (2008), which is incorporated herein by reference in its entirety. Those skilled in the art will recognize that because RNA is less stable in cells and more susceptible to attack by nucleases, experimentally RNA is usually reverse transcribed into DNA prior to sequencing.

[0162] Specific descriptions of some DNA sequencing approaches include fluorescence-based sequencing methodologies (see, e.g., Birren et al., Genome Analysis: Analyzing DNA, 1, Cold Spring Harbor, NY, which is incorporated herein by reference in its entirety); automated sequencing approaches; parallel sequencing of split amplicons (PCT Publication WO2006084132 to Kevin McKernan et al., which is incorporated herein by reference in its entirety; and sequencing by parallel oligonucleotide extension (see, e.g., U.S. Pat. No. 5,750,341 to Macevicz et al. and U.S. Pat. No. 6,306,597 to Macevicz et al., both of which are incorporated herein by reference in their entirety). Further descriptions of sequencing approaches include the Church polony technique (Mitra et al., 2003, Analytical Biochemistry 320, pp. 55-65; Shendure et al., 2005 Science 10, 103-105, which are incorporated herein by reference in their entirety). 309, pp. 1728-1732; U.S. Pat. No. 6,432,360, U.S. Pat. No. 6,485,944, U.S. Pat. No. 6,511,803; 454 Picotiter pyrosequencing technology (Margulies et al., 2005 Nature 811, which is incorporated by reference in its entirety). 437, pp. 376-380; U.S. Patent Application Publication No. 2005 / 0130173; Solexa single base addition technology (Bennett et al., 2005, Pharmacogenomics, 6, pp. 373-382; U.S. Patent No. 6,787,308; U.S. Patent No. 6,833,246, which are incorporated by reference in their entirety); Lynx massively parallel signature sequencing technology (Brenner et al., (2000) Nat. Biotechnol. 18:630-634; U.S. Patent No. 5,695,934; U.S. Patent No. 5,714,330, which are incorporated by reference in their entirety; and Adessi PCR colony technology (Adessi et al. (2000) Nucleic Acid Res. 28, p. E87; WO00018957, which are incorporated by reference in their entirety).See also, e.g., Voelkerding et al., Clinical Chem, 55:641-658, 2009; MacLean et al., Nature Rev. Microbiol. 7:287-296, each of which is incorporated by reference in its entirety.

[0163] Sequence analysis In some embodiments, metagenomic nucleotide sequence data is analyzed to characterize the microbial population (e.g., microbial consortium) from which the metagenomic nucleic acid was obtained. For example, in some embodiments, taxonomic units in a microbial population are taxonomically classified and / or identified by obtaining metagenomic nucleotide sequence data from the microbial population and using an algorithm that associates short genomic subsequences (k-mers) in the metagenomic nucleotide sequence data with least common ancestor (LCA) taxa (e.g., using a curated database). See, for example, Wood (2014) "Kraken: ultrafast metagenomic sequence classification using exact alignments" Genome Biology 15:R46 and Wood (2019) "Improved metagenomic analysis with Kraken 2" Genome Biology 20:257, each of which is incorporated herein by reference. In some embodiments, BLAST is used to identify microbial taxonomic units present in a microbial population (e.g., microbial consortium). See, e.g., Altschul (1990) "Basic local alignment search tool," J Mol Biol 215:403-410, incorporated herein by reference.Other tools for identifying taxonomic units in a microbial community using metagenomic sequence data from the microbial community include, for example, MEGAN (see, e.g., Huson (2007) "MEGAN analysis of metagenomic data," Genome Res 17:377-386, which is incorporated by reference herein); PhymmBL (see, e.g., Brady (2009) "Phymm and PhymmBL: metagenomic phylogenetic classification with interpolated Markov models," Nat Methods 6:673-676; and Brady (2011) "PhymmBL expanded: confidence scores, custom databases, parallelization and more," Nat Methods 8:367, each of which is incorporated by reference herein); and the Naive Bayes Classifier (NBC) (see, e.g., Rosen (2008) "Metagenome fragment classification using N-mer frequency profiles," Adv Bioinformatics 2008:1-12, which is incorporated by reference herein). In some embodiments, ribosomal RNA sequences are taxonomically identified using average nucleotide identity (ANI), provided in the GTDB-tk package.See, e.g., Chaumeil (2019) "GTDB-Tk: A toolkit to classify genomes with the Genome Taxonomy Database," Bioinformatics, btz848; Parks (2019) "A complete domain-to-species taxonomy for Bacteria and Archaea," Nat Biotechnol. 38:1079-86; and Parks (2018) "A standardized bacterial taxonomy based on genome phylogeny substantially revises the tree of life," Nat. Biotechnol 36:996-1004, each of which is incorporated by reference herein.

[0164] In some embodiments, characterizing a microbial population includes identifying, in absolute and / or relative terms, the taxonomic units (e.g., strains, subspecies, species, genus, family) of organisms present in the microbial population. In some embodiments, characterizing a microbial population includes identifying, in relative terms, for example, the taxonomic units (e.g., strains, subspecies, species, genus, family) of organisms enriched in a particular passage relative to a previous passage or an earlier environmental sample.

[0165] In some embodiments, the organism is identified by comparing the nucleotide sequence to a database of ribosomal RNA gene sequences, such as nucleotides of the 5S, 16S, and / or 23S ribosomal RNA genes. See, e.g., Tringe and Hugenholtz (2008) "A renaissance for the pioneering 16S rRNA gene," Curr Opin Microbiol 11:442-46; and Ju and Zhang (2015) "16S rRNA gene high-throughput sequencing data mining of microbial diversity and interactions," Appl Microbiol Biotechnol 99:4119-29, each of which is incorporated herein by reference. See also the Examples herein. In some embodiments, photosynthetic organisms are identified by comparing the nucleotide sequence to a database of plastid 23S ribosomal DNA sequences (see, e.g., Djemiel (2020) "μgreen-db: a reference database for the 23S rRNA gene of eukaryotic plastids and cyanobacteria," Sci Rep. 10:5915, incorporated herein by reference).

[0166] Those skilled in the art understand that there is no clear consensus on bacterial taxonomy. Hundreds of thousands of bacterial genomes are sequenced every year, including genomes that may only be found as members of a community. As a result, new taxonomic classifications of these organisms continue to evolve. See, for example, Coleman (2021) "A rooted phylogeny resolves early bacterial evolution," Science 372:(6542):eabe0511, which is incorporated herein by reference. Multiple analysis tools (e.g., ANI and BLAST) are used herein to determine the species designation of rRNA nucleotide sequences and to identify the associated taxonomy. Bacterial taxonomy provided by sequence database hits using BLAST and NCBI's nucleotide sequence database may not match taxonomy provided by other methods. For example, the genus Burkholderia is included in Betaproteobacteria in NCBI, but in Gammaproteobacteria in other databases. In all cases, the species assigned was correct and the assignment to a broader taxonomic level incorporated information from the multiple analysis methods used.

[0167] Further software packages for producing and analyzing nucleotide sequences are fastp, bowtie2, and FATQC for quality trimming and assessment; metaSPADES and MEGAHIT2 for assembly; QUAST for assessment of assemblies; METABAT2 for binning; GTDB-tk for taxonomic classification; and Barrnap for prediction of ribosomal RNA. See Example 20.

[0168] Group and community analysis In some embodiments, the stability of a microbial population and / or microbial consortium is measured, for example, by deriving a measure of similarity (or dissimilarity) between a first culture and a second culture inoculated with a portion of the first culture, and optionally following the measure of similarity upon subsequent inoculation. In some embodiments, the taxonomic classification and / or identification of the organisms in the microbial population (e.g., provided by a taxonomic classifier (e.g., Kraken2) described above) can provide input to such a stability measure. In some embodiments, the functional capabilities or functionality provided by and / or present in the microbial population (e.g., genes, gene products, functional capabilities, and / or activities) provide input to the stability measure.

[0169] A variety of measures can be used to compare the similarity (or dissimilarity) of microbial groups, including estimates of microbial group richness and diversity (see, e.g., Hughes (2001) "Counting the uncountable: statistical approaches to estimating microbial diversity" Appl. Environ. Microbiol. 67:4399-4406; and Ley (2005) "Obesity alters gut microbial ecology" Proc. Natl. Acad. Sci. USA 102:11070-11075, each of which is incorporated herein by reference), as well as estimates of alpha or beta diversity, such as the Bray-Curtis Dissimilarity Index (Bray and Curtis (1957) "An Ordination of the Upland Forest Communities of Southern Wisconsin" Ecol. Monogr. 27:325-349, incorporated herein by reference). Bray-Curtis distances can be calculated using the bcdist function in the ecodist package (Goslee (2007) "The ecodist package for dissimilarity-based analysis of ecological data" J Stat Softw 22:1-19, incorporated herein by reference). Bray-Curtis distance matrices for community data, geographic distances, and correlations between environmental variables can be calculated using the mantel function in the vegan package (Oksanen, vegan: Community Ecology Package for R); see, e.g., Legendre, P. and Legendre, L. (2012) Numerical Ecology. English 3rd ed., Elsevier, incorporated herein by reference.

[0170] During the development of embodiments of the technology described herein, the stability of the microbial consortium was evaluated using the Bray-Curtis dissimilarity index (see Examples). The Bray-Curtis dissimilarity index is bounded by 0 and 1, with a score of 0 indicating no difference between the two cultures, groups, communities, sites, passages, or other sets of compared microorganisms. A score of 1 indicates that the two cultures, groups, communities, sites, passages, or other sets of compared microorganisms have no common species. Thus, the Bray-Curtis dissimilarity index provides a useful measure of the amount of change that occurs between each individual passage. When the Bray-Curtis dissimilarity score begins to stabilize between two passages (e.g., when a plot of the Bray-Curtis dissimilarity score as a function of passage and / or time approaches a horizontal asymptote), the consortium is approaching maximum stability, and therefore there is minimal or no change from one passage to the next and / or between an inoculum and subsequent cultures produced from that inoculum.

[0171] Some context for interpreting the values ​​of the Bray-Curtis dissimilarity scores can be gained by comparing well-studied microbiota, e.g., those associated with the human body. In one comparative study using a reference set of human microbiota data, the Bray-Curtis dissimilarity between distinguishable microbiota (fecal and nasal samples) was 0.79 ± 0.08 (SD) compared to the reference dataset, with individual fecal microbiota having a mean Bray-Curtis dissimilarity of 0.24 ± 0.17 (SD) (see, e.g., Maziarz (2018) “Using standard microbiome reference groups to simplify beta-diversity analyses and facilitate independent validation,” Bioinformatics 34:3249-57, incorporated herein by reference). That is, low measurements represent variability of the microbiota in fecal samples, while high measurements indicate variation between the intestinal and respiratory systems. It should be understood that these values ​​are intended to be descriptive, and that measures of Bray-Curtis dissimilarity between other samples will differ and may vary more widely between 0.00 and 1.00.

[0172] Several tools are available that provide these and other inferences of microbial community structure (e.g., describing the abundance of community members), such as LIBSHUFF (Schloss (2004) "Integration of microbial ecology and statistics: a test to compare gene libraries" Appl. Environ. Microbiol. 70:5485-5492; and Singleton (2001) "Quantitative comparisons of 16S rRNA gene sequence libraries from environmental samples" Appl. Environ. Microbiol. 67:4374-4376, each of which is incorporated herein by reference), TreeClimber (Martin (2002) "Phylogenetic approaches for describing and comparing the diversity of microbial communities" Appl. Environ. Microbiol. 68:3673-3682; and Schloss (2006) "Introducing TreeClimber, a test to compare microbial communities" Appl. Environ. Microbiol. 68:3673-3682, each of which is incorporated herein by reference), and ELISA (Schloss (2006) "Introducing TreeClimber, a test to compare microbial communities" Appl. Environ. Microbiol. 68:3673-3682, each of which is incorporated herein by reference). Lozupone (2005) "UniFrac: a new phylogenetic method for comparing microbial communities" Appl. Environ. Microbiol. 72:2379-2384; UniFrac (incorporated herein by reference)71:8228-8235, and analysis of molecular variance (AMOVA) (Excoffier (1992) "Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data," Genetics 131:479-491; and Martin (2002) "Phylogenetic approaches for describing and comparing the diversity of microbial communities," Appl. Environ. Microbiol. 68:3673-3682, each of which is incorporated herein by reference; DOTUR (Schloss (2005) "Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness," Appl. Environ. Microbiol. 71:1501-1506, each of which is incorporated herein by reference; and SONS (Schloss (2006) "Introducing SONS,a Tool for Operational Taxonomic Unit-Based Comparisons of Microbial Community Memberships and Structures” Appl Environ Microbiol.72:6773-6779 (which includes several measures including membership measures (e.g., the incidence-based Sorenson similarity index), group structure using abundance (e.g., Clayton's theta (see, e.g., Yue (2001) "A nonparametric estimator of species overlap," Biometrics 57:743-9, each of which is incorporated herein by reference), and group richness (see, e.g., Chao (1984) "Non-parametric estimation of the number of classes in a population," Scand. J. Stat. 11:265-270; Chao (2005) "A new statistical approach for assessing similarity of species composition with incidence and abundance data," Ecol. Lett. 8:148-159; Chao (2000) "Estimating the number of shared species in two communities," Stat. Sinica 10:227-246; Chao (1992) "Estimating the number of classes via sample coverage" J. Am. Stat. Assoc. 87:210-217; and Chao (2006) "The applications of Laplace's boundary-mode approximations to estimate species richness and shared species richness" Aust. NZJ Stat. 48:117-128).

[0173] sample The present technology is not limited to the type of sample, including samples (e.g., environmental samples) containing microorganisms used as starting materials (e.g., input samples) on which the methods described herein (e.g., methods for selecting microbial consortia and / or methods for screening to identify microbial consortia) are performed. In some embodiments, the input sample used may be an environmental sample from any source, such as naturally occurring or manmade air, water systems and water sources, soil, or any other sample of interest. In some embodiments, environmental samples may be obtained, for example, from indoor or outdoor air or airborne particle collection systems, indoor surfaces, and surfaces of machines, devices, or equipment. In some embodiments, the sample is obtained from an ecosystem (e.g., in some embodiments, the sample is an environmental sample taken from an ecosystem). The ecosystem may be of the Earth, including all known terrestrial environments, including, but not limited to, soil, surface, and above-surface environments. The ecosystem includes ecosystems classified in the Food and Agriculture Organization's Land Cover Classification System (LCCS) and the Forest-Range Environmental Study Ecosystems (FRES) developed by the U.S. Forest Service. Exemplary ecosystems include forests such as tropical rainforests, temperate rainforests, temperate sclerophyll forests, boreal forests, taiga, and montane forests; grasslands including savannas and steppes; deserts; wetlands including marshes, swamps, bogs, estuaries, and sloughs; coastal ecosystems, alpine, and tundra ecosystems. Ecosystems further include ecosystems associated with aquatic environments such as lakes, running water, springs, coral reefs, beaches, estuaries, seamounts, trenches, and intertidal zones. Ecosystems also include soils, humus soils, mineral soils, and aquifers. Ecosystems further encompass subterranean environments such as mines, oil fields, caves, fault and fracture zones, geothermal fields, and aquifers. Ecosystems further include microflora associated with plants, animals, and humans. Exemplary plant-associated microbiota include ecosystems found in or near roots, bark, stems, leaves, and flowers.The microbiota associated with animals and humans includes ecosystems found in the gastrointestinal tract, respiratory system, nasal passages, urogenital tract, mammary glands, oral cavity, Eustachian tube, feces, urine, and skin. In some embodiments, the sample can be any type of clinical or medical sample. For example, the sample can be from the blood, urine, feces, nasal passages, lungs, or intestines of a mammal.

[0174] In some embodiments, one or more environmental samples are collected. If a single environmental sample is collected, the method includes homogenizing the environmental sample to provide an input sample. If multiple environmental samples are collected, the method includes blending the multiple environmental samples to provide a blended environmental sample and homogenizing the blended environmental sample to provide an input sample.

[0175] In embodiments involving the step of using multiple environmental samples to generate an input sample, collecting and mixing multiple environmental samples may serve not only to maximize the statistical sample space of microorganisms to be screened therefrom, but also to maximize the combinations of microorganisms present in the microbial consortium identified and / or generated using the techniques described herein applied to the input sample. Furthermore, collecting and mixing multiple environmental samples to generate an input sample to which the techniques described herein are applied may generate novel microbial consortia not present in nature by combining microorganisms that do not normally live in the same environment in nature. In some embodiments, various environmental samples from geographically disparate areas may be mixed to further increase the statistical sample space of microbial consortium combinations. For example, embodiments provide that multiple environmental samples are obtained, each environmental sample taken from a different ecosystem, habitat, and / or ecological niche. Embodiments further provide that the multiple environmental samples are obtained from locations that are 1 m, 10 m, 100 m, 1000 m, 10,000 m, or more than 10,000 m away from each other. In some embodiments, samples are obtained from two or more locations anywhere on Earth, including above and below the surface of the Earth's land and water bodies.

[0176] In some cases, multiple input samples may be created during collection. Each input sample of the multiple input samples may include a different combination of individual environmental samples mixed together. For example, environmental samples A, B, and C (from one or more different ecosystems, habitats, and / or ecological niches) may be mixed to provide an input sample that includes A and B, B and C, or A and C. As a further example, environmental samples A, B, C, and D (from one or more different ecosystems, habitats, and / or ecological niches) may be mixed to provide an input sample that includes A, B, and C; A, B, and D; A, C, and D; or B, C, and D. As another example, environmental samples A, B, C, D, and E (from one or more different ecosystems, habitats, and / or ecological niches) may be mixed to provide an input sample including A and B; A and C; A and D; A and E; B and C; B and D; B and E; C and D; C and E; D and E; A, B, and C; A, B, and D; A, B, and E; A, C, and D; A, C, and E; A, D, and E; B, C, and D; B, C, and E; B, D, and E; C, D, and E; A, B, C, and D; A, B, C, and E; A, B, D, and E; A, C, D, and E; B, C, D, and E; or A, B, C, D, and E. Each input sample of the multiple input samples can include a range of fraction compositions of any two individual environmental samples of the multiple individual samples that are mixed together to provide the input sample. For example, any two individual environmental samples may be mixed together to provide an input sample that comprises a fractional composition of the first environmental sample in the range of 0.01 to 0.99 (e.g., 0.01, 0.05, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 0.95, or 0.99 of the first environmental sample) and a fractional composition of the second environmental sample in the range of 0.99 to 0.01 (e.g., 0.99, 0.95, 0.90, 0.80, 0.70, 0.60, 0.50, 0.40, 0.30, 0.20, 0.10, 0.05, or 0.01 of the second environmental sample).

