Microbial consortia for soil improvement

Abstract

Provided herein are techniques relating to functional microbial consortia, and in particular, but not exclusively, to methods and systems for producing microbial consortia having a desired function and microbial consortia produced according to such methods. The microbial consortia can be used to improve soil, for example, for agricultural use.

Description

[0001] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under P30CA91842 and UL1TR002345 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0003] Sequence Listing

[0004] Attached is the text of the computer readable sequence listing filed, entitled "PLUTON-40093-601_SQL," created on March 20, 2023, with a file size of 300,854 bytes, which is hereby incorporated by reference in its entirety. Technical Field

[0005] Provided herein are technologies related to functional microbial consortia, and particularly, but not exclusively, relate to methods and systems for producing microbial consortia having desired functions and microbial consortia produced according to such methods. Background Art

[0006] Agricultural practice generally includes methods for improving soil to maintain or increase crop yield. Specifically, plant nutrients (e.g., carbon (C), nitrogen (N), phosphorus (P) and other nutrients) are generally provided to the soil in the form of chemical or biological soil conditioners. Chemical conditioners include fertilizers containing ammonia that provides fixed nitrogen and phosphates that provide phosphorus for excavation. In addition, farmers generally plant cover crops to return nitrogen, carbon and other nutrients to the soil. When growing, the microbial symbionts of cover crops fix the nitrogen released into the soil, and the termination and cultivation of cover crops return organic carbon and other nutrients to the soil. Higher soil carbon fixation is associated with better nutrient and moisture retention, which generally makes the soil healthier and increases crop yield. Since agriculture has emerged and is increasing, soil carbon fixation worldwide has been increasingly released into the atmosphere because cultivation has destroyed the soil and promoted the decomposition of carbonaceous materials by microorganisms. Returning carbon to the soil is beneficial to soil health and can remove greenhouse gases in the atmosphere.

[0007] Regarding nitrogen, approximately 1 / 3 of fixed nitrogen for agricultural use is produced through biological nitrogen fixation (BNF) using cover crops or crop rotations with legumes, and the remaining 2 / 3 is applied in the chemical form of ammonia produced by the Haber-Bosch process. 2 H mainly comes from methane in natural gas 2 The reaction generates NH 3(ammonia). Although the Haber-Bosch process has been successful in fixing nitrogen for agricultural use, it consumes up to 5% of the natural gas produced worldwide each year as a source of hydrogen and energy. The Haber-Bosch process is therefore a major contributor to the greenhouse gas CO 2 In addition, inorganic N applied to soils (such as ammonia or ammonium nitrate) is easily converted to nitrogen oxides (NOx) in a process called denitrification. Nitrogen oxides, especially N 2 O, as a greenhouse gas, is CO 2 Hundreds of times more nitrogen than fertilizers. Therefore, chemical fertilizers cause a lot of nitrogen waste, and their production is accompanied by the generation of a large amount of greenhouse gases and climate impacts. Plants provide nitrogen to the soil through biological nitrogen fixation, which is carried out by specific microorganisms associated with specific plants. However, planting certain types of plants is not all possible or feasible for all fields, soil types and locations. For example, soybean plants use nodules colonized by microorganisms to fix a large amount of nitrogen, while corn has a much more limited interaction with rhizosphere diazotrophs (nitrogen-fixing microorganisms) and requires a lot of fertilization to grow economically. Summary of the invention

[0008] Thus, in some embodiments, the technology provided herein involves the use of microbial consortia to fix carbon and nitrogen and deliver the carbon and nitrogen to the soil (or other plant growth medium) along with phosphorus and other essential nutrients. In some embodiments, the technology provides nutrients to the soil with a much lower greenhouse gas impact than other current agricultural practices.

[0009] In some embodiments, the technology provides a microbial soil amendment (e.g., including a microbial consortium) that delivers carbon (C) and nitrogen (N) to a plant's growth medium (e.g., soil or a synthetic growth medium). In some embodiments, the microbial soil amendment further delivers other nutrients (e.g., phosphorus) to a plant's growth medium (e.g., soil or a synthetic growth medium).

[0010] Specifically, the technology described herein provides biofertilizers including microbial consortia, and methods for producing the biofertilizers and / or microbial consortia, which are optimized for effectively delivering one or more of N, C, P and / or other essential nutrients to the soil. In some embodiments, the technology provides microbial consortia grown in bioreactors and applied to fields. In some embodiments, the microbial consortia provide a source of, for example, one or more of fixed nitrogen, organic carbon, phosphorus and other nutrients. In some embodiments, the microbial consortia continue to grow after being applied to the soil, and provide carbon and nitrogen to the soil after being applied to the soil. Therefore, in some embodiments, the microbial consortia remove carbon and / or nitrogen from the air through photosynthesis-driven fixation. Therefore, embodiments of the technology provide an effective source of biological nitrogen fixation, which can replace the production of CO 2 In addition, embodiments of the technology remove carbon from the air and deposit it in the soil, thereby improving soil quality while reducing atmospheric greenhouse gases. In some embodiments, the technology provides a bioavailable source of phosphorus and other nutrients in the soil for plant growth.

[0011] In some embodiments, the technology provides a microbial consortium providing a specific function. In some embodiments, technology as herein described includes providing a sample comprising many microorganisms (for example, from one or more environmental samples) and / or microbial consortium (for example, comprising one or more microorganisms from natural consortium and / or from one or more microorganisms of different environments, ecosystems, habitats and / or niches), and produces a new consortium comprising the new combination of synergistic microorganisms. By testing functional variables, the microorganism and microbial consortium providing the desired function can then be sequenced, and cultured from succession until identifying and / or separating the desired microorganism and microbial consortium. In some embodiments, the data of statistically significant amounts are used to develop a machine learning model to help select microorganisms and microbial consortiums. In some embodiments, the machine learning model is supplemented with phenotypic data for forming microorganisms.

[0012] As described herein, embodiments of the technology (e.g., methods of generating a microbial consortium) include evaluating or confirming the functionality of a culture and / or consortium (e.g., by evaluating a variable in a test) rather than focusing on the phenotype of the microorganisms. In some embodiments, functionality can include measurements of carbon sequestration, nitrogen fixation, survival time, and / or microbial persistence.

[0013] Thus, the technology provides a method for accumulating carbon and nitrogen in an agricultural medium. For example, in some embodiments, the method comprises applying a composition comprising photosynthetic nitrogen-fixing microorganisms to an agricultural medium; and 2 ) and carbon dioxide (CO2) and incubate the agricultural medium under the irradiation of a light source. In some embodiments, the agricultural medium includes soil, a greenhouse growth medium or a hydroponic growth medium. In some embodiments, the photosynthetic nitrogen-fixing microorganism is a bacterium. In some embodiments, the photosynthetic nitrogen-fixing microorganism is a member of the cyanobacteria. In some embodiments, the photosynthetic nitrogen-fixing microorganism is a member of the cyanobacteria. In some embodiments, the photosynthetic nitrogen-fixing microorganism is a member of the cyanobacteria. In some embodiments, the photosynthetic nitrogen-fixing microorganism is a member of the genus Acaryochloris, Aliinostoc, Aliterella, Amazonocrinis, Anabaena, Anabaenopsis, Aphanizomenon, Atlanticothrix, Aulosira, Calenema, Calothrix, Camptylonemopsis, Chlorogloeopsis, Chroococcidiopsis, Chrysosporum, Compactonostoc, Constrictifilum, Crocosphaera, Cyanobacterium, Cyano cohniella, Cylindrospermopsis, Cylindrospermum, Dendronalium, Desikacharya, Desmonostoc, Dolichospermum, Erythrobacter, Fischerella, Fortiea, Fremyella, Geitlerinema, Gloeocapsa, Gloeocapsopsis, Gloeothece, Gloeotrichia, Goleter, Hapalosiphon, Hydrocoryne, Jaaginema, Johanseniella, Kamptonema,Komarekiella, Leptolyngbya, Lyngbya, Macrochaete, Mastigocoleus, Microchaete, Nodosilinea, Nodularia, Nostoc, Nostocaceae, Nostochopsis, Oligotropha, Oscillatoria, Pantanalinema, Pelatocladus, Planktothrichoides, Planktothrix, Polymorphum, Porphyrobacter, Prochlorococcus In some embodiments, the photosynthetic nitrogen-fixing microorganism is a species of the genus Nostoc. In some embodiments, the photosynthetic 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 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.

[0014] In some embodiments, the agricultural medium and the composition together include a first microbial biomass; and the incubation is performed until a second microbial biomass is produced that is at least twice as large as the first microbial biomass. In some embodiments, the agricultural medium includes a first microbial biomass; and the incubation is performed until a second microbial biomass is produced that is at least twice as large as the first microbial biomass.

[0015] In some embodiments, the composition further comprises microorganisms capable of metabolizing carbon-containing and nitrogen-containing compounds produced by photosynthetic nitrogen-fixing microorganisms. In some embodiments, the composition comprises a consortium comprising the photosynthetic nitrogen-fixing microorganisms. In some embodiments, the composition comprises a consortium comprising the photosynthetic nitrogen-fixing microorganisms; and a first microorganism capable of metabolizing carbon-containing compounds produced by photosynthetic nitrogen-fixing microorganisms and / or capable of metabolizing nitrogen-containing compounds produced by photosynthetic nitrogen-fixing microorganisms. In some embodiments, the consortium comprises a second microorganism capable of metabolizing carbon-containing compounds produced by the photosynthetic nitrogen-fixing microorganisms and / or capable of metabolizing nitrogen-containing compounds produced by the photosynthetic nitrogen-fixing microorganisms. In some embodiments, the consortium comprises a second microorganism capable of metabolizing carbon-containing compounds produced by the first microorganism and / or capable of metabolizing nitrogen-containing compounds produced by the first microorganism. In some embodiments, the consortium comprises a second microorganism, and the first microorganism is capable of metabolizing carbon-containing compounds produced by the second microorganism and / or capable of metabolizing nitrogen-containing compounds produced by the second microorganism. In some embodiments, the consortium produces durable carbon compounds that provide carbon sinks in soil. In some embodiments, the consortium produces melanin. In some embodiments, the consortium produces pheomelanin, eumelanin, and / or pyomelanin.

[0016] 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 a bacterium that is a member of the genus Nodularia, Chrysosporium, Globularia, Phytophyte, Trichoderma, Hose Algae, Glue Algae, Acaryochloris, Kamptonema, Acrocephalum, Crocodile Algae, Macrochaete, Thermosynechococcus, Pseudo-Anabaena, Pseudo-Chromococcus, Prochlorococcus, Anabaena, Sclerotium, Browsing Algae, Pseudo-Cylindrospermum, Longisporium, Pseudo-Cladophora, Sclerotium, Monophyllium, Flytella, Voltiella, Aliterella , Aquatica, Prochlorococcus, Pseudohygrophila, Ghesina, Heterococcus, Jasmine, Pseudohygrophila, Pantanalinema, Oscillatoria, Spirulina, Pelatocladus, Knotweed, Aphanizomenon, Pseudohygrophila, Myxococcus, Calenema, Glechoma, Trichophyton, Synechococcus, Synechocystis, Cylindrosporium, Pseudohygrophila, Bosea, Shinella, Novosphingobium, or Rubidibacter.

[0017] In some embodiments, the algae is a member of the genus Tetraselmis, Chlorella, Coleochaete, Gonium, Paradoxia, Cymbomonas, Palmaria, Characiochloris, Marvania, Chlamydomonas, Parachlorella, Coccomyxa, Trebouxia, Cyanidium, Tetraselmis, Cymbomonas, Palmaria, Characiochloris, Marvania, Chlamydomonas, Parachlorella, Coccomyxa, Trebouxia, Cyanidium, In some embodiments, the consortium includes a second nitrogen fixing microorganism.

[0018] In some embodiments, the consortium further comprises an organism from the genus Variovorax, an organism from the phylum Proteobacteria, an organism from the genus Bosella, an organism from the genus Caulobacter, and / or an organism from the genus Pseudomonas.

[0019] In some embodiments, the light source provides light having a wavelength of from about 380 nm to 750 nm. In some embodiments, the light source is an artificial light source.

[0020] In some embodiments, the technology also provides a method for accumulating carbon and nitrogen in an agricultural medium, the method comprising applying a composition comprising photosynthetic microorganisms and nitrogen-fixing microorganisms to the agricultural medium; and 2 ) and carbon dioxide (CO 2 ) and incubate the agricultural medium under the irradiation of a light source. In some embodiments, the agricultural medium comprises soil, a greenhouse growth medium, or a 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.

[0021] In some embodiments, the agricultural medium and the composition together include a first microbial biomass; and the incubation is performed until a second microbial biomass is produced that is at least twice as large as the first microbial biomass. In some embodiments, the agricultural medium includes a first microbial biomass; and the incubation is performed until a second microbial biomass is produced that is at least twice as large as the first microbial biomass. In some embodiments, the algae is a member of the genus: Tetradinium, Chlorella, Chaetocystis, Disk, Paradoxia, Cymbomonas, Palmitomorpha, Characiochloris, Marvania, Chlamydomonas, Pseudochlorella, Gumballium, Coccochloris, Green Algae, Tetraspinosa, Dangeardinia, Galdiella, Nyholmiella, Porphyra, Chara, Fiber Algae, Chlorella, Cyanobacteria, Chondrus, Pseudochloris, Interfilum, Gelidium, Symphyogyna, Backpack Algae, or Blue Rod Algae.

[0022] In some embodiments, the bacterium is a member of the genus Nodularia, Chrysosporium, Globularia, Phytophyte, Trichoderma, Hose, Myxoplasma, Acaryochloris, Kamptonema, Acrocephalum, Crocodile, Macrochaete, Thermosynechococcus, Pseudo-Anabaena, Pseudo-Chromococcus, Prochlorococcus, Anabaena, Scleroderma, Browworm, Pseudo-Cylindrospermum, Longisporium, Pseudo-Cladophora, Scleroderma, Monodimorpha, Flytella, and Voltiella. , Aliterella, Aquatica, Prochlorococcus, Pseudohygrophila, Ghesina, Heterococcus, Jasmine, Pseudohygrophila, Pantanalinema, Oscillatoria, Spirulina, Pelatocladus, Knotweed, Aphanizomenon, Pseudohygrophila, Myxococcus, Calenema, Glechoma, Trichophyton, Synechococcus, Synechocystis, Cylindrosporium, Pseudohygrophila, Bosella, Sheinella, Neosphingobacterium, or Rubidibacter.

[0023] In some embodiments, the bacterium is a member of the genus: Glechoma, Amazonocrinis, Anabaena, Anabaena, Atlanticothrix, Hypophthalmium ...

[0024] In some embodiments, the bacterium is a member of the genus Acaryochloris, Aliterella, Amazonocrinis, Anabaena, Anabaena, Aphanizomenon, Atlanticothrix, Calenema, Calenema, Pseudo-Cyanocyanobacterium, Pseudo-Chromococcus, Chrysosporium, Compactonostoc, Constrictifilum, Crocodile Glomerella, Cyanobacteria, Cyanocohniella, Pseudo-Cylindrosporium, Cylindrosporium, Dendronalium, Desikacharya, Desmonostoc, Longisporium, Flytella, Voltiella, Fremyella, Ghesinia, Glucococcoma, Globose ... Gelatochaete, Goleter, Hose, Water, Jasmine, Johanseniella, Kamptonema, Komarekiella, Sclerotium, Sclerotium, Macrochaete, Flagellate, Microchaete, Knot, Nodularia, Nodulariaceae, Nodularia, Oscillatoria, Pantanalinema, Pelatocladus, Pseudo-Planatum, Planatum, Prochlorococcus, Prochlorococcus, Pseudo-Anabaena, Pseudo-Planatum, Acrocephalum, Phytophyta, Gelatochaete, Roholtiella, Rubidibacter, Pseudo-branched algae, Spirulina, Synechococcus, Synechocystis, Thermosynechococcus, Monochamus, Trichophyton, Violetonostoc, Lumenella or Heterococcus. In some embodiments, the bacteria has a ribosomal RNA gene nucleotide sequence that is at least 90%, 95%, 97% or 99% identical to at least one of 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.

[0025] In some embodiments, the composition further comprises the following microorganisms: capable of metabolizing carbon-containing and nitrogen-containing compounds produced by photosynthetic microorganisms and by 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; and a first microorganism capable of metabolizing carbon-containing compounds produced by the photosynthetic microorganisms and the nitrogen-fixing microorganisms and / or capable of metabolizing nitrogen-containing compounds produced by the photosynthetic microorganisms and by the nitrogen-fixing microorganisms. In some embodiments, the consortium comprises a second microorganism capable of metabolizing carbon-containing compounds produced by the photosynthetic microorganisms and by the nitrogen-fixing microorganisms and / or capable of metabolizing nitrogen-containing compounds produced by the photosynthetic microorganisms and by the nitrogen-fixing microorganisms. In some embodiments, the consortium comprises a second microorganism capable of metabolizing carbon-containing compounds produced by the first microorganism and / or capable of metabolizing nitrogen-containing compounds produced by the first microorganism. In some embodiments, the consortium comprises a second microorganism, and the first microorganism is capable of metabolizing carbon-containing compounds produced by the second microorganism and / or capable of metabolizing nitrogen-containing compounds produced by the second microorganism. In some embodiments, the consortium produces durable 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.

[0026] In some embodiments, the consortium further comprises an organism from the genus Vulgaris, an organism from the phylum Proteobacteria, an organism from the genus Bosella, an organism from the genus Caulobacter, and / or an organism from the genus Pseudomonas.

[0027] In some embodiments, the light source provides light having a wavelength of from about 380 nm to 750 nm. In some embodiments, the light source is an artificial light source. In some embodiments, the light source is the sun.

[0028] In addition, in some embodiments, the technology provides a method of producing a microbial consortium, the method comprising providing a water-based, non-fixed carbon and non-fixed nitrogen culture medium; inoculating the culture medium with a sample comprising microorganisms to produce an inoculated culture medium; and exposing the inoculated culture medium to a medium comprising N 2 and CO 2 In some embodiments, the light comprises a wavelength from about 380 nm to 750 nm. In some embodiments, the light comprises N 2 and CO 2 The gas is air. In some embodiments, the 2 and CO 2 The gas is made of purified N 2and CO 2 Provided. In some embodiments, the inoculated culture medium includes a first microbial biomass; and the exposure is performed until a second microbial biomass is produced that is at least twice as large as the first microbial biomass. In some embodiments, the consortium produces durable 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 pyomelanocin. In some embodiments, the consortium includes a microorganism having a ribosomal RNA gene nucleotide sequence that is at least 90%, 95%, 97% or 99% identical to one or more of 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.

[0029] In some embodiments, the technology provides a composition comprising a microbial consortium, the microbial consortium comprising a photosynthetic nitrogen-fixing microorganism or comprising a photosynthetic microorganism and a nitrogen-fixing microorganism; and a microorganism capable of metabolizing carbon- and nitrogen-containing compounds produced by a photosynthetic nitrogen-fixing microorganism. In some embodiments, the photosynthetic nitrogen-fixing microorganism is a bacterium. In some embodiments, the photosynthetic nitrogen-fixing microorganism is a member of Cyanobacteriales, Cyanobacteriales, Nostocales, Pseudo-Anabaenales, Spirulinales, or Oscillatorales. In some embodiments, the photosynthetic nitrogen-fixing microorganism is a member of Nostocales. In some embodiments, the photosynthetic nitrogen-fixing microorganism is a member of the genus Acaryochloris, Aliterella, Amazonocrinis, Anabaena, Anabaena, Aphanizomenon, Atlanticothrix, Calenema, Calenema, Pseudo-Cyanocyanobacterium, Pseudo-Chromococcus, Chrysosporium, Compactonostoc, Constrictifilum, Crocodile Glomerella, Cyanobacterium, Cyanocohniella, Pseudo-Cyanocyanobacterium, Cyanocyanobacterium, Dendronalium, Desikacharya, Desmonostoc, Longisporium, Erythrobacter, Flytella, Voltiella, Fremyella, Gastrosporium, Glucosporum, Globose ... genus, Goleter, Hose algae, Water stripe algae, Jasmine, Johanseniella, Kamptonema, Komarekiella, Thin sheath filament, Sheath filament, Macrochaete, Flagellate, Microchaete, Knot filament, Nodularia, Nodularia, Nodulariaceae, Nodularia, Oligospermum, Oscillatoria, Pantanalinema, Pelatocladus, Pseudofloxacin, Floatella, Polymorpha, Porphyromonas, Prochlorococcus, Prochlorococcus, Pseudoanabaena, Pseudofloxacin, Acrocephalus, Phytophyta, Gelatophyta, Roholtiella, Rubidibacter, Pseudobranchia, Spirulina, Synechococcus, Synechocystis, Thermosynechococcus, Monophydium, Trilocystis, Violetonostoc, Lumenella or Heterococcus. In some embodiments, the photosynthetic nitrogen-fixing microorganism is a Nostoc species.In some embodiments, the photosynthetic 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 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.

[0030] In some embodiments, the species that fix nitrogen is a microorganism from the following genus: Greedy Phage, Pseudomonas, Greedy Cuprum Bacteria (Cupriavidus), Brevundimonas (Brevundimonas), Fengyou Bacteria (Opitutus), Ancient Word-Shaped Bacteria (Runella), Taylor's Bacteria (Taylorella), Tahibacter, Polaromonas (Polaromonas), Chitinophaga (Chitinophaga), Ferruginibacter (Ferruginibacter), Enhydrobacter, Hymenobacter or Ramlibacter. In some embodiments, the species that photosynthesizes is a microorganism from the following genus: Bosella, Shen's Bacteria or Neosphingobacterium.

[0031] In some embodiments, the consortium produces durable 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.

[0032] In some embodiments, the composition includes a second microorganism capable of metabolizing a carbon-containing compound produced by the photosynthetic nitrogen-fixing microorganism and / or capable of metabolizing a nitrogen-containing compound produced by the photosynthetic nitrogen-fixing microorganism. In some embodiments, the consortium includes a second microorganism capable of metabolizing a carbon-containing compound produced by the first microorganism and / or capable of metabolizing a nitrogen-containing compound produced by the first microorganism. 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.

[0033] 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 are members of the genus Acaryochloris, Aliterella, Amazonocrinis, Anabaena, Anabaena, Aphanizomenon, Atlanticothrix, Calenema, Calenema, Pseudo-Cyanocyanobacterium, Pseudo-Chromosporium, Chrysosporium, Compactonostoc, Constrictifilum, Crocodile Glomerella, Cyanocohniella, Pseudo-Cyanocyanobacterium, Cyanocyanobacterium, Dendronalium, Desikacharya, Desmonostoc, Longisporium, Flying Glomerella, Voltiella, Fremyella, Gelatochloris ... eter, Hose algae, Water stripe algae, Jasmine algae, Johanseniella, Kamptonema, Komarekiella, Thin sheath filament, Sheath filament, Macrochaete, Flagellate, Microchaete, Knot filament, Nodularia, Nodulariaceae, Pseudomonas, Oscillatoria, Pantanalinema, Pelatocladus, Pseudo-flocculated filament, Flocculated filament, Prochlorococcus, Prochlorococcus, Pseudo-anabaena, Pseudo-flocculated filament, Acrocephalus, Phytophyte, Gelatophyte, Roholtiella, Rubidibacter, Pseudobranchium, Spirulina, Synechococcus, Synechocystis, Thermosynechococcus, Monophydium, Trichophyton, Violetonostoc, Lumen tuberculum, Bosella, Sheinella, Neosphingobacterium, or Heterococcus. In some embodiments, the algae is a member of the genus Tetrastigemum, Chlorella, Chaetoceros, Diskia, Paradoxia, Cymbomonas, Palmitochloris, Characiochloris, Marvania, Chlamydomonas, Pseudochlorella, Coccomycetes, Coccomycetes, Cyanochloris, Tetrastigem, Dangeardinia, Galdiella, Nyholmiella, Porphyra, Chara, Fibrous Algae, Chlorella, Cyanophytes, Chondrus, Pseudochloris, Interfilum, Gelidium, Symphyogyna, Kalamba, or Cyanophyceae.In some embodiments, the consortium includes a second nitrogen-fixing microorganism that is a member of the genus: Pseudomonas, Pseudomonas, Cupribothrium, Brevundimonas, Toyosporium, Archaeobacterium, Theileria, Tahibacter, Polarmonas, Chitinophaga, Freiherrella, Aquabacter, Lamellarium, Ramlibacter, Acaryochloris, Aliterella, Amazonocrinis, Anabaena, Anabaena, Aphanizomenon, Atlanticothrix, Sphenopsina, Calenema, Browworm, Pseudo-Cynoglossum, Pseudo-Chromosporium, Pseudo-Chromosporium, Chrysosporium, Compactonostoc, Constrictifilum, Crocodile, Cyanobacterium, Cyanocohniella, Pseudo-Cynosporium, Cynosporium, Dendronalium, Desikacharya, Desmonostoc, Long spore algae, Erythrobacterium, Flytella, Voltiella, Fremyella, Gastrospermum, Globular cyanobacteria, Gastrospermum, Gastrospermum, Goleter, Hose algae, Water strip algae, Jaspermum, Johanseniella, Kamptonema, Komarekiella, Thin sheathed filamentous algae, Sheathed filamentous algae, Macrochaete, Whip algae, Microhair algae, Knotted filamentous algae, Nodularia, Nostoc, Nostocaceae, Nodularia, Oligospermum , Oscillatoria, Pantanalinema, Pelatocladus, Pseudohygrophila, Hygrophila, Polymorpha, Porphyromonas, Prochlorococcus, Prochlorofilamentosa, Pseudoanabaena, Pseudohygrophila, Acrocephalum, Phytophyton, Gelatophyte, Roholtiella, Rubidibacter, Pseudobranchium, Spirulina, Synechococcus, Synechocystis, Thermosynechococcus, Monochamus, Trichoderma, Violetonostoc, Lumenalulus, or Heterococcus.

[0034] In some embodiments, the consortium further comprises an organism from the genus Vulgaris, an organism from the phylum Proteobacteria, an organism from the genus Bosella, an organism from the genus Caulobacter, and / or an organism from the genus Pseudomonas.

[0035] A further embodiment provides a system comprising a water-based, non-fixed carbon and non-fixed nitrogen culture medium; comprising N 2 and CO 2 a gas source; a light source; and a sample including microorganisms.

[0036] Additional embodiments of the system provide a system comprising a water-based, non-fixed carbon and non-fixed nitrogen culture medium; comprising N2 and CO 2A gas source; a light source; and a microbial consortium including photosynthetic nitrogen-fixing microorganisms or comprising photosynthetic microorganisms and nitrogen-fixing microorganisms; and a microorganism capable of metabolizing carbon- and nitrogen-containing compounds produced by photosynthetic nitrogen-fixing microorganisms. In some embodiments, the photosynthetic nitrogen-fixing microorganisms are bacteria. In some embodiments, the photosynthetic nitrogen-fixing microorganisms are members of the phylum Cyanobacteria. In some embodiments, the photosynthetic nitrogen-fixing microorganisms are members of the phylum Cyanobacteriales, the phylum Cyanobacteriales, the phylum Nostocales, the phylum Pseudo-Anabaenales, the phylum Spirulinales, or the phylum Oscillatoriales. In some embodiments, the photosynthetic nitrogen-fixing microorganisms are members of the phylum Nostocales. In some embodiments, the photosynthetic nitrogen-fixing microorganism is a member of the genus Acaryochloris, Aliterella, Amazonocrinis, Anabaena, Anabaena, Aphanizomenon, Atlanticothrix, Calenema, Calenema, Pseudo-Cyanocyanobacterium, Pseudo-Chromococcus, Chrysosporium, Compactonostoc, Constrictifilum, Crocodile Glomerella, Cyanobacterium, Cyanocohniella, Pseudo-Cyanocyanobacterium, Cyanocyanobacterium, Dendronalium, Desikacharya, Desmonostoc, Longisporium, Erythrobacter, Flytella, Voltiella, Fremyella, Gastrosporium, Glucosporum, Globose ... genus, Goleter, Hose algae, Water stripe algae, Jasmine, Johanseniella, Kamptonema, Komarekiella, Thin sheath filament, Sheath filament, Macrochaete, Flagellate, Microchaete, Knot filament, Nodularia, Nodularia, Nodulariaceae, Nodularia, Oligospermum, Oscillatoria, Pantanalinema, Pelatocladus, Pseudofloxacin, Floatella, Polymorpha, Porphyromonas, Prochlorococcus, Prochlorococcus, Pseudoanabaena, Pseudofloxacin, Acrocephalus, Phytophyta, Gelatophyta, Roholtiella, Rubidibacter, Pseudobranchia, Spirulina, Synechococcus, Synechocystis, Thermosynechococcus, Monophydium, Trilocystis, Violetonostoc, Lumenella or Heterococcus. In some embodiments, the photosynthetic nitrogen-fixing microorganism is a Nostoc species.In some embodiments, the photosynthetic 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 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.

[0037] In some embodiments, the species that fix nitrogen is a microorganism from the following genus: Greedy Phage, Pseudomonas, Greedy Cupric Bacteria, Brevundimonas, Fengyou Bacteria, Archaea, Theilerella, Tahibacter, Polar Mononas, Chitinophage, Freundii, Aquabacter, Thin Layer Bacteria or Sand Soil Bacterium. In some embodiments, the species that photosynthesize is a microorganism from the following genus: Bosella, Shen's Bacteria or Neosphingobacterium.

[0038] In some embodiments, the consortium produces durable 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.

[0039] In some embodiments, the composition includes a second microorganism capable of metabolizing a carbon-containing compound produced by the photosynthetic nitrogen-fixing microorganism and / or capable of metabolizing a nitrogen-containing compound produced by the photosynthetic nitrogen-fixing microorganism. In some embodiments, the consortium includes a second microorganism capable of metabolizing a carbon-containing compound produced by the first microorganism and / or capable of metabolizing a nitrogen-containing compound produced by the first microorganism. 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.

[0040] 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 are members of the genus Acaryochloris, Aliterella, Amazonocrinis, Anabaena, Anabaena, Aphanizomenon, Atlanticothrix, Calenema, Calenema, Pseudo-Cyanocyanobacterium, Pseudo-Chromococcus, Chrysosporium, Compactonostoc, Constrictifilum, Crocodile Glomerella, Cyanocohniella, Pseudo-Cyanocyanobacterium, Cyanocyanobacterium, Dendronalium, Desikacharya, Desmonostoc, Longisporium, Erythrobacterium, Flytella, Voltiella, Fremyella, Gaseomycetes, Globularia, Globularia, Glechoma, Glechoma, Goleter, Hose, Aqua, Jasmine, Johanseniella, Kamptonema, Komarekiella, Sclerotium, Sclerotium, Macrochaete, Flagellate, Microchaete, Knot, Nodularia, Nodulariaceae, Nodularia, Oligospermum, Oscillatoria, Pantanalinema, Pelatocladus, Pseudo-Planatum, Planatum, Polymorpha, Porphyromonas, Prochlorococcus, Prochlorococcus, Pseudo-Anabaena, Pseudo-Granite, Acrocephalus, Phytophyta, Gelatophyta, Roholtiella, Rubidibacter, Pseudobranchia, Spirulina, Synechococcus, Synechocystis, Thermosynechococcus, Monophydium, Trichophyton, Violetonostoc, Lumen, Bosella, Sheinella, Neosphingobacterium, or Heterococcus. In some embodiments, the algae is a member of the genus Tetrastigemum, Chlorella, Chaetoceros, Diskia, Paradoxia, Cymbomonas, Palmitochloris, Characiochloris, Marvania, Chlamydomonas, Pseudochlorella, Coccomycetes, Coccomycetes, Cyanochloris, Tetrastigem, Dangeardinia, Galdiella, Nyholmiella, Porphyra, Chara, Fibrous Algae, Chlorella, Cyanophytes, Chondrus, Pseudochloris, Interfilum, Gelidium, Symphyogyna, Kalpa, or Cyanophyceae.In some embodiments, the consortium includes a second nitrogen-fixing microorganism from the genus: Pseudomonas, Pseudomonas, Cupribotium, Brevundimonas, Toyosporium, Archaeobacterium, Theileria, Tahibacter, Polarmonas, Chitinophaga, Freudenbergia, Aquabacter, Thin-layered Microorganism, or Soilbacter.

[0041] In some embodiments, the consortium further comprises an organism from the genus Vulgaris, an organism from the phylum Proteobacteria, an organism from the genus Bosella, an organism from the genus Caulobacter, and / or an organism from the genus Pseudomonas.

[0042] In some embodiments, the technology provides a composition comprising a synthetic growth medium; and a microbial organism that performs photosynthesis and nitrogen fixation together. In some embodiments, the first microbial species performs photosynthesis. In some embodiments, the first microbial species that performs photosynthesis is a microorganism from the following genus: Bosella, Shen's Bacillus or Neosphingobacterium. In some embodiments, the second microbial species performs nitrogen fixation. In some embodiments, the second microbial species that performs nitrogen fixation is a microorganism from the following genus: Greedy Phage, Pseudomonas, Greedy Cupribotium, Brevundimonas, Fengyou Bacillus, Archaeobacterium, Theilerella, Tahibacter, Polar Mononas, Chitinophage, Freundii, Aquabacterium, Thin Layer Bacillus or Sandy Soil Bacillus.

