CN116648143A - Composition and method for producing bicarbonate and minerals - Google Patents

Composition and method for producing bicarbonate and minerals Download PDF

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Publication number
CN116648143A
CN116648143A CN202180084753.3A CN202180084753A CN116648143A CN 116648143 A CN116648143 A CN 116648143A CN 202180084753 A CN202180084753 A CN 202180084753A CN 116648143 A CN116648143 A CN 116648143A
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bacillus
plant
seed
composition
bacteria
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贡萨洛·富恩萨利达
塔尼亚·蒂默曼
比约恩·塔格
莉娜·利昂
纳雷什·潘迪
拉古维尔·辛格
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Andean Agriculture Corp
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Andean Agriculture Corp
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Priority claimed from PCT/US2021/056083 external-priority patent/WO2022087289A1/en
Publication of CN116648143A publication Critical patent/CN116648143A/en
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Abstract

Methods and compositions for improving carbon dioxide conversion and sequestration in soil using microorganisms integrated with plants are described herein.

Description

Composition and method for producing bicarbonate and minerals
Cross reference
The present application claims the benefit of U.S. provisional application Ser. Nos. 63/094,870 and 63/257,079, filed on Ser. No. 18/10/2021, 10/21, 2020, which are incorporated herein by reference.
Technical Field
Human activities, including the burning of fossil fuels and the deforestation, have greatly led to an increase in major greenhouse gases. Combustion of coal produces the highest amounts of CO compared to other uses of fossil fuels 2 About 2.5 tons of CO are produced per ton of coal burned 2 . Heretofore, CO in the environment 2 The concentration has reached more than 400ppm from 280ppm at the beginning of the industrial revolution in the mid 19 th century. It is expected that by the middle of this century, CO 2 The concentration may reach 600ppm and by the end of the century, it is most likely to reach 700ppm. By 2050, global temperature is reported to rise above 2 ℃, and the next 50-100 years will rise about 3-5 ℃. Such global temperature increases have worsened climate change and global consequences of climate change have occurred, including severe australian jungle fires that destroy wild animals, repeated wildfires in california forests, increased frequency and duration of heat waves, prolonged drought periods, elevated sea level, tsunami, etc. These natural disasters are not common prior to industrialization, these are CO 2 Results of elevated levels.
In addition to the known CO 2 There are also some unexplored sources, besides emissions sources, which are not only continuously increasing CO 2 And may become a critical source depending on the severity of the climate change. Soil is the largest carbon reservoir because it contains carbon (1 m deep at 1500Pg C,2m at 2500Pg C;1Pg = 1 x 10) 15 g) Far more carbon is present in vegetation and twice as much as in the atmosphere (750 Pg of C). It is estimated that 3.66 tons of CO are released per ton of organic carbon of soil 2 . The organic carbon in the soil is increased by the plants by: root death, root secretions, or other rooted organic material released by rhizosphere and root respiration. It is well documented that during photosynthesis, plants use CO 2 And converts it to sugars, however during respiration a greater amount of non-immobilized CO 2 Mainly released by the roots of the plants. Of the 120Pg carbon captured by the plants, 50% was lost to the atmosphere by respiration of the plants. Soil inhabiting organisms and microorganisms living closer to roots (rhizosphere) release CO during their respiration 2 This further exacerbates the situation. CO produced by rhizobia community 2 10 times higher than plants without rhizosphere. Soil-dwelling microorganisms are either fed by root secretions or survive by decomposing complex materials present in the soil. The effect of soil microorganisms on climate change has been previously studied and it has been shown that global warming may accelerate the rate of heterotrophic microbial activity, leading to CO in the soil 2 The outflow increases and eventually will be released to the environment. Because soil temperature can increase soil respiration, global climate change is expected to increase net transfer of carbon from the soil to the atmosphere. Although soil is a good source of storage carbon (3.3 times the size of the atmospheric reservoir (7600 hundred tons)), global warming may exacerbate depletion of reservoir C. Although it is necessary to prevent CO 2 Released into the atmosphere but via effective CO 2 Sealing and storing CO 2 Permanent storage into the soil is highly desirable. The sequestration of carbon in soil for agricultural, forestry and land reclamation has been considered as a potential option to mitigate global changes.
CO 2 Immobilization, whether biological or non-biological, begins early in the earth's history, because of CO 2 Is far above today. About 150000×10 12 High volume of CO per metric ton 2 Is fixed into carbonate minerals such as calcite, aragonite, dolomite and limestone. Generally, CO 2 Can be naturally converted to solids, including carbonate minerals such as calcium carbonate and magnesium carbonate, howeverFormation of bicarbonate-forming CO 2 Hydration is a very slow process (-1.3X10) -1 s -1 )。
Disclosure of Invention
One aspect of the present disclosure is a composition comprising a plant or plant seed and one or more microorganisms associated with the plant or plant seed, wherein the one or more microorganisms are or are derived from one or more microorganisms selected to produce or promote bicarbonate, carbonate, or one or more mineral formations. In some embodiments, the plant is a commercial plant, a fruit tree plant, a nut tree plant, a shrub plant, a bulb plant, a grassland plant, a lawn plant, or any combination thereof. In some embodiments, the plant seed is a commercial plant seed, a fruit tree seed, a nut tree seed, a shrub seed, a bulb seed, a grassland seed, a lawn plant seed, or any combination thereof. In some embodiments, one or more microorganisms associated with the plant seed are disposed in a gap between the seed coat and the seed embryo of the plant seed. In some embodiments, the one or more microorganisms associated with the plant seed are disposed as a coating of the plant seed. In some embodiments, one or more microorganisms associated with the plant seed are applied to the plant seed by an irrigation system. In some embodiments, the irrigation system includes in-furrow treatment techniques. In some embodiments, the irrigation system comprises spray technology. In some embodiments, bicarbonate sequesters carbon. In some embodiments, the carbon is gaseous carbon. In some embodiments, the gaseous carbon is carbon dioxide. In some embodiments, the carbonate sequesters carbon. In some embodiments, the carbon is gaseous carbon. In some embodiments, the gaseous carbon is carbon dioxide. In some embodiments, one or more minerals sequester carbon. In some embodiments, the carbon is gaseous carbon. In some embodiments, the gaseous carbon is carbon dioxide. In some embodiments, one or more microorganisms associated with the plant seed are disposed in a gap between a seed pericarp (seed) of the plant seed and a seed aleurone cell layer. In some embodiments, the one or more microorganisms comprise one or more carbonic anhydrases. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases from the class α. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases from class β. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases from class γ. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases from class δ. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases from the zeta class. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases from class η. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrase from the iota class.
In some embodiments, the one or more microorganisms comprise bacteria, archaebacteria, fungi, or viruses. In some embodiments, the one or more microorganisms comprise bacteria. In some embodiments, the bacteria comprise endospore-forming bacteria. In some embodiments of the present invention, in some embodiments, the bacteria include bacteria derived from Acetobacter (Acetonem sp.), actinomyces (Actinomyces sp.), bacillus (Alkalibacillus sp.), acidophilia (Ammoniphilia sp.), bisporium (Amanibacillus sp.), anaerobacter (Anaerobacter sp.), anaerobacter (Anaerobiospora sp.), thiobacillus (Aneubrinellus sp.), anaerobic bacillus sp.), bacillus (Bacillus sp.), brevibacterium sp, thermoanaerobacter (Caldanella sp.), thermoanaerobacter sp.), and other bacteria derived from Acetobacter (Acetobacter sp.), and the bacteria including Acetobacter sp, acetobacter sp. And Acetobacter sp. The bacteria are preferably used as bacteria. Cherry bacillus (Cerasibacillus sp.), clostridium (Clostridium sp.), clostridium (Clostridium sp.), ke Enshi (Cohnella sp.), ke Kesi (Coxiella sp.), bacillus (Dendrosporum sp.), desulfoenterobacter (Desulfomaculolimsp.), bacillus (Desulfolobus sp.), bacillus (Cohnella sp.) and Bacillus (Cohnella sp.) Desulfosporisomus genus (Desulfospormus sp.), desulphularia genus (Desulfospormus sp.), desulfospormus genus (Desulfospormus sp.), desulphuspormus genus (Desulfospormus sp.), producing strain (Filifactor sp.), bacillus (Filobulus sp.), gelria genus (Gelr) ia sp.), geobacillus sp, geoporobacter sp, bacillus gracillus sp, halobacillus sp, haloatron sp, solar bacterium sp, heliobacterium sp, lacefillium sp, laceyella sp, and Bacillus chrous (Lentibacillus sp.), bacillus lysii (Lysinibacillus sp.), mahela (Mahela sp.), metacter (Metacter sp.), morella sp Natroniella (Natroniella sp.), bacillus (Ocenobacillus sp.), orenica (Orenica sp.), bacillus ornithine (Ornithini bacillus sp.), bacillus megaterium, and Bacillus megaterium oxalic acid bacteria (Oxiphigus sp.), acetobacter (Oxobacillus sp.), paenibacillus (Paenibacillus sp.), bacillus marinus (Paraliobacillus sp.), pelospora (Pelospora sp.), enteromorpha (Pelotoculum sp.), pelotomorpha (Piscibacillus sp.), platymonas (Planiflum sp.), bacillus (Bacillus sp.), bacillus (Pontibacter sp.), propionibacterium (Propionibacterium sp.), salinibacillus sp, salinibacillus (Salinibacillus sp.), wild bacillus (Serinella), zosterum (Shimamzula), zosterum (Sporobacter sp.), bacillus (Sporobacter sp.), bacillus sp.sp.) Sporobacterium (Sporobacterium sp.), bacillus (Sporobactylobacter sp.), lactobacillus (Sporobactylodes sp.), banana (Sporobusta sp.), sporobactylococcus (Sporobactylocina sp.), sporobactylodes (Sporobactylodes sp.) Sporotalea (Sporotalea sp.), enterobacter sp., sporotomoaculum sp.): comrophomonas sp, syntrophospora Bacillus (Tenuigecillus sp.), thermomyces (Tepidialacterium sp.), geobacillus (Terryibacillus sp.), thermomyces (Thealabasophilus sp.), thermoacetobacter (Thermoacetogenius sp.), thermoactinomyces (Thermoactinomyces sp.), thermokallibacillus (Thermokallibacillus sp.), thermoanaerobacter (Thermoanaerobacter sp.), thermoanaerobacter (Thermom) Bacteria of oaanomonas sp.), thermoyellow genus (thermoflavobacterium sp.), thermonanobulum genus (thermobubblum sp.), bacillus megaterium genus (turnibacillus sp.), bacillus (virginacillus sp.), vulcanobacillus genus (Vulcanobacillus sp.) or a combination thereof. In some embodiments, the bacteria comprise bacteria belonging to the phylum Firmicutes (Firmicutes). In some embodiments, the bacteria comprise rhizobacteria. In some embodiments, the rhizobacteria comprise bacillus, paenibacillus, or both. In some embodiments, the bacteria comprise bacillus amyloliquefaciens (b.amycolatopsis), bacillus laterosporus (b.laborocus), bacillus licheniformis (b.lichenifermis), bacillus macerans (b.macerans), bacillus cereus (b.cereus), bacillus circulans (b.circulans), bacillus firmus (b.firmus), bacillus subtilis (b.subtitis), bacillus megaterium (b.megaterium), bacillus coagulans (b.agaricus), bacillus brevis (b.breve), bacillus sphaericus (b.sphaericus), bacillus thuringiensis (b.thuringiensis), bacillus mycoides (b.myces), bacillus cucumber (b.curcus), bacillus plantarum (b.endomycosis), bacillus pumilus (b.puus), bacillus bailii (b.bacillus subtilis), bacillus megaterium (b.methyl) or any combination thereof. In some embodiments, the bacteria comprise bacillus subtilis S3C23, bacillus subtilis MP2, bacillus subtilis RO2C15, bacillus subtilis RO2C22, bacillus megaterium 6, bacillus megaterium S3C21, bacillus megaterium RO2C12, bacillus cucumber S3C14, bacillus plantarum 5, or any combination thereof. In some embodiments, the bacteria comprise bacillus subtilis S3C23. In some embodiments, the bacteria comprise bacillus subtilis MP2. In some embodiments, the bacteria comprise Paenibacillus polymyxa (Paenibacillus polymyxa), paenibacillus persicae (Paenibacillus taohuashanense), paenibacillus pocheonensis, paenibacillus aceris, paenibacillus catalpa (Paenibacillus catalpa), paenibacillus wetland (Paenibacillus rigui), and Paenibacillus forage Bacillus (Paenibacillus pabuli), paenibacillus brazil (Paenibacillus brasiliensis) or any combination thereof. In some embodiments, the bacteria comprise paenibacillus polymyxa RO3C16, paenibacillus persicae TY4D5, paenibacillus pocheonensis S2C3, paenibacillus aceris VF2D2, paenibacillus catalpa TY2B5, paenibacillus wetland TY2D5, paenibacillus feed PG2A8, or any combination thereof. In some embodiments, the bacteria comprise non-endosporium forming bacteria. In some embodiments, the bacteria comprise bacteria belonging to the phylum Proteus (Proteus). In some embodiments of the present invention, in some embodiments, the bacteria include Klebsiella sp, rhizobium sp, klebsiella sp, rhizobium sp, ochrobium sp, sinorhizobium sp, xanthobacter sp, methylobacillus sp, actinomyces, kosakura sp, azotobacter sp, klebsiella sp, and Klebsiella sp Acetobacter (Acetobacter sp.), carpesium (Herbapirillum sp.), pseudomonas (Pseudomonas sp.), parabrukholderia sp.), ralstonia (Ralstonia sp.), geobacillus (Geobacillus sp.), serratia (Serratia sp.), pantoea (Pantoea sp.), swordaria (Ensifer sp.), enterobacter (Enterobacter sp.) or any combination thereof. In some embodiments, the bacteria comprise a sabia (Ensifer adhaerens) S3C10. In some embodiments, the bacteria comprise bacteria belonging to the phylum actinomycetes (actionobacteria). In some embodiments, the bacteria comprise Streptomyces sp, ke Kesi, frank (Frankia sp). In some embodiments, the bacteria comprise bacteria belonging to the phylum Cyanobacteria (Cyanobacteria). In some embodiments, the bacteria comprise Cyanobacteria (Cyanobacteria sp.). In some embodiments, the bacteria comprise bacteria belonging to the phylum Cloroflexi (Cloroflexi). In some embodiments, the one or more microorganisms comprise one or more fungi associated with the plant seed. In some embodiments, one or more fungi associated with the plant seed is disposed in the gap between the seed coat and the seed embryo of the plant seed. In some embodiments One or more fungi associated with the plant seed are arranged as a coating of the plant seed. In some embodiments, the one or more fungi associated with the plant seed are applied to the plant seed using in-furrow techniques. In some embodiments, the one or more fungi associated with the plant seed are applied to the plant seed using a spray technique. In some embodiments, the one or more fungi associated with the plant seed are applied to the plant seed by irrigation. In some embodiments, one or more fungi associated with the plant seed are disposed in the gap between the seed pericarp and the seed aleurone cell layer of the plant seed. In some embodiments, the one or more fungi comprise arbuscular mycorrhizal (Arbuscular Mycorrhizal) fungi. In some embodiments, the one or more fungi comprise Ectomycorrhizal (Ectomycorrhizal) fungi. In some embodiments, the one or more fungi comprise a fungus from the genus Trichoderma (Trichoderma). In some embodiments, the one or more fungi comprise a fungus from the genus Penicillium (Penicillium). In some embodiments, the one or more minerals comprise calcite, aragonite, dolomite, limestone, or any combination thereof. In some embodiments, the one or more minerals comprise CaCO 3 、MgCO 3 、CaMg(CO 3 ) 2 Or any combination thereof. In some embodiments, promoting the production of one or more minerals includes the production of ammonia and the resulting increase in pH in a medium in which plants derived from plant seeds are grown. In some embodiments, the one or more microorganisms are not naturally present in the gap between the seed pericarp and the seed aleurone cell layer of the plant seed. In some embodiments, the plant seed is a monocot seed or a dicot seed. In some embodiments, the commercial plant seed is corn seed, wheat seed, rice seed, sorghum seed, barley seed, rye seed, sugarcane seed, millet seed, oat seed, soybean seed, cotton seed, alfalfa seed, bean seed, quinoa seed, lentil seed, peanut seed, sunflower seed, canola seed, cassava seed, oil palm seed, potato seed, sugar beet seed, cocoa seed, coffee bean, lettuce seedA chicory seed, a tomato seed, a pea seed or a cabbage seed.
Another aspect of the present disclosure is a composition comprising a plant or part thereof and one or more microorganisms associated with the plant or part thereof, wherein the one or more microorganisms are or are derived from one or more microorganisms selected to produce or promote bicarbonate, carbonate, or one or more mineral formations. In some embodiments, the plant or portion thereof is a plant root, a plant stem, a plant leaf, a plant seed, a plant fruit, a plant tuber, or a plant nodule. In some embodiments, the plant or portion thereof comprises a commercial plant or portion thereof. In some embodiments, the commercial plant or portion thereof is corn, wheat, rice, sorghum, barley, rye, sugarcane, millet, oat, soybean, cotton, alfalfa, bean, quinoa, lentils, peanut, sunflower, canola, cassava, oil palm, potato, sugar beet, cocoa, coffee, lettuce, tomato, pea, cabbage, fruit trees, nut trees, forest, grasslands, or turf grasses. In some embodiments, the portion thereof is a plant seed, and wherein the one or more microorganisms associated with the plant seed are disposed in a gap between a seed coat and a seed embryo of the plant seed. In some embodiments, the one or more microorganisms associated with the plant or portion thereof are disposed as a coating of the plant or portion thereof. In some embodiments, one or more microorganisms associated with the plant or portion thereof are applied to the plant seed by an irrigation system. In some embodiments, the irrigation system includes in-furrow treatment techniques. In some embodiments, the irrigation system comprises spray technology. In some embodiments, bicarbonate sequesters carbon. In some embodiments, the carbon is gaseous carbon. In some embodiments, the gaseous carbon is carbon dioxide. In some embodiments, the carbonate sequesters carbon. In some embodiments, the carbon is gaseous carbon. In some embodiments, the gaseous carbon is carbon dioxide. In some embodiments, one or more minerals sequester carbon. In some embodiments, the carbon is gaseous carbon. In some embodiments, the gaseous carbon is carbon dioxide. In some embodiments, the portion thereof is a plant seed, and the portion thereof One or more microorganisms associated with the plant seed are disposed in the interstices between the seed pericarp and seed aleurone cell layer of the plant seed. In some embodiments, the one or more microorganisms comprise one or more carbonic anhydrases. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases belonging to class α. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases that belong to the class β. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases belonging to the class γ. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases that belong to class delta. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases belonging to the zeta class. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases that belong to class η. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases belonging to the iota class. In some embodiments, the one or more microorganisms comprise bacteria, archaebacteria, fungi, or viruses. In some embodiments, the one or more microorganisms comprise bacteria. In some embodiments, the bacteria comprise endospore-forming bacteria. In some embodiments of the present invention, in some embodiments, the bacteria include bacteria from the genus Acetobacter, actinomycetes, bacillus alcaligenes, aminophilia, bacillus bifidus, anaerobic bacillus, thiobacillus, anaerobic bacillus, bacillus, brevibacterium, thermoanaerobacter, caminiella, cherry-like bacillus, clostridium, ke Enshi, ke Kesi, acidogenes, bacillus treonam, desulphurispora, desulfosporomula, campylobacter Desulfosporulation genus, desulfurated sporulation genus, line producing genus, gelria genus, georgia genus, georrobacter genus, cellularomyces genus, halophilous bacillus genus, haloatron genus, solar bacillus genus, solar philic bacteria genus, leishmaniella genus, chromosporulation genus, lysine bacillus genus, mahela genus, metabacterium genus, mushroom genus, natroniella genus, oceanic bacillus genus, oreneia genus, ornithine bacillus genus, oxalic acid bacillus genus, Acetobacter, paenibacillus, bacillus marinus, pelospora, anaerobic Enterobacter, bacillus, phanerochaete, bacillus, propionibacterium, bacillus salina, bacillus, thermus, bacillus, and Bacillus genus Qinghai, genus Shimadzu, genus Acetobacter, genus anaerobacter, genus Sporobacter Sporobacterium, bacillus, lactobacillus, banana spore, sporobococcus, bacillus, being the same, being different from those of the same, and being different from each other Sporotalea, enterobacter, co-cultured monad, bacillus, warm bacillus, geobacillus, deep sea bacillus, thermophilic acetogenic bacteria, high temperature actinomyces bacteria of the genus Thermoalallibacillus, thermoanaerobacter, thermobacillus, thermoflavobacterium, thermovenabalum, bacillus megaterium, bacillus, vulcanobacillus, or combinations thereof. In some embodiments, the bacteria comprise bacteria belonging to the phylum firmicutes. In some embodiments, the bacteria comprise rhizobacteria. In some embodiments, the rhizobacteria comprise bacillus, paenibacillus, or both. In some embodiments, the bacteria comprise bacillus amyloliquefaciens, bacillus laterosporus, bacillus licheniformis, bacillus macerans, bacillus cereus, bacillus circulans, bacillus firmus, bacillus subtilis, bacillus megaterium, bacillus coagulans, bacillus brevis, bacillus sphaericus, bacillus thuringiensis, bacillus mycoides, bacillus cucumber, bacillus plantarum, bacillus pumilus, bacillus bailii, bacillus mucilaginosus, bacillus tefraxins, bacillus methylotrophicus, or any combination thereof. In some embodiments, the bacteria comprise bacillus subtilis S3C23, bacillus subtilis MP2, bacillus subtilis RO2C15, bacillus subtilis RO2C22, bacillus megaterium 6, bacillus megaterium S3C21, bacillus megaterium RO2C12, bacillus cucumber S3C14, bacillus plantarum 5, or any combination thereof. In some embodiments, the bacteria comprise bacillus subtilis S3C23. In some embodiments, the bacteria comprise bacillus subtilis MP2. In some embodiments, the bacteria Comprises Paenibacillus polymyxa, paenibacillus persicae, paenibacillus pocheonensis, paenibacillus aceris, paenibacillus catalpa, paenibacillus wetland, paenibacillus forage, paenibacillus brazil or any combination thereof. In some embodiments, the bacteria comprise paenibacillus polymyxa RO3C16, paenibacillus persicae TY4D5, paenibacillus pocheonensis S2C3, paenibacillus aceris VF2D2, paenibacillus catalpa TY2B5, paenibacillus wetland TY2D5, paenibacillus feed PG2A8, or any combination thereof. In some embodiments, the bacteria comprise non-endosporium forming bacteria. In some embodiments, the bacteria comprise bacteria belonging to the phylum Proteus. In some embodiments, the bacteria comprise klebsiella, rhizobium, bradyrhizobium, pallidobacter, sinorhizobium, xanthobacter, methylobacterium, actinomyces, coxsackie, azotobacter, acetobacter, rhodospirillum, pseudomonas, paraburkholderia, rochello, geobacillus, serratia, pantoea, swordlike, enterobacter, or any combination thereof. In some embodiments, the bacteria comprise a C3C 10 bacterium. In some embodiments, the bacteria comprise bacteria belonging to the phylum actinomycetes. In some embodiments, the bacteria comprise streptomyces, ke Kesi, frank, and the like. In some embodiments, the bacteria comprise bacteria belonging to the phylum cyanobacteria. In some embodiments, the bacteria comprise cyanobacteria. In some embodiments, the bacteria comprise bacteria belonging to the phylum green-forming. In some embodiments, the one or more microorganisms comprise one or more fungi associated with the plant or part thereof. In some embodiments, one or more fungi associated with the plant or part thereof is disposed in the gap between the seed coat and the seed embryo of the plant or part thereof. In some embodiments, the one or more fungi associated with the plant or part thereof are disposed as a coating of the plant or part thereof. In some embodiments, the one or more fungi associated with the plant or part thereof are applied to the plant or part thereof using in-furrow techniques. In some embodiments, the one or more of the plant or portion thereof is/are associated with the plant using a spray technique The fungus is applied to the plant or part thereof. In some embodiments, one or more fungi associated with the plant or portion thereof is applied to the plant or portion thereof by an irrigation system. In some embodiments, one or more fungi associated with the plant or portion thereof is disposed in the gap between the seed pericarp and the seed aleurone cell layer of the plant or portion thereof. In some embodiments, the one or more fungi comprise arbuscular mycorrhizal fungi. In some embodiments, the one or more fungi comprise ectomycorrhizal fungi. In some embodiments, the one or more fungi comprise fungi from the genus trichoderma. In some embodiments, the one or more fungi comprise a fungus from the genus penicillium. In some embodiments, the one or more minerals comprise calcite, aragonite, dolomite, limestone, or any combination thereof. In some embodiments, the one or more minerals comprise CaCO 3 、MgCO 3 、CaMg(CO 3 ) 2 Or any combination thereof. In some embodiments, promoting the production of one or more minerals comprises producing ammonia and a resulting increase in pH in a medium in which plants derived from the plant or part thereof are grown. In some embodiments, the portion thereof is a plant seed, and wherein the one or more microorganisms are not naturally present in the interstices between the seed pericarp and seed aleurone cell layer of the plant or portion thereof. In some embodiments, the plant or part thereof is a monocot or dicot.