[0177] The input sample can be separated and developed with varying amounts and types of environmental samples mixed together. This is because it is recognized that combinations of microorganisms can be beneficial, but also that individual microorganisms may become ineffective or that microorganisms from foreign environmental samples may become dominant. Furthermore, embodiments of the present technology include the use of a single environmental sample that is homogenized to provide an input sample. Those skilled in the art will appreciate that a single environmental sample may contain multiple individual ecosystems or ecological niches that are not naturally mixed, but are mixed when the single sample is homogenized. For example, an environmental sample may contain multiple individual sub-samples that exist as strata, layers, or subgroups, such as a strata of cylindrical soil core samples, a strata of microbial mat samples, a strata of water column samples, a subgroup of microbial communities including biofilms, etc.

[0178] That is, embodiments of the methods provided herein include the use of a single environmental sample that is homogenized to provide an input sample, and / or include the use of multiple environmental samples that are mixed and homogenized to provide an input sample.

[0179] microbial community As described herein, the technology provides microbial consortia with identified functions. In some embodiments, the microbial consortia are produced according to the selection methods described herein. In some embodiments, the technology applies selection conditions to a sample (e.g., including a homogenized environmental sample or a plurality of environmental samples that are homogenized), where the selection conditions reduce the complexity of the microbial community and improve the function of the microbial community.

[0180] During development of embodiments of the technology described herein, embodiments of the selection method produced three microbial consortia designated BW01, BW02, and BW05. Each of the BW01 and BW02 consortia consisted of organisms taxonomically identified using 16S rRNA gene sequences as being similar to unclassified Nostoc species that may be Nostoc species or form a clade separate from other known Nostoc species. Additionally, all of the minimal consortia produced from BW01, BW02, and BW05 comprised members of the Nostocaceae family. These organisms were identified as Nostoc punctiforme PCC 73102, Nostoc NIES-4103 spp., Nostoc UAM 307 spp., and Dolichospermum flos-aquae. These members represent three Nostoc genera: Nostoc PCC-73102, Desmonostoc PCC-6302, and Desmonostoc PCC-7422 (Example 20). In an independent experiment that produced a microbial consortium and six minimal consortia (Example 21), all six of the minimal consortia contained Nostoc or photosynthetic Nostoc-like organisms, such as Anabaena species.

[0181] Nostoc or Nostoc-like organisms in the microbial consortium were present at an abundance of approximately 30% of the consortium population. The original input soil sample contained Nostoc spp. at less than 0.1% of the microbial consortium. That is, Nostoc or Nostoc-like species were present in the input sample, but at very low levels in the environment. The growth conditions applied during the development of embodiments of the present technology were chosen to select for species that grow photoautotrophically and fix nitrogen from the atmosphere. Under these conditions, Nostoc or Nostoc-like species were highly competitive and increased significantly in relative abundance in the culture, providing the major components of the BW01 and BW02 consortiums. Without being bound by theory, it is hypothesized that Nostoc or Nostoc-like species are the major energy source at the top of the energy web for the BW01, BW02, and BW05 consortiums. In particular, it is envisioned that the Nostoc species (or Nostoc-like species) in BW01, BW02, and BW05 function to 1) fix carbon (e.g., as photosynthetic output (e.g., in the form of extracellular polymeric substances (EPS))) and 2) fix nitrogen. Thus, embodiments of the present technology provide microbial consortia that include at least 30% (e.g., at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60% or more) Nostoc species.

[0182] Furthermore, during the development of embodiments of the technology described herein, experiments have shown that the BW01 and BW02 microbial consortia (e.g., containing at least 30% Nostoc species) produced by the selection method also contain some heterotrophic life forms. Without being bound by theory, it is hypothesized that the heterotrophic organisms import and metabolize organic compounds, such as polysaccharides, associated with the EPS produced by Nostoc species or Nostoc-like species. Cyanobacteria are known to release significant amounts of available carbon into the environment, which can be utilized by cyanobacteria or other bacteria in the environment. See, for example, Stuart (2016) "Cyanobacterial reuse of extracellular organic carbon in microbial mats," ISME J. 10:1240-51 (2016); Bertilsson (2005) "Release of dissolved organic matter by Prochlorococcus," Vie et Milieu 55:225-31, each of which is incorporated herein by reference.

[0183] Some genera were reduced during the passaging process. For example, data collected from the characterization of the BW01 and BW02 communities showed that the communities were composed of genera such as Bacillus spp., Bradyrhizobium spp., Streptomyces spp., and Pseudomonas spp. at low levels. Although all of these genera were present in the input samples, the abundance of each of these genera was reduced to low amounts in the BW01 and BW02 communities throughout the passaging process. Thus, these data indicated that the metabolic functions of these genera were inadequate to support the growth of these organisms in the light-promoted communities BW01 and BW02.

[0184] Some genera, such as Methylobacterium spp., were present at relatively low levels in the input samples and remained at similarly low levels throughout the selection process. These data therefore indicate that these microorganisms were not adversely affected by the selection conditions and therefore may interact with the dominant genera in the community (e.g., Nostoc spp.) or consume metabolites produced by other organisms in the community.

[0185] Several genera increased during P1, then decreased in P2, and continued to decrease in P3 and P4. These genera may be important for the selection process. Without being bound by theory, it is hypothesized that these genera may be necessary for the continuation of the population as it approaches stability and / or that these genera may temporarily compete with other species. Genera that increased during passaging included Brevundimonas spp. and Aminobacter spp., both in BW01 and BW02. Both are alphaproteobacteria, and their exact role as bottleneck resolvers is currently unknown.

[0186] Data collected during the experiments described herein indicated that phototrophic purple bacteria of the genus Rhodobacter were found among the genera represented. Purple photosynthetic bacteria have been shown to provide numerous benefits for agriculture, including phosphate solubilization, nitrogen fixation, and plant growth promotion (see, e.g., Sakarika (2020) "Purple non-sulphur bacteria and plant production: benefits for fertilization, stress resistance and the environment," Microb. Biotechnol. 13:1336-65, incorporated herein by reference). Purple bacteria perform the functions of anoxygenic photosynthesis and carbon fixation, but not aerobic photosynthesis or nitrogen fixation. These processes are exquisitely regulated in these organisms as a response to oxygen availability (see, e.g., McEwan (1994) "Photosynthetic electron transport and anaerobic metabolism in purple non-sulfur phototrophic bacteria," Antonie Van Leeuwenhoek 66:151-164, incorporated herein by reference). Thus, it is assumed that these metabolically diverse purple bacteria consume the secretions of Nostoc species in a consortium when oxygen is present. In nature, complex biofilms (e.g., in agricultural environments), non-oxygenic phototrophs are exposed to both light and anoxic conditions.Anoxygenic phototrophic organisms have been shown to constitute the bottom layer of biofilms where oxygen tension is reduced (see, e.g., Roeselers (2008) "Phototrophic biofilms and their potential applications," J. Appl. Phycol. 20:227-35; and Martinez-Alonso (2005) "Diversity of anoxygenic phototrophic sulfur bacteria in the microbial mats of the Ebro Delta: a combined morphological and molecular approach," FEMS Microbiol. Ecol. 52:339-50, each of which is incorporated herein by reference). Without being bound by theory, it is hypothesized that purple photosynthetic bacteria contribute to carbon fixation in mature biofilms by absorbing infrared light that penetrates the top layer, which contains chlorophyll-a-containing Nostoc species, and fixing carbon and nitrogen in the anaerobic bottom layer of the biofilm.

[0187] Some of the consortia were composed of bacterial genera, namely Nodularia, Chrysosporum, Gloeocapsopsis, Lychearia, Mastigocoleus, Hapalosiphon, Gloeothece, Acaryochloris, Camptonema, Raphidiopsis, Crocosphera, Macrochaete, Thermosynethococcus, Pseudoanabaena, Chroococcidiopsis, Prochlorothrix, Anabaena, Leptolingbya, Callosthrix, Cylindrospermopsis, Dolichospermum, Cytonema, Lyngbya, Tolyposthrix, Fischerella, Fortiera, Alitherella, Hydrocoryne, Prochlorococcus, Planctotrichoides, Gaitrelinema, Xenococcus, Yaginema, Nostocopsis, Pantanalinema, Oscillatoria, Spirulina, Pellatocladus, Nodococcilinea, Aphanizomenon, Chloroglossum, and Chloroglossum. and one or more further photosynthetic members from the algal genera Rhoeopsis, Gloeocapsa, Karenema, Livularia, Trichorum, Synechococcus, Synechocystis, Cylindrospermum, Planktothrix, Bosea, Cinella, Novosphingobium, or Rubidibacter, or from the algal genera Tetraselmis, Chlorella, Coreochaeta, Gonium, Paradoxia, Symbomonas, Palmaria, Calasiochloris, Marvania, Chlamydomonas, Parachlorella, Coccomyxa, Trebuxia, Cyanidium, Troybaria, Danjardinia, Gardieria, Nuformia, Porphyra, Cara, Anchistrodesmus, Picochlorum, Cyanophora, Chondrus, Pseudochloris, Interphyllum, Gelidium, Symphyogina, Chlorosarquina, or Cyanothece.

[0188] In some embodiments, the subject technology provides a microbial consortium comprising organisms having a ribosomal RNA gene sequence provided by one of SEQ ID NOs: 1, 3, 7, 10, 13, 19, 22, 26, 30, 32, 34, 35, 39, 42, 45, 51, 54, 57, 61, 63, 66, 70, 74, 84, 89, 99, 103, 108, 116, 122, 123, or 125. In some embodiments, the technology provides a microbial consortium comprising organisms having a ribosomal RNA gene sequence having 97% or more, 95% or more, 90% or more, 80% or more, or 70% or more nucleotide sequence identity to a nucleotide sequence provided by one of SEQ ID NOs: 1, 3, 7, 10, 13, 19, 22, 26, 30, 32, 34, 35, 39, 42, 45, 51, 54, 57, 61, 63, 66, 70, 74, 84, 89, 99, 103, 108, 116, 122, 123, or 125.

[0189] In some embodiments, the technology provides a microbial consortium comprising organisms having a ribosomal RNA gene sequence provided by the consensus sequence of SEQ ID NO:127.

[0190] [ka]

[0191] In some embodiments, the technology provides a microbial consortium that includes organisms having ribosomal RNA gene sequences that are 95, 96, 97, 98, or 99% identical to the consensus sequence of SEQ ID NO:127.