[0043] In some embodiments, the first microbial species carries out photosynthesis and nitrogen fixation. In some embodiments, the first microbial species carrying out photosynthesis and nitrogen fixation is a bacterium. In some embodiments, the first microbial species carrying out photosynthesis and nitrogen fixation is a member of Cyanobacteria. In some embodiments, the first microbial species carrying out photosynthesis and nitrogen fixation is a member of Cyanobacteriales, Cyanobacteriales, Nostocales, Pseudo-Anabaenales, Spirulinales or Oscillatorales. In some embodiments, the first microbial species carrying out photosynthesis and nitrogen fixation is a member of Nostocales. In some embodiments, the first microbial species that performs photosynthesis and nitrogen fixation is a member of the genus Acaryochloris, Aliterella, Amazonocrinis, Anabaena, Anabaena, Aphanizomenon, Atlanticothrix, Calenema, Calenema, Pseudo-flexilla, Pseudo-chlorococcus, Pseudo-chromococcus, Chrysosporium, Compactonostoc, Constrictifilum, Crocodile Glomerella, Cyanobacterium, Cyanocohniella, Pseudo-cylindrical spores, Cylindrical spores, Dendronalium, Desikacharya, Desmonostoc, Longisporium, Erythrobacter, Flytella, Voltiella, Fremyella, Gastrosporium, Glucosporum, Globose, and Glucosporum. Algae, Gelatophyte, Goleter, Hose algae, Water stripe algae, Jasmine, Johanseniella, Kamptonema, Komarekiella, Sclerotium, Sclerotium, Macrochaete, Flagellate, Microchaete, Knot filament, Nodularia, Nodulariaceae, Nodularia, Oligospermum, Oscillatoria, Pantanalinema, Pelatocladus, Pseudofloxacin, Floatella, Polymorpha, Porphyromonas, Prochlorococcus, Prochlorococcus, Pseudoanabaena, Pseudofloxacin, Acrocephalum, Phytophyte, Gelatophyte, Roholtiella, Rubidibacter, Pseudoclase, Spirulina, Synechococcus, Synechocystis, Thermosynechococcus, Monochamus, Trichocystis, Violetonostoc, Lumenella or Heterococcus. In some embodiments, the first microbial species that performs photosynthesis and fixes nitrogen is a Nostoc species.In some embodiments, the first microbial species that performs 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 also includes a second microbial species that performs supplemental nitrogen fixation. In some embodiments, the second microbial species that performs supplemental nitrogen fixation is a microorganism from the following genera: Gram-negative bacteria, Pseudomonas, Cupric bacteria, Brevundimonas, Fengyou bacteria, Archaeobacterium, Theileria, Tahibacter, Polarmonas, Chitinophaga, Freudenbergia, Aquabacter, Thin-layer bacteria or Soil bacillus.

[0044] In some embodiments, the composition also includes a durable carbon compound that provides a carbon sink in the soil. In some embodiments, the microbial organism produces a durable carbon compound that provides a carbon sink in the soil. In some embodiments, the microbial organism produces melanin. In some embodiments, the microbial organism produces pheomelanin, eumelanin and / or pyomelanocin. In some embodiments, the composition also includes melanin. In some embodiments, the composition also includes pheomelanin, eumelanin and / or pyomelanocin.

[0045] In some embodiments, the microbial organism comprises, consists of, or consists essentially of less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, or less than 3 species. In some embodiments, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the organisms in the microbial consortium are provided by photosynthetic nitrogen-fixing microorganisms; and a 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%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, %, 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% or more is provided by microbial organisms that jointly perform photosynthesis and nitrogen fixation.

[0046] In some embodiments, the synthetic growth medium is a medium without fixed carbon. In some embodiments, the synthetic growth medium is a medium without fixed nitrogen. In some embodiments, the synthetic growth medium is a medium without fixed carbon and a medium without fixed nitrogen. 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 and nitrogen-free medium. In some embodiments, a method of accumulating carbon and nitrogen in an agricultural medium is provided, wherein the method comprises applying to the agricultural medium a composition comprising: a synthetic growth medium; and a microbial organism that performs photosynthesis and nitrogen fixation together. In some embodiments, the method further comprises exposing the composition to nitrogen gas (N 2 ), carbon dioxide (CO 2 ) 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, a greenhouse growth medium, or a 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 pyomelanocin provide fixed carbon in the soil.

[0047] Some parts of this specification describe the implementation of the technology in terms of algorithms and symbolic representations of information operations. These algorithmic descriptions and representations are often used by technicians in the field of data processing to effectively convey the essence of their work to other technicians in the field. Although these operations are described functionally, computationally or logically, they should be understood to be implemented by computer programs or equivalent circuits, microcodes, etc. In addition, without loss of generality, it is sometimes convenient to refer to these operational arrangements as modules. The described operations and their associated modules can be embodied in software, firmware, hardware, or any combination thereof.

[0048] Certain steps, operations or processes described herein may be performed or implemented using one or more hardware or software modules, alone or in combination with other devices. In some embodiments, the software module is implemented using a computer program product that includes a computer-readable medium containing a computer program code that can be implemented by a computer processor to perform any or all of the steps, operations or processes described.

[0049] In some embodiments, the system includes a virtually provided computer and / or data storage (e.g., as a cloud computing resource). In a specific embodiment, the technology includes using cloud computing to provide a virtual computer system that includes components as described herein and / or performs the functions of a computer as described herein. Thus, in some embodiments, cloud computing provides infrastructure, applications, and software as described herein over a network and / or via the Internet. In some embodiments, computing resources (e.g., data analysis, computing, data storage, applications, file storage, etc.) are provided remotely over a network (e.g., the Internet; and / or a cellular network).

[0050] Embodiments of the technology may also relate to an apparatus for performing the operations herein. The apparatus may be specially constructed for the desired purpose and / or it may include a general-purpose computing device that is selectively activated or reconfigured by a computer program stored in a 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. In addition, any computing system mentioned in the specification may include a single processor or may be an architecture that employs a multi-processor design to increase computing power.

[0051] Other implementations will be apparent to persons skilled in the relevant arts based on the teachings contained herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with one or more color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0053] These and other features, aspects and advantages of the present technology will be better understood with reference to the following drawings.

[0054] Figure 1A Light microscopy images of BW01 cells are shown. The scale bar shows a distance of 50 μm. Structures believed to be heterocysts are marked with "hc".

[0055] Figure 1B Light microscopy images of BW02 cells are shown. The scale bar shows a distance of 50 μm. Structures believed to be heterocysts are marked with "hc".

[0056] Figure 2 is a heat map showing the relative abundance of genera in the original soil sample and in the P1-P4 passages that gave rise to the BW01, BW02, and BW05 consortia. Darker colors indicate higher read abundance as determined with Kraken2.

[0057] Figure 3A is a graph showing the relative abundance of Aminobacter in BW01, BW02, and BW05 as a function of passage from the original soil sample and through P1 to P4. Error bars represent standard deviation (original soil sample n=1; P1 n=1; P2 n=2; P3 n=4; and P4 n=4).

[0058] Figure 3B is a graph showing the relative abundance of Bradyrhizobium in BW01, BW02, and BW05 as a function of passage from the original soil sample and through P1 to P4. Error bars represent standard deviation (original soil sample n=1; P1 n=1; P2 n=2; P3 n=4; and P4 n=4).

[0059] Figure 3C is a graph showing the relative abundance of Mesorhizobium in BW01, BW02, and BW05 as a function of passage from the original soil sample and through P1 to P4. Error bars represent standard deviation (original soil sample n=1; P1 n=1; P2 n=2; P3 n=4; and P4 n=4).

[0060] Figure 3Dis a graph showing the relative abundance of Nostoc in BW01, BW02, and BW05 as a function of passage from the original soil sample and through P1 to P4. Error bars represent standard deviation (original soil sample n=1; P1 n=1; P2 n=2; P3 n=4; and P4 n=4).

[0061] Figure 3E is a graph showing the relative abundance of Pseudomonas in BW01, BW02, and BW05 as a function of passage from the original soil sample and through P1 to P4. Error bars represent standard deviation (original soil sample n=1; P1 n=1; P2 n=2; P3 n=4; and P4 n=4).

[0062] Figure 3F is a graph showing the relative abundance of the genus Vacuola in BW01, BW02, and BW05 as a function of passage from the original soil sample and through P1 to P4. Error bars represent standard deviation (original soil sample n=1; P1 n=1; P2 n=2; P3 n=4; and P4 n=4).

[0063] Figure 3G is a graph showing the relative abundance of Streptomyces in BW01, BW02, and BW05 as a function of passage from the original soil sample and through P1 to P4. Error bars represent standard deviation (original soil sample n=1; P1 n=1; P2 n=2; P3 n=4; and P4 n=4).

[0064] Figure 3H is a graph showing the relative abundance of Azospirillum in BW01, BW02, and BW05 as a function of passage from the original soil sample and through P1 to P4. Error bars represent standard deviation (original soil sample n=1; P1 n=1; P2 n=2; P3 n=4; and P4 n=4).

[0065] Fig. 3I is a graph showing the relative abundance of Rhizobium in BW01, BW02, and BW05 as a function of passage from the original soil sample and through P1 to P4. Error bars represent standard deviation (original soil sample n=1; P1 n=1; P2 n=2; P3 n=4; and P4 n=4).

[0066] Figure 4 It is a graph showing the growth curves of all P4 samples of BW01, BW02 and BW05. The growth curves were determined by measuring the dry weight (grams) of cells in the culture as a function of time (days). The dry weight is the weight of each 7.5 mL well in a standard 6-well plate.

[0067] Figure 5 Is a series of graphs showing the relative abundance of specific genes (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) identified from nucleotide sequences obtained from BW01, BW02 and BW05 consortia as they change over generations. These graphs show the presence of genes in log10 gene counts from the original soil sample to P4. EggNOG orthologous groups representing functional annotations are listed in Table 4.

[0068] Figure 6 is a series of photographs showing the morphology of colony picks ("minimal colonies") when grown in illuminated liquid media. Images were obtained from below culture flasks with overhead lighting. The morphology of the cultures is shown as "light", "dark", "filament", "restreak" or "colony". Picked colonies are identified with a "P" number. See Fig. 9B .

[0069] Fig. 7A The classification system of the minimal consortium evaluated by Phyloflash is shown. The y-axis shows the identified taxa, while the x-axis shows the sample ID and morphology.

[0070] Figure 7B-7F is a series of graphs showing changes in the relative abundance of specific genes identified in nucleotide sequences obtained from colony picks ("minimal consortia"). Figure 7B : nitrogenase subunit h (nifh); Figure 7C and Fig.7D : Photosystem II subunits (psba and psbb); Fig. 7E : ribulose-1,5-bisphosphate carboxylase / oxygenase (rubiscoL and rubiscoS); and Figure 7F : Polysaccharide biosynthesis and export protein (wza) involved in EPS biosynthesis). Table 4 lists the EggNOG orthologous groups representing functional annotations.

[0071] Figure 8 Semi-logarithmic plots of a series of growth curves for each minimal consortium grown in 12-well plates with minimal medium (no nitrogen supplementation). Dry biomass (y-axis, log2 scale) is converted to kilograms per hectare. The area of ​​the well is 3.14 cm 2Three replicates were collected for each culture at each time point. The entire well was collected at each time point and endpoint data were recorded.

[0072] Fig. 9A is a bar graph showing the saturation density of full consortia (blue bars) and minimal consortia (red bars) grown in 12-well plates in minimal medium under light. Error bars are standard errors.

[0073] Fig. 9B Shows the Fig. 9A Description of the population identified by the P number in and the values ​​plotted on the graph.

[0074] Fig.10 is a series of graphs showing the growth of three consortia in a one liter photobioreactor. The dry weight on the log2 y-axis is shown on a g / photobioreactor basis. The data points are shown in addition to the curve fit generated by the R package Growthcurver. The three cultures were grown under illumination conditions in either a nitrogen-free medium (M) or a medium containing nitrogen in the form of nitrate (N), with a sample size of n=2 photobioreactors.

[0075] Fig.11 is a series of bar graphs showing the carbon and nitrogen content of cultures grown in photobioreactors. Fixed carbon and nitrogen are shown on a gram / 1 liter photobioreactor basis. Error bars are standard deviations. Each set of bars represents the mean of two biological replicates. M represents cultures grown in minimal medium without bound nitrogen. N represents cultures grown in medium with added nitrates. In the dry cell pellet weight, C is carbon (red bars), N is nitrogen (green bars), and other (blue bars) are all other compounds.

[0076] Fig. 12A A series of red fluorescence images of three consortia grown on sterile potting soil minus a day 0 blank and images aligned by image analysis software. The excitation light was 450-nm blue light. A deep red filter was used to block the blue wavelength of the camera detector. A) BW01 on day 1. B) BW01 on day 10. C) BW02 on day 1. D) BW02 on day 10. E) BW05 on day 1. F) BW05 on day 10.

[0077] Fig. 12B Shown from Fig. 12B The same series of fluorescence images with the tones inverted and converted to grayscale to increase contrast and visibility of areas containing chlorophyll and its abundance.

[0078] Fig.13is a series of graphs showing the mean Bray-Curtis dissimilarity index as a metric for passages of BW01 and BW02. Error bars represent standard deviation (n=4).

[0079] Fig.14 is a plot of the first (PCo1) and second (PCo2) principal components derived from principal coordinates analysis of the taxonomic composition determined from shotgun sequencing of the original soil microbiome and P1-P4 passages of BW01 and BW02.

[0080] Fig.15 is a schematic diagram showing predicted growth in the field relative to laboratory conditions.

[0081] Fig.16 is a schematic diagram showing calculations for field growth capacity assuming two different models. The calculations are based on thin film liquid cultures grown in 12-well plates and assume that the cultures grow in 2 dimensions, as if grown in the field.

[0082] Fig.17 is a schematic diagram showing the metabolic functions of the consortium described herein. Red represents electron sources, carriers, and sinks.

[0083] Fig.18A Photographs of cultures at inoculation (top) and after growth (bottom) are shown.

[0084] Fig.18B is a schematic diagram showing the biosynthetic pathway for melanin production.

[0085] Fig.19 Shown are the UV-visible absorption spectra of melanin reference material (0.02%, 0.01% and 0.005% w / v) and flocculants isolated from BW02 culture (1×, 0.5×, 0.2×, 0.1× and 0.05× dilutions).

[0086] Fig. 20 Shown is a series of photographs of solid medium culture plates comprising 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 and on which BW02 cultures were grown in the light for 14 days.

[0087] Fig.21Images of culture supernatants from three independent cultures in multiwell plates are shown. Supernatants were obtained from cultures grown in minimal medium without tyrosine supplementation ("standard medium") or minimal medium with tyrosine supplementation ("+L-Tyrosine"). NaOH ("NaOH blank") and synthetic eumelanin from Sigma Aldrich ("eumelanin") were used as negative and positive controls, respectively. Uninoculated minimal medium ("-inoculant") was also used as a negative control.

[0088] Fig. 22 Shown are absorbance spectra of supernatants inoculated with a microbial consortium and grown for two weeks with tyrosine supplementation. Shown are supernatants from cultures without tyrosine supplementation, supernatants from cultures supplemented with tyrosine ("+Tyr"), and melanin controls (0.0350% w / v and 0.0063% w / v).

[0089] Fig.23 Photographs of minimal consortia MC1905, MC1909, and MC1918 generated from samples S1905, S1909, and S1918, respectively, in wells of a 96-well plate are shown. Three replicates of each consortium were grown in minimal mineral medium (control, top row), and three replicates of each consortium were grown in the same medium supplemented with 2.77 mM homogentisate (HGA) (bottom row).

[0090] Fig.24 Shows Fig.23 The absorbance spectra of the samples shown in the photographs. Synthetic eumelanin is shown as a control.

[0091] Fig.25 , Fig.26 and Fig. 27 The phylum, class, order, family, and genus identified for the organisms of the minimal consortia generated from BW01, BW02, and BW05 are shown, respectively.

[0092] Fig.28A and Fig.28B The phylum, class, order, family and genus identified for the organisms of the minimal consortium generated from the FL consortium are shown.

[0093] It should be understood that the drawings are not necessarily drawn to scale, and the objects in the drawings are not necessarily drawn to scale relative to each other. The drawings are intended to make the various embodiments of the devices, systems and methods disclosed herein clear and understandable. In all drawings, the same reference numerals are used to refer to the same or similar parts as much as possible. In addition, it should be understood that the drawings are not intended to limit the scope of the present teachings in any way. DETAILED DESCRIPTION

[0094] Provided herein are technologies related to functional microbial consortia, and particularly, but not exclusively, relate to methods and systems for producing microbial consortia having desired functions and microbial consortia produced according to such methods.

[0095] In the detailed description of various embodiments, for the purpose of explanation, many specific details are set forth to provide a thorough understanding of the disclosed embodiments. However, those skilled in the art will appreciate that these different embodiments may be practiced with or without these specific details. In other cases, structures and devices are shown in block diagram form. In addition, those skilled in the art can readily appreciate that the particular order of presentation and execution methods is illustrative, and it is contemplated that the order may vary and still remain within the spirit and scope of the various embodiments disclosed herein.

[0096] All documents 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 any purpose. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as generally understood by those of ordinary skill in the art to which the various embodiments described herein belong. When the definitions of terms in the incorporated references differ from the definitions provided in this teaching, the definitions provided in this teaching 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.

[0097] ●Definition

[0098] To facilitate understanding of the present technology, several terms and phrases are defined below. Additional definitions are set forth throughout the detailed description.

[0099] Unless otherwise defined herein, scientific and technical terms related to the present technology should have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms should include plural terms, and plural terms should include singular terms. Typically, 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 as described herein are those well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present technology are generally performed according to conventional methods well known in the art, and as described in various general and more specific references cited and discussed throughout this specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989); Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2000); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992 and Supplements to 2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4thed., 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.

[0100] As used herein, the phrase "in one embodiment" does not necessarily refer to the same embodiment, although it may refer to the same embodiment. In addition, as used herein, the phrase "in another embodiment" does not necessarily refer to a different embodiment, although it may refer to a different embodiment. Therefore, as described below, the various embodiments of the present invention can be easily combined without departing from the scope or spirit of the present invention.

[0101] In addition, 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 being based on additional factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meanings of "a", "an", and "the" include plural references. The meaning of "in..." includes "in..." and "on...".

[0102] As used herein, the terms "about," "approximately," "substantially," and "significantly" are understood by those of ordinary skill in the art and will vary to some extent depending on the context in which they are used. If the use of these terms is not clear to one of ordinary skill in the art considering the context in which they are used, "about" and "approximately" mean plus or minus less than or equal to 10% of the particular term, and "substantially" and "significantly" mean plus or minus greater than 10% of the particular term.

[0103] As used herein, the disclosure of a range includes disclosure of all values ​​within the entire range and further divided ranges (including endpoints and subranges given for the range). As used herein, the disclosure of a numerical range includes endpoints and each intermediate number with the same degree of accuracy therebetween. For example, for a range of 6-9, in addition to 6 and 9, numbers 7 and 8 are also contemplated, and for a range of 6.0-7.0, 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 clearly contemplated.

[0104] As used herein, the term "without" refers to an embodiment of the technology that omits the feature described by the term "without". That is, the term "without X" as used herein means "without X", where X is a feature of the technology omitted from the "without X" technology. For example, a "calcium-free" composition does not include calcium, a "no mixing" method does not include a mixing step, etc.

[0105] Although the terms "first", "second", "third", etc. may be used in this article to describe various steps, elements, compositions, components, regions, layers and / or parts, these steps, elements, compositions, components, regions, layers and / or parts should not be limited by these terms unless otherwise stated. These terms are used to distinguish a step, element, composition, component, region, layer and / or part from another step, element, composition, component, region, layer and / or part. Unless the context clearly indicates, the terms such as "first", "second" and other numerical terms used herein do not imply a sequence or order. Therefore, without departing from the technology, the first step, element, composition, component, region, layer or part discussed herein may be referred to as a second step, element, composition, component, region, layer or part.

[0106] 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., component, effect, element). For example, when an entity is referred to as being "present," this means that the level or amount of the entity is above a predetermined threshold; conversely, when an entity is referred to as being "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 a particular test for detecting the entity. When an entity is "detected," it is "present"; when an entity is "not detected," it is "absent."

[0107] 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 variable value, relative to a predetermined value, and / or relative to a standard control value, respectively. An increase is a positive change relative to a previously measured variable value, a predetermined value, and / or a standard control value, preferably at least 10%, more preferably 50%, still more preferably 2 times, even more preferably at least 5 times, and most preferably at least 10 times. Similarly, a decrease is a negative change relative to a previously measured variable value, a predetermined value, and / or a standard control value, preferably at least 10%, more preferably 50%, still more preferably at least 80%, and most preferably at least 90%. Other terms indicating quantitative changes or differences, such as "more" or "less", are used in the same manner as described above herein.

[0108] As used herein, the term "improved" refers to a feature of an environment that is improved compared to a control environment or compared to a known average amount associated with the feature mentioned. For example, an "improved" soil may refer to a soil that increases plant biomass production after applying beneficial microorganisms or microbial consortia to the soil relative to plant biomass produced by soil that is not treated with beneficial microorganisms or microbial consortia, and other soil characteristics are substantially and / or substantially the same in terms of the impact on plant biomass production. Alternatively, the production of plant biomass after applying beneficial microorganisms or microbial consortia to the soil can be compared with the average biomass typically produced by the soil, as described in scientific or agricultural publications known to those skilled in the art. As used herein, "improved" does not necessarily require data to be statistically significant (e.g., p < 0.05); on the contrary, any quantifiable difference indicating that one value (e.g., average treatment value) is different from another value (e.g., average control value) can be raised to the "improved" level.

[0109] As used herein, "system" refers to a plurality of real and / or abstract components that operate 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 the system interacts with and / or is related to one or more other components. In some embodiments, a system refers to a combination of components and software for controlling and directing methods. For example, a "system" or "subsystem" may include one or more or any combination of the following: mechanical devices, hardware, components of hardware, circuits, lines, logical designs, logical components, software, software modules, components of software or software modules, software processes, software instructions, software routines, software objects, software functions, software classes, software programs, files containing software, etc., to perform the functions of the system or subsystem. Therefore, the methods and devices of the embodiments, or some aspects or portions thereof, may take the form of program codes (e.g., instructions) embodied in tangible media such as floppy disks, CD-ROMs, hard drives, flash memory, or any other machine-readable storage medium, wherein when the program code is loaded into a machine such as a computer and executed by it, the machine becomes a device for practicing the embodiments. In the case where the program code is implemented on a programmable computer, the computing device generally includes a processor, a storage medium readable by the processor (e.g., volatile and non-volatile memory and / or storage element), at least one input device and at least one output device. One or more programs can implement or utilize the process described in conjunction with the embodiment, for example, by using an application programming interface (API), reusable controls, etc. Such programs are preferably implemented in a high-level process or object-oriented programming language to communicate with the computer system. However, if necessary, one or more programs can be implemented in assembly language or machine language. In any case, the language can be a compiled or interpreted language and combined with a hardware implementation.

[0110] As used herein, the term "biological system" refers to a set of genes, enzymes, activities or functions that act together to provide metabolic pathways or metabolic networks. A biological system can include genes, enzymes, activities or functions provided by many individual organisms. In other words, a biological system can be distributed in an individual organism of a microbial community or a microbial consortium. Therefore, a biological system can be described by a set of genes, enzymes, activities or functions, without the need to identify the individual organisms that provide genes, enzymes, activities or functions. A biological system can also be described from the perspectives of nutrient flux, energy flux, electrochemical gradient, metabolic input (bioreactor) and metabolic output (bioproduct), for example, it provides energy input to the energy conversion used for biological processes, the anabolism synthesis of biomolecules and the elimination of waste.

[0111] As used herein, the term "metabolic pathway" refers to a set of connected metabolic, biochemical, and physical processes that convert metabolic inputs into metabolic outputs in a series of steps and intermediates.

[0112] As used herein, the term "metabolic network" refers to a set of connected metabolic pathways. A metabolic network can convert metabolic inputs into metabolic outputs in a series of steps and intermediates.

[0113] As used herein, the terms "microbial," "microbial organism," and "microorganism" refer to organisms that exist as microscopic cells, which are included in the kingdoms Archaea, Bacteria, or Eukaryota in the three-kingdom system (see Woese (1990) Proc Natl Acad Sci USA 87:4576–79, incorporated herein by reference), the latter including yeast and filamentous fungi, protozoa, algae, or higher protists. Thus, the term is intended to encompass prokaryotic or eukaryotic cells or organisms of microscopic size, and includes bacteria, archaea, and eubacteria of all species, 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 a chemical. The terms "microbial cell" and "microbe" can be used interchangeably with the term "microorganism." The terms "bacteria" and "bacterium", "archaea" and "archaeon" refer to prokaryotic organisms of the kingdoms Bacteria and Archaea in the three-kingdom system.

[0114] The term "archaea" refers to the taxonomic kingdom of organisms that are commonly 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, Archaea consist of two phylogenetically distinct groups: Crenarchaeota and Euryarchaeota. Based on their physiology, Archaea can be divided into three types: methanogens (methane-producing prokaryotes); extreme halophiles (prokaryotes that live in very high concentrations of salt (NaCl)); and extreme (hyper)thermophiles (prokaryotes that live at extremely high temperatures). In addition to the unifying archaeal features that distinguish them from bacteria (e.g., absence of muramic acid in the cell wall, ester-linked membrane lipids, etc.), these prokaryotes also exhibit unique structural or biochemical attributes that adapt them to specific habitats. Crenarchaeota consists mainly of hyperthermophilic sulfur-dependent prokaryotes, while Euryarchaeota contains methanogens and extreme halophiles.

[0115] The term "bacteria" or "eubacteria" refers to the kingdom of prokaryotic organisms. Bacteria include at least the following 11 different groups: (1) Gram-positive (Gram+) bacteria, of which there are two major categories: (1) high G+C group (Actinomyces, Mycobacteria, Micrococcus, etc.), (2) low G+C group (Bacillus, Clostridia, Lactobacillus, Staphylococci, Streptococci, Mycoplasmas); (2) Proteobacteria, e.g. Such as purple photosynthetic + non-photosynthetic Gram-negative bacteria (including most "common" Gram-negative bacteria); (3) Cyanobacteria, for example, aerobic phototrophs; (4) Spirochetes and related species; (5) Planctomyces; (6) Bacteroides, Flavobacteria; (7) Chlamydia; (8) Green sulfur bacteria; (9) Green non-sulfur bacteria (also known as anaerobic phototrophs); (10) Radioresistant Micrococcus and related groups; (11) Thermotoga and Thermosipho thermophiles.

[0116] "Gram-negative bacteria" include cocci, non-enterobacteria and enterobacteria. The genera of gram-negative bacteria include, for example, Neisseria, Spirochaete, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, and Salmonella. The present invention also includes the genera of bacteria such as aliphatic bacteria, eukaryotic organisms, bacterial pathogens, fungi, fungi, spores, proteus, vibrio, pseudomonas, bacteroides, acetobacter, aerobacter, agrobacterium, azotobacter, spirilla, serratia, rhizobium, chlamydia, rickettsia, treponema, and fusobacterium.

[0117] "Gram-positive bacteria" include cocci, non-sporing bacilli and sporing bacilli. The genera of gram-positive bacteria include, for example, Actinomyces, Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus and Streptomyces.

[0118] As used herein, the term "genus" is defined as a taxonomic group 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).

[0119] 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 are sufficiently distinct from all other organisms to be recognized as distinct units.

[0120] As used herein, the term "strain" in reference to a microorganism herein describes an isolate of a microorganism that is considered to be the same species but has a unique genome and, if the nucleotide changes are non-synonymous, a unique proteome that is different from other strains of the same organism. The non-chromosomal genetic complement of the strains may differ. Typically, strains are the result of isolation from different hosts or at different locations and times, but multiple strains of the same organism can be isolated from the same host.

[0121] As used herein, the term "naturally occurring" as applied to nucleic acids, enzymes, cells or organisms refers to nucleic acids, enzymes, cells or organisms found in nature. For example, a polypeptide or polynucleotide sequence present in an organism is naturally occurring, which can be isolated from a source in nature and has not been intentionally modified by man in the laboratory.

[0122] 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 having at least one genetic alteration not typically found in a naturally occurring nucleic acid, enzyme, cell or organism. Genetic alterations include, for example, modifications that introduce expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and / or other functional disruptions of microbial genetic material. Such modifications include, for example, coding regions of heterologous, homologous or heterologous and homologous polypeptides of a reference species and functional fragments thereof. Additional modifications include, for example, non-coding regulatory regions, modifications of which alter the expression of a gene or operon.

[0123] As used herein, the term "in vitro" refers to an artificial environment and processes or reactions that occur in 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 processes or reactions that occur in a natural environment.

[0124] As used herein, the term "cell culture" refers to any in vitro culture of cells, including, for example, prokaryotic cells 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 cell populations maintained in vitro.

[0125] As used herein, the term "culturable organism" refers to a living organism that can be maintained and grown in the laboratory. In some embodiments, a culturable organism may not be able to be maintained and grown in a pure culture in the laboratory without other organisms, and thus may be referred to as a "non-culturable organism" relative to growth as a pure culture. However, in some embodiments, such an organism can be grown in a microbial consortium that includes at least one other organism in the laboratory, and thus may be a "culturable organism" for the consortium, but also a "non-culturable organism" for growth in a pure culture without one or more other members of the consortium.

[0126] As used herein, the terms "isolated", "isolated", "isolated microorganisms", and the like are intended to indicate that one or more microorganisms have been separated from at least one material with which it is associated in a particular environment (e.g., soil, water, or a higher multicellular organism). Thus, an "isolated microorganism" does not exist in its naturally occurring environment; rather, the microorganism has been removed from its natural environment and placed in a non-naturally occurring state of existence by the various techniques described herein. Thus, an isolated strain may exist, for example, as a biologically pure culture or as a spore (or other form of strain) in combination with a carrier composition. In certain aspects of the present disclosure, an isolated microorganism exists as an isolated and biologically pure culture. It will be understood by those skilled in the art that an isolated and biologically pure culture of a particular microorganism means that the culture is substantially free of (within scientifically reasonable limits) other living organisms and contains only the individual microorganisms mentioned. A culture may contain varying concentrations of the microorganism, and an isolated and biologically pure microorganism is generally necessarily different from less pure or impure material. In addition, in some aspects, the present disclosure provides certain quantitative measurements of concentrations or purity limits found in isolated and biologically pure microbial cultures. In certain embodiments, the presence of these purity values ​​is an additional attribute that distinguishes the presently disclosed microorganisms from those microorganisms existing in a natural state.

[0127] As used herein, the term "selected environment", "conditions" or "conditions" refers to any external characteristics in which a microbial consortium of a particular organism or microbial community grows more effectively (e.g., faster, reaches a higher amount or concentration, has a higher survival rate, etc.) than the consortium of one or more other organisms or microbial communities. Exemplary "conditions" or "environments" include, but are not limited to, specific culture media, volumes, containers, temperature, mixing, aeration, gravity, electromagnetic fields, cell density, pH, nutrients, phosphate sources, nitrogen sources, symbiosis with one or more organisms, and / or interactions with species of a single organism or species of multiple organisms (e.g., mixed populations). Also included as "conditions" or "environments" are substances that may be toxic to the consortium of one or more organisms or microbial communities, such as heavy metals, antibiotics, and chlorinated compounds. It should be understood that time can also be considered a "condition" because organisms are not static entities. Thus, growing a culture over an extended period of time (e.g., days, weeks, months, years) may result in a culture comprising a particular organism or consortium that is present in the culture in a greater proportion than the relative amount of the particular organism or consortium in the culture prior to growth for that period of time.

[0128] As used herein, the term "selection" refers to increasing the frequency of different "types" of individuals in a population by intentionally or spontaneously removing or enriching certain types of individuals more than other types. The nature of a "type" can be defined by: genetic characteristics (e.g., genes or nucleotide sequences); functional characteristics (e.g., enzymatic, metabolic capacity); taxonomic characteristics (e.g., strains, subspecies, species, genus, family, or operational taxonomic units (OTUs) based on nucleotide sequence similarity or differences); or physical characteristics. In addition, a type can include one or more individuals. A typical example of selection includes, but is not limited to, growth rate selection, in which individuals that grow and reproduce faster become more common in a population. An important consideration for selection is to determine "because of what selection" or what is "selected", that is, genetic, functional and / or physical differences that are favorable or unfavorable in a particular environment. Growth rate selection is used to select organisms whose growth rate is faster than other individuals in a population and can be passed from parental cells to their offspring.

[0129] 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 in which the number (e.g., absolute and / or relative number) of one or more microorganisms present in a culture is increased, e.g., by culturing in a suitable medium under selective conditions.

[0130] As used herein, the term "medium" or "media" refers to the chemical environment to which an organism is subjected or provided access. The organism may be immersed in the medium or in physical proximity (e.g., physical contact) to it. The medium typically contains water and other additional nutrients and / or chemicals that may contribute to the growth or maintenance of the organism. These ingredients may be pure chemicals (e.g., "defined" medium) or complex, uncharacterized mixtures of chemicals, such as extracts made from milk or blood. Standardized culture media are widely used in laboratories. Examples of culture media for bacterial growth include, but are not limited to, LB and M9 minimal media. When referring to a culture medium, the term "basic" refers to a culture medium that supports the growth of an organism but consists only of the simplest possible chemical compounds. For example, M9 minimal medium may consist of the following ingredients dissolved in water and sterilized: 48mM Na 2 HPO 4 , 22mM KH 2 PO 4 , 9 mM NaCl, 19 mM NH 4 Cl, 2 mM MgSO 4 , 0.1 mM CaCl 2 , 0.2% carbon and energy (e.g., glucose).

[0131] A growth medium is also provided for the growth of the plant. As used herein, the term "agricultural growth medium" refers to a medium suitable for supporting plant growth. The agricultural growth medium can be natural or artificial, for example, soil, potting mix, bark, vermiculite, hydroponic solution, hydroponic solution applied to a solid plant support system, and tissue culture gel. The agricultural growth medium can be used alone or in combination with one or more other media. It can also be used with or without the addition of exogenous nutrients and a physical support system for roots and leaves.