Another aspect of the present disclosure is a composition comprising one or more microorganisms, wherein the one or more microorganisms are located at the gap between the coating and the cell layer of the plant or portion thereof and are or are derived from one or more microorganisms selected to produce or promote bicarbonate, carbonate, or one or more mineral formation. In some embodiments, the plant or portion thereof is a plant root, a plant stem, a plant leaf, a plant seed, a plant fruit, a plant tuber, or a plant nodule. In some embodiments, the plant or portion thereof comprises a commercial plant or portion thereof. In some embodiments, the commercial plant or part thereof is maize, wheat, rice, sorghum, barley, rye, sugarcaneMillet, oat, soybean, cotton, alfalfa, bean, quinoa, lentils, peanut, sunflower, canola, cassava, oil palm, potato, sugar beet, cocoa, coffee, lettuce, tomato, pea, cabbage, fruit trees, nut trees, forest, grasslands, or turf grasses. In some embodiments, the portion thereof is a plant seed, and wherein the one or more microorganisms associated with the plant seed are disposed in a gap between a seed coat and a seed embryo of the plant seed. In some embodiments, the one or more microorganisms associated with the plant or portion thereof are disposed as a coating of the plant or portion thereof. In some embodiments, one or more microorganisms associated with the plant or portion thereof are applied to the plant seed by an irrigation system. In some embodiments, the irrigation system includes in-furrow treatment techniques. In some embodiments, the irrigation system comprises spray technology. In some embodiments, bicarbonate sequesters carbon. In some embodiments, the carbon is gaseous carbon. In some embodiments, the gaseous carbon is carbon dioxide. In some embodiments, the carbonate sequesters carbon. In some embodiments, the carbon is gaseous carbon. In some embodiments, the gaseous carbon is carbon dioxide. In some embodiments, one or more minerals sequester carbon. In some embodiments, the carbon is gaseous carbon. In some embodiments, the gaseous carbon is carbon dioxide. In some embodiments, the portion thereof is a plant seed, and wherein the one or more microorganisms associated with the plant seed are disposed in a gap between a seed pericarp and a seed aleurone cell layer of the plant seed. In some embodiments, the one or more microorganisms comprise one or more carbonic anhydrases. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases belonging to class α. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases that belong to the class β. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases belonging to the class γ. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases that belong to class delta. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases belonging to the zeta class. In some embodiments, the one or more carbonic anhydrases comprise Carbonic anhydrases belonging to class eta. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases belonging to the iota class. In some embodiments, the one or more microorganisms comprise bacteria, archaebacteria, fungi, or viruses. In some embodiments, the one or more microorganisms comprise bacteria. In some embodiments, the bacteria comprise endospore-forming bacteria. In some embodiments of The present invention, in some embodiments, the bacteria include bacteria from The genus Acetobacter, the genus actinomycete, the genus Bacillus, the genus Alkaleidosporium, the genus Aminophilia, the genus Bacillus, the genus anaerobic, the genus Thiobacillus, the genus anaerobic bacillus, the genus Bacillus, the genus Brevibacterium, the genus Thermoanaerobacter, the genus Caminiella, the genus Prunus, the genus Clostridium, the genus Ke Enshi, the genus Ke Kesi, the genus Acidocella, the genus Enterobacter desulphurisum, the genus Bacillus Desulfosporomula, desulfocampylobacter, desulfosporula, desulfosporium, protosporium, linear, gelria, geosporobacter, cellularomyces, halophilum, haloatron, solar, japanese, leishmania, chrysosporium, lysine bacillus, mahela, metabacterium, mushroom, mortierella, and Mortierella Desulfosporomula, desulfocampylobacter, desulfosporula, protosporium, linear, gelria, geosporium, geosporobacter, bacillus, halomatron, solar bacillus, solar bacterium, leishmania, chronic Bacillus, lysine bacillus, mahela, metabacterium, mushroom, bacillus, and Bacillus Bacteria of the genus rmobacillus, thermoxanthomonas, thermonanbulum, bacillus, vulcanofacillus, or combinations thereof. In some embodiments, the bacteria comprise bacteria belonging to the phylum firmicutes. In some embodiments, the bacteria comprise rhizobacteria. In some embodiments, the rhizobacteria comprise bacillus, paenibacillus, or both. In some embodiments, the bacteria comprise bacillus amyloliquefaciens, bacillus laterosporus, bacillus licheniformis, bacillus macerans, bacillus cereus, bacillus circulans, bacillus firmus, bacillus subtilis, bacillus megaterium, bacillus coagulans, bacillus brevis, bacillus sphaericus, bacillus thuringiensis, bacillus mycoides, bacillus cucumber, bacillus plantarum, bacillus pumilus, bacillus bailii, bacillus mucilaginosus, bacillus tefraxins, bacillus methylotrophicus, or any combination thereof. In some embodiments, the bacteria comprise bacillus subtilis S3C23, bacillus subtilis MP2, bacillus subtilis RO2C15, bacillus subtilis RO2C22, bacillus megaterium 6, bacillus megaterium S3C21, bacillus megaterium RO2C12, bacillus cucumber S3C14, bacillus plantarum 5, or any combination thereof. In some embodiments, the bacteria comprise bacillus subtilis S3C23. In some embodiments, the bacteria comprise bacillus subtilis MP2. In some embodiments, the bacteria comprise paenibacillus polymyxa, paenibacillus persicae, paenibacillus pocheonensis, paenibacillus aceris, paenibacillus catalpa, paenibacillus wetland, paenibacillus feed, paenibacillus brazil, or any combination thereof. In some embodiments, the bacteria comprise paenibacillus polymyxa RO3C16, paenibacillus persicae TY4D5, paenibacillus pocheonensis S2C3, paenibacillus aceris VF2D2, paenibacillus catalpa TY2B5, paenibacillus wetland TY2D5, paenibacillus feed PG2A8, or any combination thereof. In some embodiments, the bacteria comprise non-endosporium forming bacteria. In some embodiments, the bacteria comprise bacteria belonging to the phylum Proteus. In some embodiments, the bacteria comprise klebsiella, rhizobium, or bradykinin The genus Arthrobacter, sinorhizobium, flavobacterium, methylobacterium, actinobacillus, cosaxobacteria, azotobacter, acetobacter, carpium, pseudomonas, paraquatica, ralstonia, geobacillus, serratia, pantoea, sword, enterobacter, or any combination thereof. In some embodiments, the bacteria comprise a C3C 10 bacterium. In some embodiments, the bacteria comprise bacteria belonging to the phylum actinomycetes. In some embodiments, the bacteria comprise streptomyces, ke Kesi, frank, and the like. In some embodiments, the bacteria comprise bacteria belonging to the phylum cyanobacteria. In some embodiments, the bacteria comprise cyanobacteria. In some embodiments, the bacteria comprise bacteria belonging to the phylum green-forming. In some embodiments, the one or more microorganisms comprise one or more fungi associated with the plant or part thereof. In some embodiments, one or more fungi associated with the plant or part thereof is disposed in the gap between the seed coat and the seed embryo of the plant or part thereof. In some embodiments, the one or more fungi associated with the plant or part thereof are disposed as a coating of the plant or part thereof. In some embodiments, the one or more fungi associated with the plant or part thereof are applied to the plant or part thereof using in-furrow techniques. In some embodiments, the one or more fungi associated with the plant or part thereof are applied to the plant or part thereof using a spray technique. In some embodiments, one or more fungi associated with the plant or portion thereof is applied to the plant or portion thereof by an irrigation system. In some embodiments, one or more fungi associated with the plant or portion thereof is disposed in the gap between the seed pericarp and the seed aleurone cell layer of the plant or portion thereof. In some embodiments, the one or more fungi comprise arbuscular mycorrhizal fungi. In some embodiments, the one or more fungi comprise ectomycorrhizal fungi. In some embodiments, the one or more fungi comprise fungi from the genus trichoderma. In some embodiments, the one or more fungi comprise a fungus from the genus penicillium. In some embodiments, the one or more minerals comprise calcite, aragonite, dolomite Stone, limestone, or any combination thereof. In some embodiments, the one or more minerals comprise CaCO 3 、MgCO 3 、CaMg(CO 3 ) 2 Or any combination thereof. In some embodiments, promoting the production of one or more minerals comprises producing ammonia and a resulting increase in pH in a medium in which plants derived from the plant or part thereof are grown. In some embodiments, the portion thereof is a plant seed, and wherein the one or more microorganisms are not naturally present in the interstices between the seed pericarp and seed aleurone cell layer of the plant or portion thereof. In some embodiments, the plant or part thereof is a monocot or dicot.
Another aspect of the present disclosure is a method of promoting mineralization, the method comprising: cultivating the plant or part thereof and one or more microorganisms associated with the plant or part thereof, wherein the one or more microorganisms are or are derived from microorganisms selected to produce or promote bicarbonate, carbonate, or one or more mineral formations. In some embodiments, the plant or portion thereof is a commercial plant, plant root, plant stem, plant leaf, plant seed, plant fruit, plant tuber, or plant root nodule. In some embodiments, one or more microorganisms associated with the plant or portion thereof are disposed on the plant root or rhizosphere of the plant or portion thereof. In some embodiments, one or more microorganisms associated with the plant or portion thereof are disposed on the plant root or rhizosphere of the plant or portion thereof by an irrigation system. In some embodiments, the irrigation system includes in-furrow treatment techniques. In some embodiments, the irrigation system comprises spray technology. In some embodiments, the plant or portion thereof originates from a seedling that is integrated with the microorganism by an irrigation system to stimulate the plant or portion thereof to produce one or more minerals. In some embodiments, bicarbonate sequesters carbon. In some embodiments, the carbon is gaseous carbon. In some embodiments, the gaseous carbon is carbon dioxide. In some embodiments, the carbonate sequesters carbon. In some embodiments, the carbon is gaseous carbon. In some embodiments, the gaseous carbon is carbon dioxide. In some embodiments, one or more minerals sequester carbon. In some embodiments, the carbon is gaseous carbon. In some embodiments, the gaseous carbon is carbon dioxide. In some embodiments, the one or more microorganisms comprise bacteria, archaebacteria, fungi, or viruses. In some embodiments, the one or more microorganisms comprise bacteria. In some embodiments, the bacteria comprise endospore-forming bacteria. In some embodiments, the bacteria comprise rhizobacteria. In some embodiments, the rhizobacteria comprise bacillus, paenibacillus, or both. In some embodiments, the bacteria comprise bacillus amyloliquefaciens, bacillus laterosporus, bacillus licheniformis, bacillus macerans, bacillus cereus, bacillus circulans, bacillus firmus, bacillus subtilis, bacillus sphaericus, bacillus megaterium, bacillus coagulans, bacillus brevis, bacillus thuringiensis, bacillus mycoides, bacillus cucumber, bacillus intraphytans, bacillus pumilus, bacillus bailii, bacillus mucilaginosus, bacillus tervalicalis, bacillus methylotrophicus, or any combination thereof. In some embodiments, the bacteria comprise bacillus subtilis S3C23, bacillus subtilis MP2, bacillus subtilis RO2C15, bacillus subtilis RO2C22, bacillus megaterium 6, bacillus megaterium S3C21, bacillus megaterium RO2C12, bacillus cucumber S3C14, bacillus plantarum 5, or any combination thereof. In some embodiments, the bacteria comprise bacillus subtilis S3C23. In some embodiments, the bacteria comprise bacillus subtilis MP2. In some embodiments, the one or more microorganisms comprise one or more fungi associated with the plant or part thereof. In some embodiments, one or more fungi associated with the plant or portion thereof is disposed on the plant root or rhizosphere of the plant or portion thereof by an irrigation system. In some embodiments, the one or more fungi comprise arbuscular mycorrhizal fungi. In some embodiments, the one or more fungi comprise ectomycorrhizal fungi. In some embodiments, the one or more fungi comprise fungi from the genus trichoderma. In some embodiments, the one or more fungi comprise a fungus from the genus penicillium. In some embodiments, the one or more microorganisms produce the formation of one or more carbonic anhydrases. In some embodiments, the one or more microorganisms produce the formation of one or more carbonic anhydrases belonging to class α. In some embodiments, the one or more microorganisms produce the formation of one or more carbonic anhydrases belonging to class β. In some embodiments, the one or more microorganisms produce the formation of one or more carbonic anhydrases belonging to class γ. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases that belong to class delta. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases belonging to the zeta class. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases that belong to class η. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases belonging to the iota class. In some embodiments, the one or more minerals comprise calcite, aragonite, dolomite, limestone, or any combination thereof. In some embodiments, promoting the production of one or more minerals comprises producing ammonia and the resulting increase in pH in a medium in which the plant or portion thereof is grown. In some embodiments, the one or more microorganisms are not naturally present on the one or more roots. In some embodiments, the plant or part thereof is a monocot or dicot. In some embodiments, the plant or portion thereof comprises a commercial plant or portion thereof. In some embodiments, the commercial plant or portion thereof comprises a group consisting essentially of: corn, wheat, rice, sorghum, barley, rye, sugarcane, millet, oat, soybean, cotton, alfalfa, bean, quinoa, lentils, peanut, sunflower, canola, cassava, oil palm, potato, sugar beet, cocoa, coffee, lettuce, tomato, peas, cabbage, fruit trees, nut trees, forests, grasslands, or turf grasses.
Another aspect of the present disclosure is a method of sequestering carbon, the method comprising: cultivating the plant or part thereof and one or more microorganisms associated with the plant or part thereof; wherein the one or more microorganisms are or are derived from microorganisms selected to produce or promote the formation of one or more carbonaceous minerals to sequester carbon. In some embodiments, the plant or portion thereof is a plant root, a plant stem, a plant leaf, a plant seed, a plant fruit, a plant tuber, or a plant nodule. In some embodiments, the plant or portion thereof comprises a commercial plant or portion thereof. In some embodiments, the commercial plant or portion thereof comprises a group consisting essentially of: corn, wheat, rice, sorghum, barley, rye, sugarcane, millet, oat, soybean, cotton, alfalfa, bean, quinoa, lentils, peanut, sunflower, canola, cassava, oil palm, potato, sugar beet, cocoa, coffee, lettuce, tomato, peas, cabbage, fruit trees, nut trees, forests, grasslands, or turf grasses. In some embodiments, the one or more carbonaceous minerals comprise one or more gaseous carbonation materials. In some embodiments, the one or more gaseous carbonators comprise carbon monoxide, methane or carbon dioxide. In some embodiments, the one or more gaseous carbonators is carbon dioxide. In some embodiments, the one or more microorganisms associated with the plant are disposed on the plant root or rhizosphere of the plant or portion thereof. In some embodiments, one or more microorganisms associated with a plant are disposed on the plant root or rhizosphere of the plant or portion thereof by an irrigation system. In some embodiments, the irrigation system includes in-furrow treatment techniques. In some embodiments, the irrigation system comprises spray technology. In some embodiments, the plant or portion thereof originates from a seedling that is integrated with the microorganism by an irrigation system to stimulate the plant or portion thereof to produce one or more minerals. In some embodiments, the one or more microorganisms comprise bacteria, archaebacteria, fungi, or viruses. In some embodiments, the one or more microorganisms comprise bacteria. In some embodiments, the bacteria comprise endospore-forming bacteria. In some embodiments, the bacteria comprise rhizobacteria. In some embodiments, the rhizobacteria comprise bacillus, paenibacillus, or both. In some embodiments, the bacteria comprise bacillus amyloliquefaciens, bacillus laterosporus, bacillus licheniformis, bacillus macerans, bacillus cereus, bacillus circulans, bacillus firmus, bacillus subtilis, bacillus megaterium, bacillus coagulans, bacillus brevis, bacillus sphaericus, bacillus thuringiensis, bacillus mycoides, bacillus cucumber, bacillus plantarum, bacillus pumilus, bacillus bailii, bacillus mucilaginosus, bacillus tefraxins, bacillus methylotrophicus, or any combination thereof. In some embodiments, the bacteria comprise bacillus subtilis S3C23, bacillus subtilis MP2, bacillus subtilis RO2C15, bacillus subtilis RO2C22, bacillus megaterium 6, bacillus megaterium S3C21, bacillus megaterium RO2C12, bacillus cucumber S3C14, bacillus plantarum 5, or any combination thereof. In some embodiments, the bacteria comprise bacillus subtilis S3C23. In some embodiments, the bacteria comprise bacillus subtilis MP2. In some embodiments, the one or more microorganisms comprise one or more fungi associated with the plant or part thereof. In some embodiments, one or more fungi associated with the plant or portion thereof is disposed on the plant root or rhizosphere of the plant or portion thereof by an irrigation system. In some embodiments, the one or more fungi comprise arbuscular mycorrhizal fungi. In some embodiments, the one or more fungi comprise ectomycorrhizal fungi. In some embodiments, the one or more fungi comprise fungi from the genus trichoderma. In some embodiments, the one or more fungi comprise a fungus from the genus penicillium. In some embodiments, the one or more microorganisms are not naturally present on the one or more roots. In some embodiments, the plant or part thereof is a monocot or dicot. In some embodiments, the one or more microorganisms produce the formation of one or more carbonic anhydrases. In some embodiments, the one or more microorganisms produce the formation of one or more carbonic anhydrases belonging to class α. In some embodiments, the one or more microorganisms produce the formation of one or more carbonic anhydrases belonging to class β. In some embodiments, the one or more microorganisms produce the formation of one or more carbonic anhydrases belonging to class γ. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases that belong to class delta. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases belonging to the zeta class. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases that belong to class η. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases belonging to the iota class.
Another aspect of the present disclosure is a composition comprising one or more microorganisms, wherein the one or more microorganisms are or are derived from microorganisms selected to produce or promote bicarbonate, carbonate, or one or more mineral formations. In some embodiments, the plant or portion thereof is a plant root, a plant stem, a plant leaf, a plant seed, a plant fruit, a plant tuber, or a plant nodule. In some embodiments, the plant or portion thereof comprises a commercial plant or portion thereof. In some embodiments, the commercial plant or portion thereof is corn, wheat, rice, sorghum, barley, rye, sugarcane, millet, oat, soybean, cotton, alfalfa, bean, quinoa, lentils, peanut, sunflower, canola, cassava, oil palm, potato, sugar beet, cocoa, coffee, lettuce, tomato, pea, cabbage, fruit trees, nut trees, forest, grasslands, or turf grasses. In some embodiments, bicarbonate sequesters carbon. In some embodiments, the carbon is gaseous carbon. In some embodiments, the gaseous carbon is carbon dioxide. In some embodiments, the carbonate sequesters carbon. In some embodiments, the carbon is gaseous carbon. In some embodiments, the gaseous carbon is carbon dioxide. In some embodiments, one or more minerals sequester carbon. In some embodiments, the carbon is gaseous carbon. In some embodiments, the gaseous carbon is carbon dioxide. In some embodiments, the one or more microorganisms comprise bacteria, archaebacteria, fungi, or viruses. In some embodiments, the one or more microorganisms comprise bacteria. In some embodiments, the bacteria comprise endospore-forming bacteria. In some embodiments, the bacteria comprise rhizobacteria. In some embodiments, the rhizobacteria comprise bacillus, paenibacillus, or both. In some embodiments, the bacteria comprise bacillus amyloliquefaciens, bacillus laterosporus, bacillus licheniformis, bacillus macerans, bacillus cereus, bacillus circulans, bacillus firmus, bacillus subtilis, bacillus sphaericus, bacillus megaterium, bacillus coagulans, bacillus brevis, bacillus thuringiensis, bacillus mycoides, bacillus cucumber, bacillus intraphytans, bacillus pumilus, bacillus bailii, bacillus mucilaginosus, bacillus tervalicalis, bacillus methylotrophicus, or any combination thereof. In some embodiments, the bacteria comprise bacillus subtilis S3C23, bacillus subtilis MP2, bacillus subtilis RO2C15, bacillus subtilis RO2C22, bacillus megaterium 6, bacillus megaterium S3C21, bacillus megaterium RO2C12, bacillus cucumber S3C14, bacillus plantarum 5, or any combination thereof. In some embodiments, the bacteria comprise bacillus subtilis S3C23. In some embodiments, the bacteria comprise bacillus subtilis MP2. In some embodiments, the one or more microorganisms comprise one or more fungi associated with the plant or part thereof. In some embodiments, one or more fungi associated with the plant or portion thereof is disposed on the plant root or rhizosphere of the plant or portion thereof by an irrigation system. In some embodiments, the one or more fungi comprise arbuscular mycorrhizal fungi. In some embodiments, the one or more fungi comprise ectomycorrhizal fungi. In some embodiments, the one or more fungi comprise fungi from the genus trichoderma. In some embodiments, the one or more fungi comprise a fungus from the genus penicillium. In some embodiments, the one or more minerals comprise calcite, aragonite, dolomite, limestone, or any combination thereof. In some embodiments, promoting the production of one or more minerals comprises producing ammonia and the resulting increase in pH in a medium in which the plant or portion thereof is grown. In some embodiments, the one or more microorganisms comprise one or more carbonic anhydrases. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases belonging to class α. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases that belong to the class β. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases belonging to the class γ. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases that belong to class delta. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases belonging to the zeta class. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases that belong to class η. In some embodiments, the one or more carbonic anhydrases comprise carbonic anhydrases belonging to the iota class.
Incorporation by reference
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in this specification, this specification is intended to supersede and/or take precedence over any such contradictory material.
Drawings
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the patent office upon request and payment of the necessary fee.
The novel features of the methods and compositions described herein are set forth with particularity in the appended claims. A better understanding of the features and advantages of the methods and compositions described herein will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the methods and compositions described herein are utilized, and the accompanying drawings of which:
FIG. 1 shows high CO at the plant rhizosphere 2 Concentration represents the opportunity for carbon sequestration mediated by the carbonic anhydrase-producing microorganism.
Fig. 2 depicts a two-stage plant enhancement strategy described herein. 101 illustrates monocot seeds. The seed treatment process described herein incorporates bacteria (or endospores thereof) 106 between the aleurone layer 103 and the pericarp 102. Aleurone layer 103 separates endosperm 104 from the outer layer.
FIG. 3 shows a process depicting seed treatment (Microprime TM Seed treatment process).
FIG. 4 depicts a method forObtaining a stable seed treatment by microbiological techniques (Microprime TM ) Is not limited to the current method of (a).
FIG. 5A shows Microprime TM After seed treatment, bacteria are located in the space inside the corn seeds (Zea mays).
FIG. 5B shows Microprime TM After seed treatment, bacteria are located in an enlarged image of the space inside the corn seed (Zea mays).
FIGS. 6A-6B depict a pre-emergent Microprime, respectively TM The population of bacillus subtilis (strain S3C 23) endospores within corn seeds increases and the logarithm of the population increases. Seeds sown in agar plates containing Murashige and Skoog 50% medium initially had 164,000 Colony Forming Units (CFU) at sowing time. Three days later, the population inside the seeds reached a level of 7,200,000CFU/seed. Each dot represents the mean ± standard error of three pools, each pool having five seeds.
FIGS. 7A-7B depict a pre-emergent Microprime, respectively TM The population of the endospores in bacillus subtilis (strain S3C 23) inside the rice seeds increases and the logarithm of the population increases. Seeds sown in agar plates containing Murashige and Skoog 50% medium initially had 51,167 Colony Forming Units (CFU) at sowing time. Three days later, the population inside the seeds reached a level of 36,666,667CFU/seed. Each dot represents the mean ± standard error of three pools, each pool having five seeds.
FIGS. 8A-8B depict a pre-emergent Microprime, respectively TM The population of the endospores in bacillus subtilis (strain S3C 23) inside the soybean seeds increases and the logarithm of the population increases. Seeds sown in agar plates containing Murashige and Skoog 50% medium initially had 123,333 Colony Forming Units (CFU) at sowing time. Three days later, the population inside the seeds reached a level of 674,666,667CFU/seed. Each dot represents the mean ± standard error of three pools, each pool having five seeds.
FIG. 9 depicts Microprime TM Microprime in a time range of 1 to 18 months after seed treatment TM Viability of bacillus subtilis endospores (strain S3C 23) inside corn seeds. Each time point takes the place of Table means ± standard error of three pools, five seeds per pool. S3C23 Microprime TM The germination rate of the seeds at 18 months was 98.33% identical to the untreated seeds (n=60 seeds per treatment).
FIG. 10 depicts carbonic anhydrase activity of lysate samples prepared from liquid cultures of Bacillus subtilis (strain S3C 23) as measured by enzymatic hydrolysis of 4-nitrophenylacetate to 4-nitrophenol and acetic acid according to the protocol of Zhuang et al, 2018, which is incorporated herein by reference in its entirety. Values are given as Carbonic Anhydrase (CA) activity/total protein (mg/ml). The activity in the sample showed a sharp increase at pH 8.5 compared to pH7.5, consistent with the expected pH dependence of carbonic anhydrase.
FIG. 11 depicts carbonate and bicarbonate ions as calcium carbonate (CaCO) 3 ) Is used for the enzyme-induced precipitation. Lysates or recombinant bovine carbonic anhydrase or bovine serum albumin (BSA, negative control) prepared from Bacillus subtilis (strain S3C 23) were combined with CaCl 2 (final concentration 100 mM) and Tris pH 8 (final concentration 200 mM) and 50% CO 2 Saturated water (added last to start the reaction) was mixed. The time for calcium carbonate formation was visually assessed and recorded. Normalizing values to non-biological CaCO 3 Time of precipitation. Recombinant bovine CA rapidly induced precipitation in a dose-dependent manner. 500ul of S3C23 lysate induced precipitation significantly faster than non-biological precipitation.
Detailed Description
While various embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Many changes, modifications and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.
Land for plant cultivation provides CO 2 Ideal location for sequestration due to CO with the atmosphere 2 Horizontal comparison, CO due to group absorption of plant roots and soil microorganisms 2 The level is significantly elevated.
Plant seeds and plant seed derived sources as described hereinPlants may be modified with one or more microorganisms described herein to enhance bicarbonate production and mineral formation in a given soil plot in which the plants are cultivated. The slow and rate limiting step of bicarbonate formation can be accelerated by using an enzyme called Carbonic Anhydrase (CA). CA is a zinc-containing metalloenzyme that catalyzes CO 2 Reversible hydration to about 10 higher than non-enzymatic processes 7 Bicarbonate and protons. CA is known to catalyze CO 2 The fastest enzyme is reversibly hydrated and typical rates for different types of CA can reach 10-10 6 s -1 . CA is widely distributed in animals, plants, archaebacteria and bacteria. This includes many fungi and bacteria that are normally present in soil, which produce intracellular and extracellular CA. CA versus CO 2 The extremely high selectivity of (3) makes it a CO gas mixed with other polluted gases 2 Perfect candidates for sequestration. There is a lot of convincing evidence that CA has the potential to CO in the atmosphere 2 The sealing is bicarbonate (HCO) 3 - ) Which can precipitate by reaction with various cations to form minerals (20, 21).
In accordance with the present disclosure, HCO may be based on high CA activity or in an amount sufficient to produce the desired amount in a given soil plot 3 - Is selected for one or more microorganisms. These microorganisms may be associated with various plant seeds, plants, and/or rhizosphere as described herein. In some embodiments, microorganisms with low levels of CA expression may be genetically modified to enhance CA expression. In some embodiments, a microorganism that does not express CA may be genetically modified to express CA.
Plants derived from the compositions described herein may be cultivated in a given soil plot to enhance bicarbonate and mineral formation in the soil.
Carbonic Anhydrase (CA) -induced mineralization process from CO 2 Reversible hydration to bicarbonate ions begins (shown in equation 1).
The formation of carbonate ions from bicarbonate via hydrolysis is the second step of mineralization (shown in equation 2). These carbonate and bicarbonate ions increase the pH of the medium. The pH of the medium can be further raised to 9.2 by ammonia production by the microorganism.
The final step of mineralization involves precipitation of carbonate and bicarbonate ions by cations (equation 3).
Ca 2+ +CO 3 2- →CaCO 3 (3)
Because soil naturally presents different types of cations, CA-expressing soil microorganisms present on roots and/or in rhizosphere are well suited for sustainable CO production 2 Converted into minerals. CA can also play a secondary role in increasing supersaturation of some minerals in the liquid medium by releasing large amounts of carbonate and bicarbonate. The biomineralization process can be further accelerated by the gene cluster called lcfA (consisting of five genes lcfA, ysiA, ysiB, etfB and etfA), which plays a role in carbonate biomineralization in bacillus subtilis.
To date, no technology has been available that can ensure 100% delivery of microorganisms to CO 2 Release site (root) to effectively sequester CO 2 . The compositions described herein are combined with Microprime TM Integration in agricultural practice is highly suitable, not only to maintain productivity of the soil, but also to capture CO that may be released to the atmosphere 2 . In addition, the methods and compositions described herein are cost effective in that they cost only $0.20 per acre and use environmentally formulated environmental microorganisms that pose no threat to agricultural land or crops.
The microorganisms described herein will reduce the level of carbon output by capturing various forms of CO 2 To increase land carbon sequestration. The microorganisms described herein employ a variety of mechanisms to convert CO 2 Effectively capturing to a plurality of microbial products. One of the main products is bicarbonate and minerals (solidsA body). Minerals may include calcite, aragonite, dolomite and limestone. In addition to sequestering gaseous CO 2 In addition, one of the microbial products, limestone (CaCO) 3 ) Will benefit agricultural land. The formation of calcite in the soil may avoid the use of limestone conditioning by farmers to maintain soil productivity. Limestone improves the structure of the soil and increases its pH.