[0192] In some embodiments, the technology can be used with any of the following: Aliinostoc sp. SA22, A. nigriterrae, A. nigriterrae CENA18, A. nigriterrae CENA66, A. nigriterrae CENA67, A. nigriterrae CENA69, A. thailandica, A. thailandica NUACC02, A. thailandica NUACC03, Anabaena catenula SAG 1403-1, Anabaena oryzae Ind3, Anabaena oryzae WY07, Anabaena oscillioides str.BO HINDAK 1984 / 43, Anabaena sp. 0830-A, Anabaena sp. CCAP 1403 / 4A, Anabaena sp. HBU1, Anabaena sp. HBU10, Anabaena sp. Ind5, Anabaena sp. KVSF7, Anabaena sp. PCC 7108, Anabaena spp. SN430, Anabaena spp. YBS01, Anabaenopsis circularis (A. circularis) NIES-21, Atlanticohryx sylvestris (A. silvestris), Atlanticohryx sylvestris CENA368, Atlanticohryx sylvestris CENA564, Atlanticohryx sylvestris CENA576, Atlanticohryx sylvestris CENA579, Atlanticohryx sylvestris CENA585, Atlanticohryx sylvestris CENA590, Aulosira laxa (A. laxa) NIES-50, Aulosira spp. sp. CENA272, Aurocira sp. CENA288, Aurocira sp. CENA291, Aurocira sp. CENA295, C. brevissima IAM M-249, C. brevissima NIES-22, C. membranacea SAG 1410-1, Calothrix sp.)CCAP 1410 / 13,Caloshrix spp. CENA283,Caloshrix spp. CHAB TP201506,Caloshrix spp. CHAB TP201518,Caloshrix spp. CHAB TP201519,Caloshrix spp. CHAB TP201521,Caloshrix spp. CHAB TP201524,Caloshrix spp. CHAB TP201528,Caloshrix spp. CHAB2384,Caloshrix spp. NIES-2098,Caloshrix spp. NIES-2099,Caloshrix spp. NIES-2100,Caloshrix spp. PCC 7507,Caloshrix spp. SA4,Caloshrix spp. SA47,Camptylonemopsis spp. sp. HA4241-MV5, Chrysosporum bergii 09-02, Chrysosporum bergii ANA360D, C. shennongjiaense, C. karadense, Constrictifilum caladense MKW3, Constrictifilum sp. Assy 17, C. rudolphia, Cyanocohniella rudolphia SY-1-2-Y, Cyanocohniella sp. SY-1-2-EE, C. catenatum CCALA 999, C. muscicola ACSSI 140, Cylindrospermum musicola Ind12, Cylindrospermum musicola SAG 44.79, Cylindrospermum skujae ACSSI 112, Cylindrospermum sp. ACSSI 010, Cylindrospermum sp. ACSSI 028, Cylindrospermum sp. ACSSI 040, Cylindrospermum sp. ACSSI 041, Cylindrospermum sp. ACSSI 043, Cylindrospermum sp. CHAB2115, Cylindrospermum sp. NIES-4074, Cylindrospermum sp. YK2-01, Dendronarium phyllosphaericum (D.phyllosphericum), Dendonarium phyllosphericum CENA358, Dendonarium phyllosphericum CENA389, Dendonarium phyllosphericum CENA73, Desikacharya species sp.)PS2C, D.caucasicum, D.caucasicumMZ-C154, D.danxiaense, D.danxiaenseCHAB5868, D.danxiaenseCHAB5869, D.lechangense, D.lechangenseCF01, D.magnisporum, D.magnisporumAR6_PS, D.muscorum, D.muscorum9a, D.muscorumACSSI 091, D.muscorumACSSI 149, D.muscorumCCAP 1453 / 22, D.muscorumCCAP 1453 / 32, Desmonostoc muscorum CCAP 1453 / 8, Desmonostoc muscorum DRSCY01, Desmonostoc muscorum I, Desmonostoc muscorum Ind33, Desmonostoc muscorum SAG 57.79, Desmonostoc muscorum SERB 54, Desmonostoc muscorum UTAD_N213, Desmonostoc persicum, Desmonostoc punense, Desmonostoc punense MCC 2741, Desmonostoc salinum, Desmonostoc salinum CCM-UFV059, Desmonostoc sp.), Desmonostoc sp. CCIBT 3489, Desmonostoc sp. CCIBt3489, Desmonostoc sp. CCM-UFV020, Desmonostoc sp. CCM-UFV069, Desmonostoc sp. CCM-UFV070, Desmonostoc sp. CENA362, Desmonostoc sp. CENA363, Desmonostoc sp. CENA365, Desmonostoc sp. CENA371, Desmonostoc sp. CENA380, Desmonostoc sp. CENA383, Desmonostoc sp. CENA386, Desmonostoc sp. Ds1, Desmonostoc sp. PCC 6302, Desmonostoc sp. PCC 7422, Desmonostoc sp. PCC 7906, Desmonostoc sp. PCC 8107, Desmonostoc sp. PCC 8306, Desmonostoc sp. SA25, Desmonostoc sp. UHCC0398, Dolichospermum flos-aquae, Dolichospermum flos-aquae FACHB-245, Dolichospermum flos-aquae UTCC 64, F. laiensis HA4221-MV2, Fortiea sp. PS4G, F. diplosiphon NIES-3275, Geitlerinema sp. CHAB TP201828.1, Geitlerinema sp. CHAB TP201828.2, G. echinulata PYH14, G. apudmare HA4356-MV2, Goleter sp. CHAB TP201702.1, Goleter sp. CHAB TP201821.1, Goleter sp. CHAB TP201823.11, Goleter sp. CHAB TP201823.2, Goleter sp. CHAB TP201823.8, Johanseniella A1345, Johanseniella sp. CENA33, Komarekiella atlantica (K.atlantica, Komarequilla atlantica CCIBt 3307, Komarequilla atlantica CCIBt 3481, Komarequilla atlantica CCIBt 3483, Komarequilla atlantica CCIBt 3486, Komarequilla atlantica CCIBt 3487, Komarequilla atlantica CCIBt 3552, Komarequilla atlantica CENA107, Komarequilla globosa, Komarequilla globosa PJ104, Microchaete diplosiphon CCALA 811, Microchaete sp. CENA541, Microchaete sp. SAG 47.93, Nodularia sp. sp.) Su-A, Nodularia sphaerocarpa HKVV, Nodularia sphaerocarpa UHCC 0038, Nodularia spumigena, Nostoc caeruleum SAG 52.79, Nostoc calcicola, Nostoc calcicola 99, Nostoc calcicola AM50C, Nostoc calcicola BDU 180601, Nostoc calcicola BDU 40302, Nostoc calcicola III, Nostoc calcicola SAG 1453-1, Nostoc calcicola VI, Nostoc carneum, Nostoc carneum Cy_nb3, Nostoc carneum IAM M-35, Nostoc carneum NIES-2107, Nostoc carneum SERB 44, Nostoc carneum SN437, Nostoc carneum SU_cyano_N, Nostoc cf. commune 257-16, Nostoc cf. commune 257-20, Nostoc cf. commune KG-54, Nostoc cf. commune SO-36, Nostoc cf. edaphicum Us-2-2, Nostoc cf. indistinguendum FI5-VF12, Nostoc cf. indistinguendum FI5-VF4, Nostoc cf. lichenoides (N.cf.lichenoides JT1-VF3, Nostoc cf. verrucosum, Nostoc commune, Nostoc commune 0Brien 02011101, Nostoc commune 15CT-1.2, Nostoc commune 15CT-3.1, Nostoc commune ACSSI 030, Nostoc commune ACSSI 035, Nostoc commune ACT709, Nostoc commune AHNG0605, Nostoc commune BEA 0028, Nostoc commune CANT2, Nostoc commune CANT4, Nostoc commune CCAP 1453 / 24, Nostoc commune CCIBt 3485, Nostoc commune EV1-KK1, Nostoc commune FACHB 261, Nostoc commune HK-02, Nostoc commune KU002, Nostoc commune LSB51, Nostoc commune LSB511, Nostoc commune LSB84, Nostoc commune Mon62, Nostoc commune PS27-2.2, Nostoc commune SAG 1453-3, Nostoc commune SIK85, Nostoc commune. Nostoc commune SIK94, Nostoc commune SN444, Nostoc commune SN450, Nostoc commune UTEX 584, Nostoc commune WY1KK1, Nostoc commune YK-04, Nostoc commune var. flagelliforme CCAP 1453 / 33, Nostoc desertorum, Nostoc desertorum CM1-VF14, Nostoc edaphicum ACCS 059, Nostoc edaficum ACSSI 156, Nostoc edaficum CCNP1411, Nostoc edaficum KZ-5-4-7, Nostoc edaficum TAU-MAC 2615, Nostoc edaficum X, Nostoc ergonense QUCCCM128, Nostoc ergonense TH3S05, Nostoc ellipsosporum CCAP 1453 / 15, Nostoc ellipsosporum CCAP 1453 / 2, Nostoc ellipsosporum QUCCCM991490, Nostoc ellipsosporum V, Nostoc ellipsosporum str. Lukesova 52_91, Nostoc entophytum IAM M-267, Nostoc entophytum ISC 32, Nostoc favosum, Nostoc favosum CHAB5713, Nostoc favosum CHAB5714, Nostoc flagelliforme CCNUN1, Nostoc flagelliforme CHAB2816, Nostoc flagelliforme IMGA0408, Nostoc flagelliforme str. Sunitezuoqi, Nostoc indistinguendum, Nostoc indistinguendum CM1-VF10, Nostoc insulare SAG 54.79, Nostoc lichenoides, Nostoc lichenoides CNP-AK1, Nostoc linckialinckia), Nostoc linckia ACSSI 271, Nostoc linckia BEA 0870B, Nostoc linckia NIES-25, Nostoc linckia var. arvense IAM M-30, Nostoc microscopicum SAG 40.87, Nostoc minutum ACSSI 155, Nostoc minutum ACSSI 167, Nostoc minutum ACSSI 168, Nostoc minutum NIES-26, Nostoc mirabile, Nostoc mirabile CHAB5756, Nostoc oromo, Nostoc oromo ETH.2.4.M.5, Nostoc oryzae, Nostoc paludosum BA033, Nostoc parmelioides SAG 58.79, Nostoc piscinale BF3, Nostoc piscinale CENA21, Nostoc pruniforme SAG 62.79, Nostoc punctiforme SAG 62.79punctiforme), Nostoc punctiforme ACCS 074, Nostoc punctiforme ACSSI 037, Nostoc punctiforme ACSSI 160, Nostoc punctiforme BKP_NB59, Nostoc punctiforme BKP_NS61, Nostoc punctiforme BKP_NS62, Nostoc punctiforme BKP_NS63, Nostoc punctiforme BKP_NS64, Nostoc punctiforme BKP_NS65, Nostoc punctiforme BKP_NS66, Nostoc punctiforme BKP_ NS67, Nostoc punctiforme BKP_NS68, Nostoc punctiforme BKP_NS69, Nostoc punctiforme BKP_NS70, Nostoc punctiforme BKP_SS64, Nostoc punctiforme BKP_SS66, Nostoc punctiforme BKP_SS67, Nostoc punctiforme BKP_SS68, Nostoc punctiforme CCAP 1453 / 9, Nostoc punctiforme Jbr02, Nostoc punctiforme KZ-2-2-2, Nostoc punctiforme PCC 73102, Nostoc punctiforme SAG 60.79, Nostoc punctiforme SAG 65.79, Nostoc punctiforme SAG 68.79, Nostoc punctiforme SAG 71.79, Nostoc spp., Nostoc spp. Azolla cyanobiont, Nostoc spp. Collema crispum cyanobiont, Nostoc spp. Collema nigrescens UK197 cyanobiont, Nostoc spp. Collema spp. sp.)AR75 cyanobiont, Nostoc species Corema species UK531 cyanobiont, Nostoc species Fuscopannaria leucosticta LG:R1123 cyanobiont, Nostoc species Fuscopannaria leucosticta LG:R1124 cyanobiont, Nostoc species Fuscopannaria pacifica (F.pacifica UK179 cyanobiont, Nostoc sp. Fuscopannaria praetermissa LG:R1060 cyanobiont, Nostoc sp. Helianobakht, Nostoc sp. Kroswia crystallyfera LG:M788 cyanobiont, Nostoc sp. Leptogium gelatinosum cyanobiont, Nostoc sp. Leptogium palmatum UK176 cyanobiont, Nostoc sp. Leptogium sp. LG:R2848 cyanobiont, Nostoc sp. Leptogium sp. LG:R2850 cyanobiont, Nostoc sp. Lobaria ampulissima amplissima) cyanobiont, Nostoc sp. lobaria cyanobiont 34, Nostoc sp. Lobaria hallii (L. hallii) cyanobiont, Nostoc sp. Lobaria pulmonaria (L. pulmonaria) (5183) cyanobiont, Nostoc sp. Massalongia carnosa (Massalongia carnosa cyanobiont, Nostoc sp. Mollenhauer 1:1-064, Nostoc sp. Mollenhauer 1:1-065, Nostoc sp. Mollenhauer 1:1-066, Nostoc sp. Mollenhauer 1:1-067, Nostoc sp. Mollenhauer 1:1-088, Nostoc sp. Mollenhauer 1:1-106b2, Nostoc sp. Mollenhauer 1:1-108, Nostoc sp. Mollenhauer 1:1-115, Nostoc sp. Mollenhauer 1:1-125, Nostoc sp. Mollenhauer 1:1-150b, Nostoc sp. Mollenhauer 94.1, Nostoc sp. Nephroma arcticum arcticum)UK103 Cyanobiont, Nostoc sp. Nephroma arcticumUK48 Cyanobiont, Nostoc sp. Nephroma arcticumUK564 Cyanobiont, Nostoc sp. Nephroma verum (N.bellum) cyanobiont, Nostoc sp. Nephroma helveticum cyanobiont 33, Nostoc sp. Nephroma helveticum cyanobiont 37, Nostoc sp. Nephroma helveticum cyanobiont, Nostoc sp. Nephroma laevigatum cyanobiont 39, Nostoc sp. Nephroma pariles cyanobiont 26, Nostoc sp. Nephroma pariles cyanobiont 32, Nostoc sp. Nephroma parile cyanobiont, Nostoc sp. Nephroma lespinatum cyanobiont 38, Nostoc sp. Nephroma lespinatum cyanobiont 40, Nostoc sp. Nephroma lespinatum cyanobiont, Nostoc sp. Panaria aff. Asloophyllia cyanobiont cyanobiont NZ, Nostoc sp. Panaria aff. leproloma cyanobiont 1a Ch, Nostoc sp. Panaria aff. lepromatous cyanobiont 1a NZ, Nostoc sp. Panaria aff. lepromatous cyanobiont 1b NZ, Nostoc sp. Panaria aff. lepromatous cyanobiont 2 Ch, Nostoc sp. Panaria aff. lepromatous cyanobiont 3 Ch, Nostoc sp. Panaria aff. sphinctrina cyanobiont 1a NC, Nostoc species Panaria aff. sphincterina cyanobiont 1bNC, Nostoc species Panaria andina 1 cyanobiont Chile, Nostoc species Panaria andina cyanobiont Peru, Nostoc species Panaria araneosa cyanobiont NZ, Nostoc species Panaria conoplea cyanobiont, Nostoc species Panaria durietzii cyanobiont 1 NZ, Nostoc species Panaria elixii cyanobiont 1 NZ, Nostoc species Panaria elixii cyanobiont 2 NZ, Nostoc species Panaria euphylla cyanobiont NZ, Nostoc species Panaria isabellina (P. isabellina) cyanobiont 1 Ch, Nostoc species Panaria isabellina cyanobiont 2 Ch, Nostoc species Panaria mosenii (P.mosenii cyanobiont Mex, P. obscura cyanobiont Aus, P. pallida cyanobiont 3 Ch, P. rubiginosa LG:R1008 cyanobiont, P. rubiginosa LG:R1011 cyanobiont, P. rubiginosa cyanobiont No, Pannaria sp. cephalodia LG:R969 cyanobiont, P. sphinctrina cyanobiont 1a Ch, P. sphinctrina cyanobiont 1b Ch, Nostoc species Panaria sphinctrina cyanobiont 1c Ch, Nostoc species Panaria tabaresii (P.tavaresii) cyanobiont Ch, Nostoc species Parmeliella borbonica (Parmeliella borbonica LG:R1122 cyanobiont, P. brisbanensis LG:R1019 cyanobiont, P. brisbanensis LG:R1247 cyanobiont, P. brisbanensis LG:T3 cyanobiont, P. mariana LG:R974 cyanobiont, P. polyphyllina LG:R1021 cyanobiont, P. polyphyllina LG:R1058 cyanobiont, Parmeliella sp. LG:T6 cyanobiont. , Nostoc sp. Palmeriella stylophora LG:R979 cyanobiont, Nostoc sp. Palmeriella triptophylla LG:R979 cyanobiont, Nostoc sp. Palmeriella triptophylloides LG:R979 cyanobiont, Nostoc sp. Palmeriella triptophylloides LG:R979 cyanobiont, Nostoc sp. Peltigera aftosa LG:R979 cyanobiont, Nostoc sp. aphthosa)UK52 Cyanobiont, Nostoc sp. Pertigera aphthosaUK53 Cyanobiont, Nostoc sp. Pertigera canina (P. canina)1 Cyanobiont, Nostoc sp. Pertigera canina2 Cyanobiont, Nostoc sp. Pertigera canina3 Cyanobiont, Nostoc sp. Pertigera canina4 Cyanobiont, Nostoc sp. Pertigera caninaUK106 Cyanobiont, Nostoc sp. Pertigera canina UK121 cyanobiont, Nostoc species Pertigera collina (P. collina) UK144 cyanobiont, Nostoc species Pertigera collina UK148 cyanobiont, Nostoc species Pertigera degeni (P. degeni) cyanobiont, Nostoc species Pertigera didactyla (P. didactyla) 2 cyanobiont, Nostoc species Pertigera didactyla 3 cyanobiont, Nostoc species P. evansiana UK159 cyanobiont, P. extenuata AR4b cyanobiont, P. extenuata UK4 cyanobiont, P. frigida AR49 cyanobiont, P. horizontalis cyanobiont P. lepidophora cyanobiont, P. leucophlebia UK57 cyanobiont, P. leucophlebia UK79 cyanobiont, P. malacea UK59 cyanobiont, P. malacea cyanobiont DB3992,P. membranacea 1 cyanobiont, P. membranacea 2 cyanobiont, P. membranacea 3 cyanobiont, P. membranacea 4 cyanobiont, P. membranacea 5 cyanobiont, P. membranacea UK156 cyanobiont, P. membranacea UK194 cyanobiont, P. membranacea cyanobiont, P. membranacea cyanobiont N6, P. neopolydactyla UK60 cyanobiont , Nostoc sp. Peltigera neopolydactyla agg. UK150 cyanobiont, Nostoc sp. Peltigera praetextata cyanobiont 22, Nostoc sp. Peltigera pruinosa cyanobiont 14, Nostoc sp. Peltigera rufescens 1 cyanobiont, Nostoc sp. Peltigera rufescens 2 cyanobiont, Nostoc sp. Peltigera rufescens 3 cyanobiont, Nostoc sp. Peltigera rufescens 4 cyanobiont, Nostoc sp. Peltigera rufescens 5 cyanobiont, Nostoc sp. Peltigera rufescens UK46 cyanobiont, Nostoc sp. Peltigera sp. UK437 cyanobiont, Nostoc sp. Pertigera sp. UK521b cyanobiont, Nostoc sp. Pertigera sp. cyanobiont, Nostoc sp. Pertigera venosa (P. venosa) cyanobiont 15, Nostoc sp. Physma byrsaeum LG:R1121 cyanobiont, Nostoc sp. Physma byrsaeum LG:R2 cyanobiont, Nostoc sp. Physma byrsaeum LG:R2847 cyanobiont, Nostoc sp. Physma radians LG:T5 cyanobiont,Polychidium muscicola UK175 cyanobiont, P. pezizoides UK120 cyanobiont, P. pezizoides UK73 cyanobiont, Pseudocyphellaria gilva cyanobiont Chile, Pseudocyphellaria sp. LG:R2332 cyanobiont, P. beauvoisii cyanobiont beauvoisii cyanobiont, Nostoc sp. sticta cf. caulescens AR124 cyanobiont, Nostoc sp. sticta fuliginosa cyanobiont, Nostoc sp. muscicolous cyanobiont 21, Nostoc sp. 0GU36S01, Nostoc sp. 0GU36S02, Nostoc sp. 113.5, Nostoc sp. 117.8.2, Nostoc sp. 1189P, Nostoc sp. 1190P, Nostoc sp. 152, Nostoc sp. 159, Nostoc sp. 17, Nostoc sp. 195-A21, Nostoc sp. 195-A22, Nostoc sp. 1c, Nostoc sp. 1tu14s8, Nostoc sp. 2-07, Nostoc sp. 2LT05S03, Nostoc sp. 5N-02c, Nostoc sp. 8901:1, Nostoc sp. 8916, Nostoc sp. 8923, Nostoc sp. 8926, Nostoc sp. 8929, Nostoc sp. 8938, Nostoc sp. 8941, Nostoc sp. 8963, Nostoc sp. 8964:3, Nostoc sp. 9104, Nostoc sp. 9E-03, Nostoc sp. 9d, Nostoc sp. A15, Nostoc sp. A39, Nostoc sp. ACSSI045, Nostoc sp. ACSSI047, Nostoc sp. ACSSI056, Nostoc sp. ACSSI057, Nostoc sp. ACSSI ACSSI 066, Nostoc species ACSSI 067, Nostoc species ACSSI 082, Nostoc species ACSSI 114, Nostoc species ACSSI 115, Nostoc species ACSSI 123, Nostoc species ACSSI 143,Nostoc sp. ACSSI 161, Nostoc sp. ACSSI 164, Nostoc sp. ACSSI 231, Nostoc sp. ACSSI 310, Nostoc sp. ACSSI 314, Nostoc sp. ACSSI 325, Nostoc sp. ACSSI 329, Nostoc sp. ACT703, Nostoc sp. ACT732, Nostoc sp. AH-12, Nostoc sp. ANT.L34.1, Nostoc sp. ANT.L52B.1, Nostoc sp. ANT.L52B.8, Nostoc sp. ANT.L61.1, Nostoc sp. ANT.LG2.6, Nostoc sp. AR12, Nostoc sp. ARC 64, Nostoc sp. ARC8, Nostoc sp. ATCC 53789, Nostoc sp. AWT 203, Nostoc sp. Al1, Nostoc sp. Al3, Nostoc sp. BACA0081, Nostoc sp. BDU ARC 10101, Nostoc sp. BDU80591, Nostoc sp. BDU80701, Nostoc sp. BEA 1039B, Nostoc sp. BEA 1140B, Nostoc sp. BKP_CB62, Nostoc sp. BKP_CB63, Nostoc sp. BKP_CB64, Nostoc sp. BKP_CS63, Nostoc sp. BKP_CS64, Nostoc sp. BKP_CS65, Nostoc sp. BKP_CS66, Nostoc sp. BKP_CS67, Nostoc sp. BKP_CS68, Nostoc sp. BKP_CS69, Nostoc sp. BKP_CS70, Nostoc sp. BKP_CS71, Nostoc sp. BKP_CS72, Nostoc sp. BKP_CS73, Nostoc sp. BKP_CS74, Nostoc sp. BKP_NS59, Nostoc sp. BKP_NS60, Nostoc sp. BKP_SB57, Nostoc sp. BKP_SB58, Nostoc sp. BKP_SB59, Nostoc sp. BKP_SS65, Nostoc sp. BR36, Nostoc sp. BS363, Nostoc sp. Bahar_E, Nostoc sp. Bahar_M, Nostoc sp. C052, Nostoc sp. C057, Nostoc sp. CACIAM 19, Nostoc spp. CAVN2, Nostoc spp. CAWBG77, Nostoc spp. CCAP 1453 / 25, Nostoc spp. CCAP 1453 / 28, Nostoc spp. CCAP 1453 / 31, Nostoc spp. CCAP 1453 / 35, Nostoc spp. CENA105,CENA175, CENA216, CENA219, CENA239, CENA259, CENA261, CENA269, CENA271, CENA274, CENA278, CENA281, CENA294, CENA296, CENA356, CENA511, CENA535, CENA536, CENA543, CENA544, CENA547, CENA548, CENA551, CHAB TP201513, CHAB TP201514, Nostoc spp. CHAB TP201701.1, Nostoc spp. CHAB TP201726.1, Nostoc spp. CHAB TP201727.1, Nostoc spp. CHAB TP201728.1, Nostoc spp. CHAB TP201728.4, Nostoc spp. CHAB TP201736.2, Nostoc spp. CHAB TP201822.2, Nostoc sp. CNCH1, Nostoc sp. Cam2S01, Nostoc sp. Cc2, Nostoc sp. Cr4, Nostoc sp. DM103, Nostoc sp. De1, Nostoc sp. Den-12, Nostoc sp. Ev1, Nostoc sp. FACHB-252, Nostoc sp. FB1-KK1, Nostoc sp. FI5-VF5, Nostoc sp. GM244, Nostoc sp. GSV224, Nostoc sp. GT138, Nostoc sp. HAMTA.RF, Nostoc sp. HAN11 / 1, Nostoc HK-01, Nostoc sp. IO-102-I, Nostoc sp. KK-01, Nostoc sp. KNUA003, Nostoc sp. KU001, Nostoc sp. KU028, Nostoc sp. KU281, Nostoc sp. KVJ10, Nostoc sp. KVJ18, Nostoc sp. KVJ20, Nostoc sp. KVJ3, Nostoc sp. KVJ4, Nostoc sp. KVJF4, Nostoc sp. KVJF8, Nostoc sp. KVS1, Nostoc sp. KVS11, Nostoc sp. KVSF4, Nostoc sp. Lukesova 1 / 86,Nostoc sp. Lukesova 40 / 93, Nostoc sp. Lukesova 5 / 96, Nostoc sp. MGL001, Nostoc sp. MS1, Nostoc sp. MV6, Nostoc sp. Mau15, Nostoc sp. N107.3, Nostoc sp. NIES-2094, Nostoc sp. NIES-2110, Nostoc sp. NIES-2111, Nostoc sp. NIES-3756, Nostoc sp. NIES-4103, Nostoc sp. Ni4-C, PCC 7107, Nostoc sp. PCC 7120=FACHB-418, Nostoc sp. PCC 7423, Nostoc sp. PCC 7524, Nostoc sp. PCC 8112, Nostoc sp. PCC 8976, Nostoc sp. PCC 9229, Nostoc sp. PCC 9231, Nostoc sp. PCC 9305, Nostoc sp. PCC 9426, Nostoc sp. PCC 9709, Nostoc sp. PS33-1, Nostoc sp. Prim-3-2, Nostoc sp. RE21, Nostoc sp. SAG 2306, Nostoc sp. SAG 2409, Nostoc sp. SAG 2414, Nostoc sp. SAG 29.90, Nostoc sp. SAG 34.92, Nostoc sp. SAG 35.92, Nostoc sp. SAG 36.92, Nostoc sp. SAG 39.87, Nostoc sp. SAG 41.87, Nostoc sp. SKJ1, Nostoc sp. SKJ2, Nostoc sp. SKJ4, Nostoc sp. SKJF1, Nostoc sp. SKS2, Nostoc sp. SKS5, Nostoc sp. SKS8, Nostoc sp. SKS9, Nostoc sp. SKSF3, Nostoc sp. SKSL1, Nostoc sp. SN418, Nostoc sp. SN419, Nostoc sp. SN426, Nostoc sp. SN432, Nostoc sp. SN440, Nostoc sp. SN449, Nostoc sp. SN64, Nostoc sp. TAU-MAC 0799, Nostoc sp. TCL240-02, Nostoc sp. TCL26-01, Nostoc sp. TDI#AR94, Nostoc sp. TH1S01, Nostoc sp. TO1S01, Nostoc sp. UAM 307, Nostoc sp. UAM 308, Nostoc sp. UHCC 0702, Nostoc sp. UHCC 0870, Nostoc sp. UHCC 0926, Nostoc sp. UK18, Nostoc sp. Us-7-5, Nostoc sp. VI.5, Nostoc sp. VP2-08, Nostoc sp. YK-01, Nostoc sp. rosa 1, Nostoc sp. rosa 4, Nostoc sp. sepahi, Nostoc sp. sf 6 Calc, Nostoc sp. haericum, Nostoc sp. haeroides, Nostoc sp. haeroides ACSSI 150, Nostoc sp. haeroides CCNUC1, Nostoc sp. haeroides HBHF0604, Nostoc sp. ongiaeforme Ind42, Nostoc verrucosum, Nostocaceae cyanobacterium, Nostocaceae cyanobacterium Baduki3, Nostocaceae cyanobacterium CENA376, Nostocaceae cyanobacterium CENA388, Nostocaceae cyanobacterium DW3II-PS, Pseudoariinostoc jiangxiense, Rohorchiella edafica AR2, Rohorchiella edafica AR4, Rohorchiella edafica AR6, Rohorchiella edafica KZ-5-4-5, R. fluviatilis SN435, R. fluviatilis UAM 332, R. fluviatilis UAM 334, S. bohneri SAG 255.80, S. mirabile SAG 83.79, Scytonema sp. HAN3 / 2, T. distorta ACT712, Tolypothrix sp. 9k, Tolypothrix sp. CCM-UFV067, Tolypothrix sp. IAM M-259, Tolypothrix sp. PCC 7601, Tolypothrix sp. PCC 7712, Tolypothrix sp. PCC 7910, Tolypothrix sp. UAM 335, Tolyposthrix tenui (T. tenui) PCC 7101, Tolyposthrix tenui SAG 94.79, Tolyposthrix tenui SN436, Trichormus sp. CCM-UFV035, Trichormus sp. PS4F, Trichormus sp. SBC125, Trichormus variabilis (T.valiablis, Trichorum variabilis 0441, Trichorum variabilis ATCC 29413, Trichorum variabilis GITAM RGP, Trichorum variabilis NIES-23, Trichorum variabilis RPAN45, Trichorum variabilis SN416, Trichorum variabilis str.GREIFSWALD, Trichorum variabilis str.HINDAK 2001 / 4, Violettonostoc minutum CHAB 5840, Violettonostoc minutum CHAB 5841, W. vaginalicola RPAN22, or Cyanobacterium NIES-2102.