[0132] In some embodiments, the agricultural growth medium is a naturally occurring medium, such as soil, sand, mud, clay, humus, regolith, rock or water. In some embodiments, the agricultural growth medium is artificial. Such artificial agricultural growth medium can be constructed to simulate the conditions of a natural medium; however, this is not necessary. The artificial agricultural growth medium can be made of one or more of any number of materials and combinations thereof, including sand, minerals, glass, rocks, water, metals, salts, nutrients, water. In some embodiments, the agricultural growth medium is sterile. In some embodiments, the agricultural growth medium is not sterile.

[0133] As used herein, the term "soil" refers to a solid particulate growth medium that includes organic matter and minerals, and typically also includes water and gases.

[0134] As used herein, the term "greenhouse growth medium" refers to a soil-free growth medium commonly used for greenhouses, nursery beds and container crops of plants. Exemplary greenhouse growth mediums include peat moss and may also include perlite, vermiculite, bark and / or sand.

[0135] As used herein, the term "hydroponic medium" refers to a soilless liquid growth medium that includes nutrients and / or minerals for plant growth. The use of a hydroponic medium can be combined with the use of an inert medium such as perlite, gravel or other substrate to provide physical support for the plant.

[0136] As used herein, the term "culture" refers to a medium in a container or enclosure having at least one cell or living organism, typically a medium in which the organism can grow. As used herein, the term "continuous culture" means a liquid culture to which new medium is added at a rate equal to the rate at which the medium is removed. In contrast, as used herein, "batch culture" means a culture of a fixed size or volume in which new medium is not added or removed.

[0137] As used herein, the term "microbial consortium" (plural "microbial consortia") refers to a group of microbial species or strains of species that can be described as performing a common function, or can be described as participating in, causing, or being associated with an identifiable parameter or phenotypic trait. A consortium can contain two or more microbial taxa (e.g., a family, genus, species, or strain of a species). In some cases, microorganisms coexist symbiotically within a community. A microbial consortium can be described by describing the taxa present in the consortium (e.g., several strains, subspecies, species, genus, family, or operational taxonomic units (OTUs) based on nucleotide sequence similarity or difference); by describing the genes present in the consortium; by describing the nucleotide sequences present in the consortium; or by describing the functions present and / or provided in the consortium. A microbial consortium can be a subset of organisms found in a microbial community. A microbial consortium can be described as a set of genes, enzymes, activities, or functions provided by many individual organisms. In addition, microbial consortia can be described by collections of genes, enzymes, activities, or functions without identifying the individual organisms that provide them. Microbial consortia can also be described at the level of nutrient fluxes, energy fluxes, electrochemical gradients, metabolic inputs (bioreactors), and metabolic outputs (bioproducts), e.g., providing energy inputs to energy conversion for biological processes, anabolic synthesis of biomolecules, and elimination of waste products.

[0138] As used herein, the term "microbial community" refers to a group of microorganisms comprising two or more microbial taxa (e.g., strains of a family, genus, species, or species). Unlike a microbial consortium, a microbial community does not necessarily act in concert to perform a common function, or does not necessarily participate in or cause or be associated with an identifiable parameter or phenotypic trait. See, e.g., Doolittle (2010) "Metagenomics and the Units of Biological Organization" BioScience 60: 102-112, incorporated herein by reference.

[0139] As used herein, the term "minimal consortium" (plural "minimal consortia") refers to a microbial consortium that includes a minimal set of members that can survive under given growth conditions. A minimal consortium can be generated from another more complex microbial consortium or microbial community, for example, by streaking a microbial consortium or microbial community on a solid minimal selective growth medium to isolate individual colonies that include the minimal consortium grown on the solid minimal selective growth medium, and when it is appropriate to isolate colonies with a stable morphology, picking and restreaking the colonies on the solid minimal selective growth medium to isolate individual colonies that include the minimal consortium grown on the solid minimal selective growth medium. Thus, in this example, the minimal consortium includes a minimal set of members that survive on the minimal selective medium.

[0140] As used herein, the term "relative abundance" refers to the comparison of the microbial abundance of a specific taxon (e.g., OTU) in a first biological sample with the microbial abundance of a corresponding taxon in one or other (e.g., second) samples."Relative abundance" can be reflected in, for example, the number of isolated species corresponding to a taxon or the degree to which a biomarker (e.g., nucleotide sequence) specific to a taxon exists or is expressed in a given sample. The relative abundance of a specific taxon in a sample can be determined using a culture-based method or a non-culture-based method well known in the art. The non-culture-based method includes sequence analysis of amplified polynucleotides specific to a taxon or a comparison of a proteomics-based map in a sample, reflecting the quantity and degree of polypeptide-based, lipid-based, polysaccharide-based or carbohydrate-based biomarkers of one or more taxons present in a sample. With reference to all taxon / OTUs detected, or with reference to some unchanged taxon / OTU groups, the relative abundance or abundance of a taxon or OTU can be calculated. In some embodiments, taxa are identified using sequence-based methods, as described in, e.g., 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.

[0141] 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 taxon compared to the total microorganisms in a sample or to the number of microorganisms of a corresponding taxon present in another sample. In some embodiments, a "significant increase" or "significant decrease" in relative abundance is defined as a statistically significant increase or statistically significant decrease relative to a reference value. In some embodiments, a statistically significant increase or statistically significant decrease is an increase or decrease of two, three, or four times the standard deviation of relative abundance. In some embodiments, a statistically significant increase or statistically significant decrease is an increase or decrease with a P value equal to or less than 0.1, 0.05, 0.01, or 0.005.

[0142] 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 taxa compared to each other or to a reference species or taxa using a nonparametric 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 that employs Bayesian inference and related methods.

[0143] In certain embodiments, the increase in relative abundance reflects an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more relative to a reference value. In some embodiments, the increase in relative abundance reflects an increase of 2-fold, 3-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold relative to a reference value.

[0144] 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 of the foregoing); a metabolite (e.g., a primary metabolite, a secondary metabolite); a fatty acid; a polyketide; a nucleotide; an amino acid; an auxiliary factor; and combinations, derivatives, and / or metabolites of the foregoing). The term "expression" includes, but is not limited to, one or more of the following: transcription of a gene into a pre-mRNA; processing of the pre-mRNA to produce a mature mRNA; mRNA stability; translation of the mature mRNA into a protein (including codon usage and tRNA availability); and / or glycosylation and / or other modifications of the translation product. The term "expression" also includes transcription of a non-coding RNA, e.g., a transfer RNA, a ribosomal RNA, a microRNA, a siRNA, a piRNA, a snoRNA, a snRNA, an exRNA, a scaRNA, or a long ncRNA. The term "expression" includes the production of a functional product and the production of a non-functional product, which can be used to produce a functional product by subsequent chemical or biochemical manipulation or synthesis.

[0145] As used herein, the term "phenotype" refers to an observable characteristic of a single cell, cell culture, organism, or population of organisms (e.g., a microbial consortium) that results from the interaction between the genetic makeup (e.g., genotype) of the single cell, cell culture, organism, or population of organisms and the environment.

[0146] As used herein, the term "metagenome" is defined as "the collective genome of all microorganisms present in a given habitat" (Handelsman et al., (1998) Chem. Biol. 5: R245-R249). However, the term is also intended to include clones, including genomes or genes extracted from environmental samples.

[0147] As used herein, "metagenomic DNA" refers to intact microbial-associated genomic DNA isolated from a complex sample such as an open natural environment (e.g., soil, water) or from the microbiome of a multicellular organism (e.g., human).

[0148] As used herein, a "metagenomic library" refers to a clonal collection of complete microbial-associated genomic DNA isolated in recombinant vectors from complex samples such as an open natural environment (e.g., soil, water) or from the microbiome of a multicellular organism (e.g., human).

[0149] As used herein, "genome" refers to the genetic material (eg, chromosomes) of an organism.

[0150] As used herein, the term "gene" refers to a nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide or a non-coding RNA and an expression control sequence operably linked to the nucleic acid sequence encoding the polypeptide or the non-coding RNA. For example, a gene may include a promoter, one or more enhancers, a nucleic acid sequence encoding a polypeptide or a non-coding RNA, a downstream regulatory sequence, and other nucleic acid sequences that may be involved in regulating RNA transcription from the gene.

[0151] As used herein, the term "genetic basis" refers to the underlying genetic or genomic cause of a particular observation.

[0152] As used herein, the term "genetic" refers to the heritable information encoded in the DNA nucleotide sequence. Thus, the term "genetic trait" is intended to refer to the sequencing, genotyping, comparison, mapping or other determination of the information encoded in the DNA.

[0153] As used herein, the term "genetic material" refers to the DNA that is passed from one generation to the next in an organism. Typically, genetic material refers to the genome of an organism. Extrachromosomal elements, such as organelles or plasmid DNA, can also be part of the genetic material that determines the characteristics of an organism.

[0154] As used herein, the term "genetic change" or "genetic adaptation" refers to one or more mutations within the genome of an organism. As used herein, the term "mutation" refers to the difference in the DNA nucleotide sequence of two related organisms, including, for example, substitutions, deletions, insertions and rearrangements, or the movement of mobile genetic elements.

[0155] As used herein, the term "taxon" is a group of organisms that are considered similar enough to be considered a single unit. Taxon can include, but is not limited to, a family, genus, species, or a group within a species (e.g., strain).

[0156] As used herein, the term "operational taxonomic unit" (OTU) refers to a group of microorganisms that are considered similar enough to be considered as a separate unit. OTUs may include taxonomic families, genera, or species, but are not limited thereto. OTUs are typically defined by comparing nucleotide sequences between organisms. In some cases, OTUs may include a group of microorganisms that are considered as a unit based on, for example, ≥97%, ≥95%, ≥90%, ≥80%, or ≥70% sequence identity between at least a portion of a distinguishing biomarker (such as a 16S rRNA gene).

[0157] As used herein, the term "assessment" means the observation or measurement of the observable phenotype of an organism. Assessment generally includes analysis, interpretation and / or comparison with the phenotype of another organism. It should be understood that phenotype can be assessed at the genetic level (e.g., about nucleotide sequence) and gene product level. In addition, phenotype can be assessed according to the behavior of the organism in the environment and / or the behavior of individual molecules or groups of molecules in the organism. This comparison can be used to determine the detailed function of the mutant product caused by genetic adaptation. Consortium or community can be assessed by observing or measuring the observable phenotype of the consortium or community.

[0158] As used herein, the term "stepwise" is intended to indicate a manner of a series of events one after another in time. As used herein, the term "simultaneously" is intended to indicate occurring at the same time.

[0159] As used herein, a "kilobase" (kb) or a "kilobase pair" (kbp) refers to 1000 nucleotides or 1000 base pairs of a nucleic acid (eg, DNA or RNA).

[0160] As used herein, the term "stable", when used in reference to a microbial community (e.g., a microbial community, a microbial consortium, a microbial culture, or other group, collection, or assemblage of microorganisms), refers to a microbial community 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 when the culture conditions (including external factors (light, nutrients, temperature, aeration, etc.) of the first culture and the second culture are the same. Thus, as used herein, the term "stability", when used in reference to a microbial community (e.g., "microbial community stability"), refers to a qualitative or quantitative indicator or measurement (e.g., as measured by the similarity measures discussed above) of the change in a microbial community (e.g., a microbial community, a microbial consortium, a microbial culture, or other group, collection, or assemblage 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 when the culture conditions (including external factors (light, nutrients, temperature, aeration, etc.) of the first culture and the second culture are the same.

[0161] In addition, as used herein, the term "stable", when used in reference to one or more functions provided and / or performed by a microbial community (e.g., a microbial community, a microbial consortium, a microbial culture, or other group, collection, or collection of microorganisms), refers to one or more functions that do not vary significantly from a first culture to a second culture (e.g., as measured by examining metagenomic sequences and / or by inferring functions therefrom) when a portion of the first culture is used to inoculate a culture medium to produce a second culture and when the culture conditions (including external factors (light, nutrients, temperature, aeration, etc.)) of the first culture and the second culture are the same. Thus, as used herein, the term "stability", when used to refer to one or more functions provided by a microbial community (e.g., "functional stability"), refers to a qualitative or quantitative indicator or measurement (e.g., measured by the similarity measures discussed above) of the change in one or more functions provided by a microbial community (e.g., a microbial community, a microbial consortium, a microbial culture, or other group, set, or collection 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 when the culture conditions (including external factors (light, nutrients, temperature, aeration, etc.) of the first culture and the second culture are the same. Thus, functional stability and microbial stability may be independent, such that a microbial community may be functionally stable but have changing membership and / or member abundance, such that the microbial community does not have microbial community stability. Thus, 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); a microbial community may have microbial community stability (e.g., regardless of the state of functional stability).

[0162] In some embodiments, microorganism can be " endogenous " of environment. As used herein, if microorganism is derived from the environment of its source, then this microorganism is considered as " endogenous " of environment. That is, if microorganism is found to be associated with described environment naturally, then this microorganism is endogenous to environment. In the embodiment that endogenous microorganism is applied to environment, the application amount of endogenous microorganism is different from the level in specific natural environment. Therefore, if microorganism is present in environment with non-natural level and / or if microorganism is applied to environment together with other organisms that are exogenous to environment and / or endogenous to environment but exist with non-natural level, then given environment endogenous microorganism can still improve environment.

[0163] In some embodiments, a microorganism can be "exogenous" (also referred to as "heterologous") to an environment. As used herein, a microorganism is considered "exogenous" to an environment if it is not derived from the environment from which it is derived. 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 can be considered exogenous to a second environment that naturally lacks the microorganism.

[0164] As used herein, "environmental sample" refers to a sample collected or obtained from any part of the environment (e.g., an ecosystem, an ecological niche, a habitat, etc.). Environmental samples may include liquid samples from rivers, lakes, ponds, oceans, glaciers, icebergs, rainwater, snow water, sewage, reservoirs, tap water, drinking water, etc.; solid samples from soil, compost, sand, rocks, concrete, wood, bricks, dirt, etc.; and gaseous samples from air, underwater radiators, industrial waste gas, vehicle exhaust, etc.

[0165] As used herein, the term "photosynthetic nitrogen-fixing microorganism" refers to a microorganism that can both photosynthesize and fix nitrogen.

[0166] Implementation

[0167] Provided herein is a technology related to a microbial consortium, the microbial consortium includes a microorganism capable of producing and / or delivering nutrients to the soil, for example, for agricultural purposes. In some embodiments, the microbial consortium includes many microorganisms. In some embodiments, the microbial consortium includes many biological systems, the biological system including components (e.g., enzymes, multi-enzyme complexes, metabolic pathways) provided by the microorganisms of the microbial consortium. In some embodiments, the biological system provides many biological functions, for example, photosynthesis (e.g., oxygenic photosynthesis and / or anoxygenic photosynthesis), nitrogen fixation, carbon fixation, and soil nutrients (e.g., phosphorus) are converted (e.g., dissolved) into a bioavailable form for plant use. In some embodiments, the microbial consortium includes microorganisms and / or biological systems that use photosynthetic energy to fix N and / or C from the air and produce microbial biomass. In some embodiments, the biological system includes a variety of enzymes provided by a variety of microorganisms. In some embodiments, the biological system includes an enzyme provided by a microorganism. In some embodiments, the mass (e.g., dry cell weight) of the microbial consortium is about 50% carbon and about 5% nitrogen.

[0168] In some embodiments, the technology includes methods for producing and / or optimizing microbial consortia to be applied directly (e.g., by spraying) on ​​crop fields, and growing microbial consortia with or in place of traditional plant-based cover crops. In some embodiments, the method includes growing the microbial consortium in a bioreactor. In some embodiments, the bioreactor includes a growth pond, and the microbial consortium grows in the growth pond under controlled conditions (e.g., controlled composition of the growth pond growth medium, controlled temperature, controlled light conditions (e.g., one or more incident wavelengths of light and light flux)). In some embodiments, the microbial consortium grows without providing fixed nitrogen in the growth medium. In some embodiments, the microbial consortium grows without providing fixed carbon in the growth medium. In some embodiments, the microbial consortium grows without providing fixed nitrogen in the growth medium and without providing fixed carbon in the growth medium. In some embodiments, the nitrogen and / or carbon is provided by atmospheric nitrogen (e.g., N in the atmosphere). 2 gases) and / or atmospheric carbon (e.g., atmospheric CO 2 That is, in some embodiments, the technology includes providing nitrogen and / or carbon from atmospheric nitrogen (e.g., atmospheric N 2 gases) and / or atmospheric carbon (e.g., atmospheric CO 2 The microbial consortium grows under conditions provided by gas.

[0169] In some embodiments, the controlled conditions include providing a controlled wavelength of light, a controlled spectrum, and / or a controlled photon flux of light. In some embodiments, a broad spectrum (e.g., "white") light is provided, e.g., light having a wavelength spectrum 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).

[0170] In some embodiments, the technology provides methods for preparing a composition comprising a microbial consortium and applying the composition directly (e.g., by spraying) to a crop field. In some embodiments, the microbial consortium continues to grow after being applied to a crop field. In some embodiments, the technology provides methods for preparing a composition comprising a microbial consortium, applying the composition directly (e.g., by spraying) to a crop field, and growing the microbial consortium with or instead of a traditional plant-based cover crop. Thus, applying a composition comprising a microbial consortium to a field provides N to crops growing in the field while also removing CO from the air. 2and deposits the fixed carbon in the soil. Higher soil carbon sequestration is associated with higher soil performance, including the ability to retain nutrients and stability against runoff. Therefore, in some embodiments, the technology can be used to provide growers with a low-cost carbon and nitrogen fixation technology, such as during the shoulder season.

[0171] As described herein, embodiments of the technology deliver fixed N and C to the field and reduce greenhouse gases. However, in addition to N and C, crops require additional nutrients such as phosphorus (P), potassium (K), manganese, magnesium, calcium, iron, boron, sodium, etc.

[0172] Regarding phosphorus, phosphorus cannot be captured from the air like N. Therefore, phosphorus is provided by mining phosphate minerals from the soil and adding them to fertilizers in this form. The availability of free P in the soil can be a problem because P tends to be tightly bound to soil components and is therefore difficult for plants to access and utilize. The availability of soil P to plants depends on the pH and is greatest at pH values ​​of approximately 6 to 7. When soil pH is below 6, phosphates tend to bind to metal ions such as iron or aluminum; above pH 7, phosphorus tends to complex with calcium. In addition, excessive application of phosphorus to fields has caused deterioration of water supplies and fisheries due to eutrophication due to hypoxia. Therefore, there is a need for technologies that more efficiently deliver phosphorus so that it can be accessed by crops without entering waterways.

[0173] Therefore, specific embodiments of the technology provide P in biomass (e.g., present in the biomass of a microbial consortium in a biologically useful form). In some embodiments, the microbial consortium includes microorganisms that easily accumulate phosphorus and bind phosphorus to biomass. In some embodiments, the microbial consortium includes microorganisms that accumulate polyphosphates (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 nutrient requirements of microbial consortia applied to fields and related crops. In some embodiments, the microbial consortium includes microorganisms that migrate bound phosphates from the soil and provide them to crops or other microorganisms in a bioavailable form.

[0174] As described herein, the technology provides a biological method for producing nitrogen fertilizers, which can partially or completely replace the chemical production of fertilizers. In addition, 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, for example, because it can reduce greenhouse gases, reduce the use of fossil fuels (e.g., by reducing or minimizing the use of the Haber-Bosch process), increase the efficiency of phosphorus use, and reduce denitrification in agricultural fields.

[0175] During the development of embodiments of the technology, experiments were conducted that showed that methods for producing microbial consortia using selective conditions reduced the complexity of the microbial community while selecting for carbon and nitrogen fixation functions. Although most environmental samples included phototrophic organisms that were able to survive without supplementation of culture media containing nitrogen or carbon compounds, three cultures (BW01, BW02, and BW05) performed particularly strongly in early passages. The data also showed that these cultures were capable of high productivity levels in liter-scale liquid cultures and were capable of large-scale growth in sterile soil. In addition, experiments showed that simplified versions of these consortia grew well in liquid culture.

[0176] Microbial consortium function

[0177] As described herein, the technology relates to producing, providing and / or using a microbial consortium comprising a plurality of microorganisms and providing a plurality of functions. In some embodiments, the microbial consortium comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more microorganisms. In some embodiments, the microbial consortium comprises about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 or more microorganisms.

[0178] In some embodiments, the microbial consortium includes a plurality of enzymes, multi-enzyme complexes, biological systems, biological pathways, or biological functions. In some embodiments, the enzymes, multi-enzyme complexes, biological systems, biological pathways, or biological functions provide photosynthesis (e.g., oxygenic photosynthesis and / or anoxygenic photosynthesis), nitrogen fixation, carbon fixation, and conversion of nutrients into plant-usable biological forms.

[0179] For example, embodiments of the technology provide a microbial consortium that performs photosynthesis (e.g., oxygenic photosynthesis and / or anoxygenic photosynthesis) and nitrogen fixation, and transfers carbon and electrons between metabolic functions of the consortium and / or microbial cells ( Fig.17). In particular, in some embodiments, the microbial consortium performs photosynthesis (e.g., oxygenic photosynthesis), for example, by capturing light energy (e.g., from the sun when grown in a field, or from illumination when grown in a photobioreactor), and using that energy to remove electrons from water, drive the Calvin-Benson Cycle, and fix carbon from atmospheric carbon dioxide. Fixed carbon shuttles produce molecules, including simple and complex carbohydrates. Microbial consortia utilize the energy, electrons, and protons produced by photosynthesis to fix nitrogen (e.g., biological nitrogen fixation). Nitrogen fixation can be performed in phototrophic organisms (as in the case of Nostoc species) or in separate organisms that consume sugars or other carbon compounds secreted by phototrophic organisms. Microbial consortia include the flow and / or movement of nitrogen (e.g., in the form of fixed nitrogen compounds between metabolic functions and / or cells in the consortium). Microbial consortia include the flow and / or movement of carbon between metabolic functions and / or cells in the consortium (e.g., in the form of fixed carbon compounds (e.g., carbon compounds including four or more carbons)). Microbial consortia include the flow and / or movement of electrons between metabolic functions and / or cells in the consortium (e.g., carried by electron shuttle molecules). Anoxygenic phototrophs form the main source of electrons for the consortium, for example, by using sunlight energy to remove electrons from water in the reaction center of photosystem II. These electrons are transferred by various carbon-containing compounds (e.g., sugars and polysaccharides) secreted by phototrophs. Nitrogen fixation requires reducing equivalents (electrons) and energy carried by ATP, both of which can be obtained by oxidation of these carbon-containing compounds. In addition, electrons can be recovered from the hydrogen released during nitrogen fixation. This hydrogen can also serve as a source of electrons for anoxygenic photosynthesis in the anaerobic zone of the biofilm.

[0180] Therefore, without being bound by theory, it is expected that the microorganisms of the microbial consortium capture light energy (for example, in a field or a photobioreactor) by photosynthesis (for example, oxygenic photosynthesis), and use this energy to transfer electrons from water to atmospheric carbon dioxide (for example, by the Calvin-Benson cycle) to fix carbon, for example in the form of simple and complex carbohydrates. In addition, it is expected that the energy, electrons and protons produced by photosynthesis or by the photosynthetic organisms (for example, Nostoc species) of the consortium, or by consuming another organism of the fixed carbon compounds (for example, simple carbohydrates, complex carbohydrates, other carbon compounds) secreted from the phototrophic organism, are used for biological nitrogen fixation (BNF). The fixed carbon compounds secreted by the consortium but not consumed by the members of the consortium provide soil organic carbon (SOC) or humus, thereby improving soil and isolating carbon from the atmosphere. Therefore, in some embodiments, the technology relates to a consortium that produces carbon compounds, which sequesters carbon from the atmosphere. In some embodiments, the technology provides a consortium including consortium members that produce carbon compounds, which sequesters 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 durable carbon compounds that provides a stable carbon sink to remove atmospheric carbon (e.g., CO 2 ) and stores carbon in durable carbon compounds (e.g., melanin) in the soil. Embodiments provide a microbial organism that provides a stable carbon sink to remove atmospheric carbon (e.g., CO 2 ) and store carbon in durable carbon compounds (e.g., melanin) in the soil.

[0181] In some embodiments, it is contemplated that the consortium includes microorganisms that dissolve and / or mobilize phosphate; or the consortium includes enzymes, multi-enzyme complexes, biological systems, biological pathways, or biological functions for dissolving and / or mobilizing phosphate. It is contemplated that the phosphate dissolving and / or mobilizing microorganisms consume fixed carbon compounds (e.g., simple carbohydrates, complex carbohydrates, other carbon compounds) secreted from phototrophs.

[0182] In some embodiments, technology provided herein relates to providing, producing and / or using a microbial consortium comprising photosynthetic organisms, nitrogen-fixing organisms and / or carbon-fixing microorganisms. In some embodiments, the photosynthetic organisms of the consortium also fix nitrogen and / or also fix carbon. For example, in some embodiments, the microbial consortium comprises cyanobacteria (for example, Nostoc species). In addition, in the development process of this technology embodiment, the data collected show that some organisms (for example, Nostoc species) grow faster than these organisms (for example Nostoc species) in the microbial consortium (combined with other organisms) alone (when not associated with other organisms in the microbial consortium). It is expected that some organisms (non-photosynthetic heterotrophs) in the consortium process organic molecules and hydrogen, which increases the growth rate of other organisms (for example, photosynthetic organisms such as Nostoc species). In addition, in some embodiments, the microbial consortium comprises nitrogen-fixing microorganisms from genera such as Rhizobium, Bradyrhizobium or Azospirillum. While Rhizobium and Bradyrhizobium are known to fix nitrogen when associated with legumes, Azospirillum is a free-living nitrogen-fixing microorganism.

[0183] In the development process of the embodiments of the technology described herein, experiments were conducted to apply controlled selection conditions to environmental samples, thereby reducing the complexity of microbial communities while selecting strong carbon and nitrogen fixation activities. Although most environmental samples appear to contain phototrophic organisms that can survive without combined nitrogen or carbon, three cultures (referred to herein as "BW01", "BW02" and "BW05") performed particularly strongly. See below and Examples, such as Example 20. In addition, in the development process of the embodiments of the technology described herein, experiments were conducted to produce minimal consortia from additional environmental samples using the methods described herein for producing minimal consortia. See, for example, Example 21.

[0184] method

[0185] In some embodiments, a selection method is used to produce a microbial consortium. In some embodiments, the method includes a method for producing a functional microbial consortium, as described in U.S. Patent Application Serial No. 17 / 544,879, the entire content of which is incorporated herein by reference. For example, in some embodiments, a method for screening a culture by evaluating the nucleotide sequence (for example, metagenome sequence) present in the culture after growth and / or by testing the functional characteristics of the culture is used to produce a microbial consortium. In some embodiments, one or more environmental samples (for example, environmental samples with a high organic matter content) can be collected. If a single environmental sample is collected, the method includes homogenizing the environmental sample to provide an input sample. If a plurality of environmental samples are collected, the method includes mixing a plurality of environmental samples to provide a mixed environmental sample and homogenizing the mixed environmental sample to provide an input sample.

[0186] In the embodiment comprising using multiple environmental samples to produce input samples, collecting and mixing multiple environmental samples can not only be used to maximize the statistical sample space of the microorganism to be screened, but also can be used to maximize the microbial combination present in the microbial consortium, which is identified and / or produced using the technology described herein for use in the input sample. In addition, collecting and mixing multiple environmental samples to produce the input sample of application technology described herein can produce novel microbial consortiums that do not exist in nature by combining microorganisms that do not usually live in the same environment in nature. In some embodiments, various environmental samples from different geographic regions can be mixed to further increase the statistical sample space of the microbial consortium combination. For example, the embodiment provides that multiple environmental samples can be obtained, wherein each environmental sample is taken from different ecosystems, habitats and / or niches. The embodiment further provides that multiple environmental samples can be obtained from locations separated from each other by 1m, 10m, 100m, 1000m, 10,000m or more than 10,000m. In some embodiments, the sample is obtained from two or more points anywhere on the earth, including above and below the surface of the earth's land and waters.

[0187] In some cases, multiple input samples can be produced during collection. Each input sample in multiple input samples can include the different combinations of individual environmental samples mixed together. For example, environmental samples A, B and C (from one or more different ecosystems, habitats and / or niches) can be mixed to provide an input sample comprising 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 niches) can be mixed to provide an input sample comprising 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) can be mixed to provide an input sample comprising 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 of the plurality of input samples can include a range of fractional compositions of any two of the plurality of individual environmental samples mixed together to provide the input sample. For example, any two individual environmental samples can be mixed together to provide an input sample comprising a portion of the composition of the first environmental sample ranging from 0.01 to 0.99 (e.g., a first environmental sample comprising 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) and comprising a portion of the composition of the second environmental sample ranging from 0.99 to 0.01 (e.g., a second environmental sample comprising 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).

[0188] The amount of mixed environmental samples and the variation of types can be used to separate and develop input samples. This is because people recognize that the combination of microorganisms may not only be beneficial, but may also cause individual microorganisms to become less effective or be dominated by microorganisms from external environmental samples. In addition, the embodiment of this technology includes the use of a single environmental sample that is homogenized to provide an input sample. Those of ordinary skill in the art understand that a single environmental sample can comprise a plurality of single ecosystems or niches that are not mixed in nature but become mixed when a single sample is homogenized. For example, an environmental sample may comprise a plurality of independent subsamples, and these subsamples exist as independent strata (strata), layers or sub-communities, for example, the strata of a cylindrical soil core sample, the strata of a microbial mat sample, the strata of a water column sample, the sub-community of a microbial community comprising a biofilm, etc.

[0189] Thus, embodiments of the methods provided herein include using a single environmental sample, homogenizing it to provide an input sample and / or include using multiple environmental samples, mixing and homogenizing them to provide an input sample.

[0190] In some embodiments, selection of an input sample (e.g., an environmental sample or a mixed environmental sample of a plurality of mixed environmental samples) based on one or more criteria can be performed. The culture of the input sample can be grown under one or more environmental conditions. In some embodiments, the culture medium is provided without nitrogen compounds (e.g., without a fixed nitrogen source) or carbon compounds (e.g., without a fixed carbon source). Thus, in some embodiments, the culture medium is described as a nitrogen-free and carbon-free culture medium or a "C / N-free culture medium". By supplying nitrogen from ambient air concentrations or by supplying nitrogen with anoxic N 2 The input sample can be nitrogen fixed by bubbling and supplying salts and other nutrients required by known microorganisms for nitrogen fixation. The input sample can also be subjected to CO 2 , for example, by ambient air concentration or by CO 2 Bubbling.

[0191] In some embodiments, the culture medium is a freshwater replacement medium (e.g., BG-11; see Examples). In some embodiments, the method includes the use of a saltwater or seawater culture medium (or a saltwater replacement culture medium or a seawater replacement culture medium), wherein nitrogen-fixing and carbon-sealing microorganisms adapted to seawater or other saltwater sources are selected according to the methods described herein. In some embodiments, the saltwater or seawater culture medium (or a saltwater replacement culture medium or a seawater replacement culture medium) provides trace nutrients; in some embodiments, the saltwater or seawater culture medium (or a saltwater replacement culture medium or a seawater replacement culture medium) is supplemented with trace nutrients. In some embodiments, a culture medium is produced from evaporated seawater by adding water and other nutrients (e.g., iron). In some embodiments, trace elements (e.g., compounds comprising one or more of boron, manganese, zinc, molybdenum, copper, cobalt and / or iron) are added to the culture medium.

[0192] After incubation and timing, the culture can be tested based on one or more variables, for example, for increased carbon and nitrogen or fixed CO 2And / or the activity of the increase of nitrogen. It can be measured by mass. In some embodiments, the metagenome and / or genomic DNA of the microorganism including the candidate microbial consortium is separated and sequenced for identification. In some embodiments, biomarkers are used to identify one or more microorganisms. In some embodiments, the culture is tested on nitrogen-free and carbon culture medium (liquid culture medium or solid culture medium) to measure survival time and / or persistence. In some embodiments, the selection of one or more microbial cultures and / or the specific part of one or more microbial cultures is carried out to provide the culture for testing. In some embodiments, the test includes assessing the culture for carbon capture, nitrogen fixation and / or persistence. In some cases, additives are provided to the culture to promote the absorption of the environment (such as soil) or culture medium to the microbial consortium. For example, the microbial consortium may need carbon, energy, nitrogen, trace nutrients and reducing equivalents. The above process can be iterated several times by multiple iterations, and each iteration further separates and generates the identification information of the microorganisms and specific microbial consortiums that realize the desired results in the selected variables (such as climate variables, carbon sequestration, nitrogen fixation and survival time / persistence). In some embodiments, statistical models and computational methods (including machine learning) help select microorganisms and microbial consortia for further testing.See, for example, U.S. Patent Application Serial No. 17 / 544,879, the entire contents of which are incorporated herein by reference. For example, embodiments provide data development into a machine learning model, which associates microorganisms and biomarkers and microbial combinations with the variables in the test. The selection of the microbial consortia for testing and / or the selection of environmental sample characteristics are suggested by the machine learning model as the result based on the variable in the test. In some embodiments, the expected phenotype and the expected result about the variable in the test are input, and the machine learning model can suggest related microorganisms for further testing.

[0193] In some embodiments, the technology provides machine learning techniques to identify microbial species and other information relevant to one or more variables. In some embodiments, the process includes a series of operations that can be implemented with hardware, software or a combination thereof. In the context of software, the method includes computer executable instructions, and when implemented by one or more processors, these instructions perform the operations. Usually, computer executable instructions can include routines, code segments, programs, objects, components, data structures, etc. that perform specific functions or realize specific abstract data types. The order of describing the operations is not intended to be interpreted as a restriction, and any number of described frames can be combined in any order and / or in parallel to realize the process.