Composition comprising plant seeds and bacteria
In certain aspects, disclosed herein is a composition comprising a plant seed and one or more microorganisms associated with the plant seed, wherein the one or more microorganisms are or are derived from a microorganism selected to produce or promote the formation of one or more minerals.
Biological priming and seed treatment methods: microprime TM
A typical seed priming protocol includes the following steps: the seeds are immersed in any solution containing the desired initiator (inorganic and organic salts, nanoparticles, plant growth regulating substances and/or plant growth promoting bacteria) and subsequently the seeds are dried. This results in the initiation of the germination process except for the appearance of radicles, such as heydeker et al, 1973; mahakham et al, 2017; mcDonald,1999; song et al, 2017; wright et al, 2003, which is incorporated herein by reference in its entirety. Seed priming (osmotic priming) using osmotic solutions has been done for decades (heydeker et al, 1973) and is now a common commercial practice in the selection of high value horticultural seeds. The concept also extends to water priming in cereals and legumes, and the "on-farm" priming technique has been restored (Harris et al, 2001). In recent years, several metal and carbon based nanoparticles (e.g., agNPs16, auNPs5, cuNPs17, cuNPs18, znNPs17, znNPs18, fullerenes 22, and carbon 23 nanotubes, etc.) have been used as seed initiators for promoting seed germination, seedling growth, and stress tolerance in some crops (Mahakham, et al, 2017). In different priming techniques (e.g., water priming, osmotic priming, nano priming, etc.), when using microbial cells for this procedure, the interior space within the seed has potentially ideal conditions for bacterial inoculation and colonization (McQuilken et al, 1998; ashraf and Foolad,2005; bennett et al, 2009; tabasum et al, 2018; wright et al, 2003, which is incorporated herein by reference in its entirety).
Biological initiation methods have been widely used for a variety of crops since the beginning of the 90 s and are certainly considered an environmentally friendly agricultural technique (O' Calaghan, 2016; taylor and Harman,1990, which is incorporated herein by reference in its entirety). Sometimes, the bio-priming technique is erroneously defined as the application of the whole microorganism, its secretion or some bioactive compound to the outside of the seed, which is described by El-Mougy and Abdel-Kader,2008; muller and Berg,2008; song et al, 2017; saber et al, 2012, which is incorporated herein by reference in its entirety. More precisely, by promoting the speed and uniformity of emergence and improving plant traits, bio-priming incorporates both organisms (seed inoculated with beneficial microorganisms) and physiological elements (seed hydration) into the seeds. The seed treated with the microorganism is fundamentally different from other biological seed treatments in that cells may be living when seed treated with the microorganism, and thus colonization and proliferation of added microorganisms must occur within the seed. However, most documents from the prior art are not strict in explaining the differences in detail. In particular, no results or studies have been reported regarding: 1) Survival and/or proliferation of biological agents within seeds over a relevant time frame (several months); 2) Shelf life and effective germination of seeds several months after treatment, 3) effective microbial inoculant and plant interaction after the relevant time of storage of the seeds, and 4) an economically viable method (taking into account the time, effort and effort involved in seed treatment) which has the potential to be scalable and thus can be implemented in traditional seed business models. Furthermore, when relevant to bacterial coating procedures, biological penetration initiation only demonstrated significant enhancement of the uniformity of germination and plant growth traits (Bennett et al, 2009; raj et al, 2004; shifti, 2011; shifti et al, 2012, which is incorporated herein by reference in its entirety). Several researchers have reported incubation times of 20 minutes to several days (Bennett et al, 2009; bennett and whisps, 20088 b, 2009 a; murunde and Wainwright,2018, which is incorporated by reference in its entirety The body is incorporated herein). Also, the cell suspension was broadly 10 per gram of seed 5 To 10 9 Within a range of individual cells, and depending on the type of biological agent (i.e., spores, endospores, or vegetative cells) (Wright et al, 2003; saber et al, 2012; raj et al, 2004; murunde and Wainwright, 2018). Indeed, biological priming has been practiced and explained in several ways by different researchers, but is still an ambiguous approach to be explored and discussed (Bennett et al, 2009; callan et al, 1990,1991; chakraborty et al, 2011; mirshekari et al, 2012; moeinzadeh et al, 2010; raj et al, 2004; reddy,2013; shifti, 2011; shifti et al, 2012, which is incorporated herein by reference in its entirety).
According to the prior art, song et al (Song et al 2017) explain the reason for triggering cucumber plant immunity using bacillus secretions. This method has several misleading results both in terms of method and scope because it 1) does not use bacterial inoculants or plant growth promoters derived therefrom, but instead biologically triggers seeds with peptide-based compounds; 2) Early after dormancy of seed germination, no viable microorganisms are incorporated into the seeds to bring them or their secretions into contact with the embryo; 3) It was not demonstrated whether a biologic (e.g., cyclic dipeptide) had entered the seed and triggered PGP effects (changes in gene expression) early in the embryo of the plant (before the pericarp breaks); and 4) the stability of the initiator of the plant immune trigger over time is not informed. This last problem is particularly important because commercially viable agricultural microbiological technology must be stable over a relatively long period of time (e.g., over six months) in order to be compatible with current agricultural distribution systems. In addition, the photoinitiators used in this reference work are particularly unstable over time and are susceptible to alteration by abiotic and biological environmental factors (e.g., temperature, pH, biodegradation activity of other microorganisms, etc.).
Serratia marcescens (Serratia plymuthica) strain HRO-C48 was reported as a biological agent for seed inoculation procedures (Muller and Berg, 2008). This work has attempted to compare three different techniques, such as granulation, film coating and bio-osmotic initiation. Although the cell number of each seed was determined immediately after seed treatment and storage, the authors failed to accurately quantify the shelf life of the product to make it commercially viable in the agricultural industry. In fact, strain HRO-C48 viability was determined only in a very short shelf-life (30 days). Another ambiguous topic reported by the authors relies on a bioengineering optimization procedure because 1) a high initial cell density is adjusted for seed soaking, and 2) a long seed incubation time in the presence of a biologic is used (reported as 12 hours). Of course, all of these aspects are not generally viable parameters for both industrial and commercial implementation of the process (Muller and Berg, 2008).
Some other efforts have been reported in the prior art to indicate the incorporation of synthetic microbial agents within seeds. For example, U.S. patent 2016/0338360A1 and 2016/0330976A1 mention seeds containing beneficial bacteria. The methods proposed in both of these references are based on direct inoculation of different parts of flowers and plants in order to finally obtain seeds containing the desired microorganism (Mitter et al, 2016a,2016b, which is incorporated herein by reference in its entirety).
Disclosed herein is a new, effective and reliable alternative to conventional bio-priming techniques that directly address the foregoing problems. The proposed seed treatment method is named Microprime TM It is a well-designed, calculated, performed and controlled process for obtaining commercial seeds with the desired microorganisms. Specifically, disclosed herein is a stable microbial seed treatment method by which single microorganisms and/or synthetic consortia of microorganisms (confetti) and/or secretions thereof and/or individual biomolecules thereof are incorporated into seeds by an industrial scale process, which takes into account process costs, time and effort, technical stability over time (for plant embryos and inoculants), multi-soil compatibility, stability under different environmental conditions and compatibility with traditional distribution chains of agricultural inputs. The method involves controlled, economical and rapid imbibition of seeds in an aqueous solution of an osmotically active liquid medium in addition to a surfactant that enhances the endo-permeability of the seeds and/or a set of nutrients that enhance the colonization of microorganisms within the seeds and/or supplemental agents for enhancing bacterial endosporulation It is also supplemented with specific amounts of beneficial microorganisms or synthetic consortia of microorganisms and/or secretions thereof and/or personalized biomolecules thereof. Microprime TM The seed technology ensures survival of the biological agent and extended shelf life of the treated seeds.
The compositions and methods disclosed herein present new strategies to achieve plant traits and increase yield. The strategy is based on two effects in plant seeds by implementing the specific seed treatment method explained below (Microprime TM ) To realize:
1. loading functional bacteria into seeds: endospore-forming bacteria and/or endospores are loaded into seeds by performing current seed treatment methods. As shown in fig. 2 and 5A and 5B, as Microprime TM As a result of the seed treatment, endospores forming bacteria and/or endospores are distributed into the interstices between the seed pericarp and its aleurone cell layer of the seed. The endospore-forming bacteria and/or endospores incorporated into the seed correspond to having the ability to effectively colonize the plant rhizosphere and have the ability to be produced by the CO 2 Conversion to bicarbonate and ultimately to carbonate minerals to sequester CO 2 Is a strain capable of being used for the strain. The endospore transformation capacity of the selected bacteria and their distribution within the seeds ensure Microprime TM Stability after seed treatment and throughout commercial storage. This process is demonstrated by counting bacterial cells in time.
In order for the methods and compositions disclosed herein to be valuable and truly suitable for industrial scale, the methods must be cost-effective and scalable. In the seed treatment process (as disclosed herein), there are several steps that require time, effort and effort. The methods and compositions disclosed herein have a first priority that minimizes seed treatment processing costs and time. Microprime when performed at room temperature (between 20 ℃ and 24 ℃) TM The method aims to make the seed treatment process effective while seed imbibition is performed in less than 20 minutes to 16 hours. The latter implementation is important, since it is not only in Microprime TM The seed treated seed has a minimum required number of bacteria within the seedBesides, endospore-forming bacteria and/or endospores, the bacteria must remain stable and viable over time, so that the seeds (as product) can be kept unaffected by storage, packaging, logistics and sowing processes, e.g. without Microprime TM Conventional seed treatment.
The proposed method involves the aspiration of previously sterilized seeds into a seed treatment medium (hereinafter Microprime TM Solution). Fig. 3 shows a flow chart depicting a seed treatment process.
Stability of bacterial seed treatment
The stability of bacteria within seeds over time is neither simple nor a significant problem to be solved. When using the seed treatment methods disclosed herein (Microprime TM Seed treatment) the bacteria are incorporated into the seed, the location of the bacteria within the seed is shown in fig. 5A and 5B for corn seed.
In FIG. 5A, it can be appreciated that in Microprime TM After seed treatment, bacteria labeled with Red Fluorescent Protein (RFP) remained in the space (pink filaments) inside the corn seed. This location is the gap between the seed pericarp and seed aleurone cell layer, which separates the endosperm and embryo from the outer layer. This is where some microorganisms may be comfortable for a limited period of time, after which the cells will necessarily die due to the exhaustion of the available nutrients, or in the case of some specific microorganisms, initiate the endospores formation process (from the bacteria of the phylum firmicutes, the phylum Proteus and the phylum Actinomyces). The benefit of being placed in the above-described location is to protect the microorganism from external factors that may affect its direct integrity, such as other microorganisms or dehydration.
To solve the problem of viability over a longer period of time due to the lack of nutrients, the method will depend on the type of bacteria that is handling it. The bacteria present have the ability to stop multiplying and enter a physical state called endospores under certain conditions (mainly in the case where they sense a risk of feasibility). As endospores, the bacteria go into a dormant state where it may be during a longer period of timeThe space lacks nutrients. For bacteria mainly from the phylum Thick-walled, proteobacteria and actinomycetes, which have the ability to produce endospores, microprime TM The solution is supplemented with salts that, once incorporated into the seed, push the bacteria into this inactive state. By doing the latter, the viability of the bacteria within the seed over time is ensured. When the endospore is again in favorable moisture and nutrient conditions (e.g., when the seed is sown), it reverts to an active bacterial state (vegetative cells) and begins normal function and vegetative propagation.
Another strategy is to supplement Microprime directly with endospores TM The solution, rather than pushing bacterial transformation while the seed treatment process is in progress. In Microprime TM The latter showed better yields in terms of endospores per seed that could be found after seed treatment.
Table 1 below shows some bacterial genera that are of particular interest for the proposed novel seed treatment due to their ability to convert to endospores:
TABLE 1
Bacteria of the genus bacillus are one of the most abundant bacteria with endospore formation ability. In order to sufficiently proliferate bacteria inside seeds, it is necessary to supplement microprimes with nutrients having specific compatibility with selected bacteria TM Solution, or alternatively, microprime of the desired bacteria is directly added TM The solution endospores are incorporated into the seeds.
Biological initiation of embryos
The methods of the present disclosure contemplate treating plant seeds with a bacterial composition designed as previously explained (Microprime TM Seed treatment). Such treatments may employ osmotic penetration of the seed to allow bacterial incorporation, the treatment initially described by Smith et al to introduce chemical initiators into the seed is currently referred to as osmotic priming. However, the methods of the present disclosure are suitable for treating bacterial populationsThe body is incorporated into dormant seeds, intended to induce the first stage of germination by means of the immediate biological initiation of the embryo, or early conditions that promote the formation of new plantlets, once dormancy is completed, appropriate environmental or agronomic conditions. This novel approach offers unprecedented advantages over previously disclosed bacterial formulations designed to produce or promote bioelected formation, because the incorporated bacteria are protected within dormant seeds and conveniently located to produce or promote permanent elicitation of embryo formation from the earliest possible developmental stage by itself or by the action of its secretions. In addition, the treated seeds are readily subjected to conventional transportation, storage, coating granulation and sowing treatments in accordance with standard agronomic practices without any additional requirements regarding handling, nutritional additives, preservatives or irrigation, and without the incompatibility limitations associated with pests or plant disease control agents. Thus, the method described herein (Microprime TM Seed treatment) also provides significant advantages over seed bio-initiation (mahood et al 2016) because that approach involves pre-germination and dormancy termination of the seeds, which reduces storage survival and limits handling and processing feasibility.
Furthermore, the methods of the present disclosure differ from previously reported methods of seeding seeds using the parent plant as a reactor for microbial growth or by seeding the plant sexual organ (Mitter et al, 2016a; mitter et al, 2016 b). This approach implies an inherent bias in the type of bacteria that can ultimately be incorporated into the seed, as a successful inoculum must be able to survive in the plant target organ or tissue to compete with the endogenous microorganisms and enter the seed interior space by itself. Microprime presented herein TM Strategies are not hampered by the ability of plants to grow or tissue survival, as artificially incorporated bacteria are not necessarily endosymbionts, they need not face the defensive response of mature plants nor need to overcome plant endophyte microbiota, but need only survive long enough or their secretions can reach plant embryos to produce or promote molecular initiated formation of plants.
The unique advantages and distinguishing features described above make the methods of the present disclosure not apparent to those skilled in the art related to bacterial plant growth stimulation. In fact, in order for this strategy to be successful, the candidate bacteria for the treatment composition must meet certain critical conditions that are not necessarily considered in the standard formulation of plant growth promoting microorganisms. First, bacterial compositions must be designed using the above-described criteria of effectiveness and compatibility to avoid competition and/or antagonism within the seed. The lack of these effects must be assessed experimentally before the composition is formulated. The seed internalization of each bacterium contained in the designed composition must be evaluated to determine the saturation curve, as well as the survival of the bacterium during storage time and seed treatment procedure. In addition, the internal seed tissue must be analyzed to assess the presence and viability of the desired bacteria and the relative abundance of each component strain relative to the other component strains (FIG. 4).
Plant material must also be conditioned prior to treatment with the particular bacterial composition. Seeds can be sterilized to eliminate any background noise while Microprime is determined TM Effectiveness of seed treatment.
Once Microprime occurs TM Transcriptional analysis of marker genes associated with defense, abiotic stress tolerance and development in pathogens must be determined to confirm the effect of beneficial bacteria on the treated seed. This analysis must be performed after the seed dormancy stage, before the seed pericarp and endosperm break and radicle appear. Assessment of transcriptional changes in developing embryos due to previous bacterial treatment of dormant seeds is also a key step in the validation of the method, as it provides a quick confirmation of the effectiveness of the initiation and the results are not yet affected by external factors that occur after seed rupture, including other microorganisms that enter the developing plant tissue from outside the seeds and/or the chemical composition of the surrounding soil or growth matrix.
The methods and compositions disclosed herein can be summarized as Microprime TM Seed treatment process wherein seeds are incorporated into an aqueous salt solution containing a seed-compatible bacterial composition, a bacterial-compatible nutrient (in the case of non-endospores forming bacteria), a seed soak at room temperature and for a short period of time A surfactant to increase bacterial cell loading into the seed, and a supplemental mineral to increase the conversion of the endospores forming bacteria into endospores.
Fig. 3 depicts the current method for achieving stable microbiological seed treatment.
Modified plant seeds
In one aspect, provided herein is a modified plant seed comprising a microorganism or microorganism secretion incorporated into the seed. In some embodiments, the microorganisms or secretions sequester the CO2 by converting the CO2 to bicarbonate and ultimately to carbonate minerals. In some embodiments, CO2 is sequestered by forming bicarbonate. In some embodiments, CO2 is sequestered by forming one or more carbonate minerals. In some preferred embodiments, the microorganism is an endospore-forming bacterium or an endospore thereof.
In some embodiments, the microorganism or secretion is incorporated into the interior of the seed. In some embodiments, the microorganism or secretion is incorporated into the seed under the pericarp. In some embodiments, the microorganism or secretion is incorporated into the seed between the pericarp and aleurone cell layer. In some embodiments, the microorganism or secretion contacts the embryo of the seed. In some embodiments, the microorganism or secretion does not contact the embryo of the seed. In some embodiments, the microorganism or secretion contacts the endosperm of the seed. In some embodiments, the microorganism or secretion does not contact the endosperm of the seed. In some embodiments, the microorganism or secretion is incorporated into the interstice between the seed coat and the embryo of the seed. In some embodiments, the microorganism or secretion is incorporated into the gap between the seed pericarp and the seed aleurone cell layer.
The modified plant seed may be any type of plant seed. In some embodiments, the modified seed is a monocot seed. In some embodiments, the plant seed is a maize, wheat, rice, barley, rye, sugarcane, millet, oat, or sorghum seed. In some embodiments, the plant seed is a maize seed. In some embodiments, the plant seed is a maize (Zea mail) seed. In some embodiments, the seed is a soybean seed. In some embodiments, the seed is soybean (Glycine max) seed. In some embodiments, the seed is a rice seed. In some embodiments, the seed is a rice (Oryza sativa) seed. In some embodiments, the modified seed is a dicot seed. In some embodiments, the seed is a soybean, cotton, alfalfa, bean, quinoa, lentil, peanut, sunflower, canola, cassava, oil palm, potato, sugar beet, cocoa, coffee, lettuce, tomato, or cabbage seed. In some embodiments, the seed is a Genetically Modified Organism (GMO) seed. In some embodiments, the seed is a non-GMO seed.
The amount of microorganisms or secretions incorporated into the seeds must reach a sufficient level in order to effectively sequester the CO2 in the soil. In some embodiments, the amount of microorganism incorporated into the seed is from about 250 Colony Forming Units (CFU) to about 5,000CFU. In some embodiments, the amount of microorganism incorporated into the seed is from about 250CFU to about 500CFU, from about 250CFU to about 750CFU, from about 250CFU to about 1,000CFU, from about 250CFU to about 2,000CFU, from about 250CFU to about 3,000CFU, from about 250CFU to about 4,000CFU, from about 250CFU to about 5,000CFU, from about 500CFU to about 750CFU, from about 500CFU to about 1,000CFU, from about 500CFU to about 2,000CFU, from about 500CFU to about 3,000CFU, from about 500CFU to about 4,000CFU, from about 500CFU to about 5,000CFU, from about 750CFU to about 1,000CFU, from about 750CFU to about 2,000CFU, from about 750CFU to about 3,000CFU, from about 750CFU to about 4,000CFU, from about 750CFU to about 5,000CFU, from about 1,000CFU to about 2,000CFU, from about 1,000CFU to about 3,000CFU, from about 1,000CFU to about 4,000CFU, from about 1,000CFU to about 5,000CFU, from about 37 to about 37, or from about 37 to about 5,000CFU. In some embodiments, the amount of microorganism incorporated into the seed is about 250CFU, about 500CFU, about 750CFU, about 1,000CFU, about 2,000CFU, about 3,000CFU, about 4,000CFU, or about 5,000CFU. In some embodiments, the amount of microorganism incorporated into the seed is at least about 250CFU, about 500CFU, about 750CFU, about 1,000CFU, about 2,000CFU, about 3,000CFU, or about 4,000CFU. In some embodiments, the amount of microorganism incorporated into the seed is up to about 500CFU, about 750CFU, about 1,000CFU, about 2,000CFU, about 3,000CFU, about 4,000CFU, or about 5,000CFU. In some embodiments, at least about 500CFU is incorporated into the seed. In some embodiments, at least about 1000CFU is incorporated into the seed.
In some embodiments, the microorganism or secretion incorporated into the seed is shelf stable for a prolonged period of time. In some embodiments, the modified seed is shelf stable for about 3 months to about 36 months. In some embodiments of the present invention, in some embodiments, the modified seed is in the range of about 3 months to about 6 months, about 3 months to about 9 months, about 3 months to about 12 months, about 3 months to about 15 months, about 3 months to about 18 months, about 3 months to about 21 months, about 3 months to about 24 months, about 3 months to about 30 months, about 3 months to about 36 months, about 6 months to about 9 months, about 6 months to about 12 months, about 6 months to about 15 months, about 6 months to about 18 months, about 6 months to about 21 months, about 6 months to about 24 months, about 6 months to about 30 months, about 6 months to about 36 months, about 9 months to about 12 months, about 9 months to about 15 months, about 9 months to about 18 months, about 9 months to about 21 months, about 9 months to about 24 months, about 9 months to about 30 months the storage is stable for from about 9 months to about 36 months, from about 12 months to about 15 months, from about 12 months to about 18 months, from about 12 months to about 21 months, from about 12 months to about 24 months, from about 12 months to about 30 months, from about 12 months to about 36 months, from about 15 months to about 18 months, from about 15 months to about 21 months, from about 15 months to about 24 months, from about 15 months to about 30 months, from about 15 months to about 36 months, from about 18 months to about 21 months, from about 18 months to about 24 months, from about 18 months to about 30 months, from about 18 months to about 36 months, from about 21 months to about 24 months, from about 21 months to about 30 months, from about 21 months to about 36 months, from about 24 months to about 30 months, from about 24 months to about 36 months, or from about 30 months to about 30 months. In some embodiments, the modified seed is shelf stable for about 3 months, about 6 months, about 9 months, about 12 months, about 15 months, about 18 months, about 21 months, about 24 months, about 30 months, or about 36 months. In some embodiments, the modified seed is shelf stable for at least about 3 months, about 6 months, about 9 months, about 12 months, about 15 months, about 18 months, about 21 months, about 24 months, or about 30 months. In some embodiments, the modified seed is shelf stable for up to about 6 months, about 9 months, about 12 months, about 15 months, about 18 months, about 21 months, about 24 months, about 30 months, or about 36 months.
In some embodiments, the microorganism incorporated into the seed is stable after incorporation. In some embodiments, the microorganism is stable for more than 30 days, more than six months, more than one year, or more than two years. In some embodiments, the microorganism is stable for more than 30 days. In some embodiments, the microorganism is stable for more than six months. In some embodiments, the microorganism is stable for more than one year. In some embodiments, the microorganism is stable for more than two years.
The microorganism or secretion thereof incorporated into the plant seed may be any one of the microorganisms provided herein or any other microorganism. In some embodiments, the microorganism is a microorganism (microbe). In some embodiments, the microorganism is an endospore-forming microorganism. In some embodiments, the microorganism is an endospore-forming microorganism or an endospore thereof. In some embodiments, the microorganism is an endospore of a microorganism provided herein. In some embodiments, the microorganism is an endospore-forming bacterium or an endospore thereof.
Methods of incorporating bacteria
In one aspect, provided herein are methods of incorporating one or more microorganisms or secretions thereof into one or more plant seeds. In some embodiments, the method comprises sterilizing plant seeds. In some embodiments, the method comprises contacting the seed with a solution comprising one or more microorganisms or secretions thereof. In some embodiments, the solution further comprises a salt. In some embodiments, the method comprises incubating the seed with the solution for a period of time. In some embodiments, the period of time is sufficient to allow a desired amount of microorganisms or secretions thereof to enter the plant seeds. In some embodiments, the method incorporates a desired amount of a microorganism or secretion thereof into the seed.
In some embodiments, the method comprises contacting the seed with a solution comprising a salt. Any salt may be used. In some preferred embodiments, the salt is NaCl. In some embodiments, the salt is NaCl, liCl, KCl, mgCl 2 、CaCl 2 、NaBr、LiBr、KBr、MgBr 2 、CaBr 2 、NaI、LiI、KI、MgI 2 Or CaI 2 . In some embodiments, the salt comprises sodium, lithium, or potassium ions. In some embodiments, the salt comprises an alkali metal ion. In some embodiments, the salt comprises an alkaline earth metal ion. In some embodiments, the salt comprises a halide ion. In some embodiments, the salt is an alkali or alkaline earth metal halide salt. In some embodiments, the salt comprises chloride, bromide, or iodide. In some embodiments, the salt is a sulfate, phosphate, carbonate, or nitrate.
The salt may be present in the solution at any suitable concentration. In some embodiments, the solution comprises about 0.85% salt (w/v). In some embodiments, the solution comprises from about 0.1% to about 1.25% salt (w/v). In some embodiments, the solution comprises from about 0.1% to about 2.0% salt (w/v). In some embodiments of the present invention, in some embodiments, the solution comprises about 0.1% to about 0.25%, about 0.1% to about 0.5%, about 0.1% to about 0.6%, about 0.1% to about 0.7%, about 0.1% to about 0.75%, about 0.1% to about 0.8%, about 0.1% to about 0.85%, about 0.1% to about 0.9%, about 0.1% to about 0.95%, about 0.1% to about 1%, about 0.1% to about 1.25%, about 0.25% to about 0.5%, about 0.25% to about 0.6%, about 0.25% to about 0.7%, about 0.25% to about 0.75% >; about 0.25% to about 0.8%, about 0.25% to about 0.85%, about 0.25% to about 0.9%, about 0.25% to about 0.95%, about 0.25% to about 1%, about 0.25% to about 1.25%, about 0.5% to about 0.6%, about 0.5% to about 0.7%, about 0.5% to about 0.75%, about 0.5% to about 0.8%, about 0.5% to about 0.85%, about 0.5% to about 0.9%, about 0.5% to about 0.95%, about 0.5% to about 1%, about 0.5% to about 1.25%, about 0.6% to about 0.7% >. About 0.25% to about 0.8%, about 0.25% to about 0.85%, about 0.25% to about 0.9%, about 0.25% to about 0.95%, about 0.25% to about 1%, about 0.25% to about 1.25%, about 0.5% to about 0.6%, about 0.5% to about 0.7%, about about 0.5% to about 0.75%, about 0.5% to about 0.8%, about 0.5% to about 0.85%, about 0.5% to about 0.9%, about 0.5% to about 0.95%, about 0.5% to about 1%, about 0.5% to about 1.25%, about 0.6% to about 0.7%, about, about 0.95% to about 1%, about 0.95% to about 1.25%, or about 1% to about 1.25% salt (w/v). In some embodiments, the solution comprises about 0.1%, about 0.25%, about 0.5%, about 0.6%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, about 1% or about 1.25% salt (w/v). In some embodiments, the solution comprises at least about 0.1%, about 0.25%, about 0.5%, about 0.6%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, or about 1% salt (w/v). In some embodiments, the solution comprises up to about 0.25%, about 0.5%, about 0.6%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, about 1% or about 1.25% salt (w/v). In some embodiments, the solution comprises about 0.85% salt (w/v). In some embodiments, the solution comprises from about 0.8% to about 0.9% salt (w/v). In some embodiments, the solution comprises from about 0.75% to about 0.95% salt (w/v). In some embodiments, the solution comprises from about 0.7% to about 1% salt (w/v). In some embodiments, the solution comprises from about 0.5% to about 1.25% salt (w/v). In some embodiments, the solution comprises from about 0.5% to about 2% salt (w/v). In some embodiments, the solution comprises 0.1% -0.2%, 0.2% -0.3%, 0.3% -0.4%, 0.4% -0.5%, 0.5% -0.6%, 0.6% -0.7%, 0.7% -0.8%, 0.8% -0.9%, 0.9% -1.0%, 1.0% -1.1%, 1.1% -1.2%, 1.2% -1.3%, 1.3% -1.4%, or 1.4% -1.5% salt (w/v).