[0193] In some embodiments, the technology provides a method for the treatment of a variety of fungi including Acaryochloris, Alliinostoc, Alitella, Amazonocrinis, Anabaena, Anabaenopsis, Aphanizomenon, Atlanticothrix, Aurocilla, Karenema, Calloshrix, Camptilonemopsis, Chlorogloeopsis, Chroococcidiopsis, Chrysosporum, Compactostoc, Constrictiphyllum, Crocosphera, Cyanobacterium, Cyanocorniella, Cylindrospermopsis, Cylindrospermum, Dendronarium, Desicacharia, Desmonostoc, Dolichospermum, Erythrobacter, Fischerella, Fortiera, Fremiella, Gaitrelinema, Gloeocapsa, Gloeocapsopsis, Gloeothece, Gloeotrichia, Goleter, Hapalosiphon, Hydrocoryne, Yaginema, Yo The present invention provides a microbial consortium that includes organisms from a taxonomic group (e.g., genus) that is Hanseniella, Camptonema, Komarekiella, Leptoryngbia, Lyngbia, Macrochaete, Mastigocoleus, Microchaete, Nodocilinea, Nodularia, Nostoc, Nostocaseae, Nostocoptsis, Oligotropha, Oscillatoria, Pantanalinema, Pellatocladus, Planctotrichoides, Planktothrix, Polymorpham, Porphyrobacter, Prochlorococcus, Prochlorothrix, Pseudoanabaena, Pseudoariinostoc, Raphidiopsis, Richeria, Livularia, Loforchiella, Rubisibacter, Cytonema, Spirulina, Synechococcus, Synechocystis, Thermosynechococcus, Tolyposthrix, Trichorum, Bioletnostoc, Wallea, or Xenococcus.

[0194] In some embodiments, the microbial consortium further includes organisms from the genus Variovorax (e.g., Variovorax sp. PMC12), organisms from the phylum Proteobacteria (classes Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria), organisms from the genus Bosea (e.g., Bosea sp. R-45681), organisms from the genus Caulobacter (e.g., Caulobacter segnis ATCC 21756, Caulobacter ginsengisoli), and / or organisms from the genus Pseudomonas (e.g., Pseudomonas sp. NFR16).

[0195] Community stability During the development of embodiments of the technology described herein, stability was used as a measure to identify consortia that can be produced at larger production scales and exhibit repeatable behavior as a predictor of consortia that have application in the field as a product. As used herein, stability of a microbial population or consortium refers to the amount of change in the microbial population as a function of successive passages of a culture containing the microbial population. Thus, the alpha diversity of a microbial population, defined by the number of species represented in the microbial population, is less important. In particular, increased stability indicates a decrease in the change in the microbial population as a function of successive passages of a culture containing the microbial population. That is, by using a portion of a stable consortium (e.g., a portion of a culture containing a stable consortium) to inoculate a new culture medium and growing the culture under defined conditions suitable for the production of the consortium, a culture containing the same consortium as the culture used for inoculation is expected to be produced. An increased measure of stability increases the predictability that the same consortium that was present in the inoculum will be produced in the new culture. In other words, a stable culture produces a predictable population of microorganisms as a result, given a defined set of external factors (e.g., light, nutrients, temperature, aeration, other culture conditions). That is, increased stability indicates increased predictability or reliability that the resulting microbial culture will contain a desired defined group or consortium of microorganisms.

[0196] Data collected while developing embodiments of the technology described herein showed that the Bray-Curtis dissimilarity scores for BW01 and BW02 changed rapidly between the original soil input samples and P1, and between P1 and P2. See FIG. 13. However, the Bray-Curtis dissimilarity scores for BW01 and BW02 between P2 and P3, and between P3 and P4, approached the limits of approximately 0.14-0.18. These data indicated that after two passages, the BW01 and BW02 communities began to approach an equilibrium state where differences between passages were minimal and / or disappeared in subsequent passages.

[0197] Furthermore, experiments conducted while developing the techniques described herein demonstrated that the selection process was effective in producing microbial consortia with desired functions. Principal coordinate analysis of shotgun sequencing data obtained from the original soil sample and the P1 to P4 passage samples showed that the original soil samples that produced BW01 and BW02 clustered together fairly tightly (Figure 14). After passage, the P4 BW02 cluster was more variable than the P4 BW01 cluster (Figure 14). Cultures of P4 BW01 and BW01 produced two separate clusters, but the separate clusters share common components (Figure 14). Finally, no significant changes were observed in cluster formation between early and intermediate passages (e.g., between P1 and P2) within a given sample, indicating that the populations within the P1 culture began to have increased stabilization. However, both the early and intermediate passages are significantly divergent from the initial soil sample.

[0198] Thus, technology is provided herein that provides an effective method for selective cultivation. Furthermore, selective cultivation methods have been used to produce consortia that can grow effectively on mineral media without supplementation with a combination of carbon sources other than nitrogen or atmospheric CO2. The technology described herein has application, for example, to providing treatment of agricultural soils.

[0199] Presumption of isolation During development of embodiments of the technology described herein, carbon and nitrogen fixation data from photobioreactor experiments and biofilm density measurements from vertically illuminated multiwell plate cultures were used to estimate the carbon sequestration provided by the microbial consortia described herein. The microbial consortia offset carbon dioxide emissions in two ways.

[0200] First, microorganisms absorb carbon dioxide from the air and sequester the carbon in biological molecules that persist in the soil. For example, the technology described herein provides embodiments of carbon compound-producing microbial consortia that sequester carbon from the atmosphere into persistent and stable biological molecules, such as melanins (e.g., pheomelanin, eumelanin, and / or pyomelanin), that persist in the soil. That is, the embodiments relate to sustainable carbon compound-producing consortia that provide a stable carbon sink that removes carbon (e.g., CO2) from the atmosphere and stores the carbon in sustainable carbon compounds (e.g., melanin) in the soil. Since 73% of the amount of carbon dioxide molecules is provided by oxygen atoms, 3.67 tons of carbon dioxide gas are sequestered per ton of elemental carbon contained in the microbial mat.

[0201] The second way microbial consortia offset carbon dioxide emissions is by reducing agricultural demand for nitrogen-based fertilizers. The Haber-Bosch process consumes vast amounts of natural gas to produce the hydrogen needed to fix nitrogen into ammonia. Notably, for every ton of ammonia produced, 2.16 tonnes of carbon dioxide are emitted (see, for example, Ghavam (2021) “Sustainable Ammonia Production Processes,” Front. Energy Res. 9:580-808, incorporated herein by reference). Furthermore, less than 20% of nitrogen applied in the field reaches the final crop. Much of the rest is leached or denitrified, producing nothing for the grower but causing environmental damage (see, for example, Royal Society Policy Briefing “Ammonia: zero-carbon fertiliser, fuel and energy store,” February 2020, available at royalsociety.org / green-ammonia and incorporated herein by reference). In some embodiments, the microbial consortia and / or compositions comprising the microbial consortia described herein double this efficiency by providing a sustained release of this nitrogen instead of a single application of nitrogen fertilizer use.

[0202] Cultures grown in the field from the consortium described herein (e.g., BW01) at the same rate per square centimeter as grown in shallow multi-well plates filled with nitrogen-free medium will reach saturation in approximately one month of growth. A saturated culture will produce 150 kg / acre of dry biomass containing 6.2% nitrogen and 45.2% carbon. This biomass will correlate to a carbon dioxide offset of 0.30 metric tons over this one-month growth period. This estimate assumes saturation and associated maximum accumulation (Figure 15).

[0203] However, cultures are not expected to grow to saturation in the field. Instead, once the cultures reach 100% surface coverage, they begin to grow in a Z-dimensional growth pattern as the biofilm or soil surface becomes covered with a hard coating (Figure 16). New layers of microorganisms form on top of previous layers, shading the layers below and reducing further growth. This one-dimensional growth can proceed with the doubling time calculated for the standard culture, but for each new generation that forms, one older generation will stop dividing. Thus, growth is expected to proceed linearly beyond the point of full coverage. Thus, a carbon dioxide offset of 1.66 metric tons per acre is expected in 60 days.

[0204] Additionally, in some embodiments, growth of the microbial consortium occurs in large open ponds located in locations with abundant sunlight and space (e.g., desert areas). The annual global production of Haber-Bosch ammonia for synthetic fertilizer is 120 Mt, which contains 99 Mt of elemental nitrogen. Growing the microbial consortium described herein in a space that is 17% the area of ​​Arizona would offset the entire global production of nitrogen from the Haber-Bosch process.

[0205] Similarly, producing microbial fertilizer (e.g., as nitrogen-slow-release green manure) on one million acres of land using the technology described herein replaces 100 pounds of chemical fertilizer per acre per year on 100 million acres of land. Increasing the growth of microbial consortia in the field by 100 times reduces the land required to replace 100 pounds of chemical fertilizer per acre per year on 100 million acres of land to 7221 acres. See Table 7.

[0206] [Table 1]

[0207] Compositions for agricultural applications In some embodiments, the present technology provides compositions for agricultural applications and related methods of using the compositions to improve agricultural media (e.g., soil, greenhouse growth media, or hydroponic media). In some embodiments, the compositions include a microbial consortium as described herein (e.g., photosynthetic and nitrogen fixing microorganisms, heterotrophic microorganisms that consume nitrogen-containing and / or carbon-containing compounds produced by the photosynthetic and nitrogen fixing microorganisms, and optionally one or more additional photosynthetic and / or heterotrophic life forms). In some embodiments, the consortium grows in vitro and produces nitrogen-containing and carbon-containing compounds using nitrogen and carbon from the atmosphere. In some embodiments, the composition including the consortium is applied to the agricultural medium as a "green manure", e.g., a composition including the consortium and the nitrogen-containing and carbon-containing compounds produced by the consortium. In some embodiments, the green manure consortium performs or does not perform minimal nitrogen fixation and / or carbon sequestration after the composition including the consortium is applied to the agricultural medium.

[0208] In some embodiments, the composition comprising the consortium is applied to an agricultural medium to inoculate the agricultural medium with the composition comprising the consortium. After the composition comprising the consortium is applied to the agricultural medium, the consortium continues to fix nitrogen and / or sequester carbon, and the nitrogen-containing and carbon-containing compounds produced by the consortium growing on the agricultural medium enter the agricultural medium to improve the agricultural medium and / or nourish the plant (e.g., crop) growing on the agricultural medium, in addition to the nitrogen-containing and carbon-containing compounds produced by the consortium before applying the composition to the agricultural medium. In some embodiments, the consortium produces carbon compounds that sequester carbon from the atmosphere. In some embodiments, members of the consortium produce carbon compounds that sequester carbon from the atmosphere. In some embodiments, embodiments provide a consortium that produces melanin (e.g., pheomelanin, eumelanin, and / or pyomelanin). That is, embodiments provide a consortium that produces sustainable carbon compounds that remove carbon (e.g., CO2) from the atmosphere and provide a stable carbon sink that stores the carbon in sustainable carbon compounds (e.g., melanin) in the soil.

[0209] In some embodiments, the compositions comprising the consortium are combined into agricultural compositions. In some embodiments, the agricultural compositions include one or more of a wetting agent, a compatibilizer (also referred to as a "compatibilizer"), a defoamer, a detergent, a sequestering agent, a drift reducing agent, a neutralizing agent, a buffer, a corrosion inhibitor, a dye, an odorant, a spreading agent (also referred to as a "spreader"), a penetration aid (also referred to as a "penetrating agent"), a tackifier (also referred to as a "sticker" or "binder"), a dispersant, a thickener (also referred to as a "thickener"), a stabilizer, an emulsifier, a freezing point depressant, or an antimicrobial agent.

[0210] In some embodiments, the agricultural composition is a solid. In some embodiments, the agricultural composition comprises a carrier material, such as silica, silica gel, silicates, talc, kaolin, atta clay, limestone, chalk, loess, clay, dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetic materials, fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate, thiourea, and urea, products of plant origin such as cereal meal, bark meal, wood meal, and nut meal, cellulose powder, attapulgite, montmorillonite, mica, vermiculite, synthetic silica, and synthetic calcium silicate, or combinations thereof.

[0211] In some embodiments, the agricultural composition is a liquid, i.e., in some embodiments, the agricultural composition comprises a compound or salt such as monoethanolamine salt, sodium sulfate, potassium sulfate, sodium chloride, potassium chloride, sodium acetate, ammonium hydrogen sulfate, ammonium chloride, ammonium acetate, ammonium formate, ammonium oxalate, ammonium carbonate, ammonium hydrogen carbonate, ammonium thiosulfate, ammonium dihydrogen phosphate, ammonium dihydrogen monophosphate, ammonium sodium hydrogen phosphate, ammonium thiocyanate, ammonium sulfamate, or ammonium carbamate.

[0212] In some embodiments, the agricultural composition comprises a binder, such as polyvinylpyrrolidone, polyvinyl alcohol, partially hydrolyzed polyvinyl acetate, carboxymethylcellulose, starch, vinylpyrrolidone / vinyl acetate copolymers and polyvinyl acetate, or combinations thereof; a lubricant, such as magnesium stearate, sodium stearate, talc, or polyethylene glycol, or combinations thereof; an antifoaming agent, such as a silicone emulsion, a complexing agent, such as a long chain alcohol, a phosphoric acid ester, an acetylenic diol, a fatty acid, or an organic fluorine compound, and a salt of ethylenediaminetetraacetic acid (EDTA), a salt of trinitrilotriacetic acid, or a salt of polyphosphoric acid, or combinations thereof.