[0194] In some embodiments, the technology provides additional methods for selecting a microbial consortium that provides a specific function. In some embodiments, the technology provides a method for screening microbial communities, microbial consortiums and / or multiple microorganisms to produce and / or identify a microbial consortium that provides a specific function. In some embodiments, the technology produces a microbial consortium that does not exist in nature by combining microorganisms from different environments, niches and / or habitats (e.g., microorganisms that do not exist together in nature).

[0195] In some embodiments, the method includes providing a sample comprising 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; generating 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 the set of selective conditions; generating an N+1th taxonomic classification of microorganisms in the N+1th culture; and deriving a measure of microbial community stability of the N+1th culture relative to the Nth culture using the N+1th taxonomic classification and the Nth taxonomic classification. The measure of microbial community stability is monitored to identify that 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 following steps of the method are repeated by providing the N+1th sample as the Nth sample: inoculation, growth, generating taxonomic classifications, and deriving a measure of microbial community stability. If the measure of microbial community stability has reached a plateau value, the method includes providing a stable N+1th culture as a culture comprising a consortium of microorganisms that performs a specific function. In some embodiments, the steps of inoculating, growing, generating a taxonomic classification, and deriving a measure of microbial community stability are repeated 2, 3, 4, 5, 6, 7, 8, 9, or 10 times or more.

[0196] In some embodiments, the method further comprises isolating each microorganism of the stable microbial consortium in pure culture. In some embodiments, the method further comprises obtaining the genome sequence of each microorganism of the stable microbial consortium in pure culture. In some embodiments, the method further comprises storing the stable microbial consortium and / or each microorganism of the stable microbial consortium (e.g., by freezing (e.g., at -80°C)). In some embodiments, the method further comprises measuring a specific function of the stable microbial consortium using a test substrate and a method of measuring a functional output.

[0197] In some embodiments, the technology provides an iterative method in which a portion of the first culture is used to inoculate a second volume of fresh culture medium. Thus, in some embodiments, a portion of the 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., containing the same or different growth medium as in 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 an 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 previous culture (e.g., first, second, third, fourth, fifth, sixth or seventh culture, respectively) inoculated with a culture. As used herein, the iterative culturing process by inoculating the N+1 culture with a portion of the N culture is referred to as "passaging" the culture.

[0198] In addition, the culture inoculated directly from the environmental sample may be referred to herein as a P0 (zero) culture; the first passage includes using a portion of the P0 culture to inoculate fresh culture medium to produce a P1 culture; the second passage includes using a portion of the P1 culture to inoculate fresh culture medium to produce a P2 culture; the third passage includes using a portion of the P2 culture to inoculate fresh culture medium to produce a P3 culture; the fourth passage includes using a portion of the P3 culture to inoculate fresh culture medium to produce a P4 culture; the fifth passage includes using a portion of the P4 culture to inoculate fresh culture medium to produce a P5 culture; the sixth passage includes using a portion of the P5 culture to inoculate fresh culture medium to produce a P6 culture; the seventh passage includes using a portion of the P6 culture to inoculate fresh culture medium to produce a P7 culture; the eighth passage includes using a portion of the P7 culture to inoculate fresh culture medium to produce a P8 culture; and the Nth passage includes using a portion of the P(N-1) culture to produce a PN culture. As used herein, the term "passage number" refers to a specific passage indicated by a number, for example, passage number 1 refers to the first passage, passage number 2 refers to the second passage, etc.

[0199] In some embodiments, the volume of a portion of the Nth (e.g., first) culture used to inoculate the N+1th (e.g., second) culture is 100 μl to 100 L or more, depending on the scale of the culturing 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 a culture and adding that volume to fresh culture medium. In some embodiments, the ratio of the inoculation volume to the volume of the fresh medium is about 1:10 to 1:1000. Thus, in some embodiments, the volume of the fresh medium is 1 ml to 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).

[0200] Growth of cultures

[0201] In some embodiments, the technology provides methods for reducing the complexity of a microbial community (e.g., present in an environmental sample) while selecting a microbial consortium that performs a specific function and / or identifying a microbial consortium that performs a specific function. Exemplary functions that can be selected and / or identified for a microbial consortium include, for example, photosynthesis (e.g., oxygenic photosynthesis and / or anoxygenic photosynthesis), phosphorus dissolution or mobilization, biodegradation, fermentation, generation of chemical precursors, biosensing, nitrogen fixation, carbon fixation, and / or generation of persistent carbon compounds (e.g., melanin production) for providing a carbon sink to remove carbon from the atmosphere.

[0202] In some embodiments, an environmental sample is used to inoculate a culture medium and the inoculated culture medium is grown 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 matrix (such as sand or other solid matrix)) or by selective conditions provided external to the growth medium (e.g., temperature; humidity; presence, absence, wavelength, and / or intensity of light; light / dark cycle; pressure; culture volume; culture volume material, size, or geometry; presence, absence, type, or intensity of culture agitation; presence, absence, and / or type of provided gas).

[0203] In some embodiments, the culture is inoculated and grown for a period of time, e.g., 30 to 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. , 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, 20.5, 21.0, 22.5, 23.0, 24.5, 25.0, 26.5, 27.0, 28.5, 29.0, 30.5, 31.0, 32.5, 33.0, 34.5, 35.0, 36.5, 37.0, 38.5, 39.0, 40.5, 41.0, 42.5, 43.0, 44.5, 45.0, 46.5, 47.0, 48.5, 49.0, 50.5 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 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).

[0204] In some embodiments, the empirical measurement of growth rate, time of exponential growth phase, time of culture saturation or other culture growth characteristics is measured to identify the length of time for culture growth. In some embodiments, the growth time is selected to provide a culture at the end or near the end of the exponential growth phase, thereby providing a culture with a robust type and quantity of microorganisms for further characterization and / or selection. In some embodiments, the measurement of the absolute or relative number of microorganisms in a determined culture volume is used to quantitatively and / or qualitatively measure growth. In some embodiments, by using light scattering, measuring the dry mass or wet mass of solids (such as cells) separated from the culture, using a part of the culture to count colonies grown on solid culture media, or measuring the culture or a part thereof with some other features that are related or causal to the number of microorganisms in the culture, the absolute or relative number of microorganisms in a certain volume of culture is measured. In some embodiments, the growth is characterized by determining a growth curve; in some embodiments, the growth is characterized by determining the doubling time and / or half-saturation time. In some embodiments, the growth rate is modeled using empirical data (e.g., using a logarithmic growth model).

[0205] Genetic characteristics

[0206] In some embodiments, the microorganisms in the culture are characterized by shotgun metagenomic sequencing. The techniques and systems for obtaining genetic sequences from a variety of organisms in samples such as environments or clinical samples are well known to those skilled in the art. See, for example, Aihara et al. (Appl. Environ. Microbiol. (1996) 62: 316-322) provide robust nucleic acid extraction and purification. The scheme can also be modified according to the experimental objectives and environmental sample types such as soil, sediment and groundwater. Many commercially available DNA extraction and purification kits can also be used. Samples with less than 2pg purified DNA may need to be amplified, which can be performed using conventional techniques known in the art, such as whole community genome amplification (WCGA) methods (Wu et al., Appl. Environ. Microbiol. (2006) 72, 4931-4941). The techniques and systems for obtaining purified RNA from environmental samples are also well known to those skilled in the art. For example, the method described by Hurt et al. (Appl. Environ. Microbiol. (2001) 67: 4495-4503) can be used. This method can separate DNA and RNA in the same sample at the same time. Gel electrophoresis can also be used to separate community RNA (McGrath et al., J. Microbiol. Methods (2008) 75: 172-176). Samples with less than 5pg of purified RNA may need to be amplified, which can be performed using conventional techniques known in the art to obtain cDNA, such as whole community RNA amplification method (WCRA) (Gao et al., Appl. Environ. Microbiol. (2007) 73: 563-571). In some embodiments, environmental sampling and DNA extraction are performed as described above (DeSantis et al., Microbial Ecology, 53 (3): 371-383, 2007).

[0207] Sequencing methods can be carried out to the nucleic acid (e.g., metagenomic DNA) of separation to obtain metagenomic sequencing data. Sequencing methods can be roughly divided into methods that usually use template amplification and those that do not use template amplification. The method that needs to be amplified includes pyrophosphate sequencing, Life Technologies / Ion Torrent, Solexa platform, GnuBio, and Supported Oligonucleotide Ligation and Detection (SOLiD) platform commercialized by Roche as 454 technology platforms (e.g., GS20 and GS FLX), commercialized by Illumina. Non-amplification methods, also referred to as single molecule sequencing, such as the HeliScope platform commercialized by Helicos BioSciences, and the emerging platforms commercialized by VisiGen, Oxford Nanopore Technologies Ltd., and Pacific Biosciences, respectively. Therefore, in some embodiments, metagenomic shotgun sequencing includes pyrophosphate sequencing, ligation sequencing, single molecule sequencing, sequencing by synthesis (SBS), semiconductor sequencing, nanopore sequencing, massively parallel cloning, massively parallel single molecule SBS, massively parallel single molecule real-time, massively parallel single molecule real-time nanopore technology, etc. Morozova and Marra provide a review of some such technologies in Genomics, 92:255 (2008), the entire contents of which are incorporated herein by reference. Those of ordinary skill in the art will recognize that, because RNA is less stable in cells and is more susceptible to nuclease attack, RNA is usually reverse transcribed into DNA before sequencing in experiments.

[0208] Specific descriptions of some DNA sequencing technologies include fluorescence-based sequencing methods (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 techniques; parallel sequencing of partitioned amplicons (PCT Publication No.: WO2006084132 by 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 by Macevicz et al. and U.S. Pat. No. 6,306,597 by Macevicz et al., both of which are incorporated herein by reference in their entirety). Other descriptions of sequencing technologies include Church polony technology (Mitra et al., 2003, Analytical Biochemistry 320, 55-65; Shendure et al., 2005 Science 309, 1728-1732; U.S. Pat. No. 6,432,360, U.S. Pat. No. 6,485,944, U.S. Pat. No. 6,511,803; which are incorporated herein by reference in their entireties), 454 pico-titer pyrosequencing technology (Margulies et al., 2005 Nature 437, 376-380; US20050130173; which are incorporated herein by reference in their entireties), Solexa single base addition technology (Bennett et al., 2005; et al., 2005, Pharmacogenomics, 6,373-382; U.S. Pat. No. 6,787,308; U.S. Pat. No. 6,833,246; which are incorporated herein by reference in their entireties), Lynx massively parallel signature sequencing technology (Brenner et al. (2000). Nat. Biotechnol. 18:630-634; U.S. Pat. No. 5,695,934; U.S. Pat. No. 5,714,330; which are incorporated herein by reference in their entireties), and Adessi PCR colony technology (Adessi et al. (2000). Nucleic Acid Res. 28, E87; WO00018957; which are incorporated herein by reference in their entireties). 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 herein incorporated by reference in its entirety).

[0209] Sequence analysis

[0210] In some embodiments, the metagenomic nucleotide sequence data is analyzed to characterize the microbial community (e.g., microbial consortium) from which the metagenomic nucleic acid is obtained. For example, in some embodiments, by obtaining metagenomic nucleotide sequence data from a microbial community and using an algorithm (e.g., using a selected database) that associates short genome substrings (k-mers) in the metagenomic nucleotide sequence data with the lowest common ancestor (LCA) taxonomic group, the taxonomic units in the microbial community are taxonomically classified and / or identified. 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 taxa present in a microbial community (e.g., a 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 taxa in microbial communities using metagenomic sequence data from microbial communities include, for example, MEGAN (see, e.g., Huson (2007) “MEGAN analysis of metagenomic data” Genome Res 17:377–386, incorporated herein by reference); 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 incorporated herein by reference); and Naive Bayes classifier (NBC) (see, e.g., Rosen (2008) “Metagenome fragment classification using N-mer frequency profiles” Adv Bioinformatics 2008: 1–12, incorporated herein by reference). In some embodiments, the average nucleotide identity (ANI) provided by the GTDB-tk package is used to classify ribosomal RNA sequences. See, for example, 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 herein by reference.

[0211] In some embodiments, characterizing a microbial community comprises identifying in absolute and / or relative terms the taxa (e.g., strains, subspecies, species, genus, family) of organisms present in the microbial community. In some embodiments, characterizing a microbial community comprises, for example, identifying in relative terms the taxa (e.g., strains, subspecies, species, genus, family) of organisms that are enriched in a particular passage relative to a previous passage or an initial environmental sample.

[0212] In some embodiments, an organism is identified by comparing a nucleotide sequence to a database of ribosomal RNA gene sequences (e.g., nucleotides of 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 plasmid 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).

[0213] Those of ordinary skill in the art understand that there is no clear consensus on bacterial classification systems. Thousands of bacterial genomes are sequenced every year, including genomes that can only be found as members of consortia. Therefore, new taxonomic classifications of these organisms continue to develop. See, for example, Coleman (2021) "A rooted phylogeny resolves early bacterial evolution" Science 372: (6542): eabe0511, incorporated herein by reference. A variety of analytical tools (e.g., ANI and BLAST) are used herein to determine the species name of rRNA nucleotide sequences and identify the associated taxonomy. The bacterial classification system provided by sequence database hits using BLAST and the NCBI nucleotide sequence database is sometimes inconsistent with the classification provided by others. For example, the Burkholderia genus is in the β-proteobacteria class in NCBI, but in the γ-proteobacteria class in other databases. In all cases, the species indicated are correct, and the assignment to a wider taxonomic hierarchy has combined information from the multiple analytical methods used.

[0214] Other software packages for generating and analyzing nucleotide sequences are fastp, bowtie2, and FATQC for quality fine-tuning and assessment; metaSPADES and MEGAHIT2 for assembly; QUAST for assessing assembly; METABAT2 for binning; GTDB-tk for taxonomic classification; and Barrnap for ribosomal RNA prediction. See Example 20.

[0215] Community and consortia analysis

[0216] In some embodiments, the stability of microbial communities and / or microbial consortia is measured, for example, by deriving a measure of the similarity (or dissimilarity) between a first culture and a second culture inoculated with a portion of the first culture, and, optionally, following a measure of the similarity as a function of subsequent inoculation. In some embodiments, the taxonomic classification and / or identification of organisms in microbial communities (e.g., as provided by the taxonomic classifier described above (e.g., Kraken2)) can provide input for such stability measurements. In some embodiments, functional capabilities or functions (e.g., genes, gene products, functional capabilities and / or activities) provided by microbial communities and / or present in microbial communities provide input for stability measurements.

[0217] Various measures can be used to compare the similarity (or dissimilarity) of microbial communities, including estimates of microbial community 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) and 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 distance 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). The correlation between Bray-Curtis distance matrices of community data, geographic distance, and 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. 3rd Edition English Edition. Elsevier, incorporated herein by reference).

[0218] In the development process of the embodiments of the technology described herein, the Bray-Curtis dissimilarity index is used to evaluate the stability of the microbial consortium (see Examples). The Bray-Curtis dissimilarity index is between 0 and 1, wherein a score of 0 indicates that there is no difference between two cultures, communities, consortia, places, passages or other microbial groups under comparison; a score of 1 indicates that two cultures, communities, consortia, places, passages or other microbial groups have no common species under comparison. Therefore, the Bray-Curtis dissimilarity index provides a useful metric to measure the amount of change that occurs between each individual passage. When the Bray-Curtis dissimilarity score begins to stabilize between passages (e.g., a graph of the Bray-Curtis dissimilarity score as a function of passage and / or time approaches a horizontal asymptote), the consortium approaches maximum stability, and therefore has minimal or no change from one passage to the next and / or between an inoculum and a subsequent culture produced by the inoculum.

[0219] Some context for interpreting Bray-Curtis dissimilarity scores can be obtained by comparing well-studied microbiomes, such as those associated with the human body. In a comparative study using a reference set of human microbiome data, the Bray-Curtis dissimilarity between different microbiomes (fecal and nasal samples) was 0.79 ± 0.08 (SD), and the average Bray-Curtis dissimilarity of individual fecal microbiomes compared to the reference dataset was 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). Therefore, lower measurement numbers represent the variability of the microbiome in fecal samples, while higher measurement values ​​indicate variation between the intestinal and respiratory systems. These values ​​are illustrative, and it should be understood that the measure of Bray-Curtis dissimilarity between other samples may be different and may vary more widely between 0.00 and 1.00.

[0220] Several tools are available that provide these and other estimates of microbial community structure (e.g., describing the abundance of community members). See, e.g., 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. 69:4374-4376, each of which is incorporated herein by reference), structures" Appl. Environ. Microbiol. 72:2379-2384, each of which is incorporated herein by reference), UniFrac (Lozupone (2005) "UniFrac: a new phylogenetic method for comparing microbial communities" Appl. Environ. Microbiol.71:8228-8235, incorporated herein by reference), 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, incorporated herein by reference); and SONS (Schloss (2006) "Introducing SONS, a computer program for defining operational taxonomic units and estimating species richness" Appl. Environ. Microbiol. 71:1501-1506, incorporated herein by reference); Tool for Operational Taxonomic Unit-Based Comparisons of Microbial Community Memberships and Structures" Appl Environ Microbiol. 72:6773–6779, incorporated herein by reference), which provides several metrics, including membership (e.g., based on incidence). Similarity indices), community structure using abundance (e.g., Clayton θ (see, e.g., Yue (2001) “A nonparametric estimator of species overlap” Biometrics 57:743-9, incorporated herein by reference), and community 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. NZ J Stat. 48:117-128, each of which is incorporated herein by reference).

[0221] sample

[0222] The technology is not limited to the type of sample (e.g., environmental sample) containing microorganisms used as the starting material (e.g., input sample) for performing the method as described herein (e.g., method for selecting microbial consortium and / or screening to identify microbial consortium). In some embodiments, the input sample used can be an environmental sample from any source, for example, a naturally occurring or artificial atmosphere, water system and source, soil, or any other sample of interest. In some embodiments, the environmental sample can be obtained from, for example, an indoor or outdoor air or atmospheric particle collection system; indoor surfaces and surfaces of machines, equipment, or instruments. In some embodiments, the ecosystem is sampled (e.g., in some embodiments, the sample is an environmental sample obtained from the ecosystem). The ecosystem can be terrestrial, including all known terrestrial environments, including but not limited to soil, surface, and above-surface environments. Ecosystems include ecosystems classified in the Food and Agriculture Organization Land Cover Classification System (LCCS) and the Forest Range Environmental Research Ecosystem (FRES) developed by the United States Forest Service. Exemplary ecosystems include forests, such as tropical rainforests, temperate rainforests, temperate broadleaf forests, boreal forests, coniferous forests, and mountain coniferous forests; grasslands, including savannas and grasslands; deserts; wetlands, including herbaceous swamps (quagmires), marshes, peat bogs (mud ponds), estuaries, and mires; riparian ecosystems, alpine and tundra ecosystems. Ecosystems also include ecosystems associated with aquatic environments, such as lakes, rivers, springs, coral reefs, beaches, estuaries, seamounts, trenches, and intertidal zones. Ecosystems also include soil, humus, mineral soils, and aquifers. Ecosystems also encompass underground environments, such as mines, oil fields, caves, faults and fracture zones, geothermal zones, and aquifers. Ecosystems also include microbial communities associated with plants, animals, and humans. Exemplary plant-associated microbial communities include microbial communities found in or near roots, bark, trunks, leaves, and flowers. Animal and human associated microbiomes include those found in the gastrointestinal tract, respiratory system, nares, urogenital tract, mammary glands, oral cavity, ear canal, feces, urine, and skin. In some embodiments, the sample can be any kind of clinical or medical sample. For example, the sample can be from the blood, urine, feces, nares, lungs, or intestines of a mammal.

[0223] 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 mixing the multiple environmental samples to provide a mixed environmental sample and homogenizing the mixed environmental sample to provide an input sample.

[0224] In the embodiment that comprises using multiple environmental samples to produce input sample, collecting and mixing multiple environmental samples can be used not only to maximize the statistical sample space of the microorganism to be screened, but also can be used to maximize the microorganism combination present in the microorganism consortium, which is identified and / or produced using the technology applied to the input sample described herein. In addition, collecting and mixing multiple environmental samples to produce the input sample of application technology described herein can produce novel microorganism consortium that does not exist in nature by combining microorganisms that usually do not live in the same environment in nature. In some embodiments, various environmental samples from different geographic regions can be mixed to further increase the statistical sample space of microorganism consortium combination. For example, the embodiment provides that multiple environmental samples can be obtained, wherein each environmental sample is taken from different ecosystems, habitats and / or ecological niches. The embodiment further provides that multiple environmental samples can be obtained from locations separated from each other by 1m, 10m, 100m, 1000m, 10,000m or more than 10,000m. In some embodiments, the sample is obtained from two or more points anywhere on the earth, including above and below the surface of the earth's land and waters.

[0225] In some cases, multiple input samples can be produced during collection. Each input sample in multiple input samples can include the different combinations of individual environmental samples mixed together. For example, environmental samples A, B and C (from one or more different ecosystems, habitats and / or niches) can be mixed to provide an input sample comprising 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 niches) can be mixed to provide an input sample comprising 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) can be mixed to provide an input sample comprising 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 of the plurality of input samples can include a range of partial compositions of any two of the plurality of individual environmental samples mixed together to provide the input sample. For example, any two individual environmental samples can be mixed together to provide an input sample comprising a portion of the composition of the first environmental sample ranging from 0.01 to 0.99 (e.g., a first environmental sample comprising 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) and comprising a portion of the composition of the second environmental sample ranging from 0.99 to 0.01 (e.g., a second environmental sample comprising 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).

[0226] The amount of mixed environmental samples and the variation of types can be used to separate and develop input samples. This is because it is recognized that the combination of microorganisms may not only be beneficial, but may also cause individual microorganisms to become less effective or dominated by microorganisms from external environmental samples. In addition, the embodiment of the technology includes the use of a single environmental sample that is homogenized to provide an input sample. Those of ordinary skill in the art understand that a single environmental sample may comprise a plurality of single ecosystems or niches that are not mixed in nature but become mixed when a single sample is homogenized. For example, an environmental sample may comprise a plurality of independent subsamples that exist as independent strata, layers or sub-communities, for example, strata of a cylindrical soil core sample, strata of a microbial mat sample, strata of a water column sample, sub-communities of microbial communities comprising biofilms, etc.

[0227] Thus, embodiments of the methods provided herein include using a single environmental sample, homogenizing it to provide an input sample and / or include using multiple environmental samples, mixing and homogenizing them to provide an input sample.

[0228] Microbial consortium

[0229] As described herein, the technology provides a microbial consortium with a specific function. In some embodiments, the microbial consortium is produced according to the selection method described herein. In some embodiments, the technology applies selective conditions to a sample (e.g., including a homogenized environmental sample or a plurality of homogenized environmental samples), and the selective conditions reduce the complexity of the microbial community and improve the function of the microbial community.

[0230] In the development process of the embodiments of the technology described herein, the embodiments of the selection method produced three kinds of microbial consortia, named BW01, BW02 and BW05. Each of the BW01 and BW02 consortia includes such organisms: the organism is taxonomically identified as Nostoc species or similar to unclassified Nostoc species using 16S rRNA gene sequences, and the unclassified Nostoc species can form branches different from other known Nostoc species. In addition, all minimal consortia produced from BW01, BW02 and BW05 include members of the Nostocaceae family. These organisms are identified as Nostoc punctiforme PCC 73102, Nostoc species NIES-4103, Nostoc species UAM 307 and Dolichospermum flos-aquae. These members represent three Nostoc species: Nostoc PCC-73102, Desmonostoc PCC-6302, and Desmonostoc PCC-7422. (Example 20). In independent experiments that generated microbial consortia and six minimal consortia (Example 21), all six minimal consortia included Nostoc or photosynthetic Nostoc organisms, e.g., Anabaena species.

[0231] The Nostoc or Nostoc-like organisms of the microbial consortium exist with about 30% abundance of the consortium population. The soil sample initially input includes 0.1% Nostoc species less than the microbial population. Therefore, although Nostoc species or Nostoc-like species are present in the input sample, its content in the environment is very low. The growth conditions used in the development process of the embodiment of the technology are selected to select the species of photoautotrophic growth and nitrogen fixation from the atmosphere. Under these conditions, Nostoc species or Nostoc-like species have high competitiveness in the culture and relative abundance significantly increases, thereby providing the main component of BW01 and BW02 consortiums. Not bound by theory, it is expected that Nostoc species or Nostoc-like species are the main energy suppliers at the top of the energy network of BW01, BW02 and BW05 consortiums. Specifically, it is expected that the role of Nostoc species (or Nostoc-like species) in BWOl, BW02, and BW05 is: 1) fixation of carbon (e.g., as photosynthetic output (e.g., in the form of extracellular polymeric substances (EPS))); and 2) fixation of nitrogen. Accordingly, embodiments of the technology provide a microbial consortium comprising 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.

[0235] In addition, during the development of embodiments of the technology described herein, experiments showed that the BW01 and BW02 microbial consortia (e.g., containing at least 30% Nostoc species) produced by the selection method also included many heterotrophic organisms. Without being bound by theory, it is expected that heterotrophic organisms import and metabolize organic compounds, such as polysaccharides associated with EPS produced by Nostoc species or Nostoc species. It has been shown that cyanobacteria release large amounts of available carbon into their 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.

[0236] Some genera decrease during passage. For example, data collected from characterizing the BW01 and BW02 consortia indicate that the consortia include low levels of genera such as Bacillus species, Bradyrhizobium species, Streptomyces species, and Pseudomonas species. Although these genera are present in the input sample, the number of each of these genera in the BW01 and BW02 consortia is reduced to a lower number during passage. Therefore, these data indicate that the metabolic function of these genera is not sufficient to support the growth of these organisms in the light-driven consortia BW01 and BW02.

[0237] Some genera, such as Methylobacterium species, were present at relatively low levels in the input samples and remained at similar low levels throughout the selection process. Thus, these data suggest that these microorganisms are not adversely affected by the selection conditions and can therefore interact with the dominant genera of the consortium (e.g., Nostoc) or consume metabolites produced by other organisms of the consortium.

[0238] 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 expected that these genera may be necessary for succession when the colony moves toward stability, and / or these genera may also compete with other species for a short time. The genera that increased during passage in both BW01 and BW02 include Brevundimonas species and Aminobacterium species. Both are α-Proteobacteria, and their exact role as bottleneck resolvers is still unclear.

[0239] Data collected during the experiments described herein indicate that phototropic purple bacteria of the genus Rhodobacter are found in the genus represented. Purple photosynthetic bacteria have been shown to provide numerous benefits to 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 anoxygenic photosynthesis and carbon fixation functions, and do not perform oxygenic photosynthesis or nitrogen fixation functions; these processes are precisely 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). Therefore, it is expected that these metabolically diverse purple bacteria consume the secretions of the Nostoc species in the consortium when oxygen exists. In natural, complex biofilms (e.g., in agricultural environments), anoxygenic phototrophs are exposed to light and anaerobic conditions. Anoxygenic phototrophs have been shown to constitute the lower 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 Martínez-Alonso (2005) "Diversity of anoxygenic phototrophic sulfur bacteria in the microbialmats 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 believed that the purple photosynthetic bacteria aid in carbon fixation in the mature biofilm by absorbing infrared light that passes through the upper layer comprising the Nostoc species containing chlorophyll a, and fixes carbon and nitrogen in the anaerobic lower layer of the biofilm.

[0240] Some consortia include one or more additional photosynthetic members from the following bacterial genera: Nodularia, Chrysosporium, Globularia, Phytophyte, Trichoderma, Hosecochlear, Myxobacterium, Acaryochloris, Kamptonema, Acrocephalum, Crocochlear, Macrochaete, Thermosynechococcus, Pseudoanabaena, Pseudochromococcus, Prochlorococcus, Anabaena, Sclerotium, Browsing, Pseudocylospora, Longisporium, Pseudobranchium, Myxobacterium, Monochaete, Flytella, Voltiella, Aliterella, Aquatica, Prochlorococcus, Pseudothioura, Gastrodia, Isococcus, Jasmine, Pseudothioura, Pantanalinema, Oscillatoria, Spirulina, Pelatocladus, Knotweed, Aphanizomenon, Pseudochlorococcus, Myxobacterium, Myxococcus or from the following algae genera: Tetrastomium, Chlorella, Chaetoceros, Diskia, Paradoxia, Cymbomonas, Palmitomorpha, Characiochloris, Marvania, Chlamydomonas, Pseudochlorella, Glechoma, Symcoccus, Cynochloris, Tetraspinosa, Dangeardinia, Galdiella, Nyholmiella, Porphyra, Chara, Fibrous Algae, Chlorella, Cyanophyte, Chondrus, Pseudochloris, Interfilum, Gelidium, Symphyogyna, Backpacker, or Cyanophyte.

[0241] In some embodiments, the technology provides a microbial consortium that includes an organism having a ribosomal RNA gene sequence provided by any one of 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 or 125. In some embodiments, the technology provides a microbial consortium comprising an organism having a ribosomal RNA gene sequence having ≥97%, ≥95%, ≥90%, ≥80% or ≥70% nucleotide sequence identity to a nucleotide sequence provided by any 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.

[0242] In some embodiments, the technology provides a microbial consortium comprising an organism having a ribosomal RNA gene sequence provided by the consensus sequence of SEQ ID NO: 127:

[0243] ACGGAGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGTATGCT

[0244] TAACACATGCAAGTC

[0245] GAACGGTGTCTTCGGACATAGTGGCGGACGGGTGAGTAACGCGT

[0246] GAGAATCTnGCTTCAG

[0247] GTCTGGGACAACCACTGGAAACGGTGGCTAATACCGGATGTGCC

[0248] GnAAGGTGAAAGGnTT

[0249] nCTGCCTnAAGATGAGCTCGCGTCTGATTAGCTAGTnGGTGGGGTA

[0250] AnAGnCTACCnAGG

[0251] CGACGATCAGTAGCTGGTCTGAGAGGAnGATCAGCCACACTGGG

[0252] ACTGAGACACGGCCCA

[0253] GACTCCTACGGGAGGCAGCAGTGGGGAATTTTCCGCAATGGGCG

[0254] AAAGCCTGACGGAGCA

[0255] ATACCGCGTGAGGGAGGAAGGCTCTTGGGTTGTAAAACCTCTTTTC

[0256] TCAGGGAAGAACACA

[0257] ATGACGGTACCTGAGGAATAAGCATCGGCTAACTCCGTGCCAGCA

[0258] GCCGCGGTAATACGG

[0259] AGGATGCAAGCGTTATCCGGAATGATTGGGCGTAAAGCGTCCGC

[0260] AGGTGGCnnTGTAAGT

[0261] CTGCTGTTAAAGAGTnTAGCTCAACTnnATAAAGGCAGTGGAAAC

[0262] TACAnAGCTAGAGTn

[0263] CGTTCGGGGCAGAGGGAATTCCTGGTGTAGCGGTGAAATGCGTA

[0264] GAGATCAGGAAGAACA

[0265] CCGGTGGCGAAGGCGCTCTGCTAGGCCGnAACTGACACTGAGGG

[0266] ACGAAAGCTAGGGGAG

[0267] CGAATGGGATTAGATACCCCAGTAGTCCTAGCCGTAAACGATGGA

[0268] TACTAGGCGTGGCTT

[0269] GTATCGACCCGAGCCGTGCCGTAGCTAACGCGTTAAGTATCCCGC

[0270] CTGGGGAGTACGCnC

[0271] GCAAnnGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCG

[0272] GTGGAGTATGTGGTTT

[0273] AATTCGATGCAACGCGAAGAACCTTACCAAGGCTTGACATGTCG

[0274] CGAATCCTnCTGAAAG

[0275] GnGGGAGTGCCTTCGGGAGCGCGAACACAGGTGGTGCATGGCTG

[0276] TCGTCAGCTCGTGTCG

[0277] TGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTTTTT

[0278] AGTTGCCAGCATTAA

[0279] GTTGGGCACTCTAGAGAGACTGCCGGTGACAAACCGGAGGAAG

[0280] GTGGGGATGACGTCAAG

[0281] TCAGCATGCCCCTTACGCCTTGGGCTACACACGTACTACAATGCTn

[0282] CGGACAGAGGGCAG

[0283] CAAGCnAGCGAnnGCAAGCnAATCCCnTAAACCGnnGCTCAGTTCA

[0284] GATCGCAGGCTGCA

[0285] ACTCGCCTnCGTGAAGGAGGAATCGCTAGTAATTGCAGGTCAGCA

[0286] TACTGCAGTGAATTC

[0287] GTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGAAGCTG

[0288] GnAACGCCCGAAGTCA

[0289] TTACTCCAACCTTTCGGGGAGGAGGATGCCTAAGGCAGGnCTGGT

[0290] GACTGGGGTGAAGTC

[0291] GTAACAAGGTAGCCGTACCGGAAGGTGTGGCTGGATCACCTCCTTTn

[0292] In some embodiments, the technology provides a microbial consortium that includes organisms having a ribosomal RNA gene sequence that is 95, 96, 97, 98, or 99% identical to the consensus sequence of SEQ ID NO:127.