In some embodiments, the solution comprises additional additives. In some embodiments, the solution comprises Dimethylsulfoxide (DMSO), 1-dodecylazepan-2-one, laurocapramKetones, 1-methyl-2-pyrrolidone (NMP), oleic acid, ethanol, methanol, polyethylene glycols (Brij 35, 58, 98), polyethylene glycol monolaurates (e.g., tween 20), tween 40 (polyoxyethylene sorbitol esters), tween 60, tween 80 (non-ionic), cetyl methyl ammonium bromide (CTAB), urea, lecithin (solidified fatty acids derived from soy), chitosan, poloxamer 188, poloxamer 237, poloxamer 338, poloxamer 407, or combinations thereof. In some embodiments, the solution comprises polyethylene glycol monolaurate (e.g., tween 20), poloxamer 188, poloxamer 237, poloxamer 338, poloxamer 407, or a combination thereof. In some embodiments, the solution comprises a poloxamer. In some embodiments, the solution comprises polyethylene glycol monolaurate (e.g., tween 20). The additional additives may be present in any concentration. In some embodiments, the additional additive comprises up to about 0.01%, 0.05%, 0.1%, 0.125%, 0.15%, 0.2%, 0.5%, or 1% (v/v) of the solution. In some embodiments, the additional additive comprises about 0.01% to about 1% (v/v) of the solution. In some embodiments, the additional additive comprises about 0.1% (v/v) of the solution.
In some embodiments, the solution comprises additional metal ions. In some embodiments, the solution comprises magnesium, calcium, manganese, or any combination thereof. In some embodiments, the solution comprises magnesium. In some embodiments, the solution comprises calcium. In some embodiments, the solution comprises manganese. In some embodiments, the solution comprises magnesium and calcium. In some embodiments, the solution comprises magnesium and manganese. In some embodiments, the solution comprises calcium and manganese. In some embodiments, the solution comprises magnesium, calcium, and manganese.
In some embodiments, the solution comprises one or more nutrients for the microorganism. In some embodiments, the solution comprises a bacterial growth medium. In some embodiments, the solution comprises a Lysogenic Broth (LB), a nutrient broth, or a combination thereof. In some embodiments, the solution comprises a lysogenic broth. In some embodiments, the solution comprises a nutrient broth.
In some embodiments, the solution comprises a microorganism. In some embodiments, the solution comprises about 10 3 To about 10 17 Colony Forming Units (CFU)/mL. In some embodiments, the solution comprises about 10 3 To about 10 4 About 10 3 To about 10 5 About 10 3 To about 10 6 About 10 3 To about 10 7 About 10 3 To about 10 8 About 10 3 To about 10 9 About 10 3 To about 10 10 About 10 3 To about 10 12 About 10 3 To about 10 15 About 10 3 To about 10 17 About 10 4 To about 10 5 About 10 4 To about 10 6 About 10 4 To about 10 7 About 10 4 To about 10 8 About 10 4 To about 10 9 About 10 4 To about 10 10 About 10 4 To about 10 12 About 10 4 To about 10 15 About 10 4 To about 10 17 About 10 5 To about 10 6 About 10 5 To about 10 7 About 10 5 To about 10 8 About 10 5 To about 10 9 About 10 5 To about 10 10 About 10 5 To about 10 12 About 10 5 To about 10 15 About 10 5 To about 10 17 About 10 6 To about 10 7 About 10 6 To about 10 8 About 10 6 To about 10 9 About 10 6 To about 10 10 About 10 6 To about 10 12 About 10 6 To about 10 15 About 10 6 To about 10 17 About 10 7 To about 10 8 About (about)10 7 To about 10 9 About 10 7 To about 10 10 About 10 7 To about 10 12 About 10 7 To about 10 15 About 10 7 To about 10 17 About 10 8 To about 10 9 About 10 8 To about 10 10 About 10 8 To about 10 12 About 10 8 To about 10 15 About 10 8 To about 10 17 About 10 9 To about 10 10 About 10 9 To about 10 12 About 10 9 To about 10 15 About 10 9 To about 10 17 About 10 10 To about 10 12 About 10 10 To about 10 15 About 10 10 To about 10 17 About 10 12 To about 10 15 About 10 12 To about 10 17 Or about 10 15 To about 10 17 CFU/mL of microorganism. In some embodiments, the solution comprises about 10 3 About 10 4 About 10 5 About 10 6 About 10 7 About 10 8 About 10 9 About 10 10 About 10 12 About 10 15 Or about 10 17 CFU/mL of microorganism. In some embodiments, the solution comprises at least about 10 3 About 10 4 About 10 5 About 10 6 About 10 7 About 10 8 About 10 9 About 10 10 About 10 12 Or about 10 15 CFU/mL of microorganism. In some embodiments, the solution comprises up to about 10 4 About 10 5 About 10 6 About 10 7 About 10 8 About 10 9 About 10 10 About 10 12 About 10 15 Or about 10 17 CFU/mL of microorganism. In some embodiments, the solution comprises at least about 10 6 To 10 7 CFU/mL of microorganism. In some embodiments, the solution comprises 1×10 3 Up to 1X 10 4 CFU/mL；1×10 4 Up to 1X 10 5 CFU/mL；1×10 5 Up to 1X 10 6 CFU/mL；1×10 6 Up to 1X 10 7 CFU/mL；1×10 7 Up to 1X 10 8 CFU/mL；1×10 8 Up to 1X 10 9 CFU/mL；1×10 9 Up to 1X 10 10 CFU/mL；1×10 10 Up to 1X 10 11 CFU/mL；1×10 11 Up to 1X 10 12 CFU/mL；1×10 12 Up to 1X 10 13 CFU/mL；1×10 13 Up to 1X 10 14 CFU/mL；1×10 14 Up to 1X 10 15 CFU/mL；1×10 15 Up to 1X 10 16 CFU/mL; or 1X 10 16 Up to 1X 10 17 CFU/mL of microorganism.
In some embodiments, the solution contains a desired amount of microorganisms per seed mass. In some embodiments, the solution comprises about 10 3 To about 10 17 Colony Forming Units (CFU)/gram seed. In some embodiments, the solution comprises about 10 3 To about 10 4 About 10 3 To about 10 5 About 10 3 To about 10 6 About 10 3 To about 10 7 About 10 3 To about 10 8 About 10 3 To about 10 9 About 10 3 To about 10 10 About 10 3 To about 10 12 About 10 3 To about 10 15 About 10 3 To about 10 17 About 10 4 To about 10 5 About 10 4 To about 10 6 About 10 4 To about 10 7 About 10 4 To about 10 8 About 10 4 To about 10 9 About 10 4 To about 10 10 About 10 4 To about 10 12 About 10 4 To about 10 15 About 10 4 To about 10 17 About 10 5 To about 10 6 About 10 5 To about 10 7 About 10 5 To about 10 8 About 10 5 To about 10 9 About 10 5 To about 10 10 About 10 5 To about 10 12 About 10 5 To about 10 15 About 10 5 To about 10 17 About 10 6 To about 10 7 About 10 6 To about 10 8 About 10 6 To about 10 9 About 10 6 To about 10 10 About 10 6 To about 10 12 About 10 6 To about 10 15 About 10 6 To about 10 17 About 10 7 To about 10 8 About 10 7 To about 10 9 About 10 7 To about 10 10 About 10 7 To about 10 12 About 10 7 To about 10 15 About 10 7 To about 10 17 About 10 8 To about 10 9 About 10 8 To about 10 10 About 10 8 To about 10 12 About 10 8 To about 10 15 About 10 8 To about 10 17 About 10 9 To about 10 10 About 10 9 To about 10 12 About 10 9 To about 10 15 About 10 9 To about 10 17 About 10 10 To about 10 12 About 10 10 To about 10 15 About 10 10 To about 10 17 About 10 12 To about 10 15 About 10 12 To about 10 17 Or about 10 15 To about 10 17 CFU/gram seed. In some embodiments, the solution comprises about 10 3 About 10 4 About 10 5 About 10 6 About 10 7 About 10 8 About 10 9 About 10 10 About 10 12 About 10 15 Or about 10 17 CFU/gram seed. In some embodiments, the solution comprises at least about 10 3 About 10 4 About 10 5 About 10 6 About 10 7 About 10 8 About 10 9 About 10 10 About 10 12 Or about 10 15 CFU/gram seed. In some embodiments, the solution comprises up to about 10 4 About 10 5 About 10 6 About 10 7 About 10 8 About 10 9 About 10 10 About 10 12 About 10 15 Or about 10 17 CFU/gram seed. In some embodiments, the solution comprises less than 10 10 CFU/gram seed. In some embodiments, the solution comprises less than 10 9 CFU/gram seed. In some embodiments, the solution comprises less than 10 8 CFU/gram seed. In some embodiments, the solution comprises less than 10 11 CFU/gram seed. In some embodiments, the solution comprises about 10 5 To about 10 9 CFU/gram seed.
In some embodiments, the solution comprises about 10 3 To about 10 17 Individual colony cells/gram seed. In some embodiments, the solution comprises about 10 3 To about 10 4 About 10 3 To about 10 5 About 10 3 To about 10 6 About 10 3 To about 10 7 About 10 3 To about 10 8 About 10 3 To about 10 9 About 10 3 To about 10 10 About 10 3 To about 10 12 About 10 3 To about 10 15 About 10 3 To about 10 17 About 10 4 To about 10 5 About 10 4 To about 10 6 About 10 4 To about 10 7 About 10 4 To about 10 8 About 10 4 To about 10 9 About 10 4 To about 10 10 About 10 4 To about 10 12 About 10 4 To about 10 15 About 10 4 To about 10 17 About 10 5 To about 10 6 About 10 5 To about 10 7 About 10 5 To about 10 8 About 10 5 To about 10 9 About 10 5 To about 10 10 About 10 5 To about 10 12 About 10 5 To about 10 15 About 10 5 To about 10 17 About 10 6 To about 10 7 About 10 6 To about 10 8 About 10 6 To about 10 9 About 10 6 To about 10 10 About 10 6 To about 10 12 About 10 6 To about 10 15 About 10 6 To about 10 17 About 10 7 To about 10 8 About 10 7 To about 10 9 About 10 7 To about 10 10 About 10 7 To about 10 12 About 10 7 To about 10 15 About 10 7 To about 10 17 About 10 8 To about 10 9 About 10 8 To about 10 10 About 10 8 To about 10 12 About 10 8 To about 10 15 About 10 8 To about 10 17 About 10 9 To about 10 10 About 10 9 To about 10 12 About 10 9 To about 10 15 About 10 9 To about 10 17 About 10 10 To about 10 12 About 10 10 To about 10 15 About 10 10 To about 10 17 About 10 12 To about 10 15 About 10 12 To about 10 17 Or about 10 15 To about 10 17 Individual cells/gram seed. In some embodiments, the solution comprises about 10 3 About 10 4 About 10 5 About 10 6 About 10 7 About 10 8 About 10 9 About 10 10 About 10 12 About 10 15 Or about 10 17 Individual cells/gram seed. In some embodiments, the solution comprises at least about 10 3 About 10 4 About 10 5 About 10 6 About 10 7 About 10 8 About 10 9 About 10 10 About 10 12 Or about 10 15 Individual cells/gram seed. In some embodiments, the solution comprises up to about 10 4 About 10 5 About 10 6 About 10 7 About 10 8 About 10 9 About 10 10 About 10 12 About 10 15 Or about 10 17 Individual cells/gram seed. In some embodiments, the solution comprises less than 10 10 Individual cells/gram seed. In some embodiments, the solution comprises less than 10 9 Individual cells/gram seed. In some embodiments, the solution comprises less than 10 8 Individual cells/gram seed. In some embodiments, the solution comprises less than 10 11 Individual cells/gram seed. In some embodiments, the solution comprises about 10 5 To about 10 9 Individual cells/gram seed.
In some embodiments, the seed comprises the desired amount of microorganism per seed. In some embodiments, the solution comprises about 10 3 To about 10 17 Colony Forming Units (CFU)/seed. In some embodiments, the solution comprises about 10 3 To about 10 4 About 10 3 To about 10 5 About 10 3 To about 10 6 About 10 3 To about 10 7 About 10 3 To about 10 8 About 10 3 To about 10 9 About 10 3 To about 10 10 About 10 3 To about 10 12 About 10 3 To about 10 15 About 10 3 To about 10 17 About 10 4 To about 10 5 About 10 4 To about 10 6 About 10 4 To about 10 7 About 10 4 To about 10 8 About 10 4 To about 10 9 About 10 4 To about 10 10 About 10 4 To about 10 12 About 10 4 To about 10 15 About 10 4 To about 10 17 About 10 5 To about 10 6 About 10 5 To about 10 7 About 10 5 To about 10 8 About 10 5 To about 10 9 About 10 5 To about 10 10 About 10 5 To about 10 12 About 10 5 To about 10 15 About 10 5 To about 10 17 About 10 6 To about 10 7 About 10 6 To about 10 8 About 10 6 To about 10 9 About 10 6 To about 10 10 About 10 6 To about 10 12 About 10 6 To about 10 15 About 10 6 To about 10 17 About 10 7 To about 10 8 About 10 7 To about 10 9 About 10 7 To about 10 10 About 10 7 To about 10 12 About 10 7 To about 10 15 About 10 7 To about 10 17 About 10 8 To about 10 9 About 10 8 To about 10 10 About 10 8 To about 10 12 About 10 8 To about 10 15 About 10 8 To about 10 17 About 10 9 To about 10 10 About 10 9 To about 10 12 About 10 9 To about 10 15 About 10 9 To about 10 17 About 10 10 To about 10 12 About 10 10 To about 10 15 About 10 10 To about 10 17 About 10 12 To about 10 15 About 10 12 To about 10 17 Or about 10 15 To about 10 17 CFU/seed. In some embodiments, the solution comprises about 10 3 About 10 4 About 10 5 About 10 6 About 10 7 About 10 8 About 10 9 About 10 10 About 10 12 About 10 15 Or about 10 17 CFU/seed. In some embodiments, the solution comprises at least about 10 3 About 10 4 About 10 5 About 10 6 About 10 7 About 10 8 About 10 9 About 10 10 About 10 12 Or about 10 15 CFU/seed. In some embodiments, the solution comprises up to about 10 4 About 10 5 About 10 6 About 10 7 About 10 8 About 10 9 About 10 10 About 10 12 About 10 15 Or about 10 17 CFU/seed. In some embodiments, the solution comprises less than 10 10 CFU/seed. In some embodiments, the solution comprises less than 10 9 CFU/seed. In some embodiments, the solution comprises less than 10 8 CFU/seed. In some embodiments, the solution comprises less than 10 11 CFU/seed. In some embodiments, the solution comprises about 10 5 To about 10 9 CFU/seed.
In some embodiments, the seed comprises the desired amount of microorganism per seed. In some embodiments, the solution comprises about 10 3 To about 10 17 Individual cells/seeds. In some embodiments, the solution comprises about 10 3 To about 10 4 About 10 3 To about 10 5 About 10 3 To about 10 6 About 10 3 To about 10 7 About 10 3 To about 10 8 About 10 3 To about 10 9 About 10 3 To about 10 10 About 10 3 To about 10 12 About 10 3 To about 10 15 About 10 3 To about 10 17 About 10 4 To about 10 5 About 10 4 To about 10 6 About 10 4 To about 10 7 About 10 4 To about 10 8 About 10 4 To about 10 9 About 10 4 To about 10 10 About 10 4 To about 10 12 About 10 4 To about 10 15 About 10 4 To about 10 17 About 10 5 To about 10 6 About 10 5 To about 10 7 About 10 5 To about 10 8 About 10 5 To about 10 9 About 10 5 To about 10 10 About 10 5 To about 10 12 About 10 5 To about 10 15 About 10 5 To about 10 17 About 10 6 To about 10 7 About 10 6 To about 10 8 About 10 6 To about 10 9 About 10 6 To about 10 10 About 10 6 To about 10 12 About 10 6 To about 10 15 About 10 6 To about 10 17 About 10 7 To about 10 8 About 10 7 To about 10 9 About 10 7 To about 10 10 About 10 7 To about 10 12 About 10 7 To about 10 15 About 10 7 To about 10 17 About 10 8 To about 10 9 About 10 8 To about 10 10 About 10 8 To about 10 12 About 10 8 To about 10 15 About 10 8 To about 10 17 About 10 9 To about 10 10 About 10 9 To about 10 12 About 10 9 To about 10 15 About 10 9 To about 10 17 About 10 10 To about 10 12 About 10 10 To about 10 15 About 10 10 To about 10 17 About 10 12 To about 10 15 About 10 12 To about 10 17 Or about 10 15 To about 10 17 Individual cells/seeds. In some embodiments, the solution comprises about 10 3 About 10 4 About 10 5 About 10 6 About 10 7 About 10 8 About 10 9 About 10 10 About 10 12 About 10 15 Or about 10 17 Individual cells/seeds. In some embodiments, the solution comprises at least about 10 3 About 10 4 About 10 5 About 10 6 About 10 7 About 10 8 About 10 9 About 10 10 About 10 12 Or about 10 15 Individual cells/seeds. In some embodiments, the solution comprises up to about 10 4 About 10 5 About 10 6 About 10 7 About 10 8 About 10 9 About 10 10 About 10 12 About 10 15 Or about 10 17 Individual cells/seeds. In some embodiments, the solution comprises less than 10 10 Individual cells/seeds. In some embodiments, the solution comprises less than 10 9 Individual cells/seeds. In some embodiments, the solution comprises less than 10 8 Individual cells/seeds. In some embodiments, the solution comprises less than 10 11 Individual cells/seeds. In some embodiments, the solution comprises about 10 5 To about 10 9 Individual cells/seeds.
In some embodiments, the microorganism is a bacterium. In some embodiments, the bacteria are endospore-forming bacteria. In some embodiments, the method comprises inducing endospores formation of the endospore-forming bacteria. In some embodiments, the bacteria incorporated into the seed are endospores. In some embodiments, the solution comprises one or more components that induce formation of endospores. In some embodiments, the solution comprises potassium, ferrous sulfate, calcium, magnesium, manganese, or a combination thereof.
In some embodiments, the method comprises sterilizing the seed. In some embodiments, the method comprises sterilizing the seed surface. Any method of producing seeds having a sterilized surface may be used. In some embodiments, the seed is sterilized with a bleaching solution. In some embodiments, the seed is sterilized prior to immersing the seed in the solution containing the one or more microorganisms. In some embodiments, the seed is a sterilized seed. In some embodiments, the seed has a sterilized surface. As used herein, "sterilized," "sterilized" and related terms (e.g., "sanitized" and the like) mean that there are substantially no living microorganisms on the sterilized article. In some embodiments, the seed is sterilized prior to incubating the seed in a solution comprising the microorganism. In some embodiments, the seed is sterilized after incubating the seed in a solution comprising the microorganism. In some embodiments, the fungicide is added to the seed surface.
In some embodiments, the sterilized or disinfected seed comprises substantially no viable microorganisms on the seed (e.g., seed surface). In some embodiments, the sterile or sterilized seed comprises less than 1CFU, less than 5CFU, less than 10CFU, less than 20CFU, less than 30CFU, less than 40CFU, or less than 50CFU of microorganisms on the seed.
In some embodiments, the plant seed is incubated with the solution containing the microorganism for a time sufficient to incorporate the microorganism into the seed. In some embodiments, the plant seed is incubated with the solution containing the endospores forming bacteria or endospores thereof for about 1 minute to about 960 minutes. In some embodiments, the plant seed is incubated with the solution containing the endospores forming bacteria or endospores thereof for about 1 to about 5 minutes, about 1 to about 10 minutes, about 1 to about 20 minutes, about 1 to about 60 minutes, about 1 to about 240 minutes, about 1 to about 960 minutes, about 5 to about 10 minutes, about 5 to about 20 minutes, about 5 to about 60 minutes, about 5 to about 240 minutes, about 5 to about 960 minutes, about 10 to about 20 minutes, about 10 to about 60 minutes, about 10 to about 240 minutes, about 10 to about 960 minutes, about 20 to about 60 minutes, about 20 to about 240 minutes, about 60 to about 960 minutes, or about 240 to about 960 minutes. In some embodiments, the plant seed is incubated with the solution containing the endospores forming bacteria or endospores thereof for about 1 minute, about 5 minutes, about 10 minutes, about 20 minutes, about 60 minutes, about 240 minutes, or about 960 minutes. In some embodiments, the plant seed is incubated with the solution containing the endospores forming bacteria or endospores thereof for at least about 1 minute, about 5 minutes, about 10 minutes, about 20 minutes, about 60 minutes, or about 240 minutes. In some embodiments, the plant seed is incubated with the solution containing the endospores forming bacteria or endospores thereof for up to about 5 minutes, about 10 minutes, about 20 minutes, about 60 minutes, about 240 minutes, or about 960 minutes. In some embodiments, the plant seed is incubated with the solution containing the endospores forming bacteria or endospores thereof for about 1 minute. In some embodiments, the plant seed is incubated with the solution containing the endospores forming bacteria or endospores thereof for about 5 minutes. In some embodiments, the plant seed is incubated with the solution containing the endospores forming bacteria or endospores thereof for about 10 minutes. In some embodiments, the plant seed is incubated with the solution containing the endospores forming bacteria or endospores thereof for about 20 minutes. In some embodiments, the plant seed is incubated with the solution containing the endospores forming bacteria or endospores thereof for about 60 minutes. In some embodiments, the plant seed is incubated with the solution containing the endospores forming bacteria or endospores thereof for about 240 minutes. In some embodiments, the plant seed is incubated with the solution containing the endospores forming bacteria or endospores thereof for about 960 minutes.
In some embodiments, the plant seed is incubated with the solution containing the microorganism or secretion thereof for about 1 minute to about 960 minutes. In some embodiments, the plant seed is incubated with the solution containing the microorganism or secretion thereof for about 1 to about 5 minutes, about 1 to about 10 minutes, about 1 to about 20 minutes, about 1 to about 60 minutes, about 1 to about 240 minutes, about 1 to about 960 minutes, about 5 to about 10 minutes, about 5 to about 20 minutes, about 5 to about 60 minutes, about 5 to about 240 minutes, about 5 to about 960 minutes, about 10 to about 20 minutes, about 10 to about 60 minutes, about 10 to about 240 minutes, about 10 to about 960 minutes, about 20 to about 60 minutes, about 20 to about 240 minutes, about 20 to about 960 minutes, about 60 to about 240 minutes, about 60 to about 960 minutes, or about 240 to about 960 minutes. In some embodiments, the plant seed is incubated with the solution containing the microorganism or secretion thereof for about 1 minute, about 5 minutes, about 10 minutes, about 20 minutes, about 60 minutes, about 240 minutes, or about 960 minutes. In some embodiments, the plant seed is incubated with the solution containing the microorganism or secretion thereof for at least about 1 minute, about 5 minutes, about 10 minutes, about 20 minutes, about 60 minutes, or about 240 minutes. In some embodiments, the plant seed is incubated with the solution containing the microorganism or secretion thereof for up to about 5 minutes, about 10 minutes, about 20 minutes, about 60 minutes, about 240 minutes, or about 960 minutes. In some embodiments, the plant seed is incubated with the solution containing the microorganism or secretion thereof for about 1 minute. In some embodiments, the plant seed is incubated with the solution containing the microorganism or secretion thereof for about 5 minutes. In some embodiments, the plant seed is incubated with the solution containing the microorganism or secretion thereof for about 10 minutes. In some embodiments, the plant seed is incubated with the solution containing the microorganism or secretion thereof for about 20 minutes. In some embodiments, the plant seed is incubated with the solution containing the microorganism or secretion thereof for about 60 minutes. In some embodiments, the plant seed is incubated with the solution containing the microorganism or secretion thereof for about 240 minutes. In some embodiments, the plant seed is incubated with the solution containing the microorganism or secretion thereof for about 960 minutes.
In some embodiments, the seeds are incubated with the solution at a desired temperature. In some embodiments, the seeds are incubated with the solution at a temperature of about 2 ℃ to about 40 ℃. In some embodiments, the seed is incubated with the solution at about 2 ℃ to about 4 ℃, about 2 ℃ to about 8 ℃, about 2 ℃ to about 12 ℃, about 2 ℃ to about 16 ℃, about 2 ℃ to about 25 ℃, about 2 ℃ to about 30 ℃, about 2 ℃ to about 35 ℃, about 2 ℃ to about 40 ℃, about 4 ℃ to about 8 ℃, about 4 ℃ to about 12 ℃, about 4 ℃ to about 16 ℃, about 4 ℃ to about 25 ℃, about 4 ℃ to about 30 ℃, about 4 ℃ to about 40 ℃, about 8 ℃ to about 12 ℃, about 8 ℃ to about 16 ℃, about 8 ℃ to about 25 ℃, about 8 ℃ to about 30 ℃, about 8 ℃ to about 35 ℃, about 8 ℃ to about 40 ℃, about 12 ℃ to about 16 ℃, about 12 ℃ to about 25 ℃, about 12 ℃ to about 30 ℃, about 12 ℃ to about 35 ℃, about 12 ℃ to about 40 ℃, about 16 ℃ to about 30 ℃, about 16 ℃ to about 35 ℃, about 16 ℃ to about 40 ℃, about 25 ℃ to about 25 ℃, about 25 ℃ to about 35 ℃, about 25 ℃ to about 40 ℃, about 40 ℃ or about 40 ℃ to about 40 ℃. In some embodiments, the seed is incubated with the solution at a temperature of about 2 ℃, about 4 ℃, about 8 ℃, about 12 ℃, about 16 ℃, about 25 ℃, about 30 ℃, about 35 ℃, or about 40 ℃. In some embodiments, the seed is incubated with the solution at a temperature of at least about 2 ℃, about 4 ℃, about 8 ℃, about 12 ℃, about 16 ℃, about 25 ℃, about 30 ℃, or about 35 ℃. In some embodiments, the seed is incubated with the solution at a temperature of up to about 4 ℃, about 8 ℃, about 12 ℃, about 16 ℃, about 25 ℃, about 30 ℃, about 35 ℃, or about 40 ℃.