[0213] In some embodiments, the agricultural composition comprises a surface active agent. In some embodiments, the surfactant is added to a liquid agricultural composition. In some embodiments, the surfactant is added to a solid formulation, particularly a formulation designed to be diluted with a carrier before application. That is, in some embodiments, the agricultural composition comprises a surfactant. Surfactants may be used alone or with other additives (e.g., mineral oil or vegetable oil) as adjuvants to spray tank mixtures to improve the biological function of the target community. Surfactants may be anionic, cationic, or nonionic and may be employed as emulsifiers, wetting agents, suspending agents, or for other purposes. In some embodiments, the surfactant is nonionic, such as alkyl ethoxylates, linear fatty alcohol ethoxylates, and fatty amine ethoxylates. In some embodiments, the agricultural composition may be selected from the group consisting of salts of alkyl sulfates, such as diethanolammonium lauryl sulfate; alkylaryl sulfonate salts, such as calcium dodecylbenzene sulfonate; alkylphenol-alkylene oxide adducts, such as nonylphenol-Cis ethoxylate; alcohol-alkylene oxide adducts, such as tridecyl alcohol-C16 ethoxylate; soaps, such as sodium stearate; alkylnaphthalene-sulfonate salts, such as sodium dibutyl-naphthalene sulfonate; dialkyl esters of sulfosuccinate salts, such as di(2-ethylhexyl) sorbitol esters, such as sorbitol oleate; quaternary amines, such as lauryltrimethylammonium chloride; polyethylene glycol esters of fatty acids, such as polyethylene glycol stearate; block copolymers of ethylene oxide and propylene oxide; salts of mono- and di-alkyl phosphate esters; vegetable oils, such as soybean oil, rapeseed / canola oil, olive oil, castor oil, sunflower seed oil, coconut oil, corn oil, cottonseed oil, linseed oil, palm oil, peanut oil, safflower oil, sesame oil, tung oil, etc.; and esters of the above vegetable oils, especially the methyl esters.

[0214] In some embodiments, the agricultural composition comprises a wetting agent, a dispersing agent, a polymeric surfactant, an emulsifier, a solubilizer, an organic solvent, a gelling agent, an antifoaming agent, and / or a preservative.

[0215] Additionally, in some embodiments, the agricultural composition comprises a pesticide, herbicide, bactericide, fungicide, insecticide, virucide, acaricide, nematicide, acaricide, plant growth regulator, rodenticide, algaecide, biocontrol agent, beneficial agent, known fertilizer, inert ingredient, and / or biologically active agent.

[0216] In some embodiments, the agricultural compositions and / or consortia described herein are applied to agricultural growth media as topical applications to improve crop growth, yield, and / or quality. Topical applications may be in the form of dry mixes or powders or compositions for spreading, or may be liquid-based formulations. In some embodiments, the agricultural compositions and / or consortia described herein may be formulated as solutions; wettable powders; spreading powders; soluble powders; emulsion or suspension concentrates; tablets; water-dispersible granules; water-soluble granules (slow or fast release); microencapsulated granules or suspensions; and as irrigant components. In some embodiments, the agricultural compositions and / or consortia described herein are diluted in aqueous media before spray application. In some embodiments, the agricultural compositions and / or consortia described herein are applied to soil, plants, seeds, rhizosphere, root sheath, or other areas where applying the composition may be beneficial.

[0217] While the disclosure herein refers to certain illustrated embodiments, it should be understood that these embodiments are presented by way of example and not by way of limitation. EXAMPLES

[0218] During the development of embodiments of the technology described herein, experiments were performed to produce microbial consortia from environmental samples using selection techniques applied at the population level. Data collected during these experiments demonstrated the effectiveness of novel selective culture methods to select microbial consortia that can grow effectively on mineral media and atmospheric nitrogen and carbon dioxide without supplementation with nitrogen or carbon compounds. The microbial consortia are stable if grown under defined selective conditions. The inventors have also applied these same methods to large-scale batches of several agricultural soils, which are expected to yield up to 80 additional cultures to select from. This will allow for a significant expansion of diversity and capacity as we move to field testing for the first three consortia.

[0219] Materials and Methods Environmental Sample Collection. Liquid and soil samples were collected from eight separate locations in two privately owned residential areas in and near St. Louis, Missouri. Half of the samples were collected in midwinter and the other half in spring. Liquid samples were collected in midwinter from the bottom of a nearly empty rain barrel and contained green sludge. The green sludge material was stored in a glass jar at room temperature for several weeks in low light until use. Soil samples were collected with a sterile spoon or scoop and placed into a clean plastic zip-top bag. Loose material was first removed from the sampling site with a brush. Then, a sample was placed in the soil approximately 5 cm deep to a volume of approximately 100 cm. 3 Holes were cut and soil was removed from the soil inside the holes. Soil samples were stored at 4°C in the dark until use.

[0220] Inoculation of growth medium. The environmental samples were first mechanically homogenized from the outside of the bag. Then, approximately 10 cm 3of sample material was transferred to a 50 mL conical tube. The appropriate growth medium was then added to a total volume of 40 mL. The sample was vortexed until large clumps were dispersed and the sample was essentially homogenous. A volume of 0.5-1.0 mL of the vortexed sample was then added to the growth vessel using a wide-bore pipette tip. The volume added depended on the volume of the growth vessel. Inoculation from the suspended sample was performed immediately or the suspension was stored at room temperature in low light for several days and vortexed again before use.

[0221] Growth conditions. Three conditions were used for growth of samples in various media types: 1) shake flasks; 2) still bottles with liquid; and 3) still bottles with sterile sand and liquid. The shake flasks were 125 mL standard Erlenmeyer flasks covered with metal culture caps or aluminum foil. The shakers were a) an ATS-CHILL600V (Advanced Technology Solutions, Inc.) water cooler to provide cold water to the heat exchanger; b) a pair of MRKD-40K22-40W-U 4000K 40-W adjustable intensity LED lights (Super Bright LEDs, Inc.); and c) a New Brunswick G-25R incubating floor shaker fitted with two 20-W halogen bulbs (Feit Electric). The lights were operated at full power with an 18:6 photoperiod (18 hours on and 6 hours off). The light intensity at the shaker deck level was approximately 120 μmol photons / m using an Apogee Instruments MQ-500 light meter. 2The temperature was measured in s. The temperature in the shaker was maintained at room temperature (e.g., about 23-25°C). To remove heat from the 80W lighting, chilled water (e.g., about 5°C) from an ATS water cooler was fed to an integrated heat exchanger in the shaker temperature control box to maintain the temperature of the shaker. The shaker's internal heater was set to produce a nighttime differential. 160mL square milk diluent bottles or 250mL media bottles were placed at room temperature in two racks in front of two 42W 4000K LED shop lights (e.g., SLLP-40K4-42 from Super Bright LEDs, Inc. or equivalent) and four 20W halogen bulbs (Feit Electric). Light intensity at the level of the shaker deck was measured at approximately 120 μmol photons / m using an Apogee Instruments MQ-500 light meter. 2 The incubation time was measured in s. The same 18:6 photoperiod (18 h on and 6 h off) was used for the shakers. Bottles were filled to volume with medium or three-quarters full with sterile sand (Sigma-Aldrich) and then medium was added to volume. The lids were left loose to allow gas exchange.

[0222] Growth media. Two custom media mixtures were used: media A and media B. Media A was used with and without added combined nitrogen. Media B contained 1% complete combined nitrogen. Media A was either standard UTEX BG-11(-N) or normal nitrate BG-11(+N) with two modifications: 1) slightly lower phosphate levels and 2) 10 mM TES buffer was added at pH 7.0 to maintain medium pH long term. Media B is a modified version of complete M9 medium, designated BW-M9.

[0223] Medium A(+N) was made by adding 10 mL of 100x BG11(+N) concentrate (see below), 1 mL of 1000x ferric ammonium citrate stock solution (0.6 g ferric ammonium citrate per 100 mL water; store at 4°C protected from light; check for growth before use), 1 mL of 1000x Na2CO3 stock solution (2 g Na2CO3 per 100 mL water; store at 4°C), 1 mL of 1000x K2HPO4 stock solution (3.05 g K2HPO4 in 100 mL water, store at 4°C), and 10 mL of 1 M TES (pH 8.2) to double-distilled water to make 1 liter. After autoclaving and cooling, 1 mL of 1000x Wolfe's Vitamins (ATCC) was added. 100x BG11(+N) was made by adding 149.6 g NaNO3 (17.6 mM final medium), 7.5 g MgSO4·7H2O (0.3 mM final medium), 3.6 g CaCl2·2H2O (0.24 mM final medium), 0.60 g citric acid (or 0.89 g Na salts) (0.031 mM final medium), 1.12 mL 0.25 M Na-EDTA (pH 8.0) (0.0028 mM final medium), and 100 mL 1000x trace minerals (see below) to 1 liter of water. To prevent precipitation, these additions were made from 100x stock solutions. For nitrogen-free medium A (medium A(-N)), 100×BG11(-N) stock was prepared as above, except that NaNO3 was not added, and the 100×BG11(-N) stock was used to prepare nitrogen-free medium A (medium A(-N)). 1000×trace minerals was prepared by adding 2.68 g H3BO3, 1.81 g MnCl2·4H2O, 0.22 g ZnSO4·7H2O, 0.39 g Na2MoO4·2H2O, 0.079 g CuSO4·5H2O, and 0.049 g Co(NO3)2·6H2O to 1 liter of water. The medium was stored at 4°C.

[0224] Medium B (BW-M9) was made by mixing (per 1000 ml) 100 mL of 10x low-N M9 salts (see below) and 895 mL of water. After autoclaving and cooling, low-N M9 salts solution, 0.2 mL of filter-sterilized 20% w / v sodium acetate, 0.3 mL of filter-sterilized 1 M CaCl2 (14.7 g / 100 mL), 1 mL of filter-sterilized 1 M MgSO4 (24.65 g / 100 mL), 1 mL of filter-sterilized 1000x BW-M9 trace elements (see below), and 100x filter-sterilized Wolfe Vitamins (ATCC) were added. The mixture was autoclaved at 2500°F (1210°C) for 20 minutes using slow venting for liquid media. Vitamin solution was used at 1 / 4 strength, but is optional. After each stock was added, the solution was mixed. Low-N M9 (10x) salts were made by mixing 11.33 g Na2HPO4-7H2O, 3 g KH2PO4, 0.5 g NaCl, and 0.01 g NH4Cl in distilled water to 100 mL and adjusting the pH to 7.2 with NaOH. BW-M9 trace elements (1000x) were made by mixing 0.5 g EDTA, 0.83 g FeCl3-6H2O, 22 mg ZnSO4-7H2O, 7.9 mg CuSO4-5H2O, 10.00 mg CoCl2-2H2O, 0.10 g H3BO3, 0.18 g MnCl2-4H2O, and 39.00 mg Na2MoO4-2H2O in distilled water to 100 mL. EDTA was added first and the pH was adjusted to 7.5 with NaOH, and the solution was sterilized using a 0.22 μm filter.

[0225] Subculture, cryopreservation, and sample collection. Cultures were passaged to enrich the cultures by selection and to assess the stability of the microbial population. Because the nitrogen fixing consortium grew in clumps, culture growth was not measured using standard optical density measurements. Instead, a mass-based growth rate assay was developed (see Growth Curve section below), and passages were normalized between samples using growth time rather than direct measurements of cell mass. Growth was observed visually to determine the length of time from inoculation to late exponential phase before the culture began to decline. These observations indicated that exponential growth was reached 17±1 days after inoculation, therefore a passaging period of 17±1 days was used in the experiments.

[0226] Passages were performed as follows: Culture vessels were removed from the incubator and resuspended by swirling and / or scraping if a biofilm had formed. Immediately after swirling, 5 mL of culture was drawn into a serological pipette and 5 mL of inoculum was transferred to a 250 mL Erlenmeyer flask filled with approximately 100 mL of the same growth medium used to grow the culture from which it was taken. Each passage was repeated by inoculating two new cultures with 5 mL of inoculum taken from the same previous culture. Samples of the culture were set aside for DNA extraction by pelleting approximately 1-1.5 mL of culture and storing at -20°C until use. Additionally, one or two stocks of cells in growth medium were stored at -80°C in 20% glycerol. The passage of cells from which the inoculum was taken from the initial culture inoculated with the environmental sample was designated P1. Subsequent passages were designated P2, P3, and P4. That is, P2 was inoculated with P1, P3 was inoculated with P2, and P4 was inoculated with P3.

[0227] Growth curves. As noted above, the cultures were clumpy due to the formation of biofilms and possibly the production of EPS in the cultures. Therefore, optical density measurements did not provide an accurate measure of growth, and instead, growth curves were generated using the dry mass of the cell pellet. To provide an accurate measure, each time point was grown in a single well of a multi-well plate, and the experiment was designed as an end-point mass assay for each time point. For each P4 culture (8 populations x each lineage, i.e., BW01, BW02, or BW05), three biological replicates were used for each time point. For 6-well plates, each well was filled with 7.5 mL of BG-11(-N) medium and inoculated with 50 μL of mature (stationary phase) culture as follows: A 1 mL aliquot was taken from each flask and vortexed continuously for 5 min until the cell clumps were dispersed into a uniform suspension, resulting in a culture from which a small volume (e.g., 50 μL) could be accurately removed with a pipette for inoculation. An additional 200 μL aliquot was removed from the remaining vortexed culture and saved as a time point (see below). Each plate was placed on an orbital shaker at 150 rpm under two 40-W 4000K LED shop lights.

[0228] Each time point was collected using a disposable transfer pipette. All material, including biofilm attached to the bottom and sides of untreated wells, was disrupted and pipetted. Ultrapure water was used as a rinse and all rinses were collected. All material from each well was collected in a 15 mL conical centrifuge tube and centrifuged at 5000 rpm for 10 min in a swinging bucket rotor. The supernatant was discarded and a sufficient volume of ultrapure water was added to obtain a total volume of 1.5 mL. Using a disposable transfer pipette, the sample was transferred to a pre-weighed 1.5 mL microcentrifuge tube. The sample was centrifuged at 10,000 rpm for 10 min and the supernatant was discarded. The wet mass was measured before opening the tubes and placed in a 55°C oven for 1-2 days or until the viscous pellet reduced to a dry, flaky film and separated from the tube wall. For each time point, three control tubes were also placed in the oven, and the slight decrease in mass of these tubes was used to calculate the dry tare mass change factor. The product of the tare mass and the dry tare mass change factor was used to obtain the adjusted tare mass. The sample tube containing the sample was weighed and the adjusted tare mass was subtracted to obtain the final dry mass of the sample. Dry mass was plotted using the ggplot2 package in R studio (see, for example, Wickham (2016) "gplot2: Elegant Graphics for Data Analysis," Springer-Verlag New York). Growth curves were modeled using the R package Growthcurver (see, for example, Sprouffske (2016) "Growthcurver: An R package for obtaining interpretable metrics from microbial growth curves," BMC Bioinformatics 17:172). Growth curve data for all replicates were input into an algorithm to model the lineage of BW01 or BW02. The resulting curves were used to determine maximum doubling times and other metrics. Default inputs were used unless specified. The TO dry mass was below the detection limit (<0.0001 g), so the default minimum correction was used to prevent approximation errors.

[0229] DNA extraction. DNA extraction was performed using the Lucigen MasterPure Gram Positive DNA Extraction kit MGP04100. This kit is suitable for both Gram-negative and Gram-positive bacteria, reducing concerns about extraction bias.

[0230] Library preparation. Illumina sequencing libraries were prepared using the Illumina DNA Prep library preparation kit (Illumina 20018705) according to the manufacturer's instructions. Libraries were prepared using primers containing unique double indexes of 10 bp to provide multiplexing. Library yields were measured using a Qubit Fluorometer and average library fragment size was determined with a High Sensitivity DNA kit using an Agilent Bioanalyzer. The target average fragment size for the libraries was approximately 600 bp.

[0231] Illumina sequencing. Libraries were pooled and sequenced at the Genome Technology Access Center (GTAC) at the McDonnell Genome Institute (Washington University in Saint Louis) using an Illumina NovaSeq 6000 sequencer to generate 150 bp paired-end reads. Read data were demultiplexed by GTAC.

[0232] 16S Amplicon Sequencing. 16S rRNA amplicon sequencing was performed by Genewiz. Taxonomic designations were also performed by Genewiz.

[0233] Metagenomic data analysis. Taxonomy was determined using Kraken 2 v2.1.2 and the standard Kraken 2 database created on December 2, 2020. Functional annotation was determined using Phylomagnet v0.0.1. Paired-end reads were first joined using Fastq-Join v1.3.1. Unjoined reads were interleaved using Interleafq v1.1.0. Interleaved and joined reads were processed using Phylomagnet against a curated database of functionally and phylogenetically annotated orthologs (EggNOG v5.0). Contigs produced by Phylomagnet were assigned taxonomy and functional annotation. EggNOG ortholog groups representing functional annotations are listed in Table 4.

[0234] Assessment of community stability. After each passage, the cultures were assessed to assess the stability of the microbial community present in the culture. Stability was assessed using a measure of beta diversity calculated using the Bray-Curtis dissimilarity index (see, e.g., Bray and Curtis, An Ordination of the Upland Forest Communities of Southern Wisconsin. Ecol. Monogr. 27, 325-349 (1957), incorporated herein by reference). The Bray-Curtis dissimilarity index was calculated as shown in Equation 1, where S i is the total number of samples at site i, S j is the total number of samples at site j, C ij is the sum of the lowest values ​​of each sample per site.

[0235]

number

[0236] The Bray-Curtis dissimilarity index is bordered between 0 and 1, with a score of 0 indicating no differences between the two passages and a score of 1 indicating the two passages have no species in common. For example, human microbiome samples are highly variable between body sites, with some reference datasets showing mean Bray-Curtis values ​​ranging from 0.24 ± 0.17 (SD) when comparing fecal samples from individuals to 0.79 ± 0.08 (SD) when comparing fecal and nasal samples (see, e.g., Maziarz, Using standard microbiome reference groups to simplify beta-diversity analyses and facilitate independent validation. Bioinformatics 34, pp. 3249-3257 (2018), incorporated herein by reference). Soil sampling replicates (e.g., from the same area of ​​1-2 square meters) subjected to the same DNA extraction method yield Bray-Curtis scores as low as 0.1523. However, Bray-Curtis values ​​greater than 0.5 for soil samples extracted using different DNA extraction methods have been observed.

[0237] Isolation of minimal consortia. Minimal consortia were generated by pipetting 20 μL of undiluted, vortexed culture onto BG-11(-N) plates containing 1 / 4 Wolfe Vitamins (ATCC), 1 mM sodium thiosulfate, and 1% agarose. Plates were streaked with one long streak. Approximately 100 μmol / m 2After 2–4 weeks of growth at room temperature (e.g., about 24 °C) under white light (LED) with an intensity of 100 μmol / m s, large colonies appeared. A colony was picked, placed in 1 mL of sterile ultrapure water, and vortexed until clumps were dispersed. A volume of 20 μL was pipetted onto several plates of the same type, with four duplicate streaks. The colony was picked again and restreaked in the same manner until isolated colonies with nearly uniform characteristics were observed (typically after restreaking two to three times). Finally, an isolated colony was used to inoculate 30 mL of BG-11(-N) supplemented with 1 / 4 Wolfe Vitamins in an upright disposable vented tissue culture flask, with approximately 100 μmol / m 2 The plates were then shaken at 150 rpm under near white light (LED) at an intensity of 100 s at room temperature (e.g., about 24° C.). DNA was extracted and shotgun sequencing was performed in the same manner as used for the complete consortium.