[0293] In some embodiments, the technology provides a microbial consortium comprising the following organisms: Amazonocrinis sp. SA22, Amazonocrinis nigriterrae, Amazonocrinis nigriterrae CENA18, Amazonocrinis nigriterrae CENA66, Amazonocrinis nigriterrae CENA67, Amazonocrinis nigriterrae CENA69, Amazonocrinis thailandica, Amazonocrinisthailandica NUACC02, Amazonocrinis thailandica NUACC03, Anabaena catenula SAG 1403-1, Anabaena oryzae Ind3, Anabaena oryzae WY07, Anabaena oscillarioides str.BO HINDAK 1984 / 43, Anabaena species 0830-A, Anabaena species CCAP 1403 / 4A, Anabaena species HBU1, Anabaena species HBU10, Anabaena species Ind5, Anabaena species KVSF7, Anabaena species PCC 7108, Anabaena species SN430, Anabaena species YBS01, Anabaenopsis circularis NIES-21, Atlanticothrix silvestris, Atlanticothrix silvestris CENA368, Atlanticothrix silvestris CENA564, Atlanticothrix silvestris CENA576, Atlanticothrix silvestris CENA579, Atlanticothrix silvestris CENA585, Atlanticothrix silvestris CENA590, Aulosiralaxa NIES-50, Aulosiralaxa species CENA272, Aulosiralaxa species CENA288, Aulosiralaxa species CENA291, Aulosiralaxa species CENA295, Calothrix brevissima IAM M-249, Calothrix brevissima NIES-22, Calothrix membranacea SAG 1410-1, Calothrix species CCAP 1410 / 13, Calothrix species CENA283, Calothrix species CHAB TP201506, Calothrix species CHAB TP201518, Calothrix species CHAB TP201519, Calothrix species CHAB TP201521, Calothrix species CHAB TP201524, Calothrix species CHAB TP201528, Chrysosporum species CHAB2384, NIES-2098, NIES-2099, NIES-2100, PCC 7507, SA4, SA47, HA4241-MV5, Chrysosporum bergii 09-02, ANA360D, Compactonostoc shennongjiaense, Constrictifilum karadense, Constrictifilum karadense MKW3, Constrictifilum sp.Assy 17, Cyanocohniella rudolphia, Cyanocohniella rudolphia SY-1-2-Y, Cyanocohniella sp.SY-1-2-EE, Cylindrospermum catenatum CCALA 999, Cylindrospermum muscicola ACSSI 140, Cylindrospermum muscicola Ind12, Cylindrospermum SAG44.79, Cylindrospermum skujae ACSSI 112, Cylindrospermum species ACSSI 010, Cylindrospermum species ACSSI 028, Cylindrospermum species ACSSI 040, Cylindrospermum species ACSSI 041, Cylindrospermum species ACSSI 043, Columnar spore CHAB2115, Columnar spore NIES-4074, Columnar spore YK2-01, Dendronalium phyllosphericum, Dendronalium phyllosphericum CENA358, Dendronalium phyllosphericum CENA389, Dendronalium phyllosphericum CENA73, Desikacharyasp.PS2C、Desmonostoc caucasicum、Desmonostoc caucasicum MZ-C154、Desmonostocdanxiaense、Desmonostoc danxiaense CHAB5868、Desmonostoc danxiaense CHAB5869、Desmonostoc lechangense、Desmonostoc lechangense CF01、Desmonostoc magnisporum、Desmonostoc magnisporum AR6_PS、Desmonostoc muscorum、Desmonostoc muscorum 9a、Desmonostoc muscorum ACSSI 091、Desmonostoc muscorum ACSSI 149、Desmonostocmuscorum CCAP 1453 / 22、Desmonostoc muscorum CCAP 1453 / 32、Desmonostoc muscorumCCAP 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, Desmonostocsp.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, Dolichospermum laiensis HA4221-MV2, Dolichospermum species PS4G, Fremyella diplosiphon NIES-3275, Dolichospermum species CHAB TP201828.1, Glechoma species CHAB TP201828.2, Gloeotrichiaechinulata PYH14, Goleter 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, Komarekiella atlantica CCIBt 3307, Komarekiella atlantica CCIBt 3481, Komarekiella atlantica CCIBt 3483, Komarekiella atlantica CCIBt 3486, Komarekiella atlantica CCIBt 3487, Komarekiella atlantica CCIBt 3552, Komarekiella atlantica CENA107, Komarekiella globosa, Komarekiella globosa PJ104, Microchaete diplosiphon CCALA 811, Microchaete species CENA541, Microchaete species SAG 47.93, Nodularia species 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 carneum cf. cf. commune)257-16, common Nostoc cf.257-20, common Nostoc cf.KG-54, common Nostoc cf.SO-36, Nostoc cf.edaphicum Us-2-2, Nostoc cf.indistinguendum FI5-VF12, Nostoc cf.indistinguendum FI5-VF4, Nostoc cf.lichenoides JT1-VF3, Nostoc cf.verrucosum, common Nostoc (Nostoc commune), common Nostoc 0Brien 02011101, common Nostoc 15CT-1.2. Nostoc 15CT-3.1, Nostoc ACSSI 030, Nostoc ACSSI 035, Nostoc ACT709, Nostoc AHNG0605, Nostoc BEA 0028, Nostoc CANT2, Nostoc CANT4, Nostoc CCAP 1453 / 24, Nostoc CCIBt 3485, Nostoc EV1-KK1, Nostoc FACHB 261, Nostoc HK-02, Nostoc KU002, Nostoc LSB51, Nostoc LSB511, Nostoc LSB84, Nostoc Mon62, Nostoc PS27-2.2, Nostoc SAG 1453-3, Nostoc edulis SIK85, Nostoc edulis SIK94, Nostoc edulis SN444, Nostoc edulis SN450, Nostoc edulis UTEX 584, Nostoc edulis WY1KK1, Nostoc edulis YK-04, Nostoc edulis var. flagelliforme CCAP 1453 / 33, Nostoc desertorum, Nostoc edulis CM1-VF14, Nostoc edaphicum ACCS 059, Nostoc edaphicum ACSSI 156, Nostoc edaphicum CCNP1411, Nostoc edaphicum KZ-5-4-7, Nostoc edaphicum TAU-MAC2615, Nostoc edaphicum X, Nostoc elgonense QUCCCM128, Nostoc elgonense TH3S05, Nostoc ellipsosporum CCAP 1453 / 15, Nostoc ellipsosporum CCAP 1453 / 2, Nostoc ellipsosporum QUCCCM991490, Nostoc ellipsosporum V, Nostoc ellipsosporum str.Lukesova 52_91, Nostocentophytum IAM M-267, Nostoc favosum ISC 32, Nostoc favosum, Nostoc favosum CHAB5713, Nostoc favosum CHAB5714, Nostoc flagelliforme CCNUN1, Nostoc flagelliforme CHAB2816, Nostoc flagelliforme IMGA0408, Nostoc flagelliforme Sunite Zuoqi strain, Nostoc indistinguendum, Nostoc indistinguendum CM1-VF10, Nostoc insulare SAG 54.79, Nostoc lichenoides, Nostoc lichenoides CNP-AK1, Nostoc linckia, Nostoc linckia ACSSI 271, Nostoc linckia BEA 0870B, Nostoc linckia NIES-25, Nostoc arvense 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 mirabilis, Nostoc mirabilis CHAB5756, Nostoc oromo, Nostoc oromo ETH.2.4.M.5, Nostoc oryzae, Nostoc paludosum BA033, Nostoc parmelioides SAG58.79, Nostoc piscinale BF3, Nostoc piscinale CENA21, Nostoc pruniforme SAG 62.79, Nostoc punctata, Nostoc punctata ACCS 074, Nostoc punctata ACSSI 037, Nostoc punctata ACSSI 160, Nostoc punctata BKP_NB59, Nostoc punctata BKP_NS61, Nostoc punctata BKP_NS62, Nostoc punctata BKP_NS63, Nostoc punctata BKP_NS64, Nostoc punctata BKP_NS65, Nostoc punctata BKP_NS66, Nostoc punctata BKP_NS67, Nostoc punctata BKP_NS68, Nostoc punctata BKP_NS69, Nostoc punctata BKP_NS70, Nostoc punctata BKP_SS64, Nostoc punctata BKP_SS66, Nostoc punctata BKP_SS67, Nostoc punctata BKP_SS68, Nostoc punctata CCAP 1453 / 9, Nostoc punctata Jbr02, Nostoc punctata.

[0294] KZ-2-2-2, Nostoc sp. PCC 73102, Nostoc sp. SAG 60.79, Nostoc sp. SAG 65.79, Nostoc sp. SAG 68.79, Nostoc sp. SAG 71.79, Nostoc sp., Azolla species, Collema crispum species, Collema nigrescens species UK197, Collema nigrescens species AR75, Collema nigrescens species UK531, Nostoc sp. LG:R1123, Nostoc sp. LG:R1124, Fuscopannaria species pacificaUK179Symbiotic cyanobacteria, Nostoc speciesFuscopannaria praetermissaLG:R1060Symbiotic cyanobacteria, Nostoc speciesHelianobakht, Nostoc speciesKroswia crystallyferaLG:M788Symbiotic cyanobacteria, Nostoc speciesLeptogium gelatinosumSymbiotic cyanobacteria, Nostoc speciesLeptogiumpalmatum UK176Symbiotic cyanobacteria, Nostoc speciesLeptogium sp.LG:R2848Symbiotic cyanobacteria, Nostoc speciesLeptogium sp.LG:R2850Symbiotic cyanobacteria, Nostoc speciesLobariaamplissimaSymbiotic cyanobacteria, Nostoc speciesLobariaSymbiotic cyanobacteria34, Nostoc speciesLobaria halliiSymbiotic cyanobacteria, Nostoc speciesLobaria pulmonaria(5183) symbiotic cyanobacteria, Massalongia carnosa symbiotic cyanobacteria, Massalongia carnosa symbiotic cyanobacteria, Massalongia carnosa symbiotic cyanobacteria, Massalongia carnosa symbiotic cyanobacteria, Massalongia carnosa symbiotic cyanobacteria, Massalongia carnosa symbiotic cyanobacteria, Massalongia carnosa symbiotic cyanobacteria, Massalongia carnosa symbiotic cyanobacteria, Massalongia carnosa symbiotic cyanobacteria, Massalongia carnosa symbiotic cyanobacteria, Massalongia carnosa symbiotic cyanobacteria, Massalongia carnosa symbiotic cyanobacteria, Massalongia carnosa symbiotic cyanobacteria, Massalongia carnosa symbiotic cyanobacteria, Massalongia carnosa symbiotic cyanobacteria, Massalongia carnosa symbiotic cyanobacteria, Massalongia carnosa symbiotic cyanobacteria, Massalongia carnosa symbiotic cyanobacteria, Massalongia carnosa symbiotic cyanobacteria, Massalongia carnosa symbiotic cyanobacteria, Massalongia carnosa symbiotic cyanobacteria, Massalongia carnosa symbiotic cyanobacteria, Massalongia carnosa symbiotic cyanobacteria, Massalongia carnosa symbiotic cyanobacteria, Massalongia carnosa symbiotic cyanobacteria, Massalongia carnosa symbiotic cyanobacteria, Massalongia carnosa symbiotic cyanobacteria,94.1、Symbiotic cyanobacteria of Nephroma arcticum UK103、Symbiotic cyanobacteria of Nephroma arcticum UK48、Symbiotic cyanobacteria of Nephroma arcticum UK564、Symbiotic cyanobacteria of Nephroma bellum、Symbiotic cyanobacteria of Nephroma helveticum 33、Symbiotic cyanobacteria of Nephroma helveticum 37、Symbiotic cyanobacteria of Nephroma helveticum、Symbiotic cyanobacteria of Nephroma laevigatum 39、Symbiotic cyanobacteria of Nephroma laevigatum parile) symbiotic cyanobacteria 26, Nostoc species symbiotic cyanobacteria of Nephromaresupinatum 32, Nostoc species symbiotic cyanobacteria of Nephromaresupinatum, Nostoc species symbiotic cyanobacteria of Nephromaresupinatum 38, Nostoc species symbiotic cyanobacteria of Nephromaresupinatum 40, Nostoc species symbiotic cyanobacteria of Nephromaresupinatum, Nostoc species Pannaria aff.athroophylla symbiotic cyanobacteria Symbiotic cyanobacteria NZ, Nostoc species Pannaria aff.leproloma symbiotic cyanobacteria 1a Ch, Nostoc species Pannariaaff.leproloma symbiotic cyanobacteria 1a NZ, Nostoc species Pannaria Symbiotic cyanobacteria of Pannaria aff.leproloma 2Ch, Symbiotic cyanobacteria of Pannaria aff.leproloma 3Ch, Symbiotic cyanobacteria of Pannaria aff.sphinctrina 1aNC, Symbiotic cyanobacteria of Pannaria aff.sphinctrina 1bNC, Symbiotic cyanobacteria of Pannaria andina 1 Chile, Symbiotic cyanobacteria of Pannaria Andina Peru, Symbiotic cyanobacteria of Pannaria araneosa NZ, Symbiotic cyanobacteria of Pannaria conoplea, Symbiotic cyanobacteria of Pannaria durietzii 1NZ, Symbiotic cyanobacteria of Pannaria elixii 1NZ, Symbiotic cyanobacteria of Pannaria elixii 2NZ, Symbiotic cyanobacteria of Pannaria euphylla symbiotic cyanobacteria NZ, Nostoc species Pannariaisabellina symbiotic cyanobacteria 1Ch, Pannaria isabellina symbiotic cyanobacteria 2Ch, Pannaria mosenii symbiotic cyanobacteria Mex, Pannaria obscura symbiotic cyanobacteria Aus, Pannaria pallida symbiotic cyanobacteria 3Ch, Pannaria rubiginosa LG: R1008 symbiotic cyanobacteria, Pannaria rubiginosa LG: R1011 symbiotic cyanobacteria, Pannaria rubiginosa symbiotic cyanobacteria No, Pannaria cephalodia LG: R969 symbiotic cyanobacteria, Pannaria sphinctrina symbiotic cyanobacteria 1a Ch, Pannaria sphinctrina symbiotic cyanobacteria 1b Ch, Pannaria sphinctrina symbiotic cyanobacteria 1c Ch、Nostoc species Pannaria tavaresii Symbiotic cyanobacteriaCh、Nostoc species Parmeliella borbonica LG:R1122 Symbiotic cyanobacteria、Nostoc species Parmeliella brisbanensis LG:R1019 Symbiotic cyanobacteria、Nostoc species Brisbane LG:R1247 Symbiotic cyanobacteria、Nostoc species Brisbane LG:T3 Symbiotic cyanobacteria、Nostoc species Brisbane LG:T7 Symbiotic cyanobacteria、Nostoc species Parmeliella mariana LG:R974 Symbiotic cyanobacteria、Nostoc species Parmeliella polyphyllina LG:R1021 Symbiotic cyanobacteria、Nostoc species Parmeliella polyphyllina LG:R1058 Symbiotic cyanobacteria, Nostoc species, Parmeliella species LG:T6 Symbiotic cyanobacteria, Nostoc species, Parmeliella stylophora LG:R979 Symbiotic cyanobacteria, Nostoc species, Parmeliella triptophylla Symbiotic cyanobacteria 30, Nostoc species, Parmeliella triptophylloides LG:R965 Symbiotic cyanobacteria, Nostoc species, Peltigera aphthosa UK52 Symbiotic cyanobacteria, Nostoc species, Peltigera aphthosa UK53 Symbiotic cyanobacteria, Nostoc species, Peltigeracanina)1 Symbiotic cyanobacteria, Nostoc species canine volume2 Symbiotic cyanobacteria, Nostoc species canine volume3 Symbiotic cyanobacteria, Nostoc species canine volume4 Symbiotic cyanobacteria, Nostoc species canine volumeUK106 Symbiotic cyanobacteria, Nostoc species canine volumeUK121 Symbiotic cyanobacteria, Nostoc species shield volume (Peltigera collina)UK144 Symbiotic cyanobacteria, Nostoc species shield volumeUK148 Symbiotic cyanobacteria, Nostoc species Peltigera degeni Symbiotic cyanobacteria, Nostoc species divided finger volume (Peltigeradidactyla)2 Symbiotic cyanobacteria, Nostoc species divided finger volume3 Symbiotic cyanobacteria, Nostoc species Peltigeraevansiana UK159 Symbiotic cyanobacteria, Nostoc species Peltigera extenuata AR4b Symbiotic cyanobacteria, Nostoc species Peltigera extenuata Symbiotic cyanobacteria UK4, Peltigera frigida AR49, Peltigera horizontalis, Peltigera lepidophora, Peltigera lepidophora UK57, Peltigera leucophlebia UK79, Peltigera malacea UK59, Peltigera soft cyanobacteria DB3992, Peltigera membrane membranacea)1 Symbiotic cyanobacteria, Nostoc species membrane ground volume 2 Symbiotic cyanobacteria, Nostoc species membrane ground volume 3 Symbiotic cyanobacteria, Nostoc species membrane ground volume 4 Symbiotic cyanobacteria, Nostoc species membrane ground volume 5 Symbiotic cyanobacteria, Nostoc species membrane ground volume UK156 Symbiotic cyanobacteria, Nostoc species membrane ground volume UK194 Symbiotic cyanobacteria, Nostoc species membrane ground volume Symbiotic cyanobacteria, Nostoc species membrane ground volume Symbiotic cyanobacteria N6, Nostoc species long root ground volume (Peltigera neopolydactyla) UK60 Symbiotic cyanobacteria, Nostoc species long root ground volume agg. UK150 Symbiotic cyanobacteria (cyanobiont), Nostoc species frost ground volume (Peltigera pruinosa) Symbiotic cyanobacteria 22, Nostoc species frost ground volume Symbiotic cyanobacteria 14, Nostoc species ground volume (Peltigerarufescens)1 Symbiotic cyanobacteria, Nostoc species terrestrial volume 2 Symbiotic cyanobacteria, Nostoc species terrestrial volume 3 Symbiotic cyanobacteria, Nostoc species terrestrial volume 4 Symbiotic cyanobacteria, Nostoc species terrestrial volume 5 Symbiotic cyanobacteria, Nostoc species terrestrial volume UK46 Symbiotic cyanobacteria, Nostoc species terrestrial volume species UK437 Symbiotic cyanobacteria, Nostoc species terrestrial volume species UK521b Symbiotic cyanobacteria, Nostoc species terrestrial volume species Symbiotic cyanobacteria, Nostoc species Peltigera venosa Symbiotic cyanobacteria 15, Nostoc species Physma byrsaeum LG: R1121 Symbiotic cyanobacteria, Nostoc species Physma byrsaeum LG: R2 Symbiotic cyanobacteria, Nostoc species Physma byrsaeum LG: R2847 Symbiotic cyanobacteria, Nostoc species Physma radiata radians)LG:T5Symbiotic cyanobacteria, Nostoc speciesPolychidium muscicolaUK175Symbiotic cyanobacteria, Nostoc speciesProtopannaria pezizoidesUK120Symbiotic cyanobacteria, Nostoc speciesProtopannaria pezizoidesUK73Symbiotic cyanobacteria, Nostoc speciesgilvaSymbiotic cyanobacteriaChile, Nostoc speciesPseudocyphellaria speciesLG:R2332Symbiotic cyanobacteria, Nostoc speciesSticta beauvoisiiSymbiotic cyanobacteria, Nostoc speciesSticta cf.caulescensAR124Symbiotic cyanobacteria, Nostoc speciesSticta fuliginosa symbiotic cyanobacteria, Nostoc species muscicolous symbiotic cyanobacteria 21, Nostoc species 0GU36S01, Nostoc species 0GU36S02, Nostoc species 113.5, Nostoc species 117.8.2, Nostoc species 1189P, Nostoc species 1190P, Nostoc species 152, Nostoc species 159, Nostoc species 17, Nostoc species 195-A21, Nostoc species 195-A22, Nostoc species 1c, Nostoc species 1tu14s8 , Nostoc species 2-07, Nostoc species 2LT05S03, Nostoc species 5N-02c, Nostoc species 8901:1, Nostoc species 8916, Nostoc species 8923, Nostoc species 8926, Nostoc species 8929, Nostoc species 8938, Nostoc species 8941, Nostoc species 8963, Nostoc species 8964:3, Nostoc species 9104, Nostoc species 9E-03, Nostoc species 9d, Nostoc species A15, Nostoc species A39, Nostoc species ACSSI045、Nostoc species ACSSI 047、Nostoc species ACSSI 056、Nostoc species ACSSI 057、Nostoc species ACSSI 066、Nostoc species ACSSI067、Nostoc species ACSSI 082、Nostoc species ACSSI 114、Nostoc species ACSSI 115、Nostoc species ACSSI 123、Nostoc species ACSSI 143、Nostoc species ACSSI 161、Nostoc species ACSSI164、Nostoc species ACSSI 231、Nostoc species ACSSI 310、Nostoc species ACSSI 314、Nostoc species ACSSI 325、Nostoc species ACSSI 329, Nostoc species ACT703, Nostoc species ACT732, Nostoc species AH-12, Nostoc species ANT.L34.1, Nostoc species ANT.L52B.1, Nostoc species ANT.L52B.8, Nostoc species ANT.L61.1, Nostoc species ANT.LG2.6, Nostoc species AR12, Nostoc species ARC 64, Nostoc species ARC8, Nostoc species ATCC 53789, Nostoc species AWT 203, Nostoc species Al1, Nostoc species Al3, Nostoc species BACA0081, Nostoc species BDU ARC 10101, Nostoc species BDU80591, Nostoc species BDU80701, Nostoc species BEA 1039B, Nostoc species BEA 1140B, Nostoc species BKP_CB62, Nostoc species BKP_CB63, Nostoc species BKP_CB64, Nostoc species BKP_CS63, Nostoc species BKP_CS64, Nostoc species BKP_CS65, Nostoc species BKP_CS66, Nostoc species BKP_CS67, Nostoc species BKP_CS68, Nostoc species BKP_CS69, Nostoc species BKP_CS70, Nostoc species BKP_CS71, Nostoc species BKP_CS72, Nostoc species Nostoc species BKP_CS73, Nostoc species BKP_CS74, Nostoc species BKP_NS59, Nostoc species BKP_NS60, Nostoc species BKP_SB57, Nostoc species BKP_SB58, Nostoc species BKP_SB59, Nostoc species BKP_SS65, Nostoc species BR36, Nostoc species BS363, Nostoc species Bahar_E, Nostoc species Bahar_M, Nostoc species C052, Nostoc species C057, Nostoc species CACIAM19. Nostoc species CAVN2, Nostoc species CAWBG77, Nostoc species CCAP 1453 / 25, Nostoc species CCAP 1453 / 28, Nostoc species CCAP 1453 / 31, Nostoc species CCAP 1453 / 35, Nostoc species CENA105, Nostoc species CENA175, Nostoc species CENA216, Nostoc species CENA219, Nostoc species CENA239, Nostoc species CENA259, Nostoc species CENA261, Nostoc species CENA269, Nostoc species CENA271, Nostoc species CENA274, Nostoc species CENA278, Nostoc species CENA281, Nostoc species Species of the genus CENA294, genus Nostoc species CENA296, genus Nostoc species CENA356, genus Nostoc species CENA511, genus Nostoc species CENA535, genus Nostoc species CENA536, genus Nostoc species CENA543, genus Nostoc species CENA544, genus Nostoc species CENA547, genus Nostoc species CENA548, genus Nostoc species CENA551, genus Nostoc species CHABTP201513, genus Nostoc species CHAB TP201514, Nostoc species CHAB TP201701.1, Nostoc species CHAB TP201726.1, Nostoc species CHAB TP201727.1, Nostoc species CHAB TP201728.1, Nostoc species CHAB TP201728.4, Nostoc species CHABTP201736.2, Nostoc species CHABTP201822.2, Nostoc species CNCH1, Nostoc species Cam2S01, Nostoc species Cc2, Nostoc species Cr4, Nostoc species DM103, Nostoc species De1, Nostoc species Den-12, Nostoc species Ev1, Nostoc species FACHB-252, Nostoc species FB1-KK1, Nostoc species FI5-VF5, Nostoc species GM244, Nostoc species GSV224, Nostoc species GT138, Nostoc species HAMTA.RF, Nostoc species H AN11 / 1, Nostoc species HK-01, Nostoc species IO-102-I, Nostoc species KK-01, Nostoc species KNUA003, Nostoc species KU001, Nostoc species KU028, Nostoc species KU281, Nostoc species KVJ10, Nostoc species KVJ18, Nostoc species KVJ20, Nostoc species KVJ3, Nostoc species KVJ4, Nostoc species KVJF4, Nostoc species KVJF8, Nostoc species KVS1, Nostoc species KVS11, Nostoc species KVSF4, Nostoc species Lukesova 1 / 86, Nostoc species Lukesova 40 / 93, Nostoc species Lukesova 5 / 96, Nostoc species MGL001, Nostoc species MS1, Nostoc species MV6, Nostoc species Mau15, Nostoc species N107.3, Nostoc species NIES-2094, Nostoc species NIES-2110, Nostoc species NIES-2111, Nostoc species NIES-3756, Nostoc species NIES-4103, Nostoc species Ni4-C1, Nostoc species Os-1, Nostoc species Os1-C1, Nostoc species PCC 6720, Nostoc species PCC7107, Nostoc species PCC 7120=FACHB-418, Nostoc species PCC 7423, Nostoc species PCC7524, Nostoc species PCC 8112, Nostoc species PCC 8976, Nostoc species PCC 9229, Nostoc species PCC 9231, Nostoc species PCC 9305, Nostoc species PCC 9426, Nostoc species PCC 9709, Nostoc species PS33-1, Nostoc species Prim-3-2, Nostoc species RE21, Nostoc species SAG2306, Nostoc species SAG 2409, Nostoc species SAG 2414, Nostoc species SAG 29.90, Nostoc species SAG 34.92, Nostoc species SAG35.92, Nostoc species SAG 36.92, Nostoc species SAG39.87, Nostoc species SAG 41.87, Nostoc species SKJ1, Nostoc species SKJ2, Nostoc species SKJ4, Nostoc species SKJF1, Nostoc species SKS2, Nostoc species SKS5, Nostoc species SKS8, Nostoc species SKS9, Nostoc species SKSF3, Nostoc species SKSL1, Nostoc species SN418, Nostoc species SN419, Nostoc species SN426, Nostoc species SN432, Nostoc species SN440, Nostoc species SN449, Nostoc species SN64, Nostoc species TAU-MAC 0799, Nostoc species TCL240-02, Nostoc species TCL26-01, Nostoc species TDI#AR94, Nostoc species TH1S01, Nostoc species TO1S01, Nostoc species UAM 307, Nostoc species UAM 308, Nostoc species UHCC 0702, Nostoc species UHCC0870, Nostoc species UHCC 0926, Nostoc species UK18, Nostoc species Us-7-5, Nostoc species VI.5, Nostoc species VP2-08, Nostoc species YK-01, Nostoc species roza1, Nostoc species roza4, Nostoc species sepahi, Nostoc species sf 6Calc, Nostoc spherical (Nostoc sphaericum), Nostoc sphaeroides, Nostoc sphaeroides ACSSI 150, Nostoc sphaeroides CCNUC1, Nostoc sphaeroides HBHF0604, Nostoc spongiaeforme Ind42, Nostoc verrucosum, Nostocaceae cyanobacterium, Nostocaceae cyanobacterium Baduki3, Nostocaceae cyanobacterium CENA376, Nostocaceae cyanobacterium CENA388, Nostocaceae cyanobacterium DW3II-PS, Pseudoaliinostoc jiangxiense, Roholtiella edaphica AR2, Roholtiella edaphica AR4, Roholtiella edaphica AR6, Roholtiella edaphica KZ-5-4-5, Roholtiella fluviatilis SN435, Roholtiella fluviatilis UAM 332, Roholtiella fluviatilisUAM 334, Scytonema bohneri SAG 255.80, Scytonema mirabile SAG 83.79, Scytonema sp. HAN3 / 2, Tolypothrix distorta ACT712, Tolypothrix sp. 9k, Tolypothrix sp. CCM-UFV067, Tolypothrix sp. IAM M-259, Tolypothrix sp. PCC 7601, Tolypothrix sp. PCC 7712, Tolypothrix sp. PCC7910, Tolypothrix sp. UAM 335, Tolypothrix tenuis PCC 7101, Micromonospora SAG94.79, Micromonospora SN436, Trichoderma sp. CCM-UFV035, Trichoderma PS4F, Trichoderma SBC125, Trichoderma variabilis, Trichoderma variabilis 0441, Trichoderma variabilis ATCC29413, Trichoderma variabilis GITAM RGP, Trichoderma variabilis NIES-23, Trichoderma variabilis RPAN45, Trichoderma variabilis SN416, Trichoderma variabilis str. GREIFSWALD, Trichoderma variabilis str. HINDAK 2001 / 4, Violetonostoc minutum CHAB 5840, Violetonostoc minutum CHAB 5841, Wollea vaginicola) RPAN22 or cyanobacterium NIES-2102.

[0295] In some embodiments, the technology provides a microbial consortium comprising organisms from the following taxonomic groups (e.g., genera): Acaryochloris, Aliterella, Amazonocrinis, Anabaena, Anabaena, Aphanizomenon, Atlanticothrix, Calenema, Calenema, Pseudo-Cyanocyanobacterium, Pseudo-Chromococcus, Chrysosporium, Compactonostoc, Constrictifilum, Crocodile Glomerella, Cyanophyceae, Cyanocohniella, Pseudo-Cyanocyanobacterium, Cyanophyceae, Dendronalium, Desikacharya, Desmonostoc, Longisporium, Erythrobacter, Flytella, Voltiella, Fremyella, Gastrosporium, Myxococcus, Globose, Cyanophyceae, Species: Myxochaete, Gelatochaete, Goleter, Hose, Water, Jasmine, Johanseniella, Kamptonema, Komarekiella, Sclerotium, Sclerotium, Macrochaete, Trichochaete, Knot, Nodularia, Nodulariaceae, Nodularia, Oligospermum, Oscillatoria, Pantanalinema, Pelatocladus, Pseudo-Planatum, Planatum, Polymorpha, Porphyrobacter, Prochlorococcus, Prochlorococcus, Pseudo-Anabaena, Pseudo-Galatochaete, Acrocephalum, Phytophyte, Gelatochaete, Roholtiella, Rubidibacter, Pseudo-branched algae, Spirulina, Synechococcus, Synechocystis, Thermosynechococcus, Monophydium, Trichophyton, Violetonostoc, Lumenella, or Heterococcus.

[0296] In some embodiments, the microbial consortium further includes: an organism from the genus Caulobacter (e.g., Caulobacter species PMC12), an organism from the phylum Proteobacteria (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria), an organism from the genus Bosella (e.g., Bosella species R-45681), an organism from the genus Caulobacter (e.g., Caulobacter segnis ATCC 21756, Caulobacter ginsengisoli), and / or an organism from the genus Pseudomonas (e.g., Pseudomonas species NFR16).

[0297] Consortium stability

[0298] In the development process of the embodiments of the technology described herein, stability is used as a measure for identifying consortia that exhibit repeatable behavior, as a predictor of consortia that can be produced at a larger production scale and will find use in the field as a product. As used herein, the stability of a microbial community or consortium refers to the amount of change in a microbial community that changes with the continuous passage of a culture comprising a microbial community. Therefore, the α diversity of a microbial community defined by the number of species represented in a microbial community is less important. Specifically, an increase in stability indicates a decrease in the change in a microbial community that changes with the continuous passage of a culture comprising a microbial community. Therefore, a portion of a stable consortium (e.g., a portion of a culture comprising a stable consortium) is used to inoculate a new culture medium, and the culture is grown under conditions suitable for consortium production, which is expected to produce a culture comprising the same consortium as the culture used for the inoculum. As the stability metric increases, the predictability of producing the same consortium present in the inoculum in a new culture increases. In other words, a stable culture produces a predictable resulting population of microbial organisms given a defined set of external factors (e.g., culture conditions such as light, nutrients, temperature, aeration, etc.) Thus, an increase in stability indicates an increase in the predictability or confidence that the resulting microbial culture will contain the desired, defined microbial community or consortium.

[0299] Data collected during the development of embodiments of the technology described herein indicate that the Bray-Curtis dissimilarity scores for both BW01 and BW02 change rapidly between the original soil input sample and P1, and between P1 and P2. Fig.13 However, the Bray-Curtis dissimilarity scores for both BW01 and BW02 between P2 and P3 and between P3 and P4 approached a limit of approximately 0.14 to 0.18. These data suggest that the BW01 and BW02 consortia began to reach an equilibrium state after two passages, where the differences between passages were minimized and / or eliminated with subsequent passages.

[0300] Furthermore, experiments performed during the development of the technology described herein demonstrated that the selection process was effective in generating microbial consortia with desired functionality. Principal coordinate analysis of shotgun sequencing data obtained from the original soil sample and the P1 to P4 passage samples showed that BW01 and BW02 generated from the original soil sample clustered fairly closely together ( Fig.14 After subculturing, the P4 BW02 cluster was more diverse than the P4BW01 cluster ( Fig.14 Although P4 BW01 and BW01 cultures produced two separate clusters, these separate clusters shared common components ( Fig.14Finally, within a given sample, no significant changes were observed in clustering between the initial and intermediate passages (e.g., between P1 and P2), indicating that the population in the P1 culture had begun to have increased stability. However, both the initial and intermediate passages differed greatly from the initial soil sample.

[0301] Thus, the technology provided herein provides an efficient method for selective cultivation. In addition, the selective cultivation method is used to produce plants that can grow without supplementing bound nitrogen or removing atmospheric CO. 2 The technology described herein can be used, for example, to provide treatments for agricultural soils.

[0302] Assessment of storage

[0303] During development of embodiments of the technology described herein, carbon and nitrogen fixation data from photobioreactor experiments and measurements of biofilm density from vertically illuminated multi-well plate cultures were used to assess carbon sequestration provided by the microbial consortia described herein. The microbial consortia offset carbon dioxide emissions in two ways.

[0304] In the first, microorganisms absorb carbon dioxide from the air and sequester the carbon in biomolecules that persist in the soil. For example, the technology described herein provides embodiments of microbial consortia that produce carbon compounds that sequester carbon from the atmosphere in durable and stable biomolecules that persist in the soil, such as melanins (e.g., pheomelanin, eumelanin, and / or pyomelanin). That is, embodiments relate to a consortium that produces durable carbon compounds that provides a stable carbon sink to remove atmospheric carbon (e.g., CO 2 ) and stores the carbon in durable carbon compounds (e.g., melanin) in the soil. For every ton of elemental carbon in the microbial mat, 3.67 tons of carbon dioxide gas are sequestered because 73% of the mass of a carbon dioxide molecule is provided by oxygen atoms.