In some embodiments, the method comprises drying the seed. In some embodiments, the seed is dried to about 10% of the total moisture of the seed. In some embodiments, the seed is dried to about 5% to about 25% of the total moisture of the seed. In some embodiments, the seed is dried to about 5% to about 8%, about 5% to about 10%, about 5% to about 12%, about 5% to about 15%, about 5% to about 20%, about 5% to about 25%, about 8% to about 10%, about 8% to about 12%, about 8% to about 15%, about 8% to about 20%, about 8% to about 25%, about 10% to about 12%, about 10% to about 15%, about 10% to about 20%, about 10% to about 25%, about 12% to about 15%, about 12% to about 20%, about 12% to about 25%, about 15% to about 20%, about 15% to about 25%, or about 20% to about 25% of the total moisture of the seed. In some embodiments, the seed is dried to about 5%, about 8%, about 10%, about 12%, about 15%, about 20%, or about 25% of the total moisture of the seed. In some embodiments, the seed is dried to at least about 5%, about 8%, about 10%, about 12%, about 15%, or about 20% of the total moisture of the seed. In some embodiments, the seed is dried to at most about 8%, about 10%, about 12%, about 15%, about 20%, or about 25% of the total moisture of the seed. In some embodiments, the seeds are dried to prevent germination of the seeds. In some embodiments, the seeds are dried prior to planting the seeds to prevent germination.
Formulations for incorporation of microorganisms
In one aspect, provided herein is a formulation for incorporating a microorganism, endospore or secretion thereof into a seed. In some embodiments, the formulation comprises one or more microorganisms or endospores thereof and a salt. The one or more microorganisms may be any one of the microorganisms provided herein or an endospore thereof. In some embodiments, the one or more microorganisms include one or more endospore-forming bacteria or endospores thereof. In some embodiments, the formulation comprises secretions of microorganisms. The secretions may be from any of the microorganisms provided herein.
In some embodiments, the formulation is a solution. In some embodiments, the formulation is an aqueous solution.
In some embodiments, the formulation comprises a salt. The salt may be present in the formulation in any suitable concentration. In some embodiments, the formulation comprises about 0.85% salt (w/v). In some embodiments, the formulation comprises from about 0.1% to about 1.25% salt (w/v). In some embodiments, the formulation comprises from about 0.1% to about 2.0% salt (w/v). In some embodiments of the present invention, in some embodiments, the formulation comprises from about 0.1% to about 0.25%, from about 0.1% to about 0.5%, from about 0.1% to about 0.6%, from about 0.1% to about 0.7%, from about 0.1% to about 0.75%, from about 0.1% to about 0.8%, from about 0.1% to about 0.85%, from about 0.1% to about 0.9%, from about 0.1% to about 0.95%, from about 0.1% to about 1%, from about 0.1% to about 1.25%, from about 0.25% to about 0.5%, from about 0.25% to about 0.6%, from about 0.25% to about 0.7%, from about 0.25% to about 0.75% >. About 0.25% to about 0.8%, about 0.25% to about 0.85%, about 0.25% to about 0.9%, about 0.25% to about 0.95%, about 0.25% to about 1%, about 0.25% to about 1.25%, about 0.5% to about 0.6%, about 0.5% to about 0.7%, about 0.5% to about 0.75%, about 0.5% to about 0.8%, about 0.5% to about 0.85%, about 0.5% to about 0.9%, about 0.5% to about 0.95%, about 0.5% to about 1%, about 0.5% to about 1.25%, about 0.6% to about 0.7% >. About 0.25% to about 0.8%, about 0.25% to about 0.85%, about 0.25% to about 0.9%, about 0.25% to about 0.95%, about 0.25% to about 1%, about 0.25% to about 1.25%, about 0.5% to about 0.6%, about 0.5% to about 0.7%, about about 0.5% to about 0.75%, about 0.5% to about 0.8%, about 0.5% to about 0.85%, about 0.5% to about 0.9%, about 0.5% to about 0.95%, about 0.5% to about 1%, about 0.5% to about 1.25%, about 0.6% to about 0.7%, about, about 0.95% to about 1%, about 0.95% to about 1.25%, or about 1% to about 1.25% salt (w/v). In some embodiments, the formulation comprises about 0.1%, about 0.25%, about 0.5%, about 0.6%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, about 1% or about 1.25% salt (w/v). In some embodiments, the formulation comprises at least about 0.1%, about 0.25%, about 0.5%, about 0.6%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, or about 1% salt (w/v). In some embodiments, the formulation comprises up to about 0.25%, about 0.5%, about 0.6%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, about 1% or about 1.25% salt (w/v). In some embodiments, the formulation comprises about 0.85% salt (w/v). In some embodiments, the formulation comprises from about 0.8% to about 0.9% salt (w/v). In some embodiments, the formulation comprises from about 0.75% to about 0.95% salt (w/v). In some embodiments, the formulation comprises from about 0.7% to about 1% salt (w/v). In some embodiments, the formulation comprises from about 0.5% to about 1.25% salt (w/v). In some embodiments, the formulation comprises from about 0.5% to about 2% salt (w/v). In some embodiments, the formulation comprises 0.1% -0.2%, 0.2% -0.3%, 0.3% -0.4%, 0.4% -0.5%, 0.5% -0.6%, 0.6% -0.7%, 0.7% -0.8%, 0.8% -0.9%, 0.9% -1.0%, 1.0% -1.1%, 1.1% -1.2%, 1.2% -1.3%, 1.3% -1.4% or 1.4% -1.5% salt (w/v).
Any salt can be used. In some preferred embodiments, the salt is NaCl. In some embodiments, the salt is NaCl, liCl, KCl, mgCl 2 、CaCl 2 、NaBr、LiBr、KBr、MgBr 2 、CaBr 2 、NaI、LiI、KI、MgI 2 Or CaI 2 . In some embodiments, the salt comprises sodium, lithium, or potassium ions. In some embodiments, the salt comprises an alkali metal ion. In some embodiments, the salt comprises an alkaline earth metal ion. In some embodiments, the salt comprises a halide ion. In some embodiments, the salt is an alkali or alkaline earth metal halide salt. In some embodiments, the salt comprises chloride, bromide, or iodide. In some embodiments, the salt is a sulfate, phosphate, carbonate, or nitrate.
In some embodiments, the formulation comprises additional additives. In some embodiments, the formulation comprises Dimethylsulfoxide (DMSO), 1-dodecylazepan-2-one, laurocapramKetones, 1-methyl-2-pyrrolidone (NMP), oleic acid, ethanol, methanol, polyethylene glycols (Brij 35, 58, 98), polyethylene glycol monolaurates (e.g., tween 20), tween 40 (polyoxyethylene sorbitol esters), tween 60, tween 80 (non-ionic), cetyl methyl ammonium bromide (CTAB), urea, lecithin (solidified fatty acids derived from soy), chitosan, poloxamer 188, poloxamer 237, poloxamer 338, poloxamer 407, or combinations thereof. In some embodiments, the formulation comprises polyethylene glycol monolaurate (e.g., tween 20), poloxamer 188, poloxamer 237, poloxamer 338, poloxamer 407, or a combination thereof. In some embodiments, the formulation comprises a poloxamer. In some embodiments, the formulation comprises polyethylene glycol monolaurate (e.g., tween 20). The additional additives may be present in any concentration. In some embodiments, the additional additive comprises up to about 0.01%, 0.05%, 0.1%, 0.125%, 0.15%, 0.2%, 0.5%, or 1% (v/v) of the formulation. In some embodiments, the additional additive comprises about 0.01% to about 1% (v/v) of the formulation. In some embodiments The additional additive comprises about 0.1% (v/v) of the formulation.
In some embodiments, the formulation comprises additional metal ions. In some embodiments, the formulation comprises magnesium, calcium, manganese, or any combination thereof. In some embodiments, the formulation comprises magnesium. In some embodiments, the formulation comprises calcium. In some embodiments, the formulation comprises manganese. In some embodiments, the formulation comprises magnesium and calcium. In some embodiments, the formulation comprises magnesium and manganese. In some embodiments, the formulation comprises calcium and manganese. In some embodiments, the formulation comprises magnesium, calcium, and manganese.
In some embodiments, the formulation comprises one or more nutrients for the microorganism. In some embodiments, the formulation comprises a bacterial growth medium. In some embodiments, the formulation comprises a Lysogenic Broth (LB), a nutrient broth, or a combination thereof. In some embodiments, the formulation comprises a lysogenic broth. In some embodiments, the formulation comprises a nutrient broth.
In some embodiments, the formulation comprises additional ingredients for promoting endospores formation of one or more microorganisms. In some embodiments, the formulation comprises potassium, ferrous sulfate, calcium, magnesium, manganese, or a combination thereof. In some embodiments, the formulation comprises potassium. In some embodiments, the formulation comprises ferrous sulfate. In some embodiments, the formulation comprises calcium. In some embodiments, the formulation comprises magnesium. In some embodiments, the formulation comprises manganese.
In some embodiments, the formulation comprises a microorganism. In some embodiments, the formulation comprises about 10 3 To about 10 17 Colony Forming Units (CFU)/mL. In some embodiments, the formulation comprises at least 1 x 10 6 CFU/mL of microorganism. In some embodiments, the formulation comprises about 10 3 To about 10 4 About 10 3 To about 10 5 About 10 3 To about 10 6 About 10 3 To about 10 7 About 10 3 To about 10 8 About 10 3 To about 10 9 About 10 3 To about 10 10 About 10 3 To about 10 12 About 10 3 To about 10 15 About 10 3 To about 10 17 About 10 4 To about 10 5 About 10 4 To about 10 6 About 10 4 To about 10 7 About 10 4 To about 10 8 About 10 4 To about 10 9 About 10 4 To about 10 10 About 10 4 To about 10 12 About 10 4 To about 10 15 About 10 4 To about 10 17 About 10 5 To about 10 6 About 10 5 To about 10 7 About 10 5 To about 10 8 About 105 to about 10 9 About 10 5 To about 10 10 About 10 5 To about 10 12 About 10 5 To about 10 15 About 10 5 To about 10 17 About 10 6 To about 10 7 About 10 6 To about 10 8 About 10 6 To about 10 9 About 10 6 To about 10 10 About 10 6 To about 10 12 About 10 6 To about 10 15 About 10 6 To about 10 17 About 10 7 To about 10 8 About 10 7 To about 10 9 About 10 7 To about 10 10 About 10 7 To about 10 12 About 10 7 To about 10 15 About 10 7 To about 10 17 About 10 8 To about 10 9 About 10 8 To about 10 10 About 10 8 To about 10 12 About 10 8 To about 10 15 About 10 8 To about 10 17 About 10 9 To about 10 10 About 10 9 To about 10 12 About 10 9 To about 10 15 About 10 9 To about 10 17 About 10 10 To about 10 12 About 10 10 To about 10 15 About 10 10 To about 10 17 About 10 12 To about 10 15 About 10 12 To about 10 17 Or about 10 15 To about 10 17 CFU/mL of microorganism. In some embodiments, the formulation comprises about 10 3 About 10 4 About 10 5 About 10 6 About 10 7 About 10 8 About 10 9 About 10 10 About 10 12 About 10 15 Or about 10 17 CFU/mL of microorganism. In some embodiments, the formulation comprises at least about 10 3 About 10 4 About 10 5 About 10 6 About 10 7 About 10 8 About 10 9 About 10 10 About 10 12 Or about 10 15 CFU/mL of microorganism. In some embodiments, the formulation comprises up to about 10 4 About 10 5 About 10 6 About 10 7 About 10 8 About 10 9 About 10 10 About 10 12 About 10 15 Or about 10 17 CFU/mL of microorganism. In some embodiments, the formulation comprises at least about 10 6 To 10 7 CFU/mL of microorganism. In some embodiments, the formulation comprises 1 x 10 3 Up to 1X 10 4 CFU/mL；1×10 4 Up to 1X 10 5 CFU/mL；1×10 5 Up to 1X 10 6 CFU/mL；1×10 6 Up to 1X 10 7 CFU/mL；1×10 7 Up to 1X 10 8 CFU/mL；1×10 8 Up to 1X 10 9 CFU/mL；1×10 9 Up to 1X 10 10 CFU/mL；1×10 10 Up to 1X 10 11 CFU/mL；1×10 11 Up to 1X 10 12 CFU/mL；1×10 12 Up to 1X 10 13 CFU/mL；1×10 13 Up to 1X 10 14 CFU/mL；1×10 14 Up to 1X 10 15 CFU/mL；1×10 15 Up to 1X 10 16 CFU/mL; or 1X 10 16 Up to 1X 10 17 CFU/mL of microorganism. The microorganism may be any of the microorganisms provided herein or an endospore of any of the microorganisms provided herein.
In some embodiments, the formulation is maintained at a desired temperature. In some embodiments, the formulation is maintained at a temperature of about 2 ℃ to about 40 ℃. In some embodiments, the formulation is maintained at about 2 ℃ to about 4 ℃, about 2 ℃ to about 8 ℃, about 2 ℃ to about 12 ℃, about 2 ℃ to about 16 ℃, about 2 ℃ to about 25 ℃, about 2 ℃ to about 30 ℃, about 2 ℃ to about 35 ℃, about 2 ℃ to about 40 ℃, about 4 ℃ to about 8 ℃, about 4 ℃ to about 12 ℃, about 4 ℃ to about 16 ℃, about 4 ℃ to about 25 ℃, about 4 ℃ to about 30 ℃, about 4 ℃ to about 35 ℃, about 4 ℃ to about 40 ℃, about 8 ℃ to about 12 ℃, about 8 ℃ to about 16 ℃, about 8 ℃ to about 25 ℃, about 8 ℃ to about 30 ℃, about 8 ℃ to about 35 ℃, about 8 ℃ to about 40 ℃, about 12 ℃ to about 16 ℃, about 12 ℃ to about 25 ℃, about 12 ℃ to about 30 ℃, about 12 ℃ to about 35 ℃, about 12 ℃ to about 40 ℃, about 16 ℃ to about 25 ℃, about 16 ℃ to about 35 ℃, about 16 ℃ to about 40 ℃, about 25 ℃ to about 25 ℃, about 25 ℃ to about 40 ℃, about 40 ℃ to about 40 ℃ or about 40 ℃ to about 35 ℃. In some embodiments, the formulation is maintained at a temperature of about 2 ℃, about 4 ℃, about 8 ℃, about 12 ℃, about 16 ℃, about 25 ℃, about 30 ℃, about 35 ℃, or about 40 ℃. In some embodiments, the formulation is maintained at a temperature of at least about 2 ℃, about 4 ℃, about 8 ℃, about 12 ℃, about 16 ℃, about 25 ℃, about 30 ℃, or about 35 ℃. In some embodiments, the formulation is maintained at a temperature of up to about 4 ℃, about 8 ℃, about 12 ℃, about 16 ℃, about 25 ℃, about 30 ℃, about 35 ℃, or about 40 ℃.
Microorganisms and secretions
The microorganisms provided herein, or secretions thereof, produce or promote bicarbonate and one or more mineral formations. In some embodiments, bicarbonate formation sequesters CO 2 . In some embodiments, bicarbonate formation results in mineral formation. In some embodiments, the formed minerals stably sequester carbon in the soil. In some embodiments, the microorganism is a bacterium. In some embodiments, the microorganism is an endospore-forming bacterium. In some embodiments, the microorganism is a bacterial endospore. Whenever a microorganism (e.g., a bacterium) referred to herein is capable of forming an endospore, it is intended to encompass any endospore of the microorganism as well. For example, if the plant seed treatment formulation comprises bacillus, the formulation may comprise endospores of bacillus.
In some embodiments, the microorganism is a microorganism from the phylum firmicutes, the phylum proteus, and the phylum actinomycetes. In some embodiments, the microorganism is a microorganism from the phylum firmicutes. In some embodiments, the microorganism is a microorganism from the phylum Proteus. In some embodiments, the microorganism is a microorganism from the phylum actinomycetes. In some embodiments, the microorganism is an endospore of any one of the microorganisms.
Rhizobacteria that fix atmospheric nitrogen are present on the roots of plants. These organisms are able to survive in soil and react to large amounts of CO 2 Tolerating CO 2 Is released by the roots of plants during respiration or by nearby microorganisms and soil animal communities through respiration.
Because these rhizobacteria are closer to the root, these organisms have the ability to utilize root secretions as a carbon and energy source. Many of them have evolved to possess a property that allows them to CO 2 Genes that are transformed into biomass or any metabolite for their own benefits. In some embodiments, the bacteria are not genetically modified. In some embodiments, the CO is according to bacteria 2 The ability to convert to bicarbonate and minerals to select bacteria.
Rhizobacteria can more aggressively colonize plant roots. Thus, these bacteria form stable communities that can survive in varying soil environments, secrete antimicrobial compounds to inhibit the growth of pathogens or intruders, and can form endospores that have a selective fitness for survival in harsh environments.
In addition to fixing CO 2 Among other means, rhizosphere bacteria have the ability to express the well characterized enzyme Carbonic Anhydrase (CA). Broad temperature tolerance (up to 50 ℃), broad pH range and high level expression of CA as CO in sequestered soil 2 Is a desirable candidate for a cell. The enzyme will CO 2 Converts to bicarbonate and converts bicarbonate to carbonate ions via hydrolysis. The carbonate ions will react with cations present in the soil and produce minerals. Because the soil is rich in a plurality of cations (Ca 2+ 、Mg 2+ 、Na + 、K + ) This maintains mineralization and can form a variety of minerals as a permanent capture of CO in the soil 2 Means of (3). In one placeIn some embodiments, the bacteria are selected according to the use of carbonic anhydrase.
In some embodiments, the carbonic anhydrase is α -Ca, β -CA, δ -CA, ζ -CA, η -CA, or iota-CA. In some embodiments, the CA is CA-1, CA-2, CA-3, CA-4, CA-5A, CA-5B, CA-6, CA-7, CA-8, CA-9, CA-10, CA-11, CA-12, CA-13, CA-14 or CA-15.
In some embodiments, a plurality of rhizosphere bacteria are effectively loaded into the seed. In some embodiments, the rhizobacteria include endospore-forming bacteria that enhance biological nitrogen fixation. In some embodiments, the rhizobacteria comprise bacillus, paenibacillus, or both. In some embodiments, the one or more microorganisms comprise bacillus amyloliquefaciens, bacillus laterosporus, bacillus licheniformis, bacillus macerans, bacillus cereus, bacillus circulans, bacillus firmus, bacillus subtilis, bacillus sphaericus, bacillus megaterium, bacillus coagulans, bacillus brevis, bacillus thuringiensis, bacillus mycoides, bacillus cucumber, bacillus plantarum, bacillus pumilus, bacillus bailii, bacillus mucilaginosus, bacillus tertazii, bacillus methylotrophicus, or any combination thereof. In some embodiments, the one or more microorganisms comprise bacillus subtilis S3C23, bacillus subtilis MP2, bacillus subtilis RO2C15, bacillus subtilis RO2C22, bacillus megaterium 6, bacillus megaterium S3C21, bacillus megaterium RO2C12, bacillus cucumber S3C14, bacillus plantarum 5, or any combination thereof. In some embodiments, the one or more microorganisms comprise bacillus subtilis S3C23. In some embodiments, the one or more microorganisms comprise bacillus subtilis MP2.
CO of these microorganisms 2 Sequestration may be achieved by their ability to produce or promote CA formation. These rhizobacteria can colonize the root and can replace other microbial communities in the vicinity that would otherwise use nutrients from root secretions. CO from root, soil zoon or microflora 2 Can be captured by CA by hydration to bicarbonate. Typically, to form minerals (CaCO) 3 、MgCO 3 、CaMg(CO 3 ) 2 ) Cations are required to continue the mineral production process. There are a variety of cations in the soil, which have allowed for a sustainable process. Ca in soil 2+ And Mg (magnesium) 2+ The amount of (c) may depend on the geographic location, soil type and irrigation pattern. Farmers can further adjust these cations by applying limestone to maintain high fertility of the soil. Considering the initial 15cm depth, a typical well irrigated soil averages 850kg (Ca 2+ ) Per acre and 218kg (Mg 2+ ) Per acre. According to a previously published study, CO produced in corn rhizosphere 2 The amount was about 7000 kg/acre/corn season. Taking into account the available CO 2 And the amount of cations, a large amount of CO 2 Can be stored as Ca or Mg minerals. Mathematically, 425kg CaCO can be formed 3 And 114kg MgCO 3 To form other minerals (such as Na 2 CO 3 ) Depending on the other related cations (Na + 、K + ) Is present. Because lime treatment is performed by farmers to maintain high fertility of soil, microorganisms disclosed herein can biologically produce limestone (CaCO) 3 ) To eliminate this requirement. In addition, depending on the availability of other cations in the soil, various minerals may be formed to store gaseous CO 2 . These minerals include, but are not limited to calcite, aragonite, dolomite, limestone, carbonate, magnesium carbonate, iron carbonate, magnesite, siderite (coenite), diamond, carbonate, ferrous carbonate, wollastonite, and tobermorite.
In some embodiments, the amount of minerals produced may be 50 kg/acre to 1000 kg/acre. In some embodiments, the amount of minerals produced may be from about 50 kg/acre to about 1,000 kg/acre. In some embodiments of the present invention, in some embodiments, the amount of minerals produced may be about 50 kg/acre to about 100 kg/acre, about 50 kg/acre to about 200 kg/acre, about 50 kg/acre to about 300 kg/acre, about 50 kg/acre to about 400 kg/acre, about 50 kg/acre to about 500 kg/acre, about 50 kg/acre to about 600 kg/acre, about 50 kg/acre to about 700 kg/acre, about 50 kg/acre to about 800 kg/acre, about 50 kg/acre to about 900 kg/acre, about 50 kg/acre to about 1,000 kg/acre, about 100 kg/acre to about 200 kg/acre, about 100 kg/acre to about 300 kg/acre, about 100 kg/acre to about 400 kg/acre, about 100 kg/acre to about 500 kg/acre, about 100 kg/acre to about 600 kg/acre about 100 kg/acre to about 700 kg/acre, about 100 kg/acre to about 800 kg/acre, about 100 kg/acre to about 900 kg/acre, about 100 kg/acre to about 1,000 kg/acre, about 200 kg/acre to about 300 kg/acre, about 200 kg/acre to about 400 kg/acre, about 200 kg/acre to about 500 kg/acre, about 200 kg/acre to about 600 kg/acre, about 200 kg/acre to about 700 kg/acre, about 200 kg/acre to about 800 kg/acre, about 200 kg/acre to about 900 kg/acre, about 200 kg/acre to about 1,000 kg/acre, about 300 kg/acre to about 400 kg/acre, about 300 kg/acre to about 500 kg/acre, about 300 kg/acre to about 600 kg/acre, about 300 kg/acre to about 800 kg/acre, about 300 kg/acre to about 900 kg/acre, about 300 kg/acre to about 1,000 kg/acre, about 400 kg/acre to about 500 kg/acre, about 400 kg/acre to about 600 kg/acre, about 400 kg/acre to about 700 kg/acre, about 400 kg/acre to about 800 kg/acre, about 400 kg/acre to about 900 kg/acre, about 400 kg/acre to about 1,000 kg/acre, about 500 kg/acre to about 600 kg/acre, about 500 kg/acre to about 700 kg/acre, about 500 kg/acre to about 500 kg/acre, about 500 kg/acre to about 1,000 kg/acre, about 1 kg/acre to about 600 kg/acre, about 600 kg/acre to about 600 kg/acre, about 1 kg/acre to about 600 kg/acre. In some embodiments, the amount of minerals produced may be about 50 kg/acre, about 100 kg/acre, about 200 kg/acre, about 300 kg/acre, about 400 kg/acre, about 500 kg/acre, about 600 kg/acre, about 700 kg/acre, about 800 kg/acre, about 900 kg/acre, or about 1,000 kg/acre. In some embodiments, the amount of minerals produced may be at least about 50 kg/acre, about 100 kg/acre, about 200 kg/acre, about 300 kg/acre, about 400 kg/acre, about 500 kg/acre, about 600 kg/acre, about 700 kg/acre, about 800 kg/acre, or about 900 kg/acre. In some embodiments, the amount of minerals produced may be up to about 100 kg/acre, about 200 kg/acre, about 300 kg/acre, about 400 kg/acre, about 500 kg/acre, about 600 kg/acre, about 700 kg/acre, about 800 kg/acre, about 900 kg/acre, or about 1,000 kg/acre.
In some embodiments, the CO converted by the microorganism 2 The amount of CO was 0.1 ton 2 Per acre to 2.5 tons of CO 2 Per acre. In some embodiments, the microorganism converts 2.5 to 5.3 tons of CO 2 Per acre. In some embodiments, the microorganism converts 5.3 to 7.5 tons of CO 2 Per acre. In some embodiments, the microorganism converts 7.5 to 10 tons of CO 2 Per acre. In some embodiments, the microorganism converts 10 to 15 tons of CO 2 Per acre. In some embodiments, the microbial conversion is 15 to 20 tons/acre. In some embodiments, the CO converted by the microorganism 2 The amount is from about 2 tons/acre to about 20 tons/acre. In some embodiments, the CO converted by the microorganism 2 The amount is from about 2 tons/acre to about 4 tons/acre, from about 2 tons/acre to about 6 tons/acre, from about 2 tons/acre to about 8 tons/acre, from about 2 tons/acre to about 10 tons/acre, from about 2 tons/acre to about 12 tons/acre, from about 2 tons/acre to about 14 tons/acre, from about 2 tons/acre to about 16 tons/acre, from about 2 tons/acre to about 18 tons/acre, from about 2 tons/acre to about 20 tons/acre, from about 4 tons/acre to about 6 tons/acre, from about 4 tons/acre to about 8 tons/acre, from about 4 tons/acre to about 10 tons/acre, from about 4 tons/acre to about 12 tons/acre, from about 4 tons/acre about 4 to about 14 tons/acre, about 4 to about 16 tons/acre, about 4 to about 18 tons/acre, about 4 to about 20 tons/acre, about 6 to about 8 tons/acre, about 6 to about 10 tons/acre, about 6 to about 12 tons/acre, about 6 to about 14 tons/acre, about 6 to about 16 tons/acre, about 6 to about 18 tons/acre, about 6 to about 20 tons/acre, about 8 to about 10 tons/acre, about 8 to about 12 tons/acre About 8 to about 14 tons/acre, about 8 to about 16 tons/acre, about 8 to about 18 tons/acre, about 8 to about 20 tons/acre, about 10 to about 12 tons/acre, about 10 to about 14 tons/acre, about 10 to about 16 tons/acre, about 10 to about 18 tons/acre, about 10 to about 20 tons/acre, about 12 to about 14 tons/acre, about 12 to about 16 tons/acre, about 14 to about 16 tons/acre, about 16 to about 16 tons/acre. In some embodiments, the CO converted by the microorganism 2 The amount is about 2 tons/acre, about 4 tons/acre, about 6 tons/acre, about 8 tons/acre, about 10 tons/acre, about 12 tons/acre, about 14 tons/acre, about 16 tons/acre, about 18 tons/acre, or about 20 tons/acre. In some embodiments, the CO converted by the microorganism 2 The amount is at least about 2 tons/acre, about 4 tons/acre, about 6 tons/acre, about 8 tons/acre, about 10 tons/acre, about 12 tons/acre, about 14 tons/acre, about 16 tons/acre, or about 18 tons/acre. In some embodiments, the CO converted by the microorganism 2 The amount is up to about 4 tons/acre, about 6 tons/acre, about 8 tons/acre, about 10 tons/acre, about 12 tons/acre, about 14 tons/acre, about 16 tons/acre, about 18 tons/acre, or about 20 tons/acre.
In some embodiments, promoting production of the one or more minerals comprises producing ammonia and a resulting increase in pH in a medium in which plants derived from the plant seeds are grown.