[0238] Phylogenetic analysis of minimal communities. Taxonomy was determined using Phyloflash v3.4 and the SILVA database v138.1. Taxonomy was updated using NCBI taxa API. Results were verified using the binning algorithm MetaBat v2.15, followed by read assembly using MegaHit v1.2.9. Contigs in each bin were assessed using CheckM v1.1.3.

[0239] Taxonomic analysis of photosynthetic microorganisms. Forward row sequence reads were compared to a reference database of plastid 23S ribosomal DNA sequences by the blast algorithm (see, e.g., Djemiel (2020) "μgreen-db: a reference database for the 23S rRNA gene of eukaryotic plastids and cyanobacteria," Sci Rep. 10:5915, incorporated herein by reference). Results were filtered to identify mismatches of less than 10 base pairs, alignment lengths of greater than 130 base pairs, and matches with greater than 95% identity.

[0240] Photobioreactor growth. A series of 1 liter glass flasks were used to construct the photobioreactor. Each of the flasks was placed on a stir plate and closed with a stopper containing a glass foam line and an outlet line. The growth medium was BG-11 without TES buffer. To mimic large-scale production conditions where the use of TES is costly and difficult to implement, TES buffer was omitted from the growth medium. Therefore, TES buffer is an optional component that may be used for long-term shake culture and passaging. Growth medium and stir bar were placed in flasks, the openings were covered with aluminum foil, and autoclaved. Stopper assemblies (Whatman HEPA-vent) containing inlet filters were autoclaved separately in dry packs. After cooling, the liquid medium was inoculated with 2 mL of stationary phase culture. The inoculum concentration was established using the dry mass measurement technique described for the growth curves. The inlet side of each filter was connected to the outlet line of a humidification bottle. The foam line inlet of each humidifier bottle was connected to a 10 gallon Whisper air pump protected by a check valve.

[0241] Time points were taken from the photobioreactor cultures to generate growth curves. Prior to each time point, the formed biofilm was disrupted by shaking or scraping the culture flask wall. For each time point, 1 mL of culture was removed using a 1 mL serological pipette and 1 ml of culture was added to a pre-weighed 1.7 mL plastic centrifuge tube. Centrifugation, drying, and weighing were as described herein to generate the growth curves. Despite the use of humidified bottles, some evaporation was observed during the growth period. To correct for the effect of evaporation on the growth curve data, the bioreactor volume was recorded (to the nearest 50 mL) for each sample. Curves were fitted to the points plotted of 1 mL of culture dry mass versus grams of bioreactor base scaled to the current culture volume at the time of sampling.

[0242] Carbon and nitrogen determination. Total elemental carbon and nitrogen were determined on dried cell pellets following harvest of photobioreactor cultures. Cells were separated from liquid medium at very late logarithmic or stationary phase (days 32 or 45 for light and dark experiments, respectively). Total cell pellets were harvested in a Beckman-Coulter Allegra 25R centrifuge at 5000 rpm in a swinging bucket rotor containing four 500 mL bottles. Pellets were transferred to pre-weighed disposable 50 mL conical tubes and dried in an oven at 55° C. until hardened and brittle. Pellets were analyzed by combustion in a LECO analyzer from Midwest Laboratories (Omaha, NE, USA) following protocol MWL WC ​​PROC 55.

[0243] Growth in soil. Commercially available culture mixtures were sterilized by autoclaving in glass Petri plates. Red fluorescence images of three consortia grown on sterilized culture soil were inoculated, the day 0 blank was subtracted, and the images were aligned by dedicated image analysis software. The excitation light was 450 nm blue light. A deep red filter was used to block blue wavelengths to the camera detector.

[0244] [Example 1] During the development of embodiments of the technology provided herein, experiments were conducted to select for organisms that would grow using only light, basic mineral salts, and atmospheric gases, without the addition of reduced nitrogen and carbon. Eight environmental samples collected in mid-winter or early spring were subjected to multiple selection growth conditions, providing 56 different starting samples (Table 1). Many conditions showed growth. However, the collected data showed that three cultures (populations) inoculated from winter soil had exceptional growth over a short period of time (28 days). Further experiments focused on these three cultures (BW01, BW02, and BW05). Condition 7 was a positive control for microbial growth. Growth in Table 1 was measured visually by comparing the growth of each condition 1-6 to the growth of the positive control condition 7 and assigning a number from 1 (low growth) to 8 (high growth). BW01, BW02, and BW05 were produced in BG-11 medium ("Medium A") without nitrogen (-N) under illumination. BW01 and BW02 were produced in flasks with shaking, BW05 was produced in sand.

[0245] [Table 2]

[0246] [Example 2] The initial cultures of BW01, BW02, and BW05 were used to inoculate two new flasks each containing the same medium used to grow and enrich the cultures of BW01, BW02, and BW05 during initial selection. Culture growth was monitored by visual inspection, and an inoculum taken during the late exponential phase of growth (e.g., 2.5 weeks after inoculation) was used to inoculate a new flask containing fresh selective growth medium.

[0247] Each inoculation step, growth under selection, and associated characterization of the resulting culture is referred to as a "passage". Each passage is assigned a unique identifier (e.g., "passage 1" or "P1"). Each population was tracked in duplicate flasks grown under the same selection pressure. Four passages were performed to assess the stability of the cultures from passage to passage (see below) and to generate consortia containing key components of the initial population. Passages were performed simultaneously for both (-N) and (+N) conditions, and the (+N) culture was used as a positive control to verify that the conditions were conducive to growth. The positive control supplemented with nitrogen grew extremely rapidly, suggesting that population growth could be accelerated by nutrient supplementation. For each passage, samples of exponential phase cultures were collected and retained for storage and shotgun metagenomic sequencing.

[0248] Different culture morphologies were observed at the final passage (P4). The BW01 lineage had two morphological classes, with four P4 flasks having light green small clumps and four flasks having much darker, larger clumps. BW02 contained highly clumpy cells and had a rapid growth phenotype. Upon dehydration, cultures of BW02 had a jelly-like morphology consistent with extracellular polymeric substances (EPS) secreted by cyanobacteria of the Nostocaseaceae family, e.g., Anabaena spp. (see, e.g., Moreno, Chemical and rheological properties of an extracellular polysaccharide produced by the cyanobacterium Anabaena sp. ATCC 33047. Biotechnol. Bioeng. 67, 8 (2000), incorporated herein by reference).

[0249] Both BW01 and BW02 cultures contained filaments with heterocysts evident when viewed by light microscopy (FIGS. 1A and 1B). Heterocysts are an adaptation of Nostocaseae used to fix nitrogen simultaneously with oxygenic photosynthesis (see, e.g., Kumar, Cyanobacterial Heterocysts. Cold Spring Harb. Perspect. Biol. 2, a000315-a000315 (2010), incorporated herein by reference). Because nitrogenase is highly sensitive to oxygen poisoning, the heterocyst serves to provide an anaerobic environment for nitrogen fixation while the vegetative cells are performing photosynthesis (e.g., oxygenic photosynthesis). Sugars and fixed nitrogen are exchanged between the heterocyst and the vegetative cells through microplasmodesmata. Nostocaseae also possess a second differentiated cell type, called the akinete, which is highly persistent against environmental insults and thus serves as a long-term survival mechanism for the organism.

[0250] [Example 3] During the development of embodiments of the technology described herein, cultures of BW01, BW02, and BW05 were evaluated using shotgun metagenomic sequencing of the population to assess changes in population diversity and to elucidate the composition of the population. DNA extraction and shotgun metagenomic sequencing were performed on each culture from each of the four passages. Illumina sequencing was performed. To identify the microbial genera that constitute the population, sequence data was analyzed using Kraken2 (see, e.g., Wood, Improved metagenomic analysis with Kraken 2. Genome Biol. 20, 257 (2019), incorporated herein by reference). For a readout of the overall community composition, replicate measurements from the same passage were averaged in this analysis. Additionally, the initial soil samples used were also subjected to shotgun metagenomic sequencing to provide a description of the starting sample. Sequence data derived from cultures grown under selective conditions demonstrated that some lineages with different physical characteristics developed slightly differently in each duplicate flask.

[0251] Taxonomic classification of metagenomic sequence data collected during these experiments showed that the selective growth conditions significantly altered the composition of the populations compared to the original soil samples. Interestingly, although the populations passaged from the different starting samples differed with respect to many characteristics, it was observed that the populations shared some similar features. The selection process modified the populations by changing the proportions of each genera. The data showed that selection produced four general changes (Figure 2, Figure 3A-H).

[0252] 1. The relative abundance of some genera increased steadily through the successions. For example, Nostoc spp. was present in 31 ± 17% (SD) and 33 ± 10% (SD) of the passage 4 (P4) samples for BW01 and BW02, respectively, which increased from less than 0.1% in the original soil. A similar trend was observed for BW05. The occurrence of this genus increased rapidly and remained stable through the successions in all three of BW01, BW02, and BW05. Methylibium spp. also increased steadily through the successions, but in lower abundance than Nostoc spp.

[0253] 2. The relative abundance of other genera steadily decreased with increasing succession: for example, Streptomyces spp. and Mesorhizobium spp. both appeared less adapted under the selective conditions than in the original soil, and overall their populations decreased in all cultures throughout succession.

[0254] 3. Some genera increased in occurrence in the early stages, and other genera, such as Aminobacter species and Rhizobium species, became dominant in the later stages of the succession.

[0255] 4. Other genera, such as Pseudomonas spp. and Azospirillum spp., initially decreased in occurrence during P1 compared with the original soil and increased during later successions.

[0256] [Example 4] During the development of an embodiment of the technology described herein, replicate growth curves were used to measure the growth phenotype of the population (FIG. 4). Growth curves were plotted using the dry mass of culture in grams per 7.5 mL well in a standard 6-well plate. This also provided a measure of carbon sequestration. Growth curves incorporating all replicate measurements from BW01 (n=24), BW02 (n=24), and BW05 (n=24) were modeled using a logarithmic function. The resulting metrics are shown in Table 2, including the uncertainty associated with each curve fit. The ½K values ​​to reach half-maximal growth were 19.1 and 15.8 days for BW01 and BW02, respectively, and the maximum doubling times were 4.2 and 3.6 days. Parameters for Table 2 are as follows: Unless otherwise noted, all mass units are grams (dry mass) per 7.5 mL well in a standard 6-well plate, and times are in days. k = carrying capacity, n0 = mass at the beginning of the growth phase, r = growth rate, sigma = goodness of fit, df = degrees of freedom, t_mid = time of inflection point where the number of bacteria is 1 / 2k, t_gen = fastest generation time or doubling time. SE is standard error and p is P value.

[0257] [Table 3]

[0258] Individual P4 populations were evaluated in more detail by fitting curves for the individual populations (n=3 for each population). Eight P4 populations were produced from each soil lineage BW01 and BW02. The output parameters for each of these 16 populations are shown in Table 3. Averaging the fitted curve doubling times within each soil lineage, the resulting doubling times are found to be 4.0±0.5 days and 3.5±0.7 days for BW01 and BW02, respectively, where the error is reported as one standard deviation from the mean. The parameters in Table 3 are as follows: Unless otherwise noted, all mass units are grams per 7.5 ml well in a standard 6-well plate and times are in days. k = carrying capacity, n0 = mass at the beginning of the growth phase, r = growth rate, sigma = goodness of fit, df = degrees of freedom, t_mid = time of inflection point where the number of bacteria is 1 / 2k, t_gen = fastest generation time or doubling time, auc = 1, area under the curve from the logistic equation, e is the experimental data point.

[0259] [Table 4]

[0260] [Example 5] During the development of embodiments of the technology described herein, four passages were evaluated for activity of specific genes and / or enzymes (Table 4 and Figure 5). Gene function was evaluated for the two phyla represented in the taxonomic characterization analysis, namely Cyanobacteria and Proteobacteria. Gene functions from the photosynthesis, nitrogen fixation, and EPS production pathways were selected for this analysis.

[0261] [Table 5]

[0262] [Example 6] During the development of embodiments of the technology described herein, experiments were performed to produce minimal consortia that are reduced in complexity while still providing the phototrophic and nutrient fixation functions of the more complex consortia identified in other experiments. In these experiments, cultures were diluted and streaked several times on plates to isolate individual colonies with stable shapes. Cyanobacteria produce complex extracellular matrices and often closely associate other microorganisms. As a result, cyanobacteria often carry other microorganisms when streaked, and the associates remain in the seemingly isolated colonies. In these experiments, this association was to identify colonies that grew robustly on the solid form of minimal medium used for selection, even though the colonies were separated from the complete consortium. Without being bound by theory, it was assumed that successful colonies contain community members that provide functions for survival on minimal culture medium, and that microorganisms that cannot survive on minimal medium without other community members would not produce colonies. Therefore, experiments were performed to identify colonies that contain minimal populations that can survive on minimal selection medium.

[0263] Two colonies with distinct morphology were picked from each plate. Two plates were grown from each colony. The colonies were then grown in the same liquid selective medium described above for the growth of consortia (e.g., BW01, BW02, and BW05). Morphology was predictable based on lineage, and all inoculated cultures grew stably and reliably to saturation in liquid (Figure 6). These cultures were grown to saturation (30 days) and DNA was extracted for shotgun sequencing. Sequencing data showed that the resulting cultures were not pure isolates, but rather consisted of less complex microbial communities than the BW01, BW02, and BW05 consortia (Figure 7A). The most complex consortia thus identified consisted of up to nine taxa, and the least complex consisted of two taxa. All sequenced samples consisted of the cyanobacterial genus Nostoc or the closely related Dolichospermum. The relative abundance of specific genes (nifh, psba, psbb, rubiscoL, rubiscoS, and wza) was identified in the nucleotide sequences obtained from the consortium (Figure 7B-F). EggNOG orthologs with functional annotation are listed in Table 4.

[0264] [Example 7] Classification of members of the minimal community was determined using two computational approaches. The first approach used Kraken2 to analyze the complete community. The second approach used Phyloflash 3.4, which uses an approach based on small subunit rRNA gene assemblies and is more appropriate than Kraken2 for groups of lower complexity (see, for example, Gruber-Vodicka (2020) “phyloFlash: Rapid SSU rRNA profiling and targeted assembly from metagenomes,” mSystems 5:e00920-20, incorporated herein by reference). Several taxa were identified in the samples, including Proteobacteria, Cyanobacteria, and tremellomycete fungi (Table 5). Each taxon contributes different metabolic functions important to the community. Notably, Cyanobacteria are always present, indicating that their functions are important for survival in the particular selection applied.

[0265] [Table 6]

[0266] Growth curves were generated by growing minimal consortia in 12-well plates. The growth curves showed similar performance of most minimal consortia compared to the full consortium control (Figure 8). A notable exception was the dark green colonies picked from plates of BW02, which produced both light brown, viscous colonies and dotted dark green colonies. The dark green colonies bleached uniformly midway through the experiment. Curve fit parameters are shown in Table 6. Parameters in Table 6 are as follows: Unless otherwise noted, all mass units are grams per 2 ml well in a standard clear 12-well plate and times are days; k = capacity, n0 = mass at the beginning of the growth phase, r = growth rate, sigma = goodness of fit, df = degrees of freedom, t_mid = time of inflection point where bacterial count is 1 / 2k, t_gen = fastest generation time or doubling time, auc = 1, area under the curve from the logistic equation, e is the experimental data point. Fits were performed using Growthcurver18 in R.

[0267] [Table 7]

[0268] In Table 6, the order of the rows for each condition in the lower part of the table is the same as the order of the rows for each condition in the upper part of the table.

[0269] [Example 8] In the field, saturation is not expected to be as important as in liquid cultures. Microbial cultures become saturated when they run out of resources (e.g., mineral nutrients), produce waste products and metabolites, self-shade, or quorum-sense. However, in liquid cultures (e.g., at production scale), saturation helps define an upper limit for culture density. Thus, while developing embodiments of the technology described herein, experiments were performed to determine a calculated carrying capacity that represents the maximum saturated culture density (Figures 9A and 9B). Curve-fitting data showed that reducing the complexity of the consortium does not necessarily reduce the carrying capacity of the culture.

[0270] [Example 9] In some embodiments of the present technology, growing large-scale cultures can be important for producing and using microbial consortia in the field. Therefore, while developing embodiments of the present technology, experiments were performed to test the feasibility of scaling up the BW01, BW02, and BW05 consortia. In these experiments, cultures of BW01, BW02, and BW05 were grown in 1-liter photobioreactors (PBRs). A PBR is a bioreactor, often referred to as a fermentor, designed to deliver light to the microorganisms growing in the PBR. Cultures of BW01, BW02, and BW05 in the PBR were mixed and / or agitated using a combination of magnetic stirring and bubbling of filter-sterilized air, allowing the photosynthetic microbial consortia to grow to high density at high growth rates without supplementing the medium with nitrogen and carbon compounds. Growth curves of BW01, BW02, and BW05 consortia under these conditions are shown in FIG. 10. These data showed that the PBR was highly productive with or without added nitrogen compounds to supplement the nitrogen supplied by the air.

[0271] The PBR was used to produce sufficient dry mass for elemental nitrogen and carbon analysis (Figure 11). The nitrogen and carbon analysis data were used to calculate predicted carbon sequestration and nitrogen fixation for the production pond or field cultures.

[0272] [Example 10] During the development of embodiments of the technology described herein, an experiment was conducted to test the growth of the BW01, BW02, and BW05 microbial consortia on soil. The growth of the consortia on soil was monitored using chlorophyll fluorescence and machine vision. The data showed that the BW01, BW02, and BW05 consortia colonized the soil over a 10-day growth period (FIGS. 12A and 12B). The data collected during this experiment showed that BW01, BW02, and BW05 contained photosynthetic members that produced chlorophyll.