[0305] The second way that microbial consortia offset carbon dioxide emissions is by reducing agriculture's need for nitrogen-based fertilizers. The Haber-Bosch process consumes large amounts of natural gas to produce the hydrogen needed to fix nitrogen into ammonia-specifically, for every ton of ammonia produced, 2.16 tons of carbon dioxide are emitted (see, e.g., Ghavam (2021) "Sustainable Ammonia Production Processes" Front. Energy Res. 9:580808, incorporated herein by reference). In addition, less than 20% of the nitrogen applied to the field enters the final crop. Most of the rest is leached or denitrified, causing environmental damage without any benefits to growers (see, e.g., Royal Society Policy Briefing "Ammonia: zero-carbon fertiliser, fuel and energy store" February 2020, available from royalsociety.org / green-ammonia and incorporated herein by reference). In some embodiments, the microbial consortia and / or compositions comprising microbial consortia described herein double this efficiency by slowly releasing this nitrogen rather than in a single application as with nitrogen fertilizers.

[0306] A culture grown in the field from a consortium described herein (e.g., BWOl) at the same growth rate as grown in a shallow well plate filled with nitrogen-free medium will reach saturation after approximately one month of growth. A saturated culture will produce 150 kg / acre of dry biomass, comprising 6.2% nitrogen and 45.2% carbon. This biomass will offset 0.30 metric tons of carbon dioxide in a one-month growing period. This estimate assumes saturation and the accompanying maximum accumulation ( Fig.15 ).

[0307] However, it is not predicted that the cultures will grow to saturation in the field. Instead, once the cultures reach 100% surface coverage, they will begin to grow in a Z-dimensional growth pattern of a biofilm or soil crust ( Fig.16 ). New microbial layers will form over previous ones, shading lower ones and reducing their further growth. This one-dimensional growth may proceed with the doubling time calculated for standard cultures, but for each new generation formed, the old generation will cease to divide. In this way, growth is expected to proceed linearly after the point of complete coverage. Therefore, the expected 60-day CO2 offset is 1.66 metric tons per acre.

[0308] In addition, in some embodiments, the growth of the microbial consortium occurs in large open ponds located in locations with ample sunlight and space (e.g., desert areas). The global annual production of ammonia for synthetic fertilizer by Haber-Bosch is 120 Mt, of which 99 Mt is elemental nitrogen. As described herein, growing a microbial consortium in a space 17% the size of Arizona would offset the total global nitrogen production produced by the Haber-Bosch process.

[0309] Similarly, producing microbial fertilizer (e.g., as a slow-release nitrogen green manure) on 1 million acres of land according to the techniques described herein would replace 100 pounds of chemical fertilizer per acre per year on 100 million acres of farmland. Increasing the growth of microbial consortia in fields by 100 times would reduce the amount of land to 7,221 acres to replace 100 pounds of chemical fertilizer per acre per year on 100 million acres of farmland. See Table 7.

[0310] Table 7 – Projected growing area to produce enough N to replace synthetic fertilizers

[0311]

[0312] *Assuming a 10 cm deep pond, using MCC#01, 50 lb / ac of applied organic actual N is equivalent to 100 lb / ac of inorganic actual N because the nitrogen fertilizer use efficiency is improved by 2 times.

[0313] Compositions for agricultural use

[0314] In some embodiments, the technology provides compositions for agricultural purposes and uses the compositions to improve agricultural culture medium (for example, soil, greenhouse growth medium or hydroponic culture medium). In some embodiments, the composition includes a microbial consortium as described herein (for example, including microorganisms that perform photosynthesis and nitrogen fixation, heterotrophic microorganisms that consume nitrogenous compounds and / or carbon-containing compounds produced by photosynthetic nitrogen-fixing microorganisms, and optionally, one or more other photosynthetic organisms and / or one or more other heterotrophic organisms). In some embodiments, the consortium grows in vitro, wherein it utilizes nitrogen and carbon from the atmosphere to produce nitrogenous compounds and carbon-containing compounds. In some embodiments, the composition including the consortium is applied to agricultural culture medium as "green manure", for example, including a composition of a consortium and nitrogenous compounds and carbon-containing compounds produced by the consortium. In some embodiments, after the composition including the consortium is applied to agricultural culture medium, the green manure consortium performs minimal or no nitrogen fixation and / or carbon sequestration.

[0315] In some embodiments, a composition comprising a consortium is applied to an agricultural medium to inoculate the agricultural medium with the composition comprising the consortium. In addition to the nitrogen-containing compounds and carbon-containing compounds produced by the consortium before the composition is applied to the agricultural medium, the consortium continues to fix nitrogen and / or sequester carbon after the composition comprising the consortium is applied to the agricultural medium, and the nitrogen-containing compounds and carbon-containing compounds produced by the consortium grown on the agricultural medium enter the agricultural medium to improve the agricultural medium and / or nourish the plants (e.g., crops) grown on 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 pyomelanocin). That is, embodiments provide a consortium that produces durable carbon compounds that provides a stable carbon sink to remove atmospheric carbon (e.g., CO 2 ) and store carbon in durable carbon compounds (e.g., melanin) in the soil.

[0316] In some embodiments, the composition comprising the consortium is incorporated into an agricultural composition. In some embodiments, the agricultural composition comprises one or more of the following: a wetting agent, a compatibilizer (also referred to as a "compatibilizing agent"), a defoamer, a cleaning agent, a chelating agent, a drift reducing agent, a neutralizing agent, a buffer, a corrosion inhibitor, a dye, an odorant, a spreader (also referred to as a "spreader"), a penetration aid (also referred to as a "penetrant"), a sticking agent (also referred to as an "adhesive" or "adhesive"), a dispersant, a thickener (also referred to as a "thickener"), a stabilizer, an emulsifier, a freezing point depressant, or an antimicrobial agent.

[0317] In some embodiments, the agricultural composition is a solid. In some embodiments, the agricultural composition includes a carrier material, for example, mineral soils such as silica, silica gel, silicates, talc, kaolin, attaclay, 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 grain flour, bark flour, wood flour and husk flour, cellulose flour, attapulgite, montmorillonite, mica, vermiculite, synthetic silica and synthetic calcium silicate or a combination of these.

[0318] In some embodiments, the agricultural composition is a liquid. Thus, in some embodiments, the agricultural composition includes a compound or salt such as a 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 hydrogen phosphate, ammonium monodihydrogen phosphate, ammonium sodium hydrogen phosphate, ammonium thiocyanate, ammonium sulfamate, or ammonium carbamate.

[0319] In some embodiments, the agricultural composition includes a binder, for example, polyvinyl pyrrolidone, polyvinyl alcohol, partially hydrolyzed polyvinyl acetate, carboxymethyl cellulose, starch, vinyl pyrrolidone / vinyl acetate copolymer and polyvinyl acetate, or a combination of these; a lubricant, such as magnesium stearate, sodium stearate, talc or polyethylene glycol, or a combination of these; an antifoaming agent such as a silicone emulsion, a long chain alcohol, a phosphate ester, acetylene glycol, a fatty acid or an organofluorine compound, and a complexing agent such as a salt of ethylenediaminetetraacetic acid (EDTA), a salt of trinitrotriacetic acid or a salt of polyphosphoric acid, or a combination of these.

[0320] In some embodiments, the agricultural composition includes a surfactant. In some embodiments, the surfactant is added to a liquid agricultural composition. In some embodiments, the surfactant is added to a solid formulation, particularly those designed to be diluted with a carrier before application. Therefore, in some embodiments, the agricultural composition comprises a surfactant. Surfactants are sometimes used alone or with other additives (e.g., mineral oil or vegetable oil) as adjuvants for spray tank mixtures to improve the biological performance of the consortium on the target. The surfactant can be anionic, cationic or nonionic and can be used as an emulsifier, wetting agent, suspending agent or for other purposes. In some embodiments, the surfactant is nonionic, for example, alkyl ethoxylates, linear fatty alcohol ethoxylates and fatty amine ethoxylates. In some embodiments, the agricultural composition includes alkyl sulfates, such as salts of diethanolammonium lauryl sulfate; alkylaryl sulfonates, such as calcium dodecylbenzenesulfonate; alkylphenol-alkylene oxide addition products, such as nonylphenol-cis-ethoxylate; alcohol-alkylene oxide addition products, such as tridecanol-C16 ethoxylate; soaps, such as sodium stearate; alkylnaphthalene-sulfonates, such as sodium dibutyl-naphthalenesulfonate; dialkyl esters of sulfosuccinates, such as sodium di(2-ethylhexyl)sulfosuccinate ; sorbitan esters, such as sorbitan oleate; quaternary amines, such as lauryl trimethyl ammonium 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 phosphates; vegetable oils, such as soybean oil, rapeseed / low-erucic acid canola oil, olive oil, castor oil, sunflower 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, particularly methyl esters.

[0321] In some embodiments, the agricultural composition includes a wetting agent, a dispersant, a polymer surfactant, an emulsifier, a solubilizer, an organic solvent, a gelling agent, a defoaming agent, and / or a preservative.

[0322] In addition, in some embodiments, the agricultural composition includes pesticides, herbicides, bactericides, fungicides, insecticides, viricides, miticides, nematicides, acaricides, plant growth regulators, rodenticides, anti-algae agents, biological control agents, beneficial agents, known fertilizers, inert ingredients and / or biologically active agents.

[0323] In some embodiments, the agricultural compositions and / or consortia described herein are applied as a topical application to an agricultural growth medium to improve crop growth, yield and / or quality. Topical applications may be in the form of a dry mixture or a powder or dusting composition, or may be a liquid-based formulation. In some embodiments, the agricultural compositions and / or consortia described herein may be formulated as: solutions; wettable powders; dusting powders; soluble powders; emulsions or suspension concentrates; tablets; water-dispersible granules; water-soluble granules (slow or fast release); microencapsulated granules or suspensions; and as irrigation components. In some embodiments, the agricultural compositions and / or consortia described herein are diluted in an aqueous medium prior to spray application. In some embodiments, the agricultural compositions and / or consortia described herein are applied to soil, plants, seeds, rhizosphere, rhizosheath, or other areas where application of the composition would be beneficial.

[0324] Although the disclosure herein is directed to certain exemplary embodiments, it should be understood that these embodiments are presented by way of example and not limitation.

[0325] Example

[0326] In the development process of the embodiment of the technology described herein, experiments were conducted to produce microbial consortia from environmental samples using the selection techniques applied at the community level. The data collected in these experiments showed the effectiveness of novel selective culture methods for selecting microbial consortia that can effectively grow in mineral culture media and atmospheric nitrogen and carbon dioxide without nitrogen or carbon compound supplementation. When grown under determined selection conditions, microbial consortia are stable. We also apply these same methods to several large-scale batches of agricultural soils, and we expect to produce up to 80 additional cultures for selection. This will enable us to greatly expand our diversity and capabilities when the first three consortia are tested in the field.

[0327] Materials and Methods

[0328] Environmental Sampling. Liquid and soil samples were collected from eight different locations on two private residential estates in or near St. Louis, Missouri. Half of the samples were collected in midwinter, while the other half were collected in the spring. Liquid samples were collected from the bottom of a nearly empty rain barrel in midwinter and included green sludge. The green sludge material was stored in glass jars under dim light at room temperature for several weeks before use. Soil samples were collected with a sterile spoon or shovel and placed in a clean plastic zipper-type bag. First, loose material was brushed off from the sampling site. Then, a volume of approximately 100 cm was dug into the soil at a depth of approximately 5 cm. 3 The soil samples were stored in a dark place at 4°C until use.

[0329] Inoculation of the growth medium. The environmental sample is first mechanically homogenized from the outside of the bag. Next, approximately 10 cm 3 The sample material is transferred to a 50-ml conical tube. Then, a suitable growth medium is added to provide a total volume of 40 mL. The sample is vortexed until large clumps are dispersed and the sample is substantially uniform. Next, a 0.5-1.0 mL volume of the vortexed sample is added to the growth container using a wide-bore pipette tip. The volume added depends on the volume of the growth container. The suspended sample is inoculated immediately, or the suspension is stored for a few days at room temperature under dim light and vortexed again before use.

[0330] Growth conditions. Three conditions were used for the growth of samples in various culture medium types: 1) shake flasks; 2) fixed bottles filled with liquid; and 3) fixed bottles filled with sterile sand and liquid. Shake flasks are 125-mL standard conical flasks covered with metal culture lids or aluminum foil. The shaker is a floor shaker incubated by New Brunswick G-25R, equipped with: a) ATS-CHILL600V (Advanced Technology Solutions, Inc.) water cooler to provide cooling water to the heat exchanger; b) a set of MRKD-40K22-40W-U 4000K 40-W dimmable LED lights (Super Bright LEDs, Inc.); and c) two 20-W halogen bulbs (Feit Electric). The lamps were operated at full power in a photoperiod of 18:6 (18 hours on, 6 hours off). Apogee Instruments MQ-500 photometer was used at approximately 120 μmol photons m –2 s –1The light intensity at the level of the shaking platform is measured at 150°C. The temperature in the shaking incubator is maintained at room temperature (e.g., approximately 23 to 25°C). In order to remove the heat generated by 80W lighting, cooling water (e.g., approximately 5°C) from an ATS water cooler is supplied to the integrated heat exchanger in the shaking incubator environment atmosphere control box to maintain the shaking incubator temperature. The internal heater of the shaking incubator is set to make up the difference at night. At room temperature, a square 160-mL milk dilution or 250-mL culture medium bottle is placed on two stands in front of two 42-W 4000K LED portable lamps (e.g., SLLP-40K4-42 or equivalent from Super Bright LEDs, Inc.) and four 20-W halogen bulbs (FeitElectric). Using Apogee Instruments MQ-500 photometer at approximately 120 μmol photons m –2 s –1 The light intensity was measured at the level of the shaking platform. The shaking platform used the same 18:6 light cycle (18 hours on, 6 hours off). The bottles were either filled to the top with culture medium or filled three quarters with sterile sand (Sigma-Aldrich) and then culture medium was added to fill. The lid was not tightly closed to allow for gas exchange.

[0331] Growth Media. Two custom media blends were used, Medium A and Medium B. Medium A was used with or without the addition of combined nitrogen. Medium B contained 1% full-strength combined nitrogen. Medium A was either standard UTEX BG-11 (–N) or BG-11 with normal nitrate (+N) with two modifications: 1) slightly lower phosphate content; and 2) the addition of 10 mM TES buffer at pH 7.0 to maintain the pH of the medium longer. Medium B was a modified version of complete M9 medium called BW-M9.

[0332] Medium A(+N) was prepared by mixing 10 mL of 100× BG11(+N) concentrate (see below), 1 mL of 1000× ammonium ferric citrate stock solution (0.6 g ammonium ferric citrate per 100 mL water; store at 4°C in the dark; check for growth before use), 1 mL of 1000× Na 2 CO 3 Stock solution (2 g Na in 100 mL water) 2 CO 3 ; stored at 4°C), 1mL 1000×K 2 HPO 4 Stock solution (3.05 g K in 100 mL water) 2 HPO 4; stored at 4°C) and 10 mL 1M TES (pH 8.2) were added to 1 liter of double distilled water. After autoclaving and cooling, 1 mL of 1000× Wolfe's vitamins (ATCC) was added. 100× BG11 (+N) was prepared by adding 149.6 g NaNO 3 (final medium 17.6mM), 7.5g MgSO 4 7H 2 O (final medium 0.3 mM), 3.6 g CaCl 2 ·2H 2 O (final medium 0.24 mM), 0.60 g citric acid (or 0.89 g sodium salt) (final medium 0.031 mM), 1.12 mL 0.25 M Na-EDTA (pH 8.0) (final medium 0.0028 mM), and 100 mL 1000× trace minerals (see below). To prevent precipitation, these additives were made from 100× stock solutions. For medium A without nitrogen (medium A(–N)), 100× BG11(–N) stock solution was made except that no NaNO was added 3 Prepare as above and use 100× BG11(-N) stock solution to prepare nitrogen-free Medium A (Medium A(-N)). 1000× trace minerals were added by adding 2.68 g H 3 BO 3 , 1.81 g MnCl 2 ·4H 2 O, 0.22g ZnSO 4 7H 2 O, 0.39 g Na 2 MoO 4 ·2H 2 O, 0.079 gCuSO 4 ·5H 2 O, and 0.049g Co(NO 3 ) 2 6H 2 O was added to one liter of water. The medium was stored at 4°C.

[0333] Medium B (BW-M9) was prepared by mixing (per 1000 ml) 100 mL of 10× low-N M9 salts (see below) and 895 mL of water. After autoclaving and cooling the low-N M9 salts solution, 0.2 mL of filter-sterilized 20% w / v sodium acetate, 0.3 mL of filter-sterilized 1 M CaCl 2 (14.7 g / 100 mL), 1 mL of filter-sterilized 1 M MgSO 4(24.65 g / 100 mL), 1 mL of filter-sterilized 1000× BW-M9 trace elements (see below), and 100× filter-sterilized Wolfe vitamins (ATCC). For liquid media, the mixture was autoclaved at 2500°F (1210°C) for 20 minutes using slow exhaust. Vitamin solutions were used at 1 / 4 concentration and were optional. The solutions were mixed after each addition of stock solution. Low-N M9 (10×) salts were prepared by mixing 11.33 g Na 2 HPO 4 -7H 2 O, 3g KH 2 PO 4 , 0.5 g NaCl and 0.01 g NH 4 Cl was mixed in distilled water to 100 mL, and then the pH was adjusted to 7.2 with NaOH. BW-M9 trace elements (1000×) were prepared by mixing 0.5 g EDTA, 0.83 g FeCl 3 -6H 2 , 22mg ZnSO 4 -7H 2 O, 7.9 mg CuSO 4 -5H 2 O, 10.00 mg CoCl 2 -2H 2 O, 0.10 g H 3 BO 3 、0.18gMnCl 2 -4H 2 O and 39.00 mg Na 2 MoO 4 -2H 2 O in distilled water to 100 mL. EDTA was first added and the pH was adjusted to 7.5 with NaOH. The solution was sterilized using a 0.22 μm filter.

[0334] Subculture, cryopreservation and sample collection. The culture is subcultured to enrich the culture by selection, and the stability of the microbial community is evaluated. The growth in the culture is not measured using standard optical density measurements because the nitrogen-fixing consortium grows in agglomerates. On the contrary, a weight-based growth rate assay (see the growth curve section below) was developed, and the growth time was used rather than directly measuring the number of cells to normalize the passage between samples. Visual observation of growth was used to determine the length of time from inoculation to the late exponential phase of growth before the culture began to decline. These observations showed that exponential growth was reached 17 ± 1 days after inoculation, so a passage time of 17 ± 1 days was used in the experiment.

[0335] Passaging was performed as follows: the culture vessel was removed from the incubator and any biofilm formed was resuspended by vortexing and / or scraping. Immediately after vortexing, 5 mL of the culture was aspirated into a serological pipette and transferred to a 250-mL Erlenmeyer flask containing approximately 100 mL of the same growth medium used to grow the culture from which the 5-ml inoculum was taken. Each passaging was repeated by inoculating two new cultures with a 5-mL inoculum taken from the same previous culture. Culture samples were saved for DNA extraction by pelleting approximately 1-1.5 mL of the culture and stored at -20°C until use. In addition, one or two cell stocks of the growth medium were stored in 20% glycerol at -80°C. The cell passage from the initial culture inoculated with an inoculum taken from an 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.

[0336] Growth curves. As described above, cultures clumped due to biofilm formation and possible EPS production in the cultures. Therefore, optical density measurements did not provide an accurate measure of growth, and growth curves were generated using the dry weight of the cell pellet. To provide accurate measurements, each time point was grown in a single well of a multiwell plate, and the experiment was designed as an endpoint weight determination for each time point. For each P4 culture (eight populations × each lineage BW01, BW02, or BW05), three biological replicates were used at 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 removed from each flask and vortexed continuously for 5 min until the cell clumps were dispersed into a uniform suspension to provide a culture from which small amounts (e.g., 50 μL) could be accurately pipetted for inoculation. An additional 200 μL aliquot was removed from the remaining vortexed culture and saved for time points (see below).Each plate was placed on an orbital shaker at 150 rpm under two 40-W 4000K LED portable lights.

[0337] Use a disposable pipette to collect each time point. All materials, including biofilms adhering to the bottom and sides of untreated wells, are destroyed and pipetted. Ultrapure water is used as a rinse and all rinses are collected. All materials from each well are collected in a 15-mL conical centrifuge tube and centrifuged in a swinging bucket rotor at 5000rpm for 10 minutes. Discard the supernatant and add ultrapure water in a volume sufficient to provide a total volume of 1.5mL. Using a disposable pipette, transfer the sample to a pre-weighed 1.5-mL microcentrifuge tube. The sample is centrifuged at 10,000rpm for 10 minutes and the supernatant is discarded. The wet weight is measured before the tube is opened and then placed in an oven at 55°C for 1 to 2 days, or before the sticky precipitate becomes a dry flaky film separated from the tube wall, the wet weight is measured. For each time point, three control tubes are also included in the oven, and the slight reduction in the weight of these tubes is used to calculate the coefficient of variation of the dry tare weight. The tare weight was multiplied by the dry tare weight variation coefficient to provide an adjusted tare weight. The sample tube containing the sample was weighed and the adjusted tare weight was subtracted to obtain the final dry weight of the sample. Dry weight was plotted using the ggplot2 package in R studio (see, e.g., Wickham, (2016) "gplot2: Elegant Graphics for Data Analysis" (Springer-Verlag New York)). Growth curves were simulated using the R package Growthcurver (see, e.g., Sprouffske (2016) "Growthcurver: An R package for obtaining interpretable metrics from microbial growth curves" BMC Bioinformatics 17: 172). All replicate growth curve data were input into the algorithm to simulate the BW01 or BW02 lineage. The resulting curves were used to determine the maximum doubling time and other metrics. Unless otherwise specified, default inputs were used. Because T 0 The dry weight was below the detection limit (<0.0001 g), so the default minimum calibration value was used to prevent fitting errors.

[0338] 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.

[0339] Library preparation. Use Illumina DNA Prep library preparation kit (Illumina 20018705) to prepare Illumina sequencing library according to manufacturer's instructions. Prepare library with primer containing 10-bp unique dual index to provide multiplicity. Use Qubit fluorometer to measure library yield, and use Agilent bioanalyzer equipped with high sensitivity DNA kit to measure average library fragment size. The target average fragment size of library is about 600bp.

[0340] Illumina sequencing. Libraries were pooled and sequenced on an Illumina NovaSeq 6000 sequencer at the Genome Technology Access Center (GTAC) at the McDonnell Genome Institute (Washington University in St. Louis) to obtain 150-bp paired-end reads. Read data were demultiplexed by GTAC.

[0341] 16S amplicon sequencing. 16S rRNA amplicon sequencing was performed by Genewiz. Genewiz also performed taxonomic calls.

[0342] Metagenomic data analysis. Taxonomy was determined using Kraken 2v2.1.2 and the standard Kraken 2 database created on December 2, 2020. Functional annotations were determined using Phylomagnet v0.0.1. Paired-end reads were first joined using Fastq-Joinv1.3.1. 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 generated by Phylomagnet were assigned to a taxonomy and functional annotations. Table 4 lists the EggNOG ortholog groups representing functional annotations.

[0343] Evaluation of consortium stability. After each passage, the culture was evaluated to estimate the stability of the microbial consortium present in the culture. Stability was evaluated using a beta diversity measure calculated by the Bray-Curtis dissimilarity index (see, e.g., Bray & 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 is calculated as shown in Equation 1, where S iis the total number of samples at site i, S j is the total number of samples in part j, and C ij It is the sum of the smaller values ​​of each sample in each part.

[0344]

[0345] The Bray-Curtis dissimilarity index ranges between 0 and 1, where a score of 0 indicates no difference between the two passages, and a score of 1 indicates no common samples between the two passages. For example, human microbiome samples vary greatly between body sites, with average Bray-Curtis values ​​ranging from 0.24±0.17 (SD) when comparing individual stool samples and 0.79±0.08 (SD) when comparing stool and nasal samples in one reference dataset (see, e.g., Maziarz, Using standard microbiome reference groups to simplify beta-diversity analyses and facilitate independent validation. Bioinformatics 34, 3249–3257 (2018), incorporated herein by reference). Bray-Curtis scores for soil sample replicates (e.g., from the same area of ​​1 to 2 square meters) extracted with the same DNA extraction method were as low as 0.1523. However, Bray-Curtis values ​​of soil samples extracted using different DNA extraction methods were observed to be >0.5.

[0346] Isolation of minimal consortia. Minimal consortia were generated using 20 μL of the undiluted vortexed culture, which was pipetted onto BG-11(–N) plates containing 1 / 4 Wolfe vitamins (ATCC), 1 mM sodium thiosulfate, and 1% agarose. A long line was streaked across the plate. At approximately 100 μmol m –2 s –1After 2 to 4 weeks of growth at room temperature (e.g., approximately 24°C) under white light (via LED) of approximately 100 μmol m–2 s–1 intensity, large colonies appear. Pick a colony, place in 1 mL of sterile ultrapure water, and vortex until clumps are dispersed. Pipette a 20 μL volume onto several plates of the same type with 4 overlapping lines. Pick a colony again and restreak in the same manner until isolated colonies are observed to have roughly consistent characteristics (usually after 2 or 3 restreaks). Finally, an isolated colony is used to inoculate 30 mL of BG-11(–N) and 1 / 4 Wolfe vitamins, placed in an upright disposable vented tissue culture flask, and shaken at 150 rpm at room temperature (e.g., approximately 24°C) under white light (via LED) of approximately 100 μmol m–2 s–1 intensity. Extract DNA and perform shotgun sequencing in the same manner as for the complete consortium.

[0347] Phylogenetic analysis of minimal consortia. The taxonomic system was determined using Phyloflash v3.4 and the SILVA database v138.1. The taxonomic system was updated using the NCBI taxonomic group API. The results were confirmed using the binning algorithm MetaBat v2.15 and after read assembly using MegaHit v1.2.9. Contigs in each bin were evaluated using CheckM v1.1.3.

[0348] Taxonomic analysis of photosynthetic microorganisms. The forward raw sequence reads were compared to a reference database of plasmid 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" SciRep. 10:5915, incorporated herein by reference). The results were filtered to identify matches with less than 10 base pair mismatches, greater than 130 base pair lengths, and greater than 95% identity.

[0349] Photobioreactor growth. A photobioreactor was constructed using a series of one-liter glass flasks. Each flask was placed on a stirring plate and closed with a stopper containing a glass bubbling line and an outlet line. The growth medium was BG-11 without TES buffer. TES buffer was removed from the growth medium to simulate large-scale production conditions, under which the use of TES was too costly. Therefore, TES buffer is an optional component that can be used for long-term shaking culture and passage. Growth medium and stirring rods were added to the flasks, the openings were covered with aluminum foil, and autoclaved. The stopper assembly including the inlet filter (Whatman HEPA-vent) was autoclaved separately in a dry package. After cooling, the liquid culture medium was inoculated with 2mL of stationary phase culture. The inoculum concentration was determined using the dry weight measurement technique described by the growth curve. The inlet side of each filter was connected to the outlet line of the humidifier bottle. The bubbler line inlet of each humidifier bottle was connected to a 10-gallon rated Whisper air pump protected by a check valve.

[0350] Obtain time points from photobioreactor cultures to generate growth curves. Before each time point, the biofilm formed was destroyed by shaking or scraping the culture flask wall. For each time point, 1 mL of culture was extracted using a 1-mL serological pipette, and 1 ml of culture was added to a pre-weighed 1.7 mL plastic microcentrifuge tube. Centrifugal, drying and weighing were used to generate growth curves as described herein. Although humidification bottles were used, some evaporation was observed during growth. In order to offset the impact of evaporation on growth curve data, the bioreactor volume (accurate to 50 mL) of each sample was recorded. Curve fitting was performed on the point drawn based on grams / bioreactor, and this converted 1 mL of culture dry weight into the current culture volume when sampling.

[0351] Determination of carbon and nitrogen. After harvesting the photobioreactor culture, the total elemental carbon and nitrogen of the stem cell precipitate were determined. Cells were separated from the liquid culture medium at the very late logarithmic phase or the stationary phase (32 or 45 days for strong light and weak light experiments, respectively). The whole cell precipitate was harvested in a Beckman-Coulter Allegra 25R centrifuge at a speed of 5000rpm in a bucket rotor containing four 500mL bottles. The precipitate was transferred to a pre-weighed disposable 50mL conical tube and dried in an oven at 55°C until it became hard and brittle. According to the MWL WC ​​PROC 55 protocol, the precipitate was analyzed by combustion in a LECO analyzer by Midwest Laboratories (Omaha, NE, USA).

[0352] Soil growth. Commercial potting mix was sterilized by autoclaving in glass petri dishes prior to inoculation with red fluorescence images of three consortia grown on sterile potting soil, day 0 blanks were subtracted, and images were compared by proprietary image analysis software. Excitation light was 450nm blue light. A deep red filter was used to block the blue wavelength of the camera detector.

[0353] Example 1

[0354] In the development process of the embodiment of the technology provided herein, experiments were conducted to select organisms that only use light, alkaline mineral salts and atmospheric gases to grow without adding reduced nitrogen and carbon. Eight environmental samples collected in midwinter or early spring were subjected to multiple selective growth conditions, providing 56 different starting samples (Table 1). Many conditions showed growth. However, the collected data showed that three cultures (colonies) inoculated from winter soil had abnormal growth in a short time (28 days). Further experiments focused on these three cultures (BW01, BW02 and BW05). Condition 7 is a positive control for microbial growth. The growth in Table 1 is visually estimated by comparing the growth of each condition in conditions 1 to 6 with the growth of positive control condition 7 and specifying numbers from 1 (low growth) to 8 (high growth). BW01, BW02 and BW05 are produced in BG-11 medium ("medium A") with light without nitrogen (-N). BW01 and BW02 are produced by shaking in a flask. BW05 is produced in sand.

[0355] Table 1 – Growth conditions of starting samples

[0356]

[0357] Example 2

[0358] 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 BW01, BW02 and BW05 cultures during the initial selection. The growth of the cultures was monitored visually, and an inoculum removed in the late exponential phase of growth (e.g., 2.5 weeks after inoculation) was used to inoculate new flasks containing fresh selective growth medium.

[0359] 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 is tracked in duplicate flasks grown under the same selective pressure. Four passages are performed to assess the stability of the cultures from the passages (see below) and to produce consortia that include key components of the initial population. Passages are performed simultaneously under (–N) and (+N) conditions, with the (+N) culture used as a positive control to verify conditions that favor growth. The nitrogen-supplemented positive control grew faster, suggesting that growth of the population can be accelerated by nutrient supplementation. For each passage, samples of the exponential phase culture are collected and preserved for storage and shotgun metagenomic sequencing.

[0360] Different culture morphologies were observed in the final passage (P4). The BW01 lineage had two morphological categories—four P4 flasks had light green small clumps, and four flasks had darker and larger clumps. BW02 contained very clumping cells and had a fast growth phenotype. When dehydrated, the BW02 culture had a jelly-like morphology consistent with the extracellular polymers (EPS) secreted by cyanobacteria of the Nostoc family (such as Anabaena species) (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).

[0361] When observed by light microscopy, both BW01 and BW02 cultures contained filaments with distinct heterocysts ( Figure 1A and Figure 1B ). Heterocysts are an adaptation of the Nostoc family for nitrogen fixation in conjunction 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, heterocysts provide an anaerobic environment for nitrogen fixation while reproductive cells perform photosynthesis (e.g., oxygenic photosynthesis). Sugars and fixed nitrogen are exchanged between heterocysts and reproductive cells via microplasmodesmata. The Nostoc family also has a second differentiated cell type, called akinete, which is highly tolerant to environmental damage and is therefore a long-term survival mechanism for the organism.

[0362] Example 3

[0363] During the development of embodiments of the technology described herein, shotgun metagenomic sequencing was used to evaluate BW01, BW02, and BW05 cultures to evaluate changes in population diversity and determine community composition. DNA extraction and shotgun metagenomic sequencing were performed on each culture from four generations. Illumina sequencing was performed. In order to identify the microbial genera that comprise the community, sequence data were analyzed using Kraken2. (See, e.g., Wood, Improved metagenomic analysis with Kraken 2. Genome Biol. 20, 257 (2019), incorporated herein by reference). In this analysis, replicates from the same generation were averaged to obtain overall community composition reads. In addition, shotgun metagenomic sequencing was also performed on the initial soil samples used to provide an explanation of the initial samples. Sequence data obtained from cultures grown under selective conditions showed that in each replicated flask, several lineages with different physical characteristics evolved slightly differently.

[0364] Taxonomic classification of metagenomic sequence data collected during these experiments indicated that the selective growth conditions significantly altered community composition compared to the original soil samples. Interestingly, while communities passaged from different starting samples differed in many traits, the communities were observed to share several similar features. The selection process modified the populations by altering the proportions of each genus. The data suggest that selection produced four general changes ( Figure 2 ; Figure 3A – Figure 3H ):

[0365] 1. The relative abundance of some genera increased throughout the passages. For example, Nostoc species were 31 ± 17% (SD) and 33 ± 10% (SD) in passage 4 (P4) samples of BW01 and BW02, respectively, an increase from less than 0.1% in the original soil; a similar trend was seen in BW05. The dominance of this genus increased rapidly and remained stable in all three passages of BW01, BW02, and BW05. Methylibium spp. also increased steadily throughout the passages, although at a lower abundance than Nostoc species.