The mineralization process may be initiated on the cell wall of the microorganism or EPS. The cell wall has been shown to be a site of nucleation and mineralization due to the presence of the overall negative charge. In some microorganisms (such as bacillus subtilis), EPS contains a large amount of glutamate, aspartate, histidine, arginine and lysine, which are negatively charged under alkaline conditions and can be used as CaCO 3 Nucleation sites for precipitation. This may beCan be beneficial because of the microorganisms attached to the root and CaCO thereon 3 It can be removed by pulling the plant root at the end of the season to prevent mineral growth in the soil (if not required).
In some embodiments, the one or more microorganisms comprise a genetic modification that results in the one or more microorganisms producing or promoting more carbonic anhydrase formation relative to the corresponding wild-type microorganism. In some embodiments, the genetic modification comprises a nucleic acid construct, wherein the nucleic acid construct comprises one or more promoters configured to drive expression of a Carbonic Anhydrase (CA) coding sequence. In some embodiments, the genetic modification comprises a nucleic acid construct, wherein the nucleic acid construct comprises a Carbonic Anhydrase (CA) coding sequence. In some embodiments, the CA coding sequence is heterologous to the one or more microorganisms. In some embodiments, the CA coding sequence is endogenous to the one or more microorganisms. In some embodiments, the nucleic acid construct is codon optimized. In some embodiments, the nucleic acid construct further comprises one or more promoters configured to drive expression of the CA coding sequence. In some embodiments, the one or more promoters drive constitutive expression of the CA coding sequence. In some embodiments, the one or more promoters drive inducible expression of the CA coding sequence. In some embodiments, the genetic modification comprises a carbonic anhydrase gene that has been modified by directed microbial evolution. In some embodiments, the genetic modification comprises a signal sequence. In some embodiments, the signal sequence comprises a periplasmic signal sequence or an extracellular secretion signal. In some embodiments, the periplasmic signal sequence or the extracellular secretion signal is located 5' to the carbonic anhydrase coding sequence. In some embodiments, the periplasmic signal sequence or the extracellular secretion signal is fused to the 5' end of the carbonic anhydrase coding sequence. In some embodiments, the genetic modification comprises one or more components of the general secretory pathway, and wherein the genetic modification results in secretion or subcellular targeting of carbonic anhydrase by the one or more microorganisms.
In some embodiments, the carbonic anhydrase is α -Ca, β -CA, δ -CA, ζ -CA, η -CA, or iota-CA. In some embodiments, the CA is CA-1, CA-2, CA-3, CA-4, CA-5A, CA-5B, CA-6, CA-7, CA-8, CA-9, CA-10, CA-11, CA-12, CA-13, CA-14 or CA-15.
In some embodiments, the one or more promoters are or are derived from one or more promoters comprised in bacillus or paenibacillus. In some embodiments, the one or more promoters are selected from P groES 、P 43 、P sigX 、P trnQ And P xylA . In some embodiments, the one or more promoters are or are derived from one or more housekeeping gene promoters or strongly expressed constitutive promoters. In some embodiments, the one or more housekeeping gene promoters or strong expressed constitutive promoters are selected from the group consisting of P liaG 、P lepA 、P veg 、P gsiB 、P43、P trnQ 、P lial (bacitracin-inducible) and P xylA (xylose inducible).
In some embodiments, the genetic modification comprises introducing an expression vector into one or more microorganisms. In some embodiments, the genetic modification comprises modification of a chromosome of one or more microorganisms.
In some embodiments, the microorganism can immobilize nitrogen and carbon dioxide. In some embodiments, the nitrogen-fixing microorganisms will continue to provide fixed nitrogen (ammonia) to the plants via biological nitrogen fixation, which will reduce the use of chemical fertilizers that pollute the environment. The end product (ammonia) of atmospheric nitrogen fixation can enhance CO 2 To minerals, as ammonia has been reported to increase pH, thereby accelerating mineralization. Because ammonia produced by the microorganisms disclosed herein is significantly higher than other soil-dwelling microorganisms, even in the presence of an external nitrogen source, the mineralization process of the strains disclosed herein may be faster than other microorganisms that inhabit the soil.
In some embodiments of the present invention, in some embodiments, the microorganism is selected from Acetobacter, actinobacillus, bacillus alcaligenes, aminophilia, bacillus bifidus, anaerobic Bacillus, thiobacillus, anaerobic Bacillus, rhizobium, brevibacterium, thermoanaerobacter, caminiella, prunus, clostridium, ke Enshi, ke Kesi, tree-derived Bacillus, being used as a microorganism desulphurized enterobacteria, desulphurized bacteria, actinomyces, linear bacteria, gelria, geobacillus, geosporic bacteria, bacillus, gracilis, halophiles, haloatron, solar bacteria, mesophiles, klebsiella, rice bacteria, chroogomphausbacteria, lysine bacteria desulphurized enterobacteria, desulfospormus, desulphurized campylobacter, desulfospormus, desulphurized Sheng Bacillus, desulphurized Bacillus, protomyces, linear Bacillus, gelria, geobacillus Geosporobacter, bacillus, haloatron, solar, japanese, klebsiella, leishneedles, bacillus, lysine, microorganisms of the genus Thermoactinomyces, thermokalibacillus, thermoanaerobacter, thermoflavobacterium, thermovenabaum, bacillus megaterium, cladosporium, vulcanobacillus and xanthobacter. In some embodiments, the microorganism is a microorganism selected from the group consisting of Acetobacter, actinobacillus, bacillus, chryseobacterium, ke Kesi, sword, gluconobacter, microbacterium, and Serratia. In some embodiments, the microorganism is acetobacter. In some embodiments, the microorganism is an actinomycete. In some embodiments, the microorganism is bacillus. In some embodiments, the microorganism is a chrysobacterium. In some embodiments, the microorganism is of the genus Ke Kesi. In some embodiments, the microorganism is a genus xiphoid. In some embodiments, the microorganism is a genus glutamate. In some embodiments, the microorganism is a genus microbacterium. In some embodiments, the microorganism is pantoea. In some embodiments, the microorganism is serratia. In some embodiments, the microorganism is an endospore of any one of the microorganisms.
In some embodiments, the microorganism comprises acetobacter (Acetobacter cerevisiae), bacillus cucumber, bacillus plantarum, bacillus megaterium, bacillus middlingeus (Bacillus nakamurai), bacillus subtilis, chrysobacterium lactis (Chryseobacterium lactis), bacillus clarkii, glutamic acid bacteria of the research team (Glutamicibacter arilaitensis), bacillus haloxyfop (Glutamicibacter halophytocola), microbacterium chocolate (Microbacterium chocolatum), microbacterium yannicii, pantoea allii, serratia marcescens (Serratia marcescens), or serratia ureae (Serratia ureilytica). In some embodiments, the microorganism comprises acetobacter cerevisiae, bacillus cucumber, bacillus plantarum, bacillus megaterium, bacillus middlingeus, bacillus subtilis, golden yellow lactobacillus, bacillus clathratus, bacillus halophilus, microbacterium chocolate, pantoea allii, or serratia marcescens. In some embodiments, the microorganism comprises acetobacter cerevisiae. In some embodiments, the microorganism comprises bacillus cucumber. In some embodiments, the microorganism comprises bacillus plantarum. In some embodiments, the microorganism comprises bacillus megatherium. In some embodiments, the microorganism comprises bacillus subtilis. In some embodiments, the microorganism comprises a. Lactis. In some embodiments, the microorganism comprises a sabia viscosa. In some embodiments, the microorganism comprises bacillus halogenes. In some embodiments, the microorganism comprises a chocolate microbacterium. In some embodiments, the microorganism comprises Pantoea allii. In some embodiments, the microorganism comprises Serratia viscosa. In some embodiments, the microorganism is an endospore of any one of the microorganisms.
In some embodiments, the microorganism is an endospore-forming bacterium. In some embodiments, the endospore-forming bacteria are from the genus bacillus. In some embodiments, the endospore-forming bacteria is bacillus. In some embodiments, the endospore-forming bacteria comprise bacillus cucumber, bacillus endophyte, bacillus megaterium, bacillus middlingeus, or bacillus subtilis. In some embodiments, the endospore-forming bacteria comprise bacillus cucumber, bacillus endophyte, bacillus megaterium, or bacillus subtilis. In some embodiments, the endospore-forming bacteria comprise bacillus cucumber. In some embodiments, the endospore-forming bacteria comprise bacillus megaterium. In some embodiments, the endospore-forming bacteria comprise bacillus cereus. In some embodiments, the endospore-forming bacteria comprise bacillus subtilis. In some embodiments, the microorganism is an endospore of any one of the microorganisms.
In some embodiments, the microorganism is an endospore. In some embodiments, the endospores are from bacillus. In some embodiments, the endospore is bacillus. In some embodiments, the endospores comprise bacillus cucumber, bacillus endophyte, bacillus megaterium, bacillus middlingeus, or bacillus subtilis. In some embodiments, the endospores comprise bacillus cucumber, bacillus endophyte, bacillus megaterium, or bacillus subtilis. In some embodiments, the endospores comprise bacillus cucumber. In some embodiments, the endospores comprise bacillus megatherium. In some embodiments, the endospores comprise bacillus cereus. In some embodiments, the endospores comprise bacillus subtilis.
In some embodiments, the microbial consortium is incorporated into the seed. In some embodiments of the present invention, in some embodiments, the consortium comprises a strain selected from the group consisting of Acetobacter, actinomycete, bacillus alcaligenes, aminophilia, bacillus bifidus, anaerobic bacillus, thiobacillus, anaerobic bacillus, bacillus, brevibacterium, thermoanaerobacter, caminiella, prunus, clostridium, ke Enshi, ke Kesi, saccharomyces, enterobacter desulphus, thermoanaerobacter, and Bacillus Desulfosporomula, desulfocampylobacter, desulfosporula, desulfosporium, protosporium, linear, gelria, geosporobacter, cellularomyces, halophilum, haloatron, solar, japanese, leishmania, chrysosporium, lysine bacillus, mahela, metabacterium, mushroom, mortierella, and Mortierella Desulfosporomula, desulfocampylobacter, desulfosporula, protosporium, linear, gelria, geosporium, geosporobacter, bacillus, halomatron, solar bacillus, solar bacterium, leishmania, chronic Bacillus, lysine bacillus, mahela, metabacterium, mushroom, bacillus, and Bacillus, two or more bacteria of the genera Thermoflavum, thermovelabalum, bacillus, cladosporium and Vulcanobacillus. In some embodiments, the consortium comprises two or more bacteria selected from the group consisting of acetobacter, actinomycetes, bacillus, chrysobacterium, ke Kesi body, sword, glutamate, microbacterium, and serratia. In some embodiments, the consortium comprises two, three, four, five, six, seven, eight, nine, ten or more bacteria. In some embodiments, the consortium comprises two bacteria. In some embodiments, the consortium comprises three bacteria. In some embodiments, the consortium comprises four bacteria. In some embodiments, the consortium comprises five bacteria. In some embodiments, the consortium comprises six bacteria. In some embodiments, the consortium comprises an endospore of any one of the microorganisms.
In some embodiments, the consortium comprises bacteria from the phylum bacillus and one or more bacteria. In some embodiments of the present invention, in some embodiments, the consortium comprises bacteria from the phylum Bacillus and is selected from the group consisting of Acetobacter, actinomyces, bacillus alcaligenes, aminophilia, bisporium, anaerobic bacillus, thiosporium, anaerobic bacillus, bacillus brevis, thermoanaerobacter, acidophilia, caminiella, prunella, clostridium, ke Enshi, ke Kesi, tree-derived sporophyta, desulphugoid, desulfosporula, desulphurisomum, desulfocampylobacter, desulfosporium, brevibacterium, gelria, geosporobacter, geobacillus, haliosporium, haliosporum, halonobium, solar bacillus, japanese bacillus, bacillus, lasiosporium, bacillus, leidella, makrigius, and Triplosis; metacteria, mushroom, natronella, bacillus megaterium, oreneia, ornithine bacillus, oxalic acid bacteria, acetobacter, paenibacillus, bacillus marinus, pelospora, anaerobic enterobacter, bacillus fish, phanerochaete, bacillus, propionispera, salinomyces, salmonella, qinghai, shimadzu, acetobacter anaerobic bacillus, sporobacter, bacillus, lactobacillus, banana, sporobacter, sporotalea, enteromorpha, co-cultured Monomonas, co-cultured Mortierella, paenibacillus, warm bacillus, geobacillus, deep sea bacillus, thermophilic Vinegar, thermoactinomyces, thermoalallibacillus, thermoanaerobacter, one or more bacteria of the genera Thermoanaerobonas, thermobacillus, thermoflavus, thermorenabalum, bacillus, cladosporium and Vulcanobacillus. In some embodiments, the consortium comprises two or more bacteria selected from the group consisting of acetobacter, actinomycetes, bacillus, chrysobacterium, ke Kesi body, sword, glutamate, microbacterium, and serratia. In some embodiments, the consortium comprises an endospore of any one of the microorganisms.
In some embodiments, the consortium comprises a mixture of two or more bacteria selected from the group consisting of bacillus plantarum, bacillus megaterium, bacillus midcuneatus, bacillus subtilis, golden yellow bacillus lactate, sword-like bacteria, glutamic acid bacteria of the research team, bacillus halogenes, microbacterium chocolate, microbacterium yannicii, pantoea allii, serratia marcescens, or serratia urealytica. In some embodiments, the consortium comprises a mixture of two or more bacteria selected from acetobacter cerevisiae, bacillus cucumber, bacillus plantarum, bacillus megaterium, bacillus middlingeus, bacillus subtilis, golden yellow bacillus lactis, bacillus clathratus, bacillus haloxyfomentarius, microbacterium chocolate, pantoea allii, and serratia marcescens. In some embodiments, the consortium comprises a mixture of two, three, four, five, six, seven, eight, nine, or ten bacteria. In some embodiments, the consortium comprises two bacteria. In some embodiments, the consortium comprises three bacteria. In some embodiments, the consortium comprises four bacteria. In some embodiments, the consortium comprises five bacteria. In some embodiments, the consortium comprises six bacteria. In some embodiments, the consortium comprises an endospore of any one of the microorganisms.
In some embodiments, the consortium comprises two or more bacteria selected from acetobacter cerevisiae, golden bacterium lacticum, bacillus cucumber, bacillus plantarum, bacillus megaterium, bacillus subtilis, and sword-shaped bacteria. In some embodiments, the consortium comprises two bacteria. In some embodiments, the consortium comprises three bacteria. In some embodiments, the consortium comprises four bacteria. In some embodiments, the consortium comprises five bacteria. In some embodiments, the consortium comprises six bacteria. In some embodiments, the consortium comprises seven bacteria. In some embodiments, the consortium comprises an endospore of any one of the microorganisms.
In some embodiments, the consortium comprises two or more bacteria selected from acetobacter cerevisiae, golden bacterium lacticum, bacillus plantarum, and bacillus megaterium. In some embodiments, the consortium comprises two bacteria selected from acetobacter cerevisiae, golden bacterium lacticum, bacillus plantarum, and bacillus megaterium. In some embodiments, the consortium comprises two bacteria selected from acetobacter cerevisiae, golden bacterium lacticum, bacillus plantarum, and bacillus megaterium. In some embodiments, the consortium comprises three bacteria selected from acetobacter cerevisiae, golden bacterium lacticum, bacillus within plants, and bacillus megaterium. In some embodiments, the consortium comprises a mixture of golden fungus lactate, bacillus within plants, and bacillus megaterium. In some embodiments, the consortium comprises a mixture of golden fungus lactate, bacillus within plants, and bacillus megaterium. In some embodiments, the consortium comprises an endospore of any one of the microorganisms.
In some embodiments, the consortium comprises two or more bacteria selected from bacillus subtilis, bacillus cucumber, and bacillus clarkii. In some embodiments, the consortium comprises a sword-shaped bacterium and bacillus subtilis or bacillus cucumber. In some embodiments, the consortium comprises sabia and bacillus subtilis. In some embodiments, the consortium comprises sabia and bacillus cucumber. In some embodiments, the consortium comprises an endospore of any one of the microorganisms.
In some embodiments, secretions from any of the microorganisms provided herein are incorporated into the cells. In some embodiments of the present invention, in some embodiments, the secretion is derived from Acetobacter, actinobacillus, bacillus alcaligenes, aminophilia, bacillus bifidus, anaerobic Bacillus, thiobacillus, anaerobic Bacillus, brevibacterium, thermoanaerobacter, caminiella, prunus, clostridium, ke Enshi, ke Kesi, acidocella, enterobacter of treorigin, bacillus desulphus, thermoanaerobacter, clostridium, and other bacteria Desulfosporomula, desulfocampylobacter, desulfosporium, desulfosporidium, protosporium, linear bacillus, gelria, georgia, geosporobacter, cellularomyces, halophilum, haloatron, solar bacillus, solar philia, leishmania, chrysosporium, lysine bacillus, mahela, metabacterium, mushroom, natroniella, desulfosporomula, desulfocampylobacter, desulfosporula, protosporula, linear, gelria, geosporobacter, cellularum, bacillus, pyricularia, and process for producing the same Bacillus, haloatron, solar, japanese, lei, chromobacterium, lysine, mahela, metacter, mulberry, natroniella, bacillus, and a pharmaceutical composition, bacteria of the genus Thermovenabalum, bacillus tumefaciens, bacillus, or Vulcanobacillus. In some embodiments, the secretion is from Acetobacter, actinobacillus, bacillus, chryseobacterium, ke Kesi, sword, gluconobacter, microbacterium, and Serratia. In some embodiments, the secretion is from acetobacter beer, bacillus cucumber, bacillus plantarum, bacillus megaterium, bacillus middlingeus, bacillus subtilis, golden yellow lactobacillus, bacillus clarkii, bacillus glutamicum of the research team, bacillus haloformans, microbacterium chocolate, microbacterium yannicii, pantoea allii, serratia marcescens, or serratia urealytica. In some embodiments, the secretion is from bacillus cucumber, bacillus plant, bacillus megaterium, bacillus middlingeus, or bacillus subtilis. In some embodiments, the secretion is from an endospore of any one of the microorganisms.
In some embodiments, the microorganism is selected for one or more characteristics related to its ability to interact with the plant. In some embodiments, the microorganism is selected for compatibility. In some embodiments, the microorganism is selected to ensure that predation or antagonism does not occur. In some embodiments, the microorganism is selected based on stability during storage. In some embodiments, microorganisms are selected based on rapid plant colonization and survival within the associated tissue. In some embodiments, the microorganism is selected based on optimal incorporation into one or more seeds. In some embodiments, the microorganism remains present throughout the plant life cycle.
In some embodiments, the microorganism incorporated into the seed is stable after incorporation. In some embodiments, the microorganism is stable for more than 30 days, more than six months, more than one year, or more than two years. In some embodiments, the microorganism is stable for more than 30 days. In some embodiments, the microorganism is stable for more than six months. In some embodiments, the microorganism is stable for more than one year. In some embodiments, the microorganism is stable for more than two years.
Compositions comprising plants and bacteria
In certain aspects, disclosed herein is a composition comprising a plant and one or more microorganisms associated with the plant, wherein the one or more microorganisms are or are derived from a microorganism selected to produce or promote bicarbonate and one or more mineral formations. In some embodiments, the composition is derived from culturing a plant or plant seed as described herein and one or more microorganisms associated with the plant or plant seed.
Modified plants
In one aspect, provided herein is a modified plant comprising a microorganism or microbial secretion incorporated into the plant. In some embodiments, the microorganism or secretion produces or promotes bicarbonate and one or more mineral formation. In some embodiments, bicarbonate formation sequesters CO 2 . In some embodiments, bicarbonate formation results in mineral formation. In some embodiments, the formed minerals stably sequester carbon in the soil. In some preferred embodiments, the microorganism is an endospore-forming bacterium or an endospore thereof.
In some embodiments, the microorganism or secretion is incorporated into the plant interior. In some embodiments, the microorganism or secretion is incorporated under the pericarp of the plant. In some embodiments, the microorganism or secretion is incorporated between the pericarp and aleurone cell layer of the plant. In some embodiments, the microorganism or secretion contacts the embryo of the plant. In some embodiments, the microorganism or secretion does not contact the embryo of the plant. In some embodiments, the microorganism or secretion contacts the endosperm of the plant. In some embodiments, the microorganism or secretion does not contact the endosperm of the plant. In some embodiments, the microorganism or secretion is incorporated into the gap between the plant's plant skin and plant embryo. In some embodiments, the microorganism or secretion is incorporated into the gap between the plant pericarp and the plant aleurone cell layer.
The modified plant may be any type of plant. In some embodiments, the modified plant is a monocot. In some embodiments, the plant is a maize, wheat, rice, barley, rye, sugarcane, millet, oat, or sorghum plant. In some embodiments, the plant is a maize plant. In some embodiments, the plant is a maize (Zea mail) plant. In some embodiments, the modified plant is a dicot. In some embodiments, the plant is a soybean, cotton, alfalfa, bean, quinoa, lentil, peanut, sunflower, canola, cassava, oil palm, potato, sugar beet, cocoa, coffee, lettuce, tomato, or cabbage plant. In some embodiments, the plant is a lettuce plant. In some embodiments, the plant is a lettuce (Lactuca sativa) plant. In some embodiments, the plant is a tomato plant. In some embodiments, the plant is a tomato (Solanum lycopersicum) plant. In some embodiments, the plant is a Genetically Modified Organism (GMO) plant. In some embodiments, the plant is a non-GMO plant.
The amount of microorganisms or secretions incorporated into the plant must be at a sufficient level to effectively sequester the CO 2 . In some embodiments, the amount of microorganisms incorporated into the plants is from about 250 Colony Forming Units (CFU) to about 5,000CFU. In some embodiments, the microorganism is incorporated into the plant in an amount of about 250CFU to about 500CFU, about 250CFU to about 750CFU, about 250CFU to about 1,000CFU, about 250CFU to about 2,000CFU, about 250CFU to about 3,000CFU, about 250CFU to about 4,000CFU, about 250CFU to about 5,000CFU, about 500CFU to about 750CFU, about 500CFU to about 1,000CFU, about 500CFU to about 2,000CFU, about 500CFU to about 3,000CFU, about 500CFU to about 4,000CFU, about 500CFU to about 5,000CFU, about 750CFU to about 1,000CFU, about 750CFU to about 2,000CFU, about 750CFU to about 3,000CFU, about 750CFU to about 4,000CFU, about 750CFU to about 5,000CFU, about 1,000CFU to about 2,000CFU, about 1,000CFU to about 3,000CFU, about 1,000CFU to about 4,000CFU, about 1,000CFU to about 37, about 37 to about 5,000CFU, or about 37 to about 5,000CFU. In some embodiments, the amount of microorganism incorporated into the plant is about250CFU, about 500CFU, about 750CFU, about 1,000CFU, about 2,000CFU, about 3,000CFU, about 4,000CFU, or about 5,000CFU. In some embodiments, the amount of microorganism incorporated into the plant is at least about 250CFU, about 500CFU, about 750CFU, about 1,000CFU, about 2,000CFU, about 3,000CFU, or about 4,000CFU. In some embodiments, the amount of microorganisms incorporated into the plant is up to about 500CFU, about 750CFU, about 1,000CFU, about 2,000CFU, about 3,000CFU, about 4,000CFU, or about 5,000CFU. In some embodiments, at least about 500CFU is incorporated into a plant. In some embodiments, at least about 1000CFU is incorporated into a plant.
In some embodiments, the microorganism or secretion incorporated into the plant is shelf stable for a prolonged period of time. In some embodiments, the modified plant is shelf stable for about 3 months to about 36 months. In some embodiments of the present invention, in some embodiments, the modified plant is in the range of about 3 months to about 6 months, about 3 months to about 9 months, about 3 months to about 12 months, about 3 months to about 15 months, about 3 months to about 18 months, about 3 months to about 21 months, about 3 months to about 24 months, about 3 months to about 30 months, about 3 months to about 36 months, about 6 months to about 9 months, about 6 months to about 12 months, about 6 months to about 15 months, about 6 months to about 18 months, about 6 months to about 21 months, about 6 months to about 24 months, about 6 months to about 30 months, about 6 months to about 36 months, about 9 months to about 12 months, about 9 months to about 15 months, about 9 months to about 18 months, about 9 months to about 21 months, about 9 months to about 24 months, about 9 months to about 30 months the storage is stable for from about 9 months to about 36 months, from about 12 months to about 15 months, from about 12 months to about 18 months, from about 12 months to about 21 months, from about 12 months to about 24 months, from about 12 months to about 30 months, from about 12 months to about 36 months, from about 15 months to about 18 months, from about 15 months to about 21 months, from about 15 months to about 24 months, from about 15 months to about 30 months, from about 15 months to about 36 months, from about 18 months to about 21 months, from about 18 months to about 24 months, from about 18 months to about 30 months, from about 18 months to about 36 months, from about 21 months to about 24 months, from about 21 months to about 30 months, from about 21 months to about 36 months, from about 24 months to about 30 months, from about 24 months to about 36 months, or from about 30 months to about 30 months. In some embodiments, the modified plant is shelf stable for about 3 months, about 6 months, about 9 months, about 12 months, about 15 months, about 18 months, about 21 months, about 24 months, about 30 months, or about 36 months. In some embodiments, the modified plant is shelf stable for at least about 3 months, about 6 months, about 9 months, about 12 months, about 15 months, about 18 months, about 21 months, about 24 months, or about 30 months. In some embodiments, the modified plant is shelf stable for up to about 6 months, about 9 months, about 12 months, about 15 months, about 18 months, about 21 months, about 24 months, about 30 months, or about 36 months.
In some embodiments, the microorganism incorporated into the plant is stable after incorporation. In some embodiments, the microorganism is stable for more than 30 days, more than six months, more than one year, or more than two years. In some embodiments, the microorganism is stable for more than 30 days. In some embodiments, the microorganism is stable for more than six months. In some embodiments, the microorganism is stable for more than one year. In some embodiments, the microorganism is stable for more than two years.
The microorganism or secretion thereof incorporated into the plant may be any one of the microorganisms provided herein or any other microorganism. In some embodiments, the microorganism is a microorganism (microbe). In some embodiments, the microorganism is an endospore-forming microorganism. In some embodiments, the microorganism is an endospore-forming microorganism or an endospore thereof. In some embodiments, the microorganism is an endospore of a microorganism provided herein. In some embodiments, the microorganism is an endospore-forming bacterium or an endospore thereof.
Methods of incorporating bacteria
In one aspect, provided herein are methods of incorporating one or more microorganisms or secretions thereof into one or more plants. In some embodiments, the method comprises sterilizing the plant. In some embodiments, the method comprises contacting the plant with a solution comprising one or more microorganisms or secretions thereof. In some embodiments, the solution further comprises a salt. In some embodiments, the method comprises incubating the plant with the solution for a period of time. In some embodiments, the period of time is sufficient to allow a desired amount of microorganisms or secretions thereof to enter the plant. In some embodiments, the method incorporates a desired amount of a microorganism or secretion thereof into a plant.
In some embodiments, the method comprises contacting the plant with a solution comprising a salt as described herein.
In some embodiments, the solution comprises additional additives as described herein.
In some embodiments, the solution comprises additional metal ions as described herein.
In some embodiments, the solution comprises one or more nutrients for the microorganism as described herein. In some embodiments, the solution comprises a bacterial growth medium. In some embodiments, the solution comprises a Lysogenic Broth (LB), a nutrient broth, or a combination thereof. In some embodiments, the solution comprises a lysogenic broth. In some embodiments, the solution comprises a nutrient broth.