[0273] [Example 11] During the development of embodiments of the technology provided herein, data was analyzed to determine the stability of the microbial consortium produced using the passaging described herein. In particular, beta diversity was calculated for each pass of the process and monitored as a measure of stability. The beta diversity index provides a useful metric for comparing two sites or passaging and measuring the changes that occur from one passaging to the next. As a result, the flattening of Bray-Curtis dissimilarity during the comparison of successive passaging indicates that the culture has reached a maximum level of stability during the passaging process.

[0274] The collected data showed that the Bray-Curtis dissimilarity scores changed rapidly as selection pressure was applied (Figure 13). The decrease in the Bray-Curtis score during each successive passage indicated that less dissimilarity was observed between successive subsequent passages as opposed to the previous passages. Comparing the last passage P4 to its parent P3 yielded Bray-Curtis scores of 0.17 ± 0.065 (SD) and 0.14 ± 0.011 (SD) for BW01 and BW02, respectively. This indicated that as passaging continued, these populations began to reach an equilibrium state where subsequent passages would have minimal impact on the culture and / or differences between passages would be minimal.

[0275] Throughout the passages, both cultures followed similar trends, but were not identical. However, by the fourth passage, the Bray-Curtis dissimilarity scores for both BW01 and BW02 flattened out, indicating that both cultures had reached maximum stability. Given that these cultures remained stable in small-scale laboratory experiments, it is assumed that the cultures will be stable at production scale.

[0276] The data indicate that selection was targeted and effective. Principal coordinate analysis (PCoA) of the shotgun sequencing data of the original soil samples and cultures P1-P4 shows that the original soil samples of BW01 and BW02 cluster fairly tightly together, but the final passage begins to develop into two separate clusters (Figure 14). By P4, a strong trend towards clustering is observed. However, in BW01, there appears to be a divergent set of samples. Given the complexity of these populations, it would be surprising if some differences were not observed throughout the passages. The relative tightness of the clustering indicates that selection has a predictable and consistent effect in most lineages.

[0277] [Example 12] Nitrogen- and carbon-fixing communities have the ability to improve soil and sequester CO2. However, some communities are adapted to intermediate season conditions that include high fluxes of broad-spectrum light (e.g., about 380-750 nm). Furthermore, plants transmit or reflect light at wavelengths that they do not absorb. Thus, crop plants typically utilize light at classically defined photosynthetically active wavelengths of radiation, from about 400 nm to about 700 nm. However, oxygenic photosynthetic microorganisms are adapted to use light outside this range, in particular far-red light (FRL), usually defined as wavelengths from about 700 nm to about 750 nm.

[0278] During development of embodiments of the technology described herein, experiments were conducted using filtered light (e.g., in the far-red portion of the visible spectrum) to produce consortia that maximized carbon capture during the busy season and soil stabilization during the growing season. Therefore, selection conditions were used to produce consortia that utilized far-red light (e.g., about 700-750 nm).

[0279] These selections are designed to enrich for organisms that use energy from far-red light to fix carbon, for example by using far-red absorbing pigments and antenna pigment proteins. During the development of embodiments of the technology described herein, experiments did not use conditions that select for organisms that fix nitrogen. Thus, the experiments involved the use of culture media that contained immobilized nitrogen compounds (e.g., BG-11 +N). This is the same medium described herein for the nitrogen addition experiments.

[0280] Far-red light (e.g., about 700-750 nm) can be used to select for organisms that utilize far-red wavelengths, a portion of the spectrum that is not widely used by plants. Therefore, experiments were performed in which 740 nm light (e.g., produced by LEDs) was the only light source provided to the community and the organisms in the community. In particular, several 1 watt LED chips were attached to an aluminum heat sink and emitted approximately 100 μmol / m 2 The LEDs were driven (e.g., with a Meanwell LED driver) with a current providing a photon flux of .s.

[0281] In addition to using a growth medium containing far-red light and nitrogen compounds, the experiment included using the same enrichment process described above in this specification (e.g., to produce a consortium containing photosynthetic and nitrogen-fixing microorganisms). Initial cultures were inoculated using samples from mud and water samples taken from small ponds and streams within a 100 mile radius of St. Louis, Missouri. Mud and soil samples were homogenized at a 10-fold dilution in sterile water using a 3.2 mm steel bead and a vortexer. A small volume was used to inoculate vented culture flasks, which were upright shaken at 100 rpm and 25°C under a far-red LED.

[0282] Passages were performed when the cultures were near saturation (e.g., at about 6 weeks) and four passages were performed. Each initial culture (P1) was used to inoculate four flasks to produce P2 cultures. Each of the four flasks was then used to inoculate one flask to produce P3 and each of the P3 cultures was used to inoculate one flask to produce P4. DNA was extracted and sequencing was performed as described.

[0283] Specifically, experiments were conducted with input samples taken from soil and water obtained at sites in Arkansas (150 soil and 30 water samples), Wisconsin (100 soil and 25 water samples), and Washington (250 soil and 15 water samples). Water samples were generally processed immediately; however, some soil samples were found to remain viable and stable after storage at 4°C. Samples were processed using sterile equipment.

[0284] First, the sample contents were homogenized (e.g., by hand from the outside of the bag for soil samples). Next, 7.5 ml of sterile water was added to 15 ml conical tubes labeled to match the sample identifier on the bag. 0.5 ml of sterile 3.2 mm chrome steel beads were added to each tube, and then the tubes were scraped approximately 2 cm apart using a sterile scoop. 2 of soil was placed in each tube. The samples were agitated and homogenized using a vortexer. Optionally, samples may be homogenized using a sonicator, but sonication may increase the chance of contamination or reduced population diversity. A 4420 microtip was attached to the sonicator for sonication. It can process 5-50 ml. To reduce contamination, the probe was cleaned with 70% ethanol. Approximately 3 / 4 of the probe was inserted into the liquid. The probe was not allowed to touch the tube wall. Samples were sonicated for 1 min using 5-s pulses at 10% amplitude with a 5-s interval between each 5-s ultrasonic pulse. If homogenization was incomplete, sonication was performed again. Samples were monitored to prevent overheating.

[0285] After entering the identifying information into the database, flasks were prepared for the selection process. Each sample-condition combination was assigned a unique identifier ("P#"). Flasks were labeled with the P#, parent sample number, medium type, and date. Flasks were filled with 30 mL of medium and held in an upright vertical position.

[0286] The homogenized sample was shaken and briefly vortexed to suspend particulates and prepare a thoroughly mixed sample. A 0.3 ml volume was removed from the homogenized sample within 30 seconds of vortexing and used to inoculate the appropriate flask prepared above. When multiple flasks were inoculated from the same homogenized sample, the sample was stirred and / or shaken to maintain sample mixing and prevent settling of particulates. The flask was capped and maintained in an upright vertical orientation throughout inoculation and subsequent growth.

[0287] For passages P2 and P3, 25 cm plastic filter-tipped culture flasks were filled with 30 ml of medium and labeled with the new P number. The flasks were kept in an upright vertical orientation. Three tubes were labeled to conserve samples (e.g., as cell pellets) and two tubes containing 0.5 ml of 40% glycerol were labeled for glycerol stocks with the parent P number. For each parent flask (e.g., P1 or P2), a plastic loop was used to remove the biofilm and homogenize the culture. A volume of 5 ml was immediately removed (e.g., before the culture settled). 1 ml was used to inoculate the P2 (or subsequently P3) flask. 1 ml was used to reserve a cell pellet sample and 0.5 ml was used for each of the two glycerol stocks. The glycerol stocks containing the cultures were vortexed and stored at -80°C. The cell pellet tubes were centrifuged at full speed for 5 min. After discarding the supernatant, the cell pellets were stored at -20°C for later DNA extraction and further analysis. The culture flasks were placed on a shaker illuminated with far-red light.

[0288] The P4 passage was produced from the P3 culture following the same inoculation method as above for further validation steps. Live cultures of BW01, BW02, and BW05 were inoculated in the same manner as the P4 culture and served as growth controls.

[0289] Growth curves were generated from the P3 culture used as inoculum, and the growing P4 and control (BW01, BW02, and BW05) cultures. To measure the initial g / ml of cell material (time T0), two 1.7 mL snap-cap tubes were pre-weighed and the empty weight was recorded for each culture and each control culture (BW01, BW02, and BW05). Three more tubes were then weighed to control for volatilization of plastic in the dryer. The same type and brand of tubes were used for the entire experiment. A 1 ml volume of homogenized P3 culture was dispensed into each tube and the cultures were pelleted by centrifugation (e.g., 5 minutes at full speed). The pellets were placed in a 55° C. oven overnight to dry, the dried pellets were weighed in the tubes, and the mass of the cell pellet was recorded.

[0290] Every week, a 1 ml aliquot of the P4 culture was used to obtain the cell pellet mass (in g / ml of culture) following the same process as above. The cell pellet mass (g / ml) was monitored as a function of time and the time when the growth curve began to plateau was noted. Once the cell pellet mass plateaued, a 5 ml volume was removed. 1 ml was used to reserve the cell pellet sample and 0.5 ml was used for each of the two glycerol stocks. The glycerol stocks containing the culture were vortexed and stored at -80°C. The cell pellet tube was centrifuged at full speed for 5 min. After discarding the supernatant, the cell pellets were stored at -20°C for later DNA extraction and further analysis. The R package Growthcurver was used to fit the curves and determine growth parameters.

[0291] DNA was extracted from the P4 pellet. Libraries were prepared for Illumina sequencing and sequencing was performed with a target depth of 50 million reads. Based on the sequence data obtained, unique populations and / or populations are identified. Cultures and / or frozen samples of several (e.g., 1-50) diverse populations are identified and retained for further study.

[0292] [Example 13] During the development of embodiments of the technology described herein, experiments were performed to identify photosynthetic members of the complete community. DNA was extracted from whole community samples S1170, S1172, S1174, S1176, S1178, S1180, S1181, and S1184. After obtaining nucleotide sequences by Illumina sequencing as described herein, forward row reads were compared to a reference database of plastid 23S ribosomal DNA sequences using the blast algorithm (see, e.g., Djemiel (2020) "μgreen-db: a reference database for the 23S rRNA gene of eukaryotic plastids and cyanobacteria," Sci Rep. 10:5915, incorporated herein by reference).After filtering for matches to the plastid database as described in Methods, sequences were selected from the following genera: Nodularia, Chrysosporum, Gloeocapsopsis, Richeria, Mastigocoleus, Hapalosiphon, Gloeothece, Acaryochloris, Camptonema, Raphidiopsis, Crocosphera, Macrochaete, Thermosynthecoccus, Pseudoanabaena, Chroococcidiopsis, Prochlorothrix, Anabaena, Leptolyngbya, Callosthrix, Cylindrospermopsis, Dolichospermum, Cytonema, Lyngbya, Tolyposthrix, Fischerella, Fortiera, Alitherella, Hydrocoryne, Prochlorococcus, Planctotrichoides, Gaitrelinema, Xenococcus, Yaginema, Nostocoptis, Pantanalinema, Oscillatoria, Spirulina, Peratoccus, and Spirulina. The bacteria were identified as being from the bacterial genus Ladus, Nodocilinea, Aphanizomenon, Chlorogloeopsis, Gloeocapsa, Karenema, Livularia, Trichorum, Synechococcus, Synechocystis, Cylindrospermum, Planktothrix, or Rubizibacter, or from the algal genus Tetraselmis, Chlorella, Coreochaeta, Gonium, Paradoxia, Symbomonas, Palmaria, Calasiochloris, Marvania, Chlamydomonas, Parachlorella, Coccomyxa, Trebuxia, Cyanidium, Troybaria, Danjardinia, Gardieria, Nuformia, Porphyra, Kara, Anchistrodesmus, Picochlorum, Cyanophora, Chondrus, Pseudochloris, Interphyllum, Gelidium, Symphyogina, Chlorosarquina, or Cyanothece.Thus, these data suggest that the microbial consortium is composed of Nodularia, Chrysosporum, Gloeocapsopsis, Lychearia, Mastigocoleus, Hapalosiphon, Gloeothece, Acaryochloris, Camptonema, Raphidiopsis, Crocosphera, Macrochaete, Thermosynethococcus, Pseudoanabaena, Chroococcidiopsis, Prochlorothrix, Anabaena, Leptoryngbia, Callosthrix, Cylindrospermopsis, Dolichospermum, Cytonema, Lyngbia, Tolyposthrix, Fischerella, Fortiera, Alitherella, Hydrocoryne, Prochlorococcus, Planctotrichoides, Gaitrelinema, Xenococcus, Yaginema, Nostocopsis, Pantanalinema, Oscillatoria, Spirulina, Pellatocladus, Nodococcilinea, Aphanizomenon, Chloroglossum, and Chloroglossum. and wherein the organism is a photosynthetic organism characterized by being a member of the bacterial genus Rhoeopsis, Gloeocapsa, Karenema, Livularia, Trichorum, Synechococcus, Synechocystis, Cylindrospermum, Planktothrix, or Rubisibacter, or from the algal genus Tetraselmis, Chlorella, Coreochaeta, Gonium, Paradoxia, Symbomonas, Palmaria, Calasiochloris, Marvania, Chlamydomonas, Parachlorella, Coccomyxa, Trebuxia, Cyanidium, Troybaria, Danjardinia, Gardieria, Nuformia, Porphyra, Cara, Anchistrodesmus, Picochlorum, Cyanophora, Chondrus, Pseudochloris, Interphyllum, Gelidium, Symphyogina, Chlorosarquina, or Cyanothece.

[0293] [Example 14] During development of an embodiment of the technology described herein, observation of cultures BW01, BW02, and BW05 showed the presence of dark pigmented aggregates in the cultures (FIG. 18A). Photobioreactors were constructed by pumping filtered air through tubing into stoppered Erlenmeyer flasks. The photobioreactors were inoculated with a consortium and grown for 2 weeks under constant light, agitation (using a stir bar), and constant aeration. Aggregates were evident in all cultures after a few days, and in all cases the cultures became dark in color after continued growth. The inoculated cultures at day 1 are shown in the top photograph of FIG. 18A. The inoculated cultures after 2 weeks of incubation under constant light, agitation, and aeration are shown in the bottom photograph of FIG. 18A.

[0294] After growth, experiments were performed to characterize the dark pigments, and in particular, during the development of embodiments of the technology described herein, it was assumed that the dark pigments were molecules that contained fixed carbon, e.g., a fixed, sustainable form of carbon.

[0295] One class of sustainable carbon compounds is melanin. Melanin biosynthesis uses the amino acid tyrosine to synthesize each of the three main types of melanin, namely, pyomelanin, pheomelanin, and eumelanin (Figure 18B). As shown in Figure 18B, the biosynthetic pathways for the biosynthesis of pyomelanin, pheomelanin, and eumelanin are different. In particular, the biosynthesis of pyomelanin from tyrosine produces homogentisic acid as an intermediate (Figure 18B). Homogentisic acid is not produced in the biosynthesis of pheomelanin or eumelanin. Furthermore, tyrosinase converts tyrosine into dopaquinone or L-dopa as the initial (or first) step in the biosynthesis of pheomelanin or eumelanin, respectively, while aromatic amino acid transaminase converts tyrosine into 4-hydroxyphenylpyruvate as the initial (or first) step in the biosynthesis of pyomelanin. See, for example, Lee (2022) “Melanin Biopolymer Synthesis Using a New Melanogenic Strain of Flavobacterium Kingsejongi and a Recombinant Strain of Escherichia Coli Expressing 4-Hydroxyphenylpyruvate Dioxygenase from F. Kingsejongi” Microb. Cell Factories 21(1):75, incorporated herein by reference.

[0296] Thus, during development of embodiments of the technology described herein, experiments were performed to determine whether the addition of melanin biosynthetic precursors to the growth medium affects (e.g., increases) the production of colored compounds by BW02 and / or BW05 in culture. Additionally, experiments were performed to characterize the pigments produced by cultures of BW02 and BW05.

[0297] [Example 15] During the development of embodiments of the technology provided herein, experiments were performed to characterize the physicochemical properties of pigments extracted from a minimal microbial consortium observed to have a brown morphology (sample MEL0152). Following growth in liquid culture of a minimal microbial consortium observed to have a brown morphology, a portion of the culture was processed using the melanin extraction method described in Kumar (2013) "Production of melanin pigment from Pseudomonas stutzeri isolated from red seaweed Hypnea musciformis," Letters in Applied Microbiology 57:295-302, which is incorporated herein by reference. Briefly, culture samples were collected and centrifuged. After separation of the supernatant and biomass pellet, the biomass pellet was resuspended in a solution containing proteinase K (to degrade proteins) and NaOH. Cells were lysed by vortexing the sample in the presence of stainless steel beads. The sample was centrifuged again and the supernatant was retained. The supernatant was autoclaved to prevent and / or minimize the formation of melanoidins during acid precipitation. Acid precipitation was performed by adding HCl to a final pH of 1.5 and incubating the sample at room temperature for several hours to a week. The sample was centrifuged again and the pellet was retained. The pellet was washed first with water, then with EtOH and dried.The extracted materials were characterized as described in Guo (2014) "Preparation of Water-Soluble Melanin from Squid Ink Using Ultrasound-Assisted Degradation and Its Anti-Oxidant Activity," J. Food Sci. Technol. 51(12):3680-90, and Fava (1993) "Characterization of a Pigment Produced by Pseudomonas Fluorescens during 3-Chlorobenzoate Co-Metabolism," Chemosphere 27(5):825-35, each of which is incorporated herein by reference. Table 7 below indicates which Guo or Fava method was used for each analysis. These references also describe the expected results for the characterization of eumelanin.

[0298] The extracted samples were dispensed into several individual tubes, and then analytical reagents were added to each tube to test certain properties (e.g., solubility, reactivity) as noted in Table 7. Solubility was determined as no coloring of the solvent and all of the sample settling ("insoluble"), some coloring of the solvent and some sample settling ("partially soluble"), or coloring of the solvent and no sample settling even after high speed centrifugation ("soluble"). Further confirmation of the insolubility result was performed by confirming that there was little or no change in the dry mass of the sample after recovery from the solvent. Stability was determined by subjecting the samples to various temperatures (50°C for 12 hours, 80°C for 12 hours, 100°C for 1 hour) and testing for changes in properties measured by physicochemical tests. No or minimal changes between the tests of the heated and original samples indicate that the sample is stable.

[0299] During the experiment, physicochemical data was collected on the pigment extracted from sample MEL0152, which has a black color and was grown from a sample (S4387) derived from BE02. The physicochemical properties of synthetic eumelanin (Sigma Aldrich) were used as a reference control for comparison. The data are provided in Table 7 (extracted pigments) and Table 8 (melanin reference). The data collected during the experiment indicated that the extracted pigment was melanin.