[0366] 2. The relative abundance of other genera gradually decreased as the passages progressed. For example, both Streptomyces species and Mesorhizobium species appeared to be less well adapted under the selective conditions than in the original soil, and overall, these groups decreased in all cultures throughout the passages.

[0367] 3. Some genera initially increase in dominance and are later surpassed by other genera during passage, such as Aminobacterium species and Rhizobium species.

[0368] 4. Compared with the original soil, other genera initially decreased in dominance during P1 and increased in subsequent passages, for example, Pseudomonas species and Azospirillum species.

[0369] Example 4

[0370] During the development of embodiments of the technology described herein, the growth phenotype of a population was measured using replicate growth curves ( Figure 4 ). Growth curves were plotted using the dry weight (in grams) of culture in each 7.5 mL well of a standard 6-well plate, which also provided a measure of carbon sequestration. Growth curves containing all replicates from BW01 (n=24), BW02 (n=24), and BW05 (n=24) were modeled using a logarithmic function. The resulting measures, including the uncertainties associated with each curve fit, are shown in Table 2. The values ​​of 1 / 2K to reach half of the maximum growth were 19.1 days and 15.8 days, and the maximum doubling times for BW01 and BW02 were 4.2 days and 3.6 days, respectively. The parameters of Table 2 are as follows. Unless otherwise specified, all weight units are grams (dry weight) per 7.5 ml well in a standard 6-well plate, and times are in days: k = carrying capacity, n 0 = weight at the start of the growth phase, r = growth rate, σ = goodness of fit, df = degrees of freedom, t_mid = inflection point time, when the population is 1 / 2k, t_gen = fastest generation time, or doubling time. SE is the standard error, and p is the P value.

[0371] Table 2 – Growth curve fitting parameters for BW01 and BW02

[0372] value k n0 r σ df t_mid t_gen BW01 0.016 0.001 0.165 0.002 101 19.108 4.194 BW02 0.016 0.001 0.191 0.003 101 15.79 3.623

[0373] error k_se k_p n0_se n0_p r_se r_p BW01 0.001 9.00E-22 0 5.00E-03 0.024 8.00E-10 BW02 0.001 2.00E-29 0 3.00E-02 0.034 2.00E-07

[0374] Individual P4 populations were evaluated in more detail by fitting curves to the individual populations (n=3 for each population). Eight P4 populations were generated from each soil lineage, BW01 and BW02. The output parameters for these 16 populations are shown in Table 3. When the fitted curve doubling times within each soil lineage were averaged, the doubling times for BW01 and BW02 were 4.0±0.5 days and 3.5±0.7 days, respectively, with the error recorded as one standard deviation of the mean. The parameters for Table 3 are as follows. Unless otherwise specified, all weights are in grams per 7.5 ml well in a standard 6-well plate, and times are in days: k=carrying capacity, n 0= weight at the beginning of the growth period, r = growth rate, σ = goodness of fit, df = degrees of freedom, t_mid = inflection point time, when the population is 1 / 2k, t_gen = fastest generation time, or doubling time, auc = area under the curve, where l represents the logistic equation and e represents the empirical data points.

[0375] Table 3 – Growth curve fitting parameters for all 16 tested populations

[0376]

[0377]

[0378] Example 5

[0379] During the development of embodiments of the technology described herein, four passages were evaluated for specific gene and / or enzyme activity (Tables 4 and Figure 5 ). Gene functions were evaluated in two phyla represented by the taxonomically characterized cyanobacteria and proteobacteria. Gene functions from the photosynthesis, nitrogen fixation, and EPS production pathways were selected for analysis.

[0380] Table 4 – Orthologous groups representing functional annotations

[0381]

[0382]

[0383] Example 6

[0384] In the development process of the embodiment of technology described herein, experiments were conducted to produce the minimum consortium with reduced complexity, while also providing the phototrophic and nutrient fixation functions of the more complex consortium identified in other experiments. In these experiments, cultures were repeatedly streaked and diluted on flat plates to separate individual bacterium colonies of stable morphology. Cyanobacteria produce complex extracellular matrix, and are often closely related to other microorganisms. Therefore, cyanobacteria often carry other microorganisms by streaking, and this association remains in obviously independent bacterium colonies. In these experiments, this association is to identify the bacterium colonies that thrive on the minimal medium in the solid form for selection, although these bacterium colonies are separated from the complete consortium. Not bound by theory, it is expected that successful bacterium colonies will include consortium members that provide survival functions on minimal medium, and there will be no bacterium colonies produced without the microorganisms that other consortium members cannot survive on minimal medium. Therefore, experiments were conducted to identify the bacterium colonies that are included in the minimum population that survives on basic selective medium.

[0385] Two colonies with different morphologies were picked from each plate; each colony was grown on two plates. The colonies were then grown in the same liquid selection medium as the consortium growth above (e.g., BW01, BW02, and BW05). The morphology was predictable based on the pedigree, and all inoculated cultures grew reliably to saturation in liquid ( Figure 6 These cultures were grown to saturation (30 days) and DNA was extracted for shotgun sequencing. Sequencing data indicated that the resulting cultures were not pure isolates but contained a simpler microbial community than the BW01, BW02, and BW05 consortia ( Fig. 7A ). The most complex consortia identified in this way consisted of up to nine taxa, and the least complex consisted of two taxa. All sequenced samples included the cyanobacterial genus Nostoc or the closely related genus Eusporium. The relative abundance of specific genes (nifh, psba, psbb, rubiscoL, rubiscoS, and wza) was identified in the nucleotide sequences obtained from the consortia ( Figure 7B-7F Table 4 lists the EggNOG orthologous groups representing functional annotations.

[0386] Example 7

[0387] Two computational methods were used to determine the taxonomy of the members of the minimal consortium. The first method used Kraken2 to analyze the complete consortium. The second method used Phyloflash3.4, which uses a method based on small subunit rRNA gene assembly and is more suitable for low-complexity communities than Kraken2 (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 found in the samples, including Proteobacteria, Cyanobacteria, and Tremellomycete fungi (Table 5). Each taxonomic unit contributes different metabolic functions, which are important for the consortium. It is worth noting that cyanobacteria are always present, indicating their key functions in surviving specific selections.

[0388]

[0389]

[0390] Table 5 - Genera identified in the minimal consortium

[0391] Growth curves were generated by growing the minimal consortia in 12-well plates. The growth curves showed that most minimal consortia had similar performance compared to the full consortium control ( Figure 8 ). A notable exception was the dark green colonies picked from the BW02 plate. BW02 produced light brown, slimy colonies and dotted dark green colonies. About halfway through the experiment, the dark green colonies generally faded. The curve fitting parameters are shown in Table 6. The parameters in Table 6 are as follows: Unless otherwise noted, all weight units are grams per 2 ml well in a standard clear 12-well plate, and time is in days: k = carrying capacity, n 0 = weight at the beginning of the growth phase, r = growth rate, σ = goodness of fit, df = degrees of freedom, t_mid = inflection point time, when the population is 1 / 2k, t_gen = fastest generation time, or doubling time, auc = area under the curve, where l represents the logistic equation and e represents the empirical data point. Growthcurver18 was used for fitting.

[0392] Table 6 - Curve fitting parameters for simplified consortium growth curves

[0393]

[0394] 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.

[0395] Example 8

[0396] In the field, saturation is not expected to be as important as in liquid culture. Microbial cultures saturate when microorganisms exhaust resources (e.g., mineral nutrients), produce waste and metabolites, self-shade, or quorum sensing. However, in liquid culture (e.g., at production scale), saturation helps determine the upper limit of culture density. Therefore, during the development of embodiments of the technology described herein, experiments were conducted to determine the calculated carrying capacity to represent the maximum saturated culture density ( Fig. 9A and Fig. 9B ). The curve fitting data suggest that reducing consortium complexity does not necessarily reduce the carrying capacity of the culture.

[0397] Example 9

[0398] In some embodiments of the technology, growing large-scale cultures can be important for producing and using microbial consortia in fields. Therefore, experiments were carried out in the development process of technology embodiments to test the scaling potential of BW01, BW02 and BW05 consortia. In these experiments, BW01, BW02 and BW05 cultures were grown in a liter of photobioreactor (PBR). PBR is a bioreactor, commonly referred to as a fermentation tank, and is intended to be a microorganism that grows in a PBR. The combination of BW01, BW02 and BW05 cultures in the PBR is mixed and / or stirred using a combination of magnetic agitation and bubbling of filtered sterilized air to grow photosynthetic microorganism consortia to high density with a high growth rate without supplementing the culture medium with nitrogen and carbon compounds. Fig.10 Growth curves for the BW01, BW02 and BW05 consortia under these conditions are shown. These data indicate that the PBR is highly productive regardless of whether nitrogen compounds are added to supplement nitrogen supplied by air.

[0399] Use PBR to generate sufficient dry mass for elemental nitrogen and carbon analysis ( Fig.11 ). Nitrogen and carbon analysis data are used to calculate predicted carbon sequestration and nitrogen fixation by cultures in production ponds or fields.

[0400] Example 10

[0401] During the development of embodiments of the technology described herein, experiments were conducted to test the growth of BW01, BW02, and BW05 microbial consortia on soil. Chlorophyll fluorescence and machine vision were used to monitor the growth of the consortia on soil. The data showed that the BW01, BW02, and BW05 consortia established communities in the soil within a 10-day growth period ( Fig. 12A and Fig. 12B ). The data collected in this experiment indicated that BW01, BW02, and BW05 included photosynthetic members that produced chlorophyll.

[0402] Embodiment 11

[0403] In the development process of the embodiments of the technology provided herein, data were analyzed to determine the stability of the microbial consortium produced using the passages described herein. In particular, β-diversity was calculated for each passage of the process and monitored as a measure of stability. The β-diversity index compares two locations or passages, thus providing a useful metric to measure the changes that occur from one passage to the next. Therefore, the Bray-Curtis dissimilarity between consecutive passage comparisons reaching a stable level indicates that the culture has reached a maximum level of stability during the passage process.

[0404] The data collected show that Bray-Curtis dissimilarity scores change rapidly as selective pressure is applied ( Fig.13 ). The decrease in Bray-Curtis scores between each successive passage indicates that less variation is observed between successive late passages than between earlier passages. Comparing the last passage, P4 with its parent P3, the Bray-Curtis scores for BW01 and BW02 were 0.17 ± 0.065 (SD) and 0.14 ± 0.011 (SD), respectively. This suggests that as passages continue, these populations begin to reach a state of equilibrium in which subsequent passages minimally affect the culture and / or minimize variation between passages.

[0405] Throughout the passages, the two cultures followed similar trends, but were not identical. However, by the fourth passage, the Bray-Curtis dissimilarity scores for BW01 and BW02 plateaued, indicating that both cultures reached maximum stability. Considering that these cultures remained stable in small-scale laboratory experiments, it is expected that the cultures will be stable in production scale.

[0406] The data showed that the selection was targeted and effective. Principal coordinate analysis (PCoA) of shotgun sequencing data from the original soil samples and cultures from P1 to P4 showed that the BW01 and BW02 original soil samples clustered fairly tightly together, but the final passages began to develop into two separate clusters ( Fig.14 ). For P4, a strong trend towards clustering was observed. However, in BW01, there appears to be a distinct set of samples. Given the complexity of these groups, it would be surprising if we did not observe any differences in the passages. The relative tightness of the clustering suggests that selection has had a predictable and consistent effect on most lineages.

[0407] Example 12

[0408] Nitrogen- and carbon-fixing consortia have the potential to improve soils and sequester CO 2 . However, some consortia are adapted to transitional seasonal conditions that include high fluxes of broad spectrum light (e.g., about 380 to 750 nm). In addition, plants transmit or reflect light at wavelengths that they do not absorb. Therefore, crop plants typically utilize light with wavelengths within the classically defined photosynthetically active radiation of about 400 nm to about 700 nm. However, oxygenic photosynthetic microorganisms have adapted to use light outside of this range, particularly far-red light (FRL), generally defined as wavelengths from about 700 nm to about 750 nm.

[0409] During the 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 in-season carbon capture and soil stabilization during the growing season. Thus, selection conditions were used to produce consortia that utilized far-red light (e.g., approximately 700 to 750 nm).

[0410] These selections are designed to enrich organisms that utilize the energy from far-red light to fix carbon, for example, by using pigments and antenna pigment-proteins that absorb far-red light. In the development process of some embodiments of the technology described herein, the experiment does not use conditions selected for the organisms of nitrogen fixation. Therefore, the experiment includes the use of a culture medium (for example, BG-11+N) that includes a fixed nitrogen compound. This is identical to the culture medium for nitrogen addition experiments described herein.

[0411] Far-red light (e.g., about 700 to 750 nm) can be used to select for organisms that utilize far-red wavelengths, which is a portion of the spectrum that is largely unused by plants. Therefore, experiments were conducted in which 740 nm light (e.g., produced by an LED) was the only light source provided to the consortium and the organisms in the consortium. Specifically, multiple one-watt LED chips were attached to an aluminum heat sink and were irradiated at a rate of about 100 μmol / m 2 / s photon flux (e.g., using a Meanwell LED driver).

[0412] The experiment included the use of the same enrichment process as described above (e.g., to produce a consortium including photosynthetic nitrogen-fixing microorganisms), except that far-red light and a growth medium containing nitrogen compounds were used. The initial culture was inoculated with sludge and water samples from small ponds and streams within a 100-mile radius of St. Louis, Missouri. The sludge and soil samples were homogenized in sterile water at a 10-fold dilution using a 3.2-mm steel ball and a vortexer. Aerated culture flasks were inoculated with a small volume, stood upright and shaken at 100 rpm under a far-red LED at 25°C.

[0413] When the culture is close to saturation (e.g., about 6 weeks), it is subcultured and subcultured four times. Each initial culture (P1) is used to inoculate four flasks to produce P2 cultures. Then, one flask is inoculated with P3 with each of the four flasks, and one flask is inoculated with P4 with each P3 culture. DNA is extracted and sequenced as described.

[0414] In particular, experiments were conducted using soil and water samples obtained from Arkansas (150 soil samples and 30 water samples), Wisconsin (100 soil samples and 25 water samples), and Washington (250 soil samples and 15 water samples). Water samples were usually processed immediately; however, some soil samples were found to remain viable and stable after storage at 4°C. Samples were handled using sterile tools.

[0415] First, the sample contents are homogenized (e.g., manually from outside the bag in the case of soil samples). Next, 7.5 ml of sterile water is added to a 15-ml conical tube labeled to match the sample identifier on the bag. After adding 0.5 ml of sterile 3.2-mm chrome steel beads to each tube, approximately 2 cm2 of soil is scooped into each tube with a sterile spoon. The sample is mixed until homogenized using a vortexer. An ultrasonic instrument can optionally be used to homogenize the sample, although ultrasonic treatment may increase the chance of contamination or reduce population diversity. For ultrasonic treatment, the ultrasonic instrument is equipped with a 4420 microtip that can handle 5-50 ml. The probe is cleaned with 70% ethanol to reduce contamination. Approximately 3 / 4 of the probe is inserted into the liquid; the probe is not allowed to touch the wall of the tube. The sample is ultrasonically treated for 1 minute using 5-second pulses of 10% amplitude, with 5 seconds between each 5-second ultrasonic pulse. If homogenization is incomplete, ultrasonic treatment is performed again. Monitor the sample to prevent overheating.

[0416] After entering the identification information into the database, prepare the flasks for the selection process. Each sample-condition combination is assigned a unique identifier ("P#"). The flasks are labeled with the P#, parent sample number, media type, and date. The flasks are filled with 30 mL of media and the flasks are kept in an upright vertical position.

[0417] The homogenized sample is shaken and briefly vortexed to suspend particulate matter and provide a thoroughly mixed sample. A volume of 0.3 ml is removed from the homogenized sample within 30 seconds of vortexing and used to inoculate the appropriate flask prepared above. If multiple flasks are inoculated from the same homogenized sample, the sample is stirred and / or shaken to maintain mixing of the sample and prevent sedimentation of particulate matter. The flasks are capped and maintained in an upright vertical orientation throughout the inoculation and subsequent growth process.

[0418] For passaging P2 and P3, fill 25-cm plastic filter culture flasks with 30 ml of medium and label with a new P number. Flasks are kept upright in a vertical orientation. Three tubes are labeled for saving samples (e.g., as cell pellets), and 2 tubes containing 0.5 ml of 40% glycerol are labeled for glycerol stocks using the parental P number. For each parental flask (e.g., P1 or P2), remove biofilm using a plastic loop and homogenize the culture. Remove a 5-ml volume immediately (e.g., before the culture settles) - 1 ml to inoculate the P2 (or subsequently P3) flask; 1 ml to save the cell pellet sample; 0.5 ml each for the two glycerol stocks. Vortex the glycerol stocks containing the cultures and store at –80°C. Centrifuge the cell pellet tube at full speed for 5 minutes. After discarding the supernatant, store the cell pellet at –20°C for later DNA extraction and additional analysis. Place the culture flasks on a shaker illuminated with far-red light.

[0419] P4 passages were generated from P3 cultures according to the same inoculation method described above and subjected to additional validation steps. Live cultures of BW01, BW02, and BW05 were inoculated in the same manner as P4 cultures and used as growth controls.

[0420] Growth curves were generated from P3 cultures used as inoculants, as well as P4 and control (BW01, BW02, and BW05) cultures during growth. To measure the initial g / ml of cell material (T0 time point), two 1.7-mL snap-top tubes were pre-weighed and the empty weight of each culture and each control culture (BW01, BW02, and BW05) was recorded. Next, an additional 3 tubes were weighed to control plastic volatilization in the desiccator. The same type and brand of tubes were used throughout the experiment. A 1 ml volume of homogenized P3 culture was aliquoted into each tube, and the culture was precipitated by centrifugation (e.g., 5 min at full speed). The precipitate was placed in a 55°C oven to dry overnight, the dried precipitate was weighed in the tube, and the cell pellet weight was recorded.

[0421] Each week, a 1-ml aliquot of P4 culture was used to obtain the cell pellet weight (in g / ml culture) according to the same process described above. The cell pellet weight (g / ml) was monitored as a function of time and it was noted when the growth curve began to plateau. When the cell pellet weight reached a plateau, a 5 ml volume was removed - 1 ml was used to save the cell pellet sample; 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 minutes. After discarding the supernatant, the cell pellet was stored at -20°C for later DNA extraction and additional analysis. The R package Growthcurver was used to fit the curves and determine growth parameters.

[0422] DNA was extracted from P4 precipitates. Libraries were prepared for Illumina sequencing and sequenced at a target depth of 50 million reads. Based on the sequence data obtained, unique populations and / or populations were identified. Cultures and / or frozen samples of multiple (e.g., 1 to 50) different populations were identified and retained for further study.

[0423] Embodiment 13

[0424] During the development of embodiments of the technology described herein, experiments were performed to identify photosynthetic members of the complete consortium. DNA was extracted from complete consortium samples S1170, S1172, S1174, S1176, S1178, S1180, S1181, and S1184. After obtaining the nucleotide sequence by Illumina sequencing as described herein, the forward raw reads were compared to a reference database of plasmid 23S ribosomal DNA sequences using a 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 the plasmid database for matches as described in the Methods, the sequences were identified as being from the following bacterial genera: Nodularia, Chrysosporium, Globularia, Phytozoa, Trichoderma, Hosecodon, Myxosarcina, Acaryochloris, Kamptonema, Acrocephalum, Crocodile, Macrochaete, Thermosynechococcus, Pseudoanabaena, Pseudochromococcus, Prochlorococcus, Anabaena, Sclerotium, Browsing, Pseudocylopodium, Longisporium, Pseudobranchium, Sheathing, Monochidium, Flytella, Voltiella, Aliterella, Hydrostriella, Prochlorococcus, Pseudothiocystis, Ghecochloris, Heterococcus, Jasmine, Pseudothiocystis, Pantanalinema, Oscillatoria, Spirulina, Pelatocladus, Knotweed, and Aphanizomenon. , Pseudochlorophyll, Myxococcus, Calenema, Glechoma, Trichophyton, Synechococcus, Synechocystis, Cylindrosporium, Planctomyces, or Rubidibacter; or from the following algae genera: Tetraselmis, Chlorella, Chaetoceros, Disk, Paradoxia, Cymbomonas, Palmitochloris, Characiochloris, Marvania, Chlamydomonas, Pseudochlorella, Glechoma, Symphyogyna, Cyanochloris, Tetraspinosus, Dangeardinia, Galdiella, Nyholmiella, Porphyra, Chara, Fibrous Algae, Chlorella, Cyanophyte, Chondrus, Pseudochloris, Interfilum, Gelidium, Symphyogyna, Backpacker, or Cyanophyte.Thus, these data suggest that the microbial consortium includes photosynthetic organisms characterized by being members of the following bacterial genera: Nodularia, Chrysosporium, Globularia, Phytophyte, Trichoderma, Hose, Myxobacterium, Acaryochloris, Kamptonema, Acrocephalum, Crocodile, Macrochaete, Thermosynechococcus, Pseudoanabaena, Pseudochromococcus, Prochlorococcus, Anabaena, Sclerotium, Browsing, Pseudocylopodium, Longisporium, Pseudobranchium, Sheathing, Monochidium, Flytella, Voltiella, Aliterella, Aquatica, Prochlorococcus, Pseudothiocyanobacteria, Gastrodia, Heterococcus, Jasmine, Pseudomonas, Pantanalinema, Oscillatoria, Spirulina, Pelatocladus, Knotweed, and Spindle. or from the genera Tetraselena, Chlorella, Chaetococcus, Diskia, Paradoxia, Cymbomonas, Palmitochloris, Characiochloris, Marvania, Chlamydomonas, Pseudochlorella, Coccomycetes, Symcoccus, Cyanochloris, Tetraselena, Dangeardinia, Galdiella, Nyholmiella, Porphyra, Chara, Cellulose, Chlorella, Cyanophytes, Chondrus, Pseudochloris, Interfilum, Gelidium, Symphyogyna, Backpacker, or Cyanophyte.

[0425] Embodiment 14

[0426] During the development of embodiments of the technology described herein, observations of cultures of BW01, BW02, and BW05 indicated the presence of flocculants containing dark pigments ( Fig.18A ). The photobioreactor was constructed by pumping filtered air through a pipe into a stoppered conical flask. The photobioreactor was inoculated with the consortium and grown for two weeks under constant illumination, agitation (e.g., using a stir bar), and constant aeration. Flocculants were evident in all cultures after a few days; after continued growth, the cultures darkened in all cases. The inoculated cultures on day 1 are shown in Fig.18A The inoculated culture is shown in the top photograph of FIG. 1 after two weeks of incubation under constant light, agitation and aeration. Fig.18A in the bottom photo.

[0427] After growth, experiments were performed to characterize the dark pigments. Specifically, during development of embodiments of the technology described herein, it was contemplated that the dark pigments were molecules that included fixed carbon, e.g., a fixed and persistent form of carbon.

[0428] One class of persistent carbon compounds is melanin. Melanin biosynthesis uses the amino acid tyrosine to synthesize each of the three main types of melanin: pyomelanin, pheomelanin, and eumelanin ( Fig.18B ).like Fig.18B As shown, the biosynthetic pathways for the biosynthesis of pyomelanocin, pheomelanin and eumelanin are different. In particular, the biosynthesis of pyomelanocin from tyrosine produces homogentisic acid as an intermediate ( Fig.18B ); homogentisate is not produced in the biosynthesis of pheomelanin or eumelanin. In addition, tyrosinase converts tyrosine to dopaquinone or L-dopa, as an early (or first) step in the biosynthesis of pheomelanin or eumelanin, respectively, while aromatic amino acid transaminases convert tyrosine to 4-hydroxyphenylpyruvate, as an early (or first) step in the biosynthesis of pyomelanocin. 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.

[0429] Therefore, during the development of embodiments of the technology described herein, experiments were performed to determine whether the addition of melanin biosynthetic precursors to the growth medium would affect (e.g., increase) the production of pigment compounds by BW02 and / or BW05 in culture. In addition, experiments were performed to characterize the pigments produced by BW02 and BW05 cultures.

[0430] Embodiment 15

[0431] During the development of the embodiments of the technology provided herein, experiments were conducted to characterize the physicochemical properties of the pigment extracted from the smallest microbial consortium observed to have a brown morphology (sample MEL0152). After the growth in the liquid culture of the smallest microbial consortium observed to have a brown morphology, a portion of the culture was treated with a melanin extraction method, which is described in Kumar (2013) "Production of melanin pigment from Pseudomonasstutzeriisolated from red seaweed Hypnea musciformis" Letters in AppliedMicrobiology 57:295–302, incorporated herein by reference. In short, the culture samples were collected and centrifuged. After separating the supernatant and the biomass precipitate, the biomass precipitate was resuspended in a solution containing proteinase K (for protein degradation) and NaOH. In the presence of stainless steel beads, the cells were lysed by vortexing the sample. The sample was centrifuged again, and the supernatant was retained. The supernatant was autoclaved to prevent and / or minimize the formation of protein melanin 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 precipitate was retained. The precipitate was first washed with water and then with EtOH before drying. The properties of the extracted material were determined 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 shows that the method of Guo or Fava was used for each analysis. These references also describe the expected results of the characterization of eumelanin.

[0432] The extracted samples were aliquoted into individual tubes, and analytical reagents were added to each tube to test for specific properties (e.g., solubility, reactivity), as shown in Table 7. Solubility was determined as no color imparted to the solvent and all samples precipitated ("insoluble"), some color imparted to the solvent and some samples precipitated ("partially soluble"), or color imparted to the solvent and no samples precipitated, even after high-speed centrifugation ("soluble"). The insoluble results were further confirmed by confirming that there was little or no change in the dry weight of the sample after recovery from the solvent. Stability was determined by placing the samples at different 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 little change between the tested heated samples and the original samples indicated that the sample was stable.

[0433] During the experiment, the physicochemical data of the pigment extracted from sample MEL0152 were collected. MEL0152 has black color, and is grown from the sample (S4387) that is derived from BW02. The physicochemical characteristics of synthetic true melanin (Sigma Aldrich) are used as a reference control for comparison. Table 7 (pigment extracted) and Table 8 (melanin reference) provide data. The data collected during the experiment show that the pigment extracted is melanin.

[0434] Table 7 - Physicochemical data of the extracted pigments

[0435]

[0436] Table 8 – Physicochemical data of melanin reference substances

[0437]

[0438] As shown by the data presented in Tables 7 and 8, sample MEL0152 (derived from BW02) from consortium S4387 produced a compound with the expected physicochemical characteristics of melanin compounds reported in the literature. Notably, the solubility, precipitation, thermal stability, and redox behavior of the extracted compound were consistent with previously reported data describing melanin in Guo and Fava (see above).

[0439] Previous publications have described the physicochemical properties of eumelanin. Although some publications (Fava and Guo) differ slightly from each other in some properties of the melanin (e.g., partially soluble vs. completely soluble), synthetic eumelanin from SigmaAldrich (St. Louis, MO) was also tested (Table 8) and the results did not completely match the properties of previously reported natural melanin. Synthetic eumelanin was found to have a lower molecular weight than eumelanin isolated from bacteria, and without being bound by theory, this difference is thought to be due to some of the different properties (e.g., solubility in DMSO and affinity with H 2 O 2 increased reactivity) (see, for example, 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 bacteria and synthetic pigments" Scientific Reports 11(1): 8538, each of which is incorporated herein by reference).

[0440] Example 16

[0441] During the development of embodiments of the technology described herein, experiments were performed to characterize the composition of the consortium grown in culture. BW02 cultures were grown as described above. Flocculants from the cultures were air-dried and resuspended in NaOH (1N). Dilutions were prepared using the resuspended flocculants in NaOH (1N) at a ratio of 1:1, 1:4, 1:9, and 1:19 of resuspended flocculants to NaOH to prepare 1×, 2×, 5×, 10×, and 20× dilutions. Samples were pipetted into 96-well plates (200 μL of each sample in each well, 3 replicates per sample tested), and absorbance spectra were obtained from 280-680 nm on a SpetraMax i3 plate reader. Absorption spectra from 280-680 nm were also obtained for approximately 0.02%, 0.01%, and 0.005% w / v eumelanin standards. The absorbance spectra of the flocculants prepared from the BW02 cultures paralleled the absorbance spectra of the melanin standards ( Fig.19 ).

[0442] Embodiment 17

[0443] During development of embodiments of the technology described herein, experiments were performed to evaluate the growth morphology of minimal consortia after supplementation of the growth medium with tyrosine.

[0444] The BW02 culture (S1906) was grown to stationary phase (four weeks in 30 mL of medium in a tissue culture flask) 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 cells. After decanting the supernatant, the cell pellet was dried and the dry cell biomass was weighed. Using the weight of the dry biomass, the inoculum density was calculated to be 0.0012 g (dry biomass) / 0.5 mL culture.

[0445] Prepare a minimal solid medium and add 2.5 mL 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. A well without tyrosine was also prepared as a control (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 plates were grown under continuous light for 14 days and imaged daily. Fig. 20 As shown, increasing the concentration of tyrosine supplementation resulted in increased pigmentation of the BW02 culture and the surrounding solid medium (S1906).

[0446] Embodiment 18

[0447] During the development of embodiments of the technology provided herein, experiments were performed to evaluate the pigment production of BW02 and BW05 cultures grown in minimal medium supplemented with tyrosine. BW02 and BW05 cultures were grown in 250-ml cell culture flasks containing 30 ml of medium and inoculated with 1 ml of a culture grown for 4 weeks to saturation. Cultures were either supplemented with 2.5 mM tyrosine or left untreated. Fig.21 S1905 and S1909 have brown morphology and are derived from BW02. S1918 has green morphology and is derived from BW05.

[0448] After two weeks of growth of S1905 and S1909 (BW02) and S1918 (BW05) in liquid culture, the cultures were centrifuged and 200 μL of each supernatant was added to the wells of a 96-well plate and the absorbance was read on a microplate reader at 275-525 nm. Fig. 22Absorption spectra of supernatants from BW02 (S1905, S1909) cultures grown without tyrosine supplementation, supernatants from BW02 cultures grown with tyrosine supplementation, supernatants from BW05 (S1918) cultures grown without tyrosine supplementation, supernatants from BW05 cultures grown with tyrosine supplementation, and melanin controls (0.0350% w / v and 0.0063% w / v) are shown. Data collected during these experiments ( Fig.21 , Fig. 22 ) showed that the addition of L-tyrosine to the culture medium increased the absorbance of the supernatant produced by cultures produced by BW02 grown in the supplemented medium compared to supernatants produced by BW02 cultures grown in medium without tyrosine supplementation and BW05 cultures grown in medium with or without tyrosine supplementation. The absorbance spectra were consistent with the melanin reference standard control. Therefore, these data indicate 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, which increases the production of at least one of the three downstream melanin products.

[0449] Embodiment 19

[0450] In the development process of the embodiment of the technology provided herein, experiments were conducted to evaluate the pigment yield of BW02 (S1905, S1909) and BW05 (S1918) cultures grown in minimal medium supplemented with homogentisic acid. Specifically, the minimum consortium (with brown morphology) produced from S1905 and S1909 and the minimum consortium (with green morphology) produced from S1918 were grown in 30mL of culture medium in a cell culture flask for 4 weeks to the stationary phase. Each stationary phase culture of a volume of 1mL was used to inoculate three replicates of the basic mineral medium (control) or three replicates of the same culture medium supplemented with 2.77mM homogentisic acid (HGA). After 15 hours of growth, strong pigmentation was clearly present in the sample comprising the culture medium supplemented with HGA.

[0451] Collect a 200-μL volume of each sample (triplicate as above) and add each volume to a well of a 96-well plate ( Fig.23 After taking photos of the plates, the absorbance of the supernatant was analyzed between 230-730 nm on a SpectraMax i3 microplate reader ( Fig.24). The collected data indicated that consortia 1905 and 1909 showed significant pigmentation after HGA supplementation, whereas consortia 1918 did not show enhanced pigmentation in the culture medium after HGA addition. The measured absorbance of each supernatant prepared from MC1905 and MC1909 cultures treated with HGA (brown morphology) was higher than the baseline absorbance observed in the corresponding controls. Fig.24 The absorbance of the supernatant prepared from the MC1918 (green morphology) culture was lower than the baseline absorbance observed in the corresponding control. Fig.24 Without being limited by theory, it is believed that this is due to the uptake of HGA by the cells, but the inability of the cells to convert it into pyomelanocin; the pigmentation of the blank medium supplemented with HGA may be due to the known in vitro fertilization of HGA in MnCl 2 -4H 2 O (present in the culture medium). Taken together, these data indicate that the BW02 consortium produces melanin.

[0452] HGA is an upstream intermediate in the synthesis of pyomelanocin from L-tyrosine, but not in the production of the other melanins eumelanin and pheomelanin. Fig.18B Visual interpretation of the well color strongly suggests that the pigmented substance observed in both the tyrosine and HGA supplemented experiments as well as in normal (unsupplemented) culture conditions is pyomelanopic pigment. The absorption spectrum shows that the addition of HGA to the culture medium results in an increase in absorption between 400-500 nm, which is within the absorption range of melanins (e.g., eumelanin).

[0453] Embodiment 20

[0454] During the development of embodiments of the technology described herein, 16S, 23S and 5S ribosomal RNA gene sequences were sequenced and analyzed to identify members of the consortium. Multiple minimal consortia (e.g., including about 2-10 different types of organisms) were isolated from each of the BW01, BW02 and BW05 consortia (e.g., including about hundreds of different types of organisms). Minimal consortia were generated by streaking each complete consortium isolated colony on agar medium. Each colony is a minimal consortium, which is assumed to require multiple members, each of which contributes a key function to the consortium, for example, nitrogen fixation, carbon fixation, or shuttling metabolites between consortium organisms that fix nitrogen and / or carbon. [0...