In some embodiments, the solution comprises a microorganism as described herein.
In some embodiments, the solution comprises a desired amount of microorganisms per plant mass as described herein.
In some embodiments, the plant comprises a desired amount of microorganisms per plant as described herein. In some embodiments, the microorganism is a bacterium. In some embodiments, the bacteria are endospore-forming bacteria. In some embodiments, the method comprises inducing endospores formation of the endospore-forming bacteria. In some embodiments, the bacteria incorporated into the plant are endospores. In some embodiments, the solution comprises one or more components that induce formation of endospores. In some embodiments, the solution comprises potassium, ferrous sulfate, calcium, magnesium, manganese, or a combination thereof.
In some embodiments, the method comprises sterilizing the plant. In some embodiments, the method comprises sterilizing the plant surface. Any method of producing plants having a sterilized surface may be used. In some embodiments, the plants are sterilized with a bleaching solution. In some embodiments, the plants are sterilized prior to immersing the plants in the solution containing the one or more microorganisms. In some embodiments, the plant is a sterilized plant. In some embodiments, the plant has a sterilized surface. As used herein, "sterilized," "sterilized" and related terms (e.g., "sanitized" and the like) mean that there are substantially no living microorganisms on the sterilized article. In some embodiments, the plants are sterilized prior to incubating the plants in the solution comprising the microorganism. In some embodiments, the plants are sterilized after incubating the plants in the solution comprising the microorganism. In some embodiments, the fungicide is added to the plant surface.
In some embodiments, the sterilized or disinfected plant comprises substantially no viable microorganisms on the plant (e.g., plant surface). In some embodiments, the sterile or sterilized plant comprises less than 1CFU, less than 5CFU, less than 10CFU, less than 20CFU, less than 30CFU, less than 40CFU, or less than 50CFU of microorganisms on the plant.
In some embodiments, the plant is incubated with the solution containing the microorganism for a time sufficient to incorporate the microorganism into the plant.
Microorganisms and secretions
The microorganisms provided herein, or secretions thereof, produce or promote bicarbonate and one or more mineral formations. In some embodiments, bicarbonate formation sequesters CO 2 . In some embodiments, bicarbonate formation results in mineral formation. In some embodiments, the formed minerals stably sequester carbon in the soil. In some embodiments, the microorganism is a bacterium as described herein. In some embodiments, the microorganism is an endospore-forming bacterium. In some embodiments, the microorganism is a bacterial endospore. Whenever a microorganism (e.g., a bacterium) referred to herein is capable of forming an endophyte, it is intended to also encompass the microorganismAny endospores of the species. For example, if the plant treatment formulation comprises bacillus, the formulation may comprise endospores of bacillus.
In some embodiments, the microorganism is a microorganism from the phylum firmicutes, the phylum proteus, and the phylum actinomycetes. In some embodiments, the microorganism is a microorganism from the phylum firmicutes. In some embodiments, the microorganism is a microorganism from the phylum Proteus. In some embodiments, the microorganism is a microorganism from the phylum actinomycetes. In some embodiments, the microorganism is an endospore of any one of the microorganisms.
Rhizobacteria that fix atmospheric nitrogen are present on the roots of plants. These organisms are able to survive in soil and react to large amounts of CO 2 Tolerating CO 2 Is released by the roots of plants during respiration or by nearby microorganisms and soil animal communities through respiration.
In some embodiments, the bacteria are not genetically modified. In some embodiments, the CO is according to bacteria 2 The ability to convert to bicarbonate and ultimately to minerals is the choice of bacteria. In some embodiments, the bacteria are selected according to the use of carbonic anhydrase.
In some embodiments, a plurality of rhizobacteria are effectively loaded into the seed. In some embodiments, the rhizobacteria include endospore-forming bacteria that enhance biological nitrogen fixation. In some embodiments, the rhizobacteria comprise bacillus, paenibacillus, or both. In some embodiments, the one or more microorganisms comprise bacillus amyloliquefaciens, bacillus laterosporus, bacillus licheniformis, bacillus macerans, bacillus cereus, bacillus circulans, bacillus firmus, bacillus subtilis, bacillus sphaericus, bacillus megaterium, bacillus coagulans, bacillus brevis, bacillus thuringiensis, bacillus mycoides, bacillus cucumber, bacillus plantarum, bacillus pumilus, bacillus bailii, bacillus mucilaginosus, bacillus tertazii, bacillus methylotrophicus, or any combination thereof. In some embodiments, the one or more microorganisms comprise bacillus subtilis S3C23, bacillus subtilis MP2, bacillus subtilis RO2C15, bacillus subtilis RO2C22, bacillus megaterium 6, bacillus megaterium S3C21, bacillus megaterium RO2C12, bacillus cucumber S3C14, bacillus plantarum 5, or any combination thereof. In some embodiments, the one or more microorganisms comprise bacillus subtilis S3C23. In some embodiments, the one or more microorganisms comprise bacillus subtilis MP2.
In some embodiments, the one or more microorganisms comprise a genetic modification that results in the one or more microorganisms producing or promoting more carbonic anhydrase formation relative to the corresponding wild-type microorganism. In some embodiments, the genetic modification comprises a nucleic acid construct, wherein the nucleic acid construct comprises one or more promoters configured to drive expression of a Carbonic Anhydrase (CA) coding sequence. In some embodiments, the genetic modification comprises a nucleic acid construct, wherein the nucleic acid construct comprises a Carbonic Anhydrase (CA) coding sequence. In some embodiments, the CA coding sequence is heterologous to the one or more microorganisms. In some embodiments, the CA coding sequence is endogenous to the one or more microorganisms. In some embodiments, the nucleic acid construct is codon optimized. In some embodiments, the nucleic acid construct further comprises one or more promoters configured to drive expression of the CA coding sequence. In some embodiments, the one or more promoters drive constitutive expression of the CA coding sequence. In some embodiments, the one or more promoters drive inducible expression of the CA coding sequence. In some embodiments, the genetic modification comprises a carbonic anhydrase gene that has been modified by directed microbial evolution. In some embodiments, the genetic modification comprises a signal sequence. In some embodiments, the signal sequence comprises a periplasmic signal sequence or an extracellular secretion signal. In some embodiments, the periplasmic signal sequence or the extracellular secretion signal is located 5' to the carbonic anhydrase coding sequence. In some embodiments, the periplasmic signal sequence or the extracellular secretion signal is fused to the 5' end of the carbonic anhydrase coding sequence. In some embodiments, the genetic modification comprises one or more components of the general secretory pathway, and wherein the genetic modification results in secretion or subcellular targeting of carbonic anhydrase by the one or more microorganisms.
In some embodiments, the one or more promoters are or are derived from one or more promoters comprised in bacillus or paenibacillus. In some embodiments, the one or more promoters are selected from P groES 、P 43 、P sigX 、P trnQ And P xylA . In some embodiments, the one or more promoters are or are derived from one or more housekeeping gene promoters or strongly expressed constitutive promoters. In some embodiments, the one or more housekeeping gene promoters or strong expressed constitutive promoters are selected from the group consisting of P liaG 、P lepA 、P veg 、P gsiB 、P43、P trnQ 、P lial (bacitracin-inducible) and P xylA (xylose inducible).
In some embodiments, the genetic modification comprises introducing an expression vector into one or more microorganisms. In some embodiments, the genetic modification comprises modification of a chromosome of one or more microorganisms.
In some embodiments, the microorganism can immobilize nitrogen and carbon dioxide. In some embodiments, the nitrogen-fixing microorganisms will continue to provide fixed nitrogen (ammonia) to the plants via biological nitrogen fixation, which will reduce the use of chemical fertilizers that pollute the environment. The end product (ammonia) of atmospheric nitrogen fixation can enhance CO 2 To minerals, as ammonia has been reported to increase pH, thereby accelerating mineralization. Because ammonia produced by the microorganisms disclosed herein is significantly higher than other soil-dwelling microorganisms, even in the presence of an external nitrogen source, the mineralization process of the strains disclosed herein may be faster than other microorganisms that inhabit the soil.
In some embodiments of the present invention, in some embodiments, the microorganism is selected from Acetobacter, actinobacillus, bacillus alcaligenes, aminophilia, bacillus bifidus, anaerobic Bacillus, thiobacillus, anaerobic Bacillus, brevibacillus, thermoanaerobacter, caminiella, prunus, clostridium, ke Enshi, ke Kesi, acidocella, desulphurized Enterobacter, bacillus, and Bacillus Desulfosporomula, desulfocampylobacter, desulfosporula, desulfosporium, protosporium, linear, gelria, geosporobacter, cellularomyces, halophilum, haloatron, solar, japanese, leishmania, chrysosporium, lysine bacillus, mahela, metabacterium, mushroom, mortierella, and Mortierella Desulfosporomula, desulfocampylobacter, desulfosporula, protosporium, linear, gelria, geosporium, geosporobacter, bacillus, halomatron, solar bacillus, solar bacterium, leishmania, chronic Bacillus, lysine bacillus, mahela, metabacterium, mushroom, bacillus, and Bacillus, microorganisms of the genus Thermoflavum, thermovelabalum, bacillus, cladosporium and Vulcanobacillus. In some embodiments, the microorganism is a microorganism selected from the group consisting of Acetobacter, actinobacillus, bacillus, chryseobacterium, ke Kesi, sword, gluconobacter, microbacterium, and Serratia. In some embodiments, the microorganism is acetobacter. In some embodiments, the microorganism is an actinomycete. In some embodiments, the microorganism is bacillus. In some embodiments, the microorganism is a chrysobacterium. In some embodiments, the microorganism is of the genus Ke Kesi. In some embodiments, the microorganism is a genus xiphoid. In some embodiments, the microorganism is a genus glutamate. In some embodiments, the microorganism is a genus microbacterium. In some embodiments, the microorganism is pantoea. In some embodiments, the microorganism is serratia. In some embodiments, the microorganism is an endospore of any one of the microorganisms.
In some embodiments, the microorganism is selected for one or more characteristics related to its ability to interact with the plant. In some embodiments, the microorganism is selected for compatibility. In some embodiments, the microorganism is selected to ensure that predation or antagonism does not occur. In some embodiments, the microorganism is selected based on stability during storage. In some embodiments, microorganisms are selected based on rapid plant colonization and survival within the associated tissue. In some embodiments, the microorganism is selected for optimal incorporation into one or more plants. In some embodiments, the microorganism remains present throughout the plant life cycle.
In some embodiments, the microorganism incorporated into the plant is stable after incorporation. In some embodiments, the microorganism is stable for more than 30 days, more than six months, more than one year, or more than two years. In some embodiments, the microorganism is stable for more than 30 days. In some embodiments, the microorganism is stable for more than six months. In some embodiments, the microorganism is stable for more than one year. In some embodiments, the microorganism is stable for more than two years.
Method for producing bicarbonate and minerals
In certain aspects, disclosed herein is a method of promoting bicarbonate formation and mineralization, the method comprising: (a) Cultivating a plant and one or more microorganisms associated with the plant, plant root and/or rhizosphere, wherein the one or more microorganisms are or are derived from microorganisms selected to produce or promote bicarbonate and one or more minerals.
Modified plants
In some embodiments, the one or more microorganisms associated with the plant are disposed on the root or rhizosphere of the plant. In some embodiments, the one or more microorganisms associated with the plant are disposed on the root or the rhizosphere of the plant by irrigation.
In some embodiments, the plant is derived from a seedling irrigated with a microorganism disclosed herein selected to stimulate the plant to produce the one or more minerals disclosed herein. In some embodiments, the plant is derived from culturing a plant seed as described herein and one or more microorganisms associated with the plant seed. In some embodiments, the seed is initiated by the methods described herein.
In one aspect, provided herein is a method comprising culturing a plant comprising a microorganism or microbial secretion incorporated into the plant. In some preferred embodiments, the microorganism is an endospore-forming bacterium or an endospore thereof.
In some embodiments, the amount of minerals produced may be 50 kg/acre to 1000 kg/acre. In some embodiments, the amount of minerals produced may be from about 50 kg/acre to about 1,000 kg/acre. In some embodiments of the present invention, in some embodiments, the amount of minerals produced may be about 50 kg/acre to about 100 kg/acre, about 50 kg/acre to about 200 kg/acre, about 50 kg/acre to about 300 kg/acre, about 50 kg/acre to about 400 kg/acre, about 50 kg/acre to about 500 kg/acre, about 50 kg/acre to about 600 kg/acre, about 50 kg/acre to about 700 kg/acre, about 50 kg/acre to about 800 kg/acre, about 50 kg/acre to about 900 kg/acre, about 50 kg/acre to about 1,000 kg/acre, about 100 kg/acre to about 200 kg/acre, about 100 kg/acre to about 300 kg/acre, about 100 kg/acre to about 400 kg/acre, about 100 kg/acre to about 500 kg/acre, about 100 kg/acre to about 600 kg/acre about 100 kg/acre to about 700 kg/acre, about 100 kg/acre to about 800 kg/acre, about 100 kg/acre to about 900 kg/acre, about 100 kg/acre to about 1,000 kg/acre, about 200 kg/acre to about 300 kg/acre, about 200 kg/acre to about 400 kg/acre, about 200 kg/acre to about 500 kg/acre, about 200 kg/acre to about 600 kg/acre, about 200 kg/acre to about 700 kg/acre, about 200 kg/acre to about 800 kg/acre, about 200 kg/acre to about 900 kg/acre, about 200 kg/acre to about 1,000 kg/acre, about 300 kg/acre to about 400 kg/acre, about 300 kg/acre to about 500 kg/acre, about 300 kg/acre to about 600 kg/acre, about 300 kg/acre to about 800 kg/acre, about 300 kg/acre to about 900 kg/acre, about 300 kg/acre to about 1,000 kg/acre, about 400 kg/acre to about 500 kg/acre, about 400 kg/acre to about 600 kg/acre, about 400 kg/acre to about 700 kg/acre, about 400 kg/acre to about 800 kg/acre, about 400 kg/acre to about 900 kg/acre, about 400 kg/acre to about 1,000 kg/acre, about 500 kg/acre to about 600 kg/acre, about 500 kg/acre to about 700 kg/acre, about 500 kg/acre to about 500 kg/acre, about 500 kg/acre to about 1,000 kg/acre, about 1 kg/acre to about 600 kg/acre, about 600 kg/acre to about 600 kg/acre, about 1 kg/acre to about 600 kg/acre. In some embodiments, the amount of minerals produced may be about 50 kg/acre, about 100 kg/acre, about 200 kg/acre, about 300 kg/acre, about 400 kg/acre, about 500 kg/acre, about 600 kg/acre, about 700 kg/acre, about 800 kg/acre, about 900 kg/acre, or about 1,000 kg/acre. In some embodiments, the amount of minerals produced may be at least about 50 kg/acre, about 100 kg/acre, about 200 kg/acre, about 300 kg/acre, about 400 kg/acre, about 500 kg/acre, about 600 kg/acre, about 700 kg/acre, about 800 kg/acre, or about 900 kg/acre. In some embodiments, the amount of minerals produced may be up to about 100 kg/acre, about 200 kg/acre, about 300 kg/acre, about 400 kg/acre, about 500 kg/acre, about 600 kg/acre, about 700 kg/acre, about 800 kg/acre, about 900 kg/acre, or about 1,000 kg/acre.
In some embodiments, the microorganism or secretion is incorporated into the plant interior. In some embodiments, the microorganism or secretion is incorporated under the pericarp of the plant. In some embodiments, the microorganism or secretion is incorporated between the pericarp and aleurone cell layer of the plant. In some embodiments, the microorganism or secretion contacts the embryo of the plant. In some embodiments, the microorganism or secretion does not contact the embryo of the plant. In some embodiments, the microorganism or secretion contacts the endosperm of the plant. In some embodiments, the microorganism or secretion does not contact the endosperm of the plant. In some embodiments, the microorganism or secretion is incorporated into the gap between the plant's plant skin and plant embryo. In some embodiments, the microorganism or secretion is incorporated into the gap between the plant pericarp and the plant aleurone cell layer.
The modified plant may be any type of plant. In some embodiments, the modified plant is a monocot. In some embodiments, the plant is a maize, wheat, rice, barley, rye, sugarcane, millet, oat, or sorghum plant. In some embodiments, the plant is a maize plant. In some embodiments, the plant is a maize (Zea mail) plant. In some embodiments, the modified plant is a dicot. In some embodiments, the plant is a soybean, cotton, alfalfa, bean, quinoa, lentil, peanut, sunflower, canola, cassava, oil palm, potato, sugar beet, cocoa, coffee, lettuce, tomato, or cabbage plant. In some embodiments, the plant is a lettuce plant. In some embodiments, the plant is a lettuce (Lactuca sativa) plant. In some embodiments, the plant is a tomato plant. In some embodiments, the plant is a tomato (Solanum lycopersicum) plant. In some embodiments, the plant is a Genetically Modified Organism (GMO) plant. In some embodiments, the plant is a non-GMO plant. In some embodiments, the plant is a monocot or dicot. In some embodiments, the plant is maize, wheat, rice, sorghum, barley, rye, sugarcane, millet, oat, soybean, cotton, alfalfa, bean, quinoa, lentils, peanut, sunflower, canola, cassava, oil palm, potato, sugar beet, cocoa, coffee, lettuce, tomato, pea, or cabbage.
The amount of microorganisms or secretions incorporated into the plant must be at a sufficient level to effectively sequester the CO 2 . In some embodiments, the amount of microorganisms incorporated into the plants is from about 250 Colony Forming Units (CFU) to about 5,000CFU. In some embodiments, the microorganism is incorporated into the plant in an amount of about 250CFU to about 500CFU, about 250CFU to about 750CFU, about 250CFU to about 1,000CFU, about 250CFU to about 2,000CFU, about 250CFU to about 3,000CFU, about 250CFU to about 4,000CFU, about 250CFU to about 5,000CFU, about 500CFU to about 750CFU, about 500CFU to about 1,000CFU, about 500CFU to about 2,000CFU, about 500CFU to about 3,000CFU, about 500CFU to about 4,000CFU, about 500CFU to about 5,000CFU, about 750CFU to about 1,000CFU, about 750CFU to about 2,000CFU, about 750CFU to about 3,000CFU, about 750CFU to about 4,000CFU, about 750CFU to about 5,000CFU, about 1,000CFU to about 2,000CFU, about 1,000CFU to about 3,000CFU, about 1,000CFU to about 4,000CFU, about 1,000CFU to about 37, about 37 to about 5,000CFU, or about 37 to about 5,000CFU. In some embodiments, the amount of microorganism incorporated into the plant is about 250CFU, about 500CFU, about 750CFU, about 1,000CFU, about 2,000CFU, about 3,000CFU, about 4,000CFU, or about 5,000CFU. In some embodiments, the amount of microorganism incorporated into the plant is at least about 250CFU, about 500CFU, about 750CFU, about 1,000CFU, about 2,000CFU, about 3,000CFU, or about 4,000CFU. In some embodiments, the amount of microorganisms incorporated into the plant is up to about 500CFU, about 750CFU, about 1,000CFU, about 2,000CFU, about 3,000CFU, about 4,000CFU, or about 5,000CFU. In some embodiments, at least about 500CFU is incorporated into a plant. In some embodiments, at least about 1000CFU is incorporated into a plant.
In some embodiments, the microorganism or secretion incorporated into the plant is shelf stable for a prolonged period of time. In some embodiments, the modified plant is shelf stable for about 3 months to about 36 months. In some embodiments of the present invention, in some embodiments, the modified plant is in the range of about 3 months to about 6 months, about 3 months to about 9 months, about 3 months to about 12 months, about 3 months to about 15 months, about 3 months to about 18 months, about 3 months to about 21 months, about 3 months to about 24 months, about 3 months to about 30 months, about 3 months to about 36 months, about 6 months to about 9 months, about 6 months to about 12 months, about 6 months to about 15 months, about 6 months to about 18 months, about 6 months to about 21 months, about 6 months to about 24 months, about 6 months to about 30 months, about 6 months to about 36 months, about 9 months to about 12 months, about 9 months to about 15 months, about 9 months to about 18 months, about 9 months to about 21 months, about 9 months to about 24 months, about 9 months to about 30 months the storage is stable for from about 9 months to about 36 months, from about 12 months to about 15 months, from about 12 months to about 18 months, from about 12 months to about 21 months, from about 12 months to about 24 months, from about 12 months to about 30 months, from about 12 months to about 36 months, from about 15 months to about 18 months, from about 15 months to about 21 months, from about 15 months to about 24 months, from about 15 months to about 30 months, from about 15 months to about 36 months, from about 18 months to about 21 months, from about 18 months to about 24 months, from about 18 months to about 30 months, from about 18 months to about 36 months, from about 21 months to about 24 months, from about 21 months to about 30 months, from about 21 months to about 36 months, from about 24 months to about 30 months, from about 24 months to about 36 months, or from about 30 months to about 30 months. In some embodiments, the modified plant is shelf stable for about 3 months, about 6 months, about 9 months, about 12 months, about 15 months, about 18 months, about 21 months, about 24 months, about 30 months, or about 36 months. In some embodiments, the modified plant is shelf stable for at least about 3 months, about 6 months, about 9 months, about 12 months, about 15 months, about 18 months, about 21 months, about 24 months, or about 30 months. In some embodiments, the modified plant is shelf stable for up to about 6 months, about 9 months, about 12 months, about 15 months, about 18 months, about 21 months, about 24 months, about 30 months, or about 36 months.
In some embodiments, the microorganism incorporated into the plant is stable after incorporation. In some embodiments, the microorganism is stable for more than 30 days, more than six months, more than one year, or more than two years. In some embodiments, the microorganism is stable for more than 30 days. In some embodiments, the microorganism is stable for more than six months. In some embodiments, the microorganism is stable for more than one year. In some embodiments, the microorganism is stable for more than two years.
The microorganism or secretion thereof incorporated into the plant may be any one of the microorganisms provided herein or any other microorganism. In some embodiments, the microorganism is a microorganism (microbe). In some embodiments, the microorganism is an endospore-forming microorganism. In some embodiments, the microorganism is an endospore-forming microorganism or an endospore thereof. In some embodiments, the microorganism is an endospore of a microorganism provided herein. In some embodiments, the microorganism is an endospore-forming bacterium or an endospore thereof.
Microorganisms and secretions
The microorganisms provided herein, or secretions thereof, are capable of producing or promoting bicarbonate and one or more mineral formations. In some embodiments, bicarbonate formation sequesters CO 2 . In some embodiments, bicarbonate formation results in mineral formation. In some embodiments, the formed minerals stably sequester carbon in the soil. In some embodiments, the microorganism is an endospore-forming bacterium. In some embodiments, the microorganism is a bacterial endospore. Whenever a microorganism (e.g., a bacterium) referred to herein is capable of forming an endospore, it is intended to encompass any endospore of the microorganism as well. For example, if the plant treatment formulation comprises bacillus, the formulation may comprise endospores of bacillus.
In some embodiments, the microorganism is a microorganism from the phylum firmicutes, the phylum proteus, and the phylum actinomycetes. In some embodiments, the microorganism is a microorganism from the phylum firmicutes. In some embodiments, the microorganism is a microorganism from the phylum Proteus. In some embodiments, the microorganism is a microorganism from the phylum actinomycetes. In some embodiments, the microorganism is an endospore of any one of the microorganisms.
Rhizobacteria that fix atmospheric nitrogen are present on the roots of plants. These organisms are able to survive in soil and react to large amounts of CO 2 Tolerating CO 2 Is released by the roots of plants during respiration or by nearby microorganisms and soil animal communities through respiration.
In some embodiments, the bacteria are not genetically modified. In some embodiments, the CO is according to bacteria 2 The ability to convert to bicarbonate and ultimately to minerals is the choice of bacteria. In some embodiments, the bacteria are selected according to the use of carbonic anhydrase. In some embodiments, the bacteria are selected according to the use of carbonic anhydrase. In some embodiments, a plurality of rhizobacteria are effectively loaded into the seed. In some embodiments, the rhizobacteria comprise bacillus, paenibacillus, or both. In some embodiments, the one or more microorganisms comprise bacillus amyloliquefaciens, bacillus laterosporus, bacillus licheniformis, bacillus macerans, bacillus cereus, bacillus circulans, bacillus firmus, bacillus subtilis, bacillus sphaericus, bacillus megaterium, bacillus coagulans, bacillus brevis, bacillus thuringiensis, bacillus mycoides, bacillus cucumber, bacillus plantarum, bacillus pumilus, bacillus bailii, bacillus mucilaginosus, bacillus tertazii, bacillus methylotrophicus, or any combination thereof. In some embodiments, the one or more microorganisms comprise bacillus subtilis S3C23, bacillus subtilis MP2, bacillus subtilis RO2C15, bacillus subtilis RO2C22, bacillus megaterium 6, bacillus megaterium S3C21, bacillus megaterium RO2C12, bacillus cucumber S3C14, bacillus plantarum 5, or any combination thereof. In some embodiments, the one or more microorganisms comprise bacillus subtilis S3C23. In some embodiments, the one or more microorganisms comprise a seed cake Bacillus subtilis MP2.
In some embodiments, the one or more microorganisms comprise a genetic modification that results in the one or more microorganisms producing or promoting more carbonic anhydrase formation relative to the corresponding wild-type microorganism. In some embodiments, the genetic modification comprises a nucleic acid construct, wherein the nucleic acid construct comprises one or more promoters configured to drive expression of a Carbonic Anhydrase (CA) coding sequence. In some embodiments, the genetic modification comprises a nucleic acid construct, wherein the nucleic acid construct comprises a Carbonic Anhydrase (CA) coding sequence. In some embodiments, the CA coding sequence is heterologous to the one or more microorganisms. In some embodiments, the CA coding sequence is endogenous to the one or more microorganisms. In some embodiments, the nucleic acid construct is codon optimized. In some embodiments, the nucleic acid construct further comprises one or more promoters configured to drive expression of the CA coding sequence. In some embodiments, the one or more promoters drive constitutive expression of the CA coding sequence. In some embodiments, the one or more promoters drive inducible expression of the CA coding sequence. In some embodiments, the genetic modification comprises a carbonic anhydrase gene that has been modified by directed microbial evolution. In some embodiments, the genetic modification comprises a signal sequence. In some embodiments, the signal sequence comprises a periplasmic signal sequence or an extracellular secretion signal. In some embodiments, the periplasmic signal sequence or the extracellular secretion signal is located 5' to the carbonic anhydrase coding sequence. In some embodiments, the periplasmic signal sequence or the extracellular secretion signal is fused to the 5' end of the carbonic anhydrase coding sequence. In some embodiments, the genetic modification comprises one or more components of the general secretory pathway, and wherein the genetic modification results in secretion or subcellular targeting of carbonic anhydrase by the one or more microorganisms.
In some embodiments, the one or more promotersIs or is derived from one or more promoters contained in the genus Bacillus or Paenibacillus. In some embodiments, the one or more promoters are selected from P groES 、P43、P sigX 、P trnQ And P xylA . In some embodiments, the one or more promoters are or are derived from one or more housekeeping gene promoters or strongly expressed constitutive promoters. In some embodiments, the one or more housekeeping gene promoters or strong expressed constitutive promoters are selected from the group consisting of P liaG 、P lepA 、P veg 、P gsiB 、P43、P trnQ 、P lial (bacitracin-inducible) and P xylA (xylose inducible).
In some embodiments, the genetic modification comprises introducing an expression vector into one or more microorganisms. In some embodiments, the genetic modification comprises modification of a chromosome of one or more microorganisms.