[0300] [Table 8]

[0301] [Table 9]

[0302] As shown by the data presented in Tables 7 and 8, sample MEL0152 from consortium S4387 originating from BW02 produces compounds with the expected physicochemical properties of melanin compounds reported in the literature. Notably, the solubility, precipitation, thermal stability, and redox behavior for the extracted compounds are consistent with previously reported data describing melanins in Guo and Fava, supra.

[0303] Previous publications have described the physicochemical properties of eumelanins. Some publications (Fava and Guo) disagree slightly with each other on certain properties of the melanins (e.g., partially soluble vs. fully soluble), but synthetic eumelanin from Sigma Aldrich (St. Louis, MO) was also tested (Table 8), and the results were not entirely consistent with the previously reported properties of natural melanins. Synthetic eumelanins have been found to have a lower molecular weight than those isolated from bacteria, and without being bound by theory, it is hypothesized that this difference is the reason for some of the different properties, such as increased solubility in DMSO and reactivity with HO (see, e.g., Lorquin (2022) "New insights and advances on pyomelanin production: from microbial synthesis to applications" Journal of Industrial Microbiology and Biotechnology 49(4):kuac013; Lorquin (2021) "Production and properties of non-cytotoxic pyomelanin by laccase and comparison to bacterial and synthetic pigments" Scientific Reports 11(1):8538, each of which is incorporated herein by reference).

[0304] [Example 16] During the development of embodiments of the technology described herein, experiments were performed to characterize the composition of the consortium grown in culture. Cultures of BW02 were grown as described above. Aggregates from the cultures were air-dried and resuspended in NaOH (1N). Dilutions were prepared in NaOH (1N) using the resuspended aggregates at ratios of 1:1, 1:4, 1:9, and 1:19 of resuspended aggregate to NaOH, resulting in 1x, 2x, 5x, 10x, and 20x dilutions. Samples were pipetted into a 96-well plate (200 μL per sample in each well, with three replicates per sample tested) and absorbance spectra were acquired from 280-680 nm on a SpetraMax i3 plate reader. Absorbance spectra from 280-680 nm were also acquired for eumelanin standards at approximately 0.02%, 0.01%, and 0.005% wt / vol. The absorbance spectrum of aggregates prepared from cultures of BW02 was comparable to that of the melanin standard (Figure 19).

[0305] [Example 17] During the development of embodiments of the technology described herein, experiments were performed to evaluate the growth morphology of minimal consortia following supplementation of the growth medium with tyrosine.

[0306] A BW02 culture (S1906) was grown to stationary phase (grown in tissue culture flasks for 4 weeks in 30 mL of medium) and a 1:9 dilution of the stationary phase culture was used to inoculate fresh minimal growth medium. Three 1 mL samples were taken from the inoculated fresh minimal growth medium and the 1 mL samples were centrifuged to pellet the cells. After removing the supernatant, the cell pellet was dried and the dried cell biomass was weighed. The mass of the dry biomass was used to calculate the inoculum density as 0.0012 g (dry biomass) / 0.5 mL culture.

[0307] Minimal solid medium was prepared and 2.5 mL was added to each well of a 12-well tissue culture plate. The wells were supplemented with 0.55 mM, 0.62 mM, 0.72 mM, 0.77 mM, 0.94 mM, 1.10 mM, 1.38 mM, 1.82 mM, 2.76 mM, and 5.52 mM L-tyrosine. Wells without tyrosine were also prepared as controls (0 mM). See FIG. 20. A volume of 0.5 mL of inoculated fresh minimal growth medium was added to each well of a 12-well tissue culture plate. The culture plates were grown under continuous light for 14 days and images were taken daily. As shown in FIG. 20, increasing concentrations of tyrosine supplementation increased pigment formation in both the cultures and the surrounding solid medium for BW02(S1906).

[0308] [Example 18] During development of embodiments of the technology provided herein, experiments were performed to evaluate pigment production by cultures of BW02 and BW05 grown in minimal medium supplemented with tyrosine. Cultures of BW02 and BW05 were grown in 250 ml cell culture flasks containing 30 ml of medium and inoculated with 1 ml of culture grown to saturation for 4 weeks. Cultures were supplemented with 2.5 mM tyrosine or left untreated. Figure 21. S1905 and S1909 have a brown morphology and are derived from BW02. S1918 has a green morphology and is derived from BW05.

[0309] After growing S1905 and S1909 (BW02), and S1918 (BW05) in liquid medium for 2 weeks, the cultures were centrifuged and 200 μL of each supernatant was added to a well of a 96-well plate and read on a microplate reader for absorbance from 275 to 525 nm. The absorbance spectra of the supernatants from cultures of BW02 (S1905, S1909) grown without supplementation with tyrosine, BW02 grown with supplementation with tyrosine, BW05 (S1918) grown without supplementation with tyrosine, BW05 grown with supplementation with tyrosine, and the melanin controls (0.0350% w / v and 0.0063% w / v) are shown in Figure 22. Data collected during these experiments (Figures 21, 22) showed that the addition of L-tyrosine to the culture medium increases the absorption of culture supernatants produced by BW02 cultures grown in supplemented medium compared to the supernatants produced by BW02 cultures grown in medium not supplemented with tyrosine and cultures of BW05 grown with or without supplementation with tyrosine. The absorption spectra are consistent with the melanin reference standard control. Thus, these data demonstrated that the addition of tyrosine to the culture medium in which BW02 (e.g., S1905 or S1909) is grown increases the passage of material through the melanin biosynthetic pathway, thereby increasing the production of at least one of the three downstream melanin products.

[0310] [Example 19] During the development of embodiments of the technology provided herein, experiments were performed to evaluate pigment production by cultures of BW02 (S1905, S1909) and BW05 (S1918) grown in minimal medium supplemented with homogentisic acid. Specifically, minimal consortia produced from S1905 and S1909 (having a brown morphology) and minimal consortia produced from S1918 (having a green morphology) were grown in cell culture flasks in 30 mL of medium for 4 weeks to stationary phase. 1 mL volume of each stationary phase culture was used to inoculate three replicates of minimal mineral medium (control) or three replicates of the same medium supplemented with 2.77 mM homogentisic acid (HGA). After 15 hours of growth, strong pigment formation was clearly present in the samples containing the medium supplemented with HGA.

[0311] A volume of 200 μL of each sample (three replicates as above) was collected and each volume was added to a well of a 96-well plate (FIG. 23). After taking a photograph of the plate, the supernatants were assayed for absorbance from 230-730 nm on a SpectraMax i3 microplate reader (FIG. 24). The collected data showed that consortia 1905 and 1909 showed significant pigmentation following HGA supplementation, while consortia 1918 did not show enhanced pigmentation in the medium following the addition of HGA. The measured absorbance of each of the supernatants prepared from cultures of MC1905 and MC1909 treated with HGA (brown morphology) is greater than the baseline absorbance observed in the respective controls. FIG. 24. The absorbance of the supernatant prepared from cultures of MC1918 (green morphology) is less than the baseline absorbance observed in the respective controls. FIG. 24. Without being bound by theory, it is hypothesized that this is due to uptake of HGA by the cells, which are incapable of converting it to pyomelanin. The pigmentation in the medium blank supplemented with HGA is likely due to the known ability of HGA to self-polymerize in the presence of MnCl2-4H2O (present in the medium). Collectively, these data indicate that the BW02 consortium produces melanin.

[0312] HGA is an upstream intermediate in the synthesis of pyomelanin from L-tyrosine, but not for the production of other melanins, i.e., eumelanin and pheomelanin. See Figure 18B. Visual interpretation of the well colors strongly suggested that the pigmented material observed in the experiments supplemented with tyrosine and HGA, as well as in normal (unsupplemented) culture conditions, was pyomelanin. Absorption spectra showed that the addition of HGA to the medium increased the absorption between 400 and 500 nm, which is within the absorption range of melanins (e.g., eumelanin).

[0313] [Example 20] During development of embodiments of the technology described herein, sequencing and analysis of 16S, 23S, and 5S ribosomal RNA gene sequences were performed to identify members of the community. Multiple minimal communities (e.g., consisting of about 2-10 different types of life forms) were isolated from each of the BW01, BW02, and BW05 communities (e.g., consisting of approximately several hundred different types of life forms). Minimal communities were created by streaking each complete community on an agar medium and isolating colonies. Each colony is a minimal community, and is presumed to require multiple members that each contribute to a critical function of the community, such as nitrogen fixation, carbon fixation, or shuttling metabolites between the nitrogen fixation and / or carbon fixation community organisms.

[0314] Sequencing libraries were generated from each minimal community, and nucleotide sequences were generated from the libraries using shotgun metagenomic sequencing. For quality trimming and assessment, fastp, bowtie2, and FASTQC were used. See, for example, Chen (2018) "fastp: an ultra-fast all-in-one FASTQ preprocessor" Bioinformatics 34:i884-i890, Langmead (2012) "Fast gapped-read alignment with Bowtie 2" Nature Methods 9:357-59, and www.bioinformatics.babraham.ac.uk / projects / fastqc / . For assembly, metaSPADES (default options) and MEGAHIT2 (default options) were used, and for assembly assessment, QUAST was used. See, for example, Bankevich (2012) "SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing," J Comput Biol 19: 455-77; Nurk (2017) "metaSPAdes: a new versatile metagenomic assembler," Genome Res 27: 824-34; Li (2015) "MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph," Bioinformatics 31: 1674-1676; and Gurevich (2013) "QUAST: quality assessment tool for genome assemblies," Bioinformatics 29: 1072-75, each of which is incorporated herein by reference. For binning, METABAT2 was used (default option).See, e.g., Kang (2019) "MetaBAT 2: an adaptive binning algorithm for robust and efficient genome reconstruction from metagenome assemblies," PeerJ 2019;7:e7359, incorporated herein by reference. 16S ribosomal RNA gene sequences were identified by shotgun sequencing using Barrnap (default options), and 16S ribosomal RNA sequences were taxonomically identified using average nucleotide identity (ANI) provided by the GTDB-tk package (Gtdbtk classify_wf and ani_rep using default options). See, e.g., Chaumeil (2019) "GTDB-Tk: A toolkit to classify genomes with the Genome Taxonomy Database," Bioinformatics, btz 848; Parks (2019) "A complete domain-to-species taxonomy for Bacteria and Archaea," Nat Biotechnol. 38:1079-86; and Parks (2018) "A standardized bacterial taxonomy based on genome phylogeny substantially revises the tree of life," Nat. Biotechnol 36:996-1004, each of which is incorporated herein by reference. The results of the 16S rRNA gene ANI analysis are provided in the columns labeled "Organism-ANI" in Tables 12-14.

[0315] Organisms were also identified using 16S rRNA gene sequences by searching the complete NCBI nucleotide sequence database using BLAST. Annotation data in the NCBI complete nucleotide sequence database associated with the matched sequences was used for further taxonomic identification of the 16S ribosomal RNA sequences produced from the minimal community. Tables 12-14 provide the top BLAST hits with greater than 97% identity and greater than 97% query coverage for each 16S rRNA gene nucleotide sequence in the column labeled "Organism-BLAST". All BLAST hits with greater than 97% identity and greater than 97% query coverage for each 16S rRNA gene sequence are provided in a supplementary table entitled "TABLES 12-14 SUPPLEMENT.txt" submitted with this patent application. The sequences and BLAST results in the supplementary tables are labeled as follows: ">nnnn BW## S#### L#### pppp". where nnnn is the sequence number of the 16S rRNA gene nucleotide sequence used as a query against the NCBI database, BW## and S#### refer to the complete consortium from which the minimal consortium was generated, L#### indicates the sequencing library and minimal consortium number, and pppp is the item id.

[0316] Samples, minimal communities, and sequencing libraries were generated and are named ...

Claims

1. A method for producing a microbial community that accumulates nitrogen in an agricultural culture medium, wherein the method is (a) the step of obtaining an input sample produced from an environmental sample containing multiple microorganisms; and (b) Starting with the input sample, (i) A process of inoculating the input sample into a growth medium that does not contain fixed carbon and fixed nitrogen to obtain a culture; (ii) A process of incubating the culture in the presence of nitrogen gas (N₂) and carbon dioxide (CO₂) gas and under irradiation by a light source to obtain an incubated culture; and (iii) A process for measuring the phenotype of the incubated culture; A method comprising the step of proceeding to produce a microbial community using an iterative process including, wherein a portion of the incubated culture serves as the input sample for the next iteration of the iterative process until the phenotype stabilizes, The microbial community comprises multiple living organisms, including cyanobacteria, and the abundance of cyanobacteria is greater than the abundance of any other organism in the microbial community, The aforementioned microbial community fixes nitrogen, a method.

2. The method according to claim 1, wherein the amount of cyanobacterium present is at least 30%.

3. The method according to claim 1, wherein the microbial community includes microorganisms belonging to the phylum Proteobacteria.

4. The method according to claim 1, wherein the microbial community comprises microorganisms belonging to the orders Cyanobacteriales, Cyanobacteriota, Nostocales, Pseudoanabaenales, Spirulinales, or Oscillatoriales.

5. The method according to claim 1, wherein the microbial community comprises microorganisms belonging to the genera Variovorax, Caulobacter, and / or Pseudomonas.

6. The microbial community comprises Acaryochloris, Aliinostoc, Aliterella, Amazonocrinis, Anabaena, Anabaenopsis, Aphanizomenon, Atlanticosrix, Aulosira, Calenema, Calothrix, and Camptilonemo. Camptylonemopsis, Chlorogloeopsis, Chroococcidiopsis, Chrysosporum, Compactonostoc, Constrictiphyllum, Crocosphera, Cyanobacterium, Cyanocohnierla, Cylindrospermopsis Cylindrospermopsis, Cylindrospermum, Dendronalium, Desikacharya, Desmonostoc, Dolicospermum, Erythrobacter, Fischerella, Fortia, Fremyella, Geitlerinema, Glo Gloeocapsa, Gloeocapsopsis, Gloeothece, Gloeotricia, Goleter, Hapalosiphon, Hydrocoryne, Jaaginema, Johansenierla, Camptonema, Komarekiella, Leptolingbya,Lyngbya, Macrochaete, Mastigocoleus, Microchaete, Nodosilinea, Nodularia, Nostoc, Nostocaseae, Nostochopsis, Oligotropha, Ossilatoria Oscillatoria, Pantanalinema, Pelatocladus, Planktothrichoides, Planktothrix, Polymorphum, Porphyrobacter, Prochlorococcus, Prochlorothrix (P Rochlorothrix, Pseudanabaena, Pseudoalinostoc, Raffidiopsis, Richelia, Rivularia, Roholtierla, Rubidibacter, Scytonema, Spirulina The method according to claim 1, comprising microorganisms belonging to the genus Synechococcus, Synechocystis, Thermosynechococcus, Tolypothrix, Trichormus, Violetonostoc, Wollea, or Xenococcus.

7. The method according to claim 1, wherein the microbial community has a ribosomal RNA gene nucleotide sequence that is at least 90% identical to SEQ ID NOs: 1, 3, 7, 10, 13, 19, 22, 26, 30, 32, 34, 35, 39, 42, 45, 51, 54, 57, 61, 63, 66, 70, 74, 84, 89, 99, 103, 108, 116, 122, 123, 125, or 127.

8. The method according to claim 1, wherein the microbial community sequesters carbon.

9. The method according to claim 1, wherein the microbial community comprises one microorganism that sequesters carbon.

10. The method according to claim 9, wherein the microbial community comprises two or more microorganisms that sequester carbon.

11. The method according to claim 1, wherein the microbial community includes microorganisms capable of metabolizing carbon-containing compounds produced by the cyanobacterium.

12. The method according to claim 1, wherein the microbial community includes one photosynthetic microorganism.

13. The method according to claim 12, wherein the microbial community comprises two or more photosynthetic microorganisms.

14. The method according to claim 12, wherein the microbial community includes photosynthetic microorganisms or photosynthetic algae.

15. The microbial community includes Nodularia, Chrysosporum, Gloeocappossis, Liqueria, Masticocoleus, Haparosiphon, Gloeotese, Acariochloris, Camptonema, Rhaphidiopsis, Crocosfera, Macrocaete, Thermosynechococcus, Pseudoanabaena, Chloococcidiopsis, Prochlorothrix, Anabaena, Leptoringbia, Kalothrix, Cylindrospermopsis, Dolicospermum, Cytonema, Ringbia, Tryposrix, Fischerella, Fortiera, Aliterella, Hydrocoryne, Prochlorothrix The method according to claim 1, wherein the member is a member of the genus Rolococcus, Planctotrichoides, Gaitrelinema, Xenococcus, Yarginema, Nostochopsis, Pantanalinema, Osillatoria, Spirulina, Peratocladas, Nodosilinea, Aphanizomenon, Chlorogloeopsis, Gloeocapsa, Karenema, Livularia, Trichormus, Synechococcus, Synechocystis, Cylindrospermum, Planctosricus, Bosea, Cinera, Novosphingobium, or Rubizibacter.

16. The method according to claim 14, wherein the photosynthetic alga is a member of the genus Tetraselmis, Chlorella, Coleocaete, Gonium, Paradoxia, Symbomonas, Palmaria, Characiochloris, Marvania, Chlamydomonas, Parachlorella, Coccomyxa, Trebuxia, Cyanidium, Troybarria, Danjardinia, Gardieria, Nuformiera, Porphyra, Cara, Anquistrodesmus, Picochlorum, Cyanophora, Condorus, Pseudochloris, Interfilm, Gerridium, Symphyogina, Chlorosarquina, or Cyanothece.

17. The method according to claim 1, wherein the microbial community comprises one nitrogen-fixing microorganism.

18. The method according to claim 1, wherein the light source provides light having a wavelength of about 380 nm to 750 nm.

19. The method according to claim 17, wherein the microbial community comprises two or more nitrogen-fixing microorganisms.

20. The method according to claim 1, wherein the cyanobacterium is a nostoc species.

21. The method according to claim 1, wherein the microbial community comprises alphaproteobacterium or gammaproteobacterium.

22. The method according to claim 1, wherein the phenotype is membership in a microbial community and / or abundance of a microbial community.

23. The method according to claim 1, wherein the phenotype is a functional phenotype.

24. The method according to claim 23, wherein the functional phenotype includes nitrogen fixation.

25. The method according to claim 24, wherein the functional phenotype further comprises carbon sequestration and / or phosphate mobilization.

Images