Claims

1. A method for accumulating carbon and nitrogen in an agricultural medium, the method comprising: applying a composition including a photosynthetic nitrogen-fixing microorganism to an agricultural medium; and The agricultural medium is incubated in the presence of nitrogen (N2) and carbon dioxide (CO2) and irradiated with a light source.

2. The method according to claim 1, wherein: The agricultural medium includes soil, greenhouse growth medium or hydroponic growth medium.

3. The method according to claim 1, wherein: The photosynthetic nitrogen-fixing microorganisms are bacteria.

4. The method according to claim 1, wherein: The photosynthetic nitrogen-fixing microorganisms are members of the phylum cyanobacteria.

5. The method according to claim 1, wherein: The photosynthetic nitrogen-fixing microorganism is a member of the orders Cyanobacteriale, Cyanobacteriota, Nostocale, Pseudoanabaenale, Spirulinale, or Oscillatoriale.

6. The method according to claim 1, wherein: The agricultural media include synthetic growth media.

7. The method according to claim 1, wherein: The composition also includes an organism from the genus Variovorax, an organism from the phylum Proteobacteria, an organism from the genus Bosella, an organism from the genus Caulobacter, and / or an organism from the genus Pseudomonas.

8. The method according to claim 1, wherein: The photosynthetic nitrogen-fixing microorganism is a member of the Nostocales.

9. The method according to claim 1, wherein: The photosynthetic nitrogen-fixing microorganisms are members of the genera Acaryochloris, Aliinostoc, Aliterella, Amazonocrinis, Anabaena, Anabaenopsis, Aphanizomenon, Atlanticothrix, Aulosira, Calenema, Calothrix, Camptylonemopsis, Chlorogloeopsis, Chroococcidi opsis), Chrysosporum, Compactonostoc, Constrictifilum, Crocosphaera, Cyanobacterium, Cyanocohniella, Cylindrospermopsis, Cylindrospermum, Dendronalium, Desikacharya, Desmonostoc, Dolichospermum, Erythrobacter, Fische rella), Fortiea, Fremyella, Geitlerinema, Gloeocapsa, Gloeocapsopsis, Gloeothece, Gloeotrichia, Goleter, Hapalosiphon, Hydrocoryne, Jaaginema, Johanseniella, Kamptonema, Komarekiella, Leptolyngbya, Leptolyngospermum, and Leptolyngospermum. yngbya), Macrochaete, Mastigocoleus, Microchaete, Nodosilinea, Nodularia, Nostoc, Nostocaceae, Nostochopsis, Oligotropha, Oscillatoria, Pantanalinema, Pelatocladus, Planktothrichoides, Planktothrix,Polymorphum, Porphyrobacter, Prochlorococcus, Prochlorothrix, Pseudanabaena, Pseudoaliinostoc, Raphidiopsis, Richelia, Rivularia, Roholtiella, Rubidibacter, Scytonema, Spirulina, Synechococcus, Synechocystis, Thermosynechococcus, Tolypothrix, Trichormus, Violetonostoc, Wollea or Xenococcus.

10. The method according to claim 1, wherein: The photosynthetic nitrogen-fixing microorganism is a species of the genus Nostoc.

11. The method according to claim 1, wherein: The photosynthetic nitrogen-fixing microorganism has 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.

12. The method according to claim 1, wherein: The composition produces durable carbon compounds that provide a carbon sink in the soil.

13. The method according to claim 1, wherein: The composition produces melanin.

14. The method according to claim 1, wherein: The composition produces pheomelanin, eumelanin and / or pyomelanin.

15. The method according to claim 1, wherein: The composition also includes a microorganism capable of metabolizing the carbon- and nitrogen-containing compounds produced by the photosynthetic nitrogen-fixing microorganism.

16. The method according to claim 1, wherein: The composition includes a consortium comprising the photosynthetic nitrogen-fixing microorganism.

17. The method according to claim 1, wherein: The composition includes a consortium, the consortium comprising the photosynthetic nitrogen-fixing microorganism; and a first microorganism capable of metabolizing a carbon-containing compound produced by the photosynthetic nitrogen-fixing microorganism and / or capable of metabolizing a nitrogen-containing compound produced by the photosynthetic nitrogen-fixing microorganism.

18. The method according to claim 17, wherein: The consortium produces melanin.

19. The method according to claim 17, wherein: The consortium produces pheomelanin, eumelanin and / or pyomelanin.

20. The method according to claim 17, wherein: The consortium includes a second microorganism capable of metabolizing a carbon-containing compound produced by the photosynthetic nitrogen-fixing microorganism and / or capable of metabolizing a nitrogen-containing compound produced by the photosynthetic nitrogen-fixing microorganism.

21. The method according to claim 17, wherein: The consortium includes a second microorganism capable of metabolizing a carbon-containing compound produced by the first microorganism and / or capable of metabolizing a nitrogen-containing compound produced by the first microorganism.

22. The method according to claim 17, wherein: 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.

23. The method according to claim 16, wherein: The consortium includes a second photosynthetic microorganism.

24. The method according to claim 23, wherein: The second photosynthetic microorganism is a bacterium.

25. The method according to claim 23, wherein: The second photosynthetic microorganism is algae.

26. The method according to claim 24, wherein: The second photosynthetic microorganism is a member of the genus Nodularia, Chrysosporium, Globular Cyanobacteria, Phytophyte, Trichoderma, Hose Algae, Sticky Rod Algae, Acaryochloris, Kamptonema, Aculeatus, Crocodile Algae, Macrochaete, Thermosynechococcus, Pseudo-Anabaena, Pseudo-Chromococcus, Prochlorofilamentous Algae, Anabaena, Sclerotium, Eyebrow Algae, Pseudo-Columnar Spore, Long Spore, Pseudo-branched Algae, Sheath Algae, Monophyll, Flytella, Voltiella, Aliterella, Water Strip Algae, Protochlorophyllum, Chlorochlore, Pseudohygrochlore, Gastrochlospermum, Heterococcus, Jassima, Pseudohygrochlore, Pantanalinema, Oscillatoria, Spirulina, Pelatocladus, Gnaphalium, Aphanizomenon, Pseudohygrochlore, Myxococcus, Calenema, Glechoma, Trichoderma, Synechococcus, Synechocystis, Cylindrospermum, Pseudohygrochlore, Bosea, Shinella, Novosphingobium or Rubidibacter.

27. The method according to claim 25, wherein: The algae are members of the genera Tetraselmis, Chlorella, Coleochaete, Gonium, Paradoxia, Cymbomonas, Palmaria, Characiochloris, Marvania, Chlamydomonas, Parachlorella, Coccomyxa, Trebouxia, Cyanidium, Treu baria, Dangeardinia, Galdieria, Nyholmiella, Porphyra, Chara, Ankistrodesmus, Picochlorum, Cyanophora, Chondrus, Pseudochloris, Interfilum, Gelidium, Symphyogyna, Chlorosarcina or Cyanothece.

28. The method of claim 16, wherein: The consortium includes a second nitrogen fixing microorganism.

29. The method of claim 1, wherein: The light source provides light having a wavelength from about 380 nm to 750 nm.

30. The method of claim 1, wherein: The light source is an artificial light source.

31. A method for accumulating carbon and nitrogen in an agricultural medium, the method comprising: applying a composition including a photosynthetic microorganism and a nitrogen-fixing microorganism to an agricultural medium; and The agricultural medium is incubated in the presence of nitrogen (N2) and carbon dioxide (CO2) and irradiated with a light source.

32. The method according to claim 31, wherein: The agricultural medium includes soil, greenhouse growth medium or hydroponic growth medium.

33. The method according to claim 31, wherein: The photosynthetic microorganisms are algae.

34. The method of claim 31, wherein: The photosynthetic microorganisms are bacteria.

35. The method of claim 31, wherein: The nitrogen-fixing microorganisms are bacteria.

36. The method of claim 31, wherein: The agricultural medium and the composition together include a first microbial biomass; and the incubating is performed until a second microbial biomass is produced that is at least two times greater than the first microbial biomass.

37. The method of claim 33, wherein: The algae is a member of the genus Tetrastigemum, Chlorella, Chaetoceros, Diskia, Paradoxia, Cymbomonas, Palmitochloris, Characiochloris, Marvania, Chlamydomonas, Pseudochlorella, Coccomycetes, Coccomycetes, Cyanochloris, Tetrastigem, Dangeardinia, Galdiella, Nyholmiella, Porphyra, Chara, Fibrous Algae, Chlorella, Cyanophytes, Chondrus, Pseudochloris, Interfilum, Gelidium, Symphyogyna, Kalamba or Cyanophyceae.

38. The method of claim 34, wherein: The photosynthetic bacteria are members of the genera Acaryochloris, Aliterella, Amazonocrinis, Anabaena, Anabaena, Aphanizomenon, Atlanticothrix, Calenema, Calenema, Pseudo-Cyanocyanobacterium, Pseudo-Chromococcus, Chrysosporium, Compactonostoc, Constrictifilum, Crocodile Glomerella, Cyanocohniella, Pseudo-Cyanocyanobacterium, Cyanocyanobacterium, Dendronalium, Desikacharya, Desmonostoc, Longisporium, Erythrobacter, Flytella, Voltiella, Fremyella, Gaseella, Glob ... of the genus Trichoderma, Jasmine, Johanseniella, Kamptonema, Komarekiella, Cladosporium, Cladosporium, Macrochaete, Trichosporon, Nodularia, Nodulariaceae, Nodularia, Oligosporium, Oscillatoria, Pantanalinema, Pelatocladus, Pseudosphaerella, Pseudosphaerella, Polymorpha, Porphyromonas, Prochlorococcus, Prochlorococcus, Pseudoanabaena, Pseudosphaerella, Acrocephalum, Phytosphaera, Gelatospermum, Roholtiella, Rubidibacter, Pseudoclase, Spirulina, Synechococcus, Synechocystis, Thermosynechococcus, Monophydium, Trichosporon, Violetonostoc, Lumena, Bostria, Sheinella, Neosphingobacterium, or Heterococcus; and The nitrogen-fixing microorganism is a microorganism from the following genera: Acaryochloris, Aliterella, Amazonocrinis, Anabaena, Anabaena, Aphanizomenon, Atlanticothrix, Calenema, Calenema, Pseudo-Cyanocyanobacterium, Pseudo-Chromococcus, Chrysosporium, Compactonostoc, Constrictifilum, Crocodile Glomerella, Cyanobacterium, Cyanocohniella, Pseudo-Cyanocyanobacterium, Cyanocyanobacterium, Dendronalium, Desikacharya, Desmonostoc, Longisporium, Erythrobacter, Flytella, Voltiella, Fremyella, Gaseella, Globose ... ma, Komarekiella, Sclerotium, Sclerotium, Macrochaete, Flagellate, Microchaete, Knotweed, Nodularia, Nodularia, Nodulariaceae, Nodularia, Oligospermum, Oscillatoria, Pantanalinema, Pelatocladus, Pseudofloccula, Floccula, Polymorpha, Porphyromonas, Prochlorococcus, Prochlorococcus, Pseudofloccula, Pseudofloccula, Pseudofloccula, Apicula, Phytophyta, Gelatophyta, Ro holtiella, Rubidibacter, Pseudomonas, Spirulina, Synechococcus, Synechocystis, Thermosynechococcus, Monochamus, Trilochaete, Violetonostoc, Lumenella, Heterococcus, Greedybacter, Pseudomonas, Cupricobacter, Brevundimonas, Toyou, Archaeobacterium, Theileria, Tahibacter, Polarmonas, Chitinophora, Freundii, Aquabacter, Thinlayers, or Soilbacter.

39. The method of claim 31, wherein: The composition also includes microorganisms capable of metabolizing carbon- and nitrogen-containing compounds produced by photosynthetic microorganisms and by nitrogen-fixing microorganisms.

40. The method of claim 31, wherein: The composition includes a consortium comprising the photosynthetic microorganism and the nitrogen-fixing microorganism.

41. The method of claim 40, wherein: The consortium includes organisms 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.

42. The method of claim 40, wherein: The consortium produces durable carbon compounds that provide a carbon sink in the soil.

43. The method of claim 40, wherein: The consortium produces melanin.

44. The method of claim 40, wherein: The consortium produces pheomelanin, eumelanin and / or pyomelanin.

45. The method of claim 40, wherein: The composition also includes an organism from the genus Vulgaris, an organism from the phylum Proteobacteria, an organism from the genus Bosella, an organism from the genus Caulobacter, and / or an organism from the genus Pseudomonas.

46. ​​The method of claim 40, wherein: The composition includes a consortium, the consortium comprising the photosynthetic microorganism and the nitrogen-fixing microorganism; and a first microorganism capable of metabolizing the carbon-containing compounds produced by the photosynthetic microorganism and the nitrogen-fixing microorganism and / or capable of metabolizing the nitrogen-containing compounds produced by the photosynthetic microorganism and the nitrogen-fixing microorganism.

47. The method of claim 46, wherein: The consortium includes a second microorganism capable of metabolizing carbon-containing compounds produced by the photosynthetic microorganism and by the nitrogen-fixing microorganism and / or capable of metabolizing nitrogen-containing compounds produced by the photosynthetic microorganism and by the nitrogen-fixing microorganism.

48. The method of claim 46, wherein: The consortium includes a second microorganism capable of metabolizing a carbon-containing compound produced by the first microorganism and / or capable of metabolizing a nitrogen-containing compound produced by the first microorganism.

49. The method of claim 46, wherein: 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.

50. The method of claim 31, wherein: The light source provides light having a wavelength from about 380 nm to 750 nm.

51. The method of claim 31, wherein: The light source is an artificial light source.

52. A method for producing a microbial consortium, the method comprising: Providing water-based, non-fixed carbon and non-fixed nitrogen culture media; inoculating the culture medium with a sample comprising a microorganism to produce an inoculated culture medium; The inoculated culture medium is exposed to gases including N2 and CO2 and light.

53. The method of claim 52, wherein: The light includes wavelengths from about 380 nm to 750 nm.

54. The method of claim 52, wherein: The gas including N2 and CO2 is air.

55. The method of claim 52, wherein: The gas including N 2 and CO 2 is provided by purified N 2 and CO 2 .

56. The method of claim 52, wherein: The inoculated culture medium includes a first microbial biomass; and the exposing is performed until a second microbial biomass is produced that is at least two times greater than the first microbial biomass.

57. A composition comprising a microbial consortium, the microbial consortium comprising: 1) photosynthetic nitrogen-fixing microorganisms; or photosynthetic microorganisms and nitrogen-fixing microorganisms; and 2) microorganisms capable of metabolizing carbon- and nitrogen-containing compounds produced by the photosynthetic nitrogen-fixing microorganism or produced jointly by the photosynthetic microorganism and the nitrogen-fixing microorganism.

58. The composition according to claim 57, wherein The photosynthetic nitrogen-fixing microorganisms are bacteria.

59. The composition according to claim 57, wherein The photosynthetic nitrogen-fixing microorganism is a member of the phylum Cyanobacteria.

60. The composition according to claim 57, wherein The photosynthetic nitrogen-fixing microorganism is a member of the order Cyanobacteriales, the phylum Cyanobacteriales, the order Nostocales, the order Pseudo-Anabaenales, the order Spirulinales, or the order Oscillatorales.

61. The composition according to claim 57, wherein The photosynthetic nitrogen-fixing microorganism is a member of the Nostocales.

62. The composition of claim 57, wherein: The photosynthetic microorganism is a microorganism from the genus: Bosella, Shenkel or Neosphingobacterium; The nitrogen-fixing microorganism is a microorganism from the following genera: Pseudomonas, Pseudomonas, Cupribotium, Brevundimonas, Toyosporium, Archaea, Theileria, Tahibacter, Polarmonas, Chitinophaga, Freudenbergia, Aquabacter, Thinlayer, or Soilobacterium; and The photosynthetic nitrogen-fixing microorganism is a member of the genus Acaryochloris, Aliterella, Amazonocrinis, Anabaena, Anabaena, Aphanizomenon, Atlanticothrix, Calenema, Calenema, Pseudo-Cyanocyanobacterium, Pseudo-Chromococcus, Chrysosporium, Compactonostoc, Constrictifilum, Crocodile Glomerella, Cyanocohniella, Pseudo-Cyanocyanobacterium, Cyanocyanobacterium, Dendronalium, Desikacharya, Desmonostoc, Longisporium, Erythrobacter, Flytella, Voltiella, Fremyella, Gelami ... eter, Hosecophyte, Waterstripe, Jasmine, Johanseniella, Kamptonema, Komarekiella, Sclerotium, Sclerotium, Macrochaete, Flagellate, Microchaete, Knotweed, Nodularia, Nodularia, Nodulariaceae, Nodularia, Oligospermum, Oscillatoria, Pantanalinema, Pelatocladus, Pseudofloxacin, Floatweed, Polymorpha, Porphyrobacter, Prochlorococcus, Prochlorococcus, Pseudoanabaena, Pseudofloxacin, Acrocephalus, Phytophyte, Gelatophyte, Roholtiella, Rubidibacter, Pseudobranchia, Spirulina, Synechococcus, Synechocystis, Thermosynechococcus, Monophyll, Trichophyton, Violetonostoc, Lumenella, or Heterococcus.

63. The composition according to claim 57, wherein The photosynthetic nitrogen-fixing microorganism is a species of the genus Nostoc.

64. The composition according to claim 57, wherein The photosynthetic nitrogen-fixing microorganism has 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.

65. The composition according to claim 57, wherein The consortium produces durable carbon compounds that provide a carbon sink in the soil.

66. The composition according to claim 57, wherein The consortium produces melanin.

67. The composition according to claim 57, wherein The consortium produces pheomelanin, eumelanin and / or pyomelanin.

68. The composition according to claim 57, wherein The composition includes a second microorganism capable of metabolizing a carbon-containing compound produced by the photosynthetic nitrogen-fixing microorganism and / or capable of metabolizing a nitrogen-containing compound produced by the photosynthetic nitrogen-fixing microorganism.

69. The composition according to claim 57, wherein The consortium includes a second microorganism capable of metabolizing a carbon-containing compound produced by the first microorganism and / or capable of metabolizing a nitrogen-containing compound produced by the first microorganism.

70. The composition of claim 57, wherein 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.

71. The composition of claim 57, wherein The consortium includes a second photosynthetic microorganism.

72. The composition according to claim 71, wherein The second photosynthetic microorganism is a bacterium or an algae.

73. The composition according to claim 71, wherein The composition also includes an organism from the genus Vulgaris, an organism from the phylum Proteobacteria, an organism from the genus Bosella, an organism from the genus Caulobacter, and / or an organism from the genus Pseudomonas.

74. The composition of claim 72, wherein The second photosynthetic microorganism is a member of the genus Nodularia, Chrysosporium, Globular Cyanobacteria, Phytophyte, Trichoderma, Hose Algae, Glue Rod Algae, Acaryochloris, Kamptonema, Acrocephalum, Crocodile Algae, Macrochaete, Thermosynechococcus, Pseudo-Anabaena, Pseudo-Chromococcus, Prochlorofilamentous Algae, Anabaena, Sclerotium, Eyebrow Algae, Pseudo-Cylindrical Algae, Longisporium, Pseudo-Cylindrical Algae, Sclerotium, Monophyllium, Flytella, and Voltiella. Aliterella, Aquatica, Prochlorococcus, Pseudohygrophila, Ghesina, Heterococcus, Jasmine, Pseudohygrophila, Pantanalinema, Oscillatoria, Spirulina, Pelatocladus, Knotweed, Aphanizomenon, Pseudohygrophila, Myxococcus, Calenema, Glechoma, Trichophyton, Synechococcus, Synechocystis, Cylindrospermum, Pseudohygrophila, Bosella, Sheinella, Neosphingobacterium, or Rubidibacter.

75. The composition of claim 72, wherein The algae is a member of the genus Tetrastigemum, Chlorella, Chaetoceros, Diskia, Paradoxia, Cymbomonas, Palmitochloris, Characiochloris, Marvania, Chlamydomonas, Pseudochlorella, Coccomycetes, Coccomycetes, Cyanochloris, Tetrastigem, Dangeardinia, Galdiella, Nyholmiella, Porphyra, Chara, Fibrous Algae, Chlorella, Cyanophytes, Chondrus, Pseudochloris, Interfilum, Gelidium, Symphyogyna, Kalamba or Cyanophyceae.

76. The composition of claim 57, wherein The consortium includes a second nitrogen fixing microorganism.

77. A system comprising: water-based, non-fixed carbon and non-fixed nitrogen media; Gas sources including N2 and CO2; Light source; and Includes samples of microorganisms.

78. A system comprising: water-based, non-fixed carbon and non-fixed nitrogen media; include Gas sources of N2 and CO2; light source; as well as A microbial consortium comprising: 1) photosynthetic nitrogen-fixing microorganisms; or 2) photosynthetic microorganisms and nitrogen-fixing microorganisms; and A microorganism capable of metabolizing carbon- and nitrogen-containing compounds produced by the photosynthetic nitrogen-fixing microorganism or produced jointly by the photosynthetic microorganism and the nitrogen-fixing microorganism.

79. The system of claim 78, wherein: The photosynthetic nitrogen-fixing microorganisms are bacteria.

80. The system of claim 78, wherein: The photosynthetic nitrogen-fixing microorganism is a member of the phylum Cyanobacteria.

81. The system of claim 78, wherein: The photosynthetic nitrogen-fixing microorganism is a member of the order Cyanobacteriales, the phylum Cyanobacteriales, the order Nostocales, the order Pseudo-Anabaenales, the order Spirulinales, or the order Oscillatorales.

82. The system of claim 78, wherein: The photosynthetic nitrogen-fixing microorganism is a member of the Nostocales.

83. The system of claim 78, wherein: The photosynthetic microorganism is a microorganism from the genus: Bosella, Shenkel or Neosphingobacterium; The nitrogen-fixing microorganism is a microorganism from the following genera: Pseudomonas, Pseudomonas, Cupribotium, Brevundimonas, Toyosporium, Archaea, Theileria, Tahibacter, Polarmonas, Chitinophaga, Freudenbergia, Aquabacter, Thinlayer, or Soilobacterium; and The photosynthetic nitrogen-fixing microorganism is a member of the genus Acaryochloris, Aliterella, Amazonocrinis, Anabaena, Anabaena, Aphanizomenon, Atlanticothrix, Calenema, Calenema, Pseudo-Cyanocyanobacterium, Pseudo-Chromococcus, Chrysosporium, Compactonostoc, Constrictifilum, Crocodile Glomerella, Cyanocohniella, Pseudo-Cyanocyanobacterium, Cyanocyanobacterium, Dendronalium, Desikacharya, Desmonostoc, Longisporium, Erythrobacter, Flytella, Voltiella, Fremyella, Gelami ... eter, Hosecophyte, Waterstripe, Jasmine, Johanseniella, Kamptonema, Komarekiella, Sclerotium, Sclerotium, Macrochaete, Flagellate, Microchaete, Knotweed, Nodularia, Nodularia, Nodulariaceae, Nodularia, Oligospermum, Oscillatoria, Pantanalinema, Pelatocladus, Pseudofloxacin, Floatweed, Polymorpha, Porphyrobacter, Prochlorococcus, Prochlorococcus, Pseudoanabaena, Pseudofloxacin, Acrocephalus, Phytophyte, Gelatophyte, Roholtiella, Rubidibacter, Pseudobranchia, Spirulina, Synechococcus, Synechocystis, Thermosynechococcus, Monophyll, Trichophyton, Violetonostoc, Lumenella, or Heterococcus.

84. The system of claim 78, wherein: The photosynthetic nitrogen-fixing microorganism is a species of the genus Nostoc.

85. The system of claim 78, wherein: The photosynthetic nitrogen-fixing microorganism has 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.

86. The system of claim 78, wherein: The consortium produces durable carbon compounds that provide a carbon sink in the soil.

87. The system of claim 78, wherein: The microbial consortium produces melanin.

88. The system of claim 78, wherein: The microbial consortium produces pheomelanin, eumelanin and / or pyomelanin.

89. The system of claim 78, wherein: The microbial consortium includes a second microorganism capable of metabolizing a carbon-containing compound produced by the photosynthetic nitrogen-fixing microorganism and / or capable of metabolizing a nitrogen-containing compound produced by the photosynthetic nitrogen-fixing microorganism.

90. The system of claim 78, wherein: The microbial consortium includes a second microorganism capable of metabolizing a carbon-containing compound produced by the first microorganism and / or capable of metabolizing a nitrogen-containing compound produced by the first microorganism.

91. The system of claim 78, wherein: The microbial 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.

92. The system of claim 78, wherein: The microbial consortium includes a second photosynthetic microorganism.

93. The system of claim 92, wherein: The second photosynthetic microorganism is a bacterium.

94. The system of claim 92, wherein: The second photosynthetic microorganism is algae.

95. The system of claim 92, wherein: The second photosynthetic microorganism is a member of the genus Nodularia, Chrysosporium, Globular Cyanobacteria, Phytophyte, Trichoderma, Hose Algae, Glue Rod Algae, Acaryochloris, Kamptonema, Acrocephalum, Crocodile Algae, Macrochaete, Thermosynechococcus, Pseudo-Anabaena, Pseudo-Chromococcus, Prochlorofilamentous Algae, Anabaena, Sclerotium, Eyebrow Algae, Pseudo-Cylindrical Algae, Longisporium, Pseudo-Cylindrical Algae, Sclerotium, Monophyllium, Flytella, and Voltiella. Aliterella, Aquatica, Prochlorococcus, Pseudohygrophila, Ghesina, Heterococcus, Jasmine, Pseudohygrophila, Pantanalinema, Oscillatoria, Spirulina, Pelatocladus, Knotweed, Aphanizomenon, Pseudohygrophila, Myxococcus, Calenema, Glechoma, Trichophyton, Synechococcus, Synechocystis, Cylindrospermum, Pseudohygrophila, Bosella, Sheinella, Neosphingobacterium, or Rubidibacter.

96. The system of claim 94, wherein: The algae is a member of the genus Tetrastigemum, Chlorella, Chaetoceros, Diskia, Paradoxia, Cymbomonas, Palmitochloris, Characiochloris, Marvania, Chlamydomonas, Pseudochlorella, Coccomycetes, Coccomycetes, Cyanochloris, Tetrastigem, Dangeardinia, Galdiella, Nyholmiella, Porphyra, Chara, Fibrous Algae, Chlorella, Cyanophytes, Chondrus, Pseudochloris, Interfilum, Gelidium, Symphyogyna, Kalamba or Cyanophyceae.

97. The system of claim 78, wherein: The consortium includes a second nitrogen fixing microorganism.

98. The system of claim 78, wherein: The composition also includes an organism from the genus Vulgaris, an organism from the phylum Proteobacteria, an organism from the genus Bosella, an organism from the genus Caulobacter, and / or an organism from the genus Pseudomonas.

99. A composition comprising a synthetic growth medium; and microbial organisms that jointly photosynthesize and fix nitrogen.

100. The composition according to claim 99, wherein The first microbial species to perform photosynthesis.

101. The composition according to claim 100, wherein The first photosynthetic microbial species is a microorganism from the genus Bosella, Sheinella or Neosphingobacterium.

102. The composition of claim 99, wherein The second microbial species performs nitrogen fixation.

103. The composition according to claim 101, wherein The second microbial species that performs nitrogen fixation is a microorganism from the following genera: Pseudomonas, Pseudomonas, Cupricobacter, Brevundimonas, Toyosporium, Archaea, Theileria, Tahibacter, Polarmonas, Chitinophaga, Freudenbergia, Aquabacter, Thin-layered Bacteria or Soilbacter.

104. The composition of claim 99, wherein The first microbial species performs photosynthesis and nitrogen fixation.

105. The composition according to claim 103, wherein The first microbial species that performs photosynthesis and nitrogen fixation is a bacterium.

106. The composition of claim 103, wherein The first microbial species to perform photosynthesis and nitrogen fixation was a member of the phylum Cyanobacteria.

107. The composition of claim 103, wherein The first microbial species that performs photosynthesis and nitrogen fixation is a member of the order Cyanobacteriales, the phylum Cyanobacteriales, the order Nostocales, the order Pseudo-Anabaenales, the order Spirulinales, or the order Oscillatoriales.

108. The composition of claim 103, wherein The first microbial species that performs photosynthesis and nitrogen fixation is a member of the Nostocales.

109. The composition according to claim 103, wherein The first microbial species that performs photosynthesis and fixes nitrogen is a member of the genus Acaryochloris, Aliterella, Amazonocrinis, Anabaena, Anabaena, Aphanizomenon, Atlanticothrix, Calenema, Calenema, Pseudo-Cyanocyanobacterium, Pseudo-Chromococcus, Chrysosporium, Compactonostoc, Constrictifilum, Crocodile Glomerella, Cyanocohniella, Pseudo-Cyanocyanobacterium, Cyanocyanobacterium, Dendronalium, Desikacharya, Desmonostoc, Longisporium, Erythrobacter, Flytella, Voltiella, Fremyella, Gastrosporium, Glucosporium, Globose ... Algae, Goleter, Hose algae, Water stripe algae, Jasmine, Johanseniella, Kamptonema, Komarekiella, Sclerotium, Sclerotium, Macrochaete, Flagellate, Microchaete, Knot filament, Nodularia, Nodulariaceae, Nodularia, Oligospermum, Oscillatoria, Pantanalinema, Pelatocladus, Pseudofloxacin, Floatella, Polymorpha, Porphyromonas, Prochlorococcus, Prochlorococcus, Pseudoanabaena, Pseudofloxacin, Acrocephalum, Phytophyta, Gelatophyta, Roholtiella, Rubidibacter, Pseudobranchia, Spirulina, Synechococcus, Synechocystis, Thermosynechococcus, Monophyll, Trichophyton, Violetonostoc, Lumenella, or Heterococcus.

110. The composition according to claim 103, wherein The first microbial species that performs photosynthesis and nitrogen fixation is a Nostoc species.

111. The composition according to claim 103, wherein The first microbial species that performs 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.

112. The composition of claim 103, further comprising a second microbial species that performs supplemental nitrogen fixation.

113. The composition according to claim 111, wherein The second microbial species for supplemental nitrogen fixation is a microorganism from the following genera: Pseudomonas, Pseudomonas, Cupricobacter, Brevundimonas, Fengyou, Archaea, Theileria, Tahibacter, Polarmonas, Chitinophaga, Freudenbergia, Aquabacter, Thin-layered Bacteria or Soilbacter.

114. The composition of claim 99, further comprising a durable carbon compound that provides a carbon sink in the soil.

115. The composition according to claim 99, wherein The microbial organisms produce durable carbon compounds that provide a carbon sink in the soil.

116. The composition of claim 99, wherein The microbial organism produces melanin.

117. The composition according to claim 99, wherein The microbial organism produces pheomelanin, eumelanin and / or pyomelanin.

118. The composition of claim 99, further comprising melanin.

119. The composition of claim 99, further comprising pheomelanin, eumelanin and / or pyomelanocin.

120. The composition of claim 99, wherein The microbial organisms comprise, consist of, or consist essentially of 10 species or fewer.

121. The composition of claim 99, wherein The microbial organism comprises, consists of, or consists essentially of 9 or fewer species.

122. The composition of claim 99, wherein The microbial organism comprises, consists of, or consists essentially of 8 or fewer species.

123. The composition of claim 99, wherein The microbial organism comprises, consists of or consists essentially of 7 or fewer species.

124. The composition of claim 99, wherein The microbial organism comprises, consists of, or consists essentially of 6 or fewer species.

125. The composition of claim 99, wherein The microbial organism comprises, consists of or consists essentially of 5 or fewer species.

126. The composition of claim 99, wherein The microbial organism comprises, consists of, or consists essentially of 4 or fewer species.

127. The composition of claim 99, wherein The microbial organism comprises, consists of or consists essentially of 3 or fewer species.

128. The composition of claim 99, wherein Thirty percent or more of the biomass of the composition is provided by the co-photosynthesizing and nitrogen-fixing microbial organisms.

129. The composition of claim 99, wherein 40% or more of the biomass of the composition is provided by the co-photosynthesizing and nitrogen-fixing microbial organisms.

130. The composition of claim 99, wherein 50% or more of the biomass of the composition is provided by the co-photosynthesizing and nitrogen-fixing microbial organisms.

131. The composition of claim 99, wherein 60% or more of the biomass of the composition is provided by the co-photosynthesizing and nitrogen-fixing microbial organisms.

132. The composition of claim 99, wherein 70% or more of the biomass of the composition is provided by the co-photosynthesizing and nitrogen-fixing microbial organisms.

133. The composition of claim 99, wherein 80% or more of the biomass of the composition is provided by the co-photosynthesizing and nitrogen-fixing microbial organisms.

134. The composition of claim 99, wherein 90% or more of the biomass of the composition is provided by the co-photosynthesizing and nitrogen-fixing microbial organisms.

135. The composition of claim 99, wherein The synthetic growth medium is a medium without fixed carbon.

136. The composition of claim 99, wherein The synthetic growth medium is a non-fixed nitrogen medium.

137. The composition of claim 99, wherein The synthetic growth medium is a non-fixed carbon and non-fixed nitrogen medium.

138. The composition of claim 99, wherein The synthetic growth medium is a carbon-free medium.

139. The composition of claim 99, wherein The synthetic growth medium is a nitrogen-free medium.

140. The composition of claim 99, wherein The synthetic growth medium is a carbon-free and nitrogen-free medium.

141. A method of accumulating carbon and nitrogen in an agricultural medium, the method comprising applying to the agricultural medium the composition of claim 99.

142. The method of claim 139, further comprising exposing the composition to nitrogen (N2), carbon dioxide (CO2), and light.

143. The method of claim 140, wherein: The light is sunlight.

144. The method of claim 139, wherein: The agricultural medium is a crop field.

145. The method of claim 139, wherein: The agricultural medium includes soil, greenhouse growth medium or hydroponic growth medium.

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