In some embodiments, the microorganism can immobilize nitrogen and carbon dioxide. In some embodiments, the nitrogen-fixing microorganisms will continue to provide fixed nitrogen (ammonia) to the plants via biological nitrogen fixation, which will reduce the use of chemical fertilizers that pollute the environment. The end product (ammonia) of atmospheric nitrogen fixation can enhance CO 2 To minerals, as ammonia has been reported to increase pH, thereby accelerating mineralization. Because ammonia produced by the microorganisms disclosed herein is significantly higher than other soil-dwelling microorganisms, even in the presence of an external nitrogen source, the mineralization process of the strains disclosed herein may be faster than other microorganisms that inhabit the soil.
Method for sequestering or converting carbon into bicarbonate and minerals
In certain aspects, disclosed herein is a method of sequestering carbon, the method comprising: a. cultivating a plant and one or more microorganisms associated with the plant, wherein the one or more microorganisms are or are derived from microorganisms selected to produce or promote bicarbonate and one or more mineral formations.
Modified plants
In one aspect, provided herein areA method of sequestering carbon comprising culturing a modified plant comprising a microorganism or microbial secretion incorporated into the plant. In some embodiments, the microorganism or secretion produces or promotes bicarbonate and one or more mineral formation. In some embodiments, bicarbonate formation sequesters CO 2 . In some embodiments, bicarbonate formation results in mineral formation. In some embodiments, the formed minerals stably sequester carbon in the soil. In some preferred embodiments, the microorganism is an endospore-forming bacterium or an endospore thereof.
In some embodiments, the CO converted by the microorganism 2 The amount of CO was 0.1 ton 2 Per acre to 2.5 tons of CO 2 Per acre. In some embodiments, the microorganism converts 2.5 to 5.3 tons of CO2 per acre. In some embodiments, the microorganism converts 5.3 to 7.5 tons of CO 2 Per acre. In some embodiments, the microorganism converts 7.5 to 10 tons of CO 2 Per acre. In some embodiments, the microorganism converts 10 to 15 tons of CO 2 Per acre. In some embodiments, the microbial conversion is 15 to 20 tons/acre. In some embodiments, the CO converted by the microorganism 2 The amount is from about 2 tons/acre to about 20 tons/acre. In some embodiments, the CO converted by the microorganism 2 The amount is from about 2 tons/acre to about 4 tons/acre, from about 2 tons/acre to about 6 tons/acre, from about 2 tons/acre to about 8 tons/acre, from about 2 tons/acre to about 10 tons/acre, from about 2 tons/acre to about 12 tons/acre, from about 2 tons/acre to about 14 tons/acre, from about 2 tons/acre to about 16 tons/acre, from about 2 tons/acre to about 18 tons/acre, from about 2 tons/acre to about 20 tons/acre, from about 4 tons/acre to about 6 tons/acre, from about 4 tons/acre to about 8 tons/acre, from about 4 tons/acre to about 10 tons/acre about 4 to about 12 tons/acre, about 4 to about 14 tons/acre, about 4 to about 16 tons/acre, about 4 to about 18 tons/acre, about 4 to about 20 tons/acre, about 6 to about 8 tons/acre, about 6 to about 10 tons/acre, about 6 to about 12 tons/acre, about 6 to about 14 tons/acre, about 6 to about 16 tons/acre, about 6 to about 18 tons/acre, about 6 to about 6 tons/acre 20 tons/acre, about 8 tons/acre to about 10 tons/acre, about 8 tons/acre to about 12 tons/acre, about 8 tons/acre to about 14 tons/acre, about 8 tons/acre to about 16 tons/acre, about 8 tons/acre to about 18 tons/acre, about 8 tons/acre to about 20 tons/acre, about 10 tons/acre to about 12 tons/acre, about 10 tons/acre to about 14 tons/acre, about 10 tons/acre to about 16 tons/acre, about 10 tons/acre to about 18 tons/acre, about 10 tons/acre to about 20 tons/acre, about 12 tons/acre to about 14 tons/acre, about 12 tons/acre to about 16 tons/acre, about 12 tons/acre to about 12 tons/acre, about 12 tons/acre to about 16 tons/acre, about 14 tons/acre to about 16 tons/acre, about 16 tons/acre to about 16 tons/acre. In some embodiments, the CO converted by the microorganism 2 The amount is about 2 tons/acre, about 4 tons/acre, about 6 tons/acre, about 8 tons/acre, about 10 tons/acre, about 12 tons/acre, about 14 tons/acre, about 16 tons/acre, about 18 tons/acre, or about 20 tons/acre. In some embodiments, the CO converted by the microorganism 2 The amount is at least about 2 tons/acre, about 4 tons/acre, about 6 tons/acre, about 8 tons/acre, about 10 tons/acre, about 12 tons/acre, about 14 tons/acre, about 16 tons/acre, or about 18 tons/acre. In some embodiments, the CO converted by the microorganism 2 The amount is up to about 4 tons/acre, about 6 tons/acre, about 8 tons/acre, about 10 tons/acre, about 12 tons/acre, about 14 tons/acre, about 16 tons/acre, about 18 tons/acre, or about 20 tons/acre.
In some embodiments, the microorganism or secretion is incorporated into the plant interior. In some embodiments, the microorganism or secretion is incorporated under the pericarp of the plant. In some embodiments, the microorganism or secretion is incorporated between the pericarp and aleurone cell layer of the plant. In some embodiments, the microorganism or secretion contacts the embryo of the plant. In some embodiments, the microorganism or secretion does not contact the embryo of the plant. In some embodiments, the microorganism or secretion contacts the endosperm of the plant. In some embodiments, the microorganism or secretion does not contact the endosperm of the plant. In some embodiments, the microorganism or secretion is incorporated into the gap between the plant's plant skin and plant embryo. In some embodiments, the microorganism or secretion is incorporated into the gap between the plant pericarp and the plant aleurone cell layer.
The modified plant may be any type of plant. In some embodiments, the modified plant is a monocot. In some embodiments, the plant is a maize, wheat, rice, barley, rye, sugarcane, millet, oat, or sorghum plant. In some embodiments, the plant is a maize plant. In some embodiments, the plant is a maize (Zea mail) plant. In some embodiments, the modified plant is a dicot. In some embodiments, the plant is a soybean, cotton, alfalfa, bean, quinoa, lentil, peanut, sunflower, canola, cassava, oil palm, potato, sugar beet, cocoa, coffee, lettuce, tomato, or cabbage plant. In some embodiments, the plant is a lettuce plant. In some embodiments, the plant is a lettuce (Lactuca sativa) plant. In some embodiments, the plant is a tomato plant. In some embodiments, the plant is a tomato (Solanum lycopersicum) plant. In some embodiments, the plant is a Genetically Modified Organism (GMO) plant. In some embodiments, the plant is a non-GMO plant.
The amount of microorganisms or secretions incorporated into the plant must be at a sufficient level to effectively sequester the CO 2 . In some embodiments, the amount of microorganisms incorporated into the plants is from about 250 Colony Forming Units (CFU) to about 5,000CFU. In some embodiments, the microorganism is incorporated into the plant in an amount of about 250CFU to about 500CFU, about 250CFU to about 750CFU, about 250CFU to about 1,000CFU, about 250CFU to about 2,000CFU, about 250CFU to about 3,000CFU, about 250CFU to about 4,000CFU, about 250CFU to about 5,000CFU, about 500CFU to about 750CFU, about 500CFU to about 1,000CFU, about 500CFU to about 2,000CFU, about 500CFU to about 3,000CFU, about 500CFU to about 4,000CFU, about 500CFU to about 5,000CFU, about 750CFU to about 1,000CFU, about 750CFU to about 2,000CFU, about 750CFU to about 3,000CFU, about 750CFU to about 4,000CFU, about 750CFU to about 5,000CFUAbout 1,000cfu to about 2,000CFU, about 1,000cfu to about 3,000CFU, about 1,000cfu to about 4,000CFU, about 1,000cfu to about 5,000CFU, about 2,000CFU to about 3,000CFU, about 2,000CFU to about 4,000CFU, about 2,000CFU to about 5,000CFU, about 3,000CFU to about 4,000CFU, about 3,000CFU to about 5,000CFU, or about 4,000CFU to about 5,000CFU. In some embodiments, the amount of microorganism incorporated into the plant is about 250CFU, about 500CFU, about 750CFU, about 1,000CFU, about 2,000CFU, about 3,000CFU, about 4,000CFU, or about 5,000CFU. In some embodiments, the amount of microorganism incorporated into the plant is at least about 250CFU, about 500CFU, about 750CFU, about 1,000CFU, about 2,000CFU, about 3,000CFU, or about 4,000CFU. In some embodiments, the amount of microorganisms incorporated into the plant is up to about 500CFU, about 750CFU, about 1,000CFU, about 2,000CFU, about 3,000CFU, about 4,000CFU, or about 5,000CFU. In some embodiments, at least about 500CFU is incorporated into a plant. In some embodiments, at least about 1000CFU is incorporated into a plant.
In some embodiments, the microorganism or secretion incorporated into the plant is shelf stable for a prolonged period of time. In some embodiments, the modified plant is shelf stable for about 3 months to about 36 months. In some embodiments of the present invention, in some embodiments, the modified plant is in the range of about 3 months to about 6 months, about 3 months to about 9 months, about 3 months to about 12 months, about 3 months to about 15 months, about 3 months to about 18 months, about 3 months to about 21 months, about 3 months to about 24 months, about 3 months to about 30 months, about 3 months to about 36 months, about 6 months to about 9 months, about 6 months to about 12 months, about 6 months to about 15 months, about 6 months to about 18 months, about 6 months to about 21 months, about 6 months to about 24 months, about 6 months to about 30 months, about 6 months to about 36 months, about 9 months to about 12 months, about 9 months to about 15 months, about 9 months to about 18 months, about 9 months to about 21 months, about 9 months to about 24 months, about 9 months to about 30 months the storage is stable for from about 9 months to about 36 months, from about 12 months to about 15 months, from about 12 months to about 18 months, from about 12 months to about 21 months, from about 12 months to about 24 months, from about 12 months to about 30 months, from about 12 months to about 36 months, from about 15 months to about 18 months, from about 15 months to about 21 months, from about 15 months to about 24 months, from about 15 months to about 30 months, from about 15 months to about 36 months, from about 18 months to about 21 months, from about 18 months to about 24 months, from about 18 months to about 30 months, from about 18 months to about 36 months, from about 21 months to about 24 months, from about 21 months to about 30 months, from about 21 months to about 36 months, from about 24 months to about 30 months, from about 24 months to about 36 months, or from about 30 months to about 30 months. In some embodiments, the modified plant is shelf stable for about 3 months, about 6 months, about 9 months, about 12 months, about 15 months, about 18 months, about 21 months, about 24 months, about 30 months, or about 36 months. In some embodiments, the modified plant is shelf stable for at least about 3 months, about 6 months, about 9 months, about 12 months, about 15 months, about 18 months, about 21 months, about 24 months, or about 30 months. In some embodiments, the modified plant is shelf stable for up to about 6 months, about 9 months, about 12 months, about 15 months, about 18 months, about 21 months, about 24 months, about 30 months, or about 36 months.
In some embodiments, the microorganism incorporated into the plant is stable after incorporation. In some embodiments, the microorganism is stable for more than 30 days, more than six months, more than one year, or more than two years. In some embodiments, the microorganism is stable for more than 30 days. In some embodiments, the microorganism is stable for more than six months. In some embodiments, the microorganism is stable for more than one year. In some embodiments, the microorganism is stable for more than two years.
The microorganism or secretion thereof incorporated into the plant may be any one of the microorganisms provided herein or any other microorganism. In some embodiments, the microorganism is a microorganism (microbe). In some embodiments, the microorganism is an endospore-forming microorganism. In some embodiments, the microorganism is an endospore-forming microorganism or an endospore thereof. In some embodiments, the microorganism is an endospore of a microorganism provided herein. In some embodiments, the microorganism is an endospore-forming bacterium or an endospore thereof.
Methods of incorporating bacteria
In one aspect, provided herein are methods of incorporating one or more microorganisms or secretions thereof into one or more plants. In some embodiments, the method comprises sterilizing the plant. In some embodiments, the method comprises contacting the plant with a solution comprising one or more microorganisms or secretions thereof. In some embodiments, the solution further comprises a salt. In some embodiments, the method comprises incubating the plant with the solution for a period of time. In some embodiments, the period of time is sufficient to allow a desired amount of microorganisms or secretions thereof to enter the plant. In some embodiments, the method incorporates a desired amount of a microorganism or secretion thereof into a plant.
In some embodiments, the method comprises contacting the plant with a solution comprising a salt as described herein.
In some embodiments, the solution comprises additional additives as described herein.
In some embodiments, the solution comprises additional metal ions as described herein.
In some embodiments, the solution comprises one or more nutrients for the microorganism as described herein. In some embodiments, the solution comprises a bacterial growth medium. In some embodiments, the solution comprises a Lysogenic Broth (LB), a nutrient broth, or a combination thereof. In some embodiments, the solution comprises a lysogenic broth. In some embodiments, the solution comprises a nutrient broth.
In some embodiments, the solution comprises a microorganism as described herein.
In some embodiments, the solution comprises a desired amount of microorganisms per plant mass as described herein.
In some embodiments, the plant comprises a desired amount of microorganisms per plant as described herein. In some embodiments, the microorganism is a bacterium. In some embodiments, the bacteria are endospore-forming bacteria. In some embodiments, the method comprises inducing endospores formation of the endospore-forming bacteria. In some embodiments, the bacteria incorporated into the plant are endospores. In some embodiments, the solution comprises one or more components that induce formation of endospores. In some embodiments, the solution comprises potassium, ferrous sulfate, calcium, magnesium, manganese, or a combination thereof.
In some embodiments, the method comprises sterilizing the plant. In some embodiments, the method comprises sterilizing the plant surface. Any method of producing plants having a sterilized surface may be used. In some embodiments, the plants are sterilized with a bleaching solution. In some embodiments, the plants are sterilized prior to immersing the plants in the solution containing the one or more microorganisms. In some embodiments, the plant is a sterilized plant. In some embodiments, the plant has a sterilized surface. As used herein, "sterilized," "sterilized" and related terms (e.g., "sanitized" and the like) mean that there are substantially no living microorganisms on the sterilized article. In some embodiments, the plants are sterilized prior to incubating the plants in the solution comprising the microorganism. In some embodiments, the plants are sterilized after incubating the plants in the solution comprising the microorganism. In some embodiments, the fungicide is added to the plant surface.
In some embodiments, the sterilized or disinfected plant comprises substantially no viable microorganisms on the plant (e.g., plant surface). In some embodiments, the sterile or sterilized plant comprises less than 1CFU, less than 5CFU, less than 10CFU, less than 20CFU, less than 30CFU, less than 40CFU, or less than 50CFU of microorganisms on the plant.
In some embodiments, the plant is incubated with the solution containing the microorganism for a time sufficient to incorporate the microorganism into the plant.
Microorganisms and secretions
The microorganisms provided herein, or secretions thereof, produce or promote bicarbonate and one or more mineral formations. In some embodiments, bicarbonate formation sequesters CO 2 . In some embodiments, bicarbonate formation results in mineral formation. In some embodimentsThe formed minerals stably sequester carbon in the soil. In some embodiments, the microorganism is a bacterium as described herein. In some embodiments, the microorganism is an endospore-forming bacterium. In some embodiments, the microorganism is a bacterial endospore. Whenever a microorganism (e.g., a bacterium) referred to herein is capable of forming an endospore, it is intended to encompass any endospore of the microorganism as well. For example, if the plant treatment formulation comprises bacillus, the formulation may comprise endospores of bacillus.
In some embodiments, the microorganism is a microorganism from the phylum firmicutes, the phylum proteus, and the phylum actinomycetes. In some embodiments, the microorganism is a microorganism from the phylum firmicutes. In some embodiments, the microorganism is a microorganism from the phylum Proteus. In some embodiments, the microorganism is a microorganism from the phylum actinomycetes. In some embodiments, the microorganism is an endospore of any one of the microorganisms.
Rhizobacteria that fix atmospheric nitrogen are present on the roots of plants. These microorganisms are able to survive in the soil and to produce large amounts of CO 2 Tolerating CO 2 Is released by the roots of plants during respiration or by nearby microorganisms and soil animal communities through respiration.
In some embodiments, the bacteria are not genetically modified. In some embodiments, the CO is according to bacteria 2 The ability to convert to bicarbonate and ultimately to minerals is the choice of bacteria. In some embodiments, the bacteria are selected according to the use of carbonic anhydrase. In some embodiments, the bacteria are selected according to the use of carbonic anhydrase. In some embodiments, a plurality of rhizobacteria are effectively loaded into the seed. In some embodiments, the rhizobacteria include endospore-forming bacteria that enhance biological nitrogen fixation. In some embodiments, the rhizobacteria comprise bacillus, paenibacillus, or both. In some embodiments, the one or more microorganisms comprise bacillus amyloliquefaciens, bacillus laterosporus, bacillus licheniformis, bacillus macerans, bacillus cereus, bacillus circulans Bacillus, bacillus firmus, bacillus subtilis, bacillus sphaericus, bacillus megaterium, bacillus coagulans, bacillus brevis, bacillus thuringiensis, bacillus mycoides, bacillus cucumber, bacillus intracellulare, bacillus pumilus, bacillus bailii, bacillus mucilaginosus, bacillus tefraxinus, bacillus methylotrophicus, or any combination thereof. In some embodiments, the one or more microorganisms comprise bacillus subtilis S3C23, bacillus subtilis MP2, bacillus subtilis RO2C15, bacillus subtilis RO2C22, bacillus megaterium 6, bacillus megaterium S3C21, bacillus megaterium RO2C12, bacillus cucumber S3C14, bacillus plantarum 5, or any combination thereof. In some embodiments, the one or more microorganisms comprise bacillus subtilis S3C23. In some embodiments, the one or more microorganisms comprise bacillus subtilis MP2.
In some embodiments, the one or more microorganisms comprise a genetic modification that results in the one or more microorganisms producing or promoting more carbonic anhydrase formation relative to the corresponding wild-type microorganism. In some embodiments, the genetic modification comprises a nucleic acid construct, wherein the nucleic acid construct comprises one or more promoters configured to drive expression of a Carbonic Anhydrase (CA) coding sequence. In some embodiments, the genetic modification comprises a nucleic acid construct, wherein the nucleic acid construct comprises a Carbonic Anhydrase (CA) coding sequence. In some embodiments, the CA coding sequence is heterologous to the one or more microorganisms. In some embodiments, the CA coding sequence is endogenous to the one or more microorganisms. In some embodiments, the nucleic acid construct is codon optimized. In some embodiments, the nucleic acid construct further comprises one or more promoters configured to drive expression of the CA coding sequence. In some embodiments, the one or more promoters drive constitutive expression of the CA coding sequence. In some embodiments, the one or more promoters drive inducible expression of the CA coding sequence. In some embodiments, the genetic modification comprises a carbonic anhydrase gene that has been modified by directed microbial evolution. In some embodiments, the genetic modification comprises a signal sequence. In some embodiments, the signal sequence comprises a periplasmic signal sequence or an extracellular secretion signal. In some embodiments, the periplasmic signal sequence or the extracellular secretion signal is located 5' to the carbonic anhydrase coding sequence. In some embodiments, the periplasmic signal sequence or the extracellular secretion signal is fused to the 5' end of the carbonic anhydrase coding sequence. In some embodiments, the genetic modification comprises one or more components of the general secretory pathway, and wherein the genetic modification results in secretion or subcellular targeting of carbonic anhydrase by the one or more microorganisms.
In some embodiments, the one or more promoters are or are derived from one or more promoters comprised in bacillus or paenibacillus. In some embodiments, the one or more promoters are selected from P groES 、P43、P sigX 、P trnQ And P xylA . In some embodiments, the one or more promoters are or are derived from one or more housekeeping gene promoters or strongly expressed constitutive promoters. In some embodiments, the one or more housekeeping gene promoters or strong expressed constitutive promoters are selected from the group consisting of P liaG 、P lepA 、P veg 、P gsiB 、P43、P trnQ 、P lial (bacitracin-inducible) and P xylA (xylose inducible).
In some embodiments, the genetic modification comprises introducing an expression vector into one or more microorganisms. In some embodiments, the genetic modification comprises modification of a chromosome of one or more microorganisms.
In some embodiments, the microorganism can immobilize nitrogen and carbon dioxide. In some embodiments, the nitrogen-fixing microorganisms will continue to provide fixed nitrogen (ammonia) to the plants via biological nitrogen fixation, which will reduce the use of chemical fertilizers that pollute the environment. The end product (ammonia) of atmospheric nitrogen fixation can enhance CO 2 To mineralsAs ammonia has been reported to increase pH, thereby accelerating mineralization. Because ammonia produced by the microorganisms disclosed herein is significantly higher than other soil-dwelling microorganisms, even in the presence of an external nitrogen source, the mineralization process of the strains disclosed herein may be faster than other microorganisms that inhabit the soil.
In some embodiments of the present invention, in some embodiments, the microorganism is selected from Acetobacter, actinobacillus, bacillus alcaligenes, aminophilia, bacillus bifidus, anaerobic Bacillus, thiobacillus, anaerobic Bacillus, brevibacillus, thermoanaerobacter, caminiella, prunus, clostridium, ke Enshi, ke Kesi, acidocella, desulphurized Enterobacter, bacillus, and Bacillus Desulfosporomula, desulfocampylobacter, desulfosporula, desulfosporium, protosporium, linear, gelria, geosporobacter, cellularomyces, halophilum, haloatron, solar, japanese, leishmania, chrysosporium, lysine bacillus, mahela, metabacterium, mushroom, mortierella, and Mortierella Desulfosporomula, desulfocampylobacter, desulfosporula, protosporium, linear, gelria, geosporium, geosporobacter, bacillus, halomatron, solar bacillus, solar bacterium, leishmania, chronic Bacillus, lysine bacillus, mahela, metabacterium, mushroom, bacillus, and Bacillus, microorganisms of the genus Thermoflavum, thermovelabalum, bacillus, cladosporium and Vulcanobacillus. In some embodiments, the microorganism is a microorganism selected from the group consisting of Acetobacter, actinobacillus, bacillus, chryseobacterium, ke Kesi, sword, gluconobacter, microbacterium, and Serratia. In some embodiments, the microorganism is acetobacter. In some embodiments, the microorganism is an actinomycete. In some embodiments, the microorganism is bacillus. In some embodiments, the microorganism is a chrysobacterium. In some embodiments, the microorganism is of the genus Ke Kesi. In some embodiments, the microorganism is a genus xiphoid. In some embodiments, the microorganism is a genus glutamate. In some embodiments, the microorganism is a genus microbacterium. In some embodiments, the microorganism is pantoea. In some embodiments, the microorganism is serratia. In some embodiments, the microorganism is an endospore of any one of the microorganisms.
In some embodiments, the microorganism is selected for one or more characteristics related to its ability to interact with the plant. In some embodiments, the microorganism is selected for compatibility. In some embodiments, the microorganism is selected to ensure that predation or antagonism does not occur. In some embodiments, the microorganism is selected based on stability during storage. In some embodiments, microorganisms are selected based on rapid plant colonization and survival within the associated tissue. In some embodiments, the microorganism is selected for optimal incorporation into one or more plants. In some embodiments, the microorganism remains present throughout the plant life cycle.
In some embodiments, the microorganism incorporated into the plant is stable after incorporation. In some embodiments, the microorganism is stable for more than 30 days, more than six months, more than one year, or more than two years. In some embodiments, the microorganism is stable for more than 30 days. In some embodiments, the microorganism is stable for more than six months. In some embodiments, the microorganism is stable for more than one year. In some embodiments, the microorganism is stable for more than two years.
Composition comprising bacteria
In certain aspects, disclosed herein is a composition comprising one or more microorganisms, wherein the one or more microorganisms are or are derived from microorganisms selected to produce or promote bicarbonate and one or more mineral formations.
The microorganisms provided herein, or secretions thereof, produce or promote bicarbonate and one or more mineral formations. In some embodiments, bicarbonate formation sequesters CO 2 . In some embodiments, bicarbonate formation results in mineral formation. In some embodiments, the formed minerals stably sequester carbon in the soil. In some embodiments, the microorganism is a bacterium as described herein. In some embodiments, the microorganism is an endospore-forming bacterium. In some embodiments, the microorganism is a bacterial endospore. Whenever a microorganism (e.g., a bacterium) referred to herein is capable of forming an endospore, it is intended to encompass any endospore of the microorganism as well. For example, if the plant treatment formulation comprises bacillus, the formulation may comprise endospores of bacillus.
In some embodiments, the microorganism is a microorganism from the phylum firmicutes, the phylum proteus, and the phylum actinomycetes. In some embodiments, the microorganism is a microorganism from the phylum firmicutes. In some embodiments, the microorganism is a microorganism from the phylum Proteus. In some embodiments, the microorganism is a microorganism from the phylum actinomycetes. In some embodiments, the microorganism is an endospore of any one of the microorganisms.
Rhizobacteria that fix atmospheric nitrogen are present on the roots of plants. These organisms are able to survive in soil and react to large amounts of CO 2 Tolerating CO 2 Is released by the roots of plants during respiration or by nearby microorganisms and soil animal communities through respiration.
In some embodiments, the bacteria are not genetically modified. In some embodiments, according to bacteriaCO is processed by 2 The ability to convert to bicarbonate and ultimately to minerals is the choice of bacteria. In some embodiments, the bacteria are selected according to the use of carbonic anhydrase. In some embodiments, the bacteria are not genetically modified. In some embodiments, the CO is according to bacteria 2 The ability to convert to bicarbonate and minerals to select bacteria. In some embodiments, the bacteria are selected according to the use of carbonic anhydrase. In some embodiments, the bacteria are selected according to the use of carbonic anhydrase. In some embodiments, a plurality of rhizobacteria are effectively loaded into the seed. In some embodiments, the rhizobacteria include endospores forming bacteria. In some embodiments, the rhizobacteria comprise bacillus, paenibacillus, or both. In some embodiments, the one or more microorganisms comprise bacillus amyloliquefaciens, bacillus laterosporus, bacillus licheniformis, bacillus macerans, bacillus cereus, bacillus circulans, bacillus firmus, bacillus subtilis, bacillus sphaericus, bacillus megaterium, bacillus coagulans, bacillus brevis, bacillus thuringiensis, bacillus mycoides, bacillus cucumber, bacillus plantarum, bacillus pumilus, bacillus bailii, bacillus mucilaginosus, bacillus tertazii, bacillus methylotrophicus, or any combination thereof. In some embodiments, the one or more microorganisms comprise bacillus subtilis S3C23, bacillus subtilis MP2, bacillus subtilis RO2C15, bacillus subtilis RO2C22, bacillus megaterium 6, bacillus megaterium S3C21, bacillus megaterium RO2C12, bacillus cucumber S3C14, bacillus plantarum 5, or any combination thereof. In some embodiments, the one or more microorganisms comprise bacillus subtilis S3C23. In some embodiments, the one or more microorganisms comprise bacillus subtilis MP2.
In some embodiments, the microorganism can immobilize nitrogen and carbon dioxide. In some embodiments, the nitrogen-fixing microorganisms will continue to provide fixed nitrogen (ammonia) to the plants via biological nitrogen fixation, which will reduce the use of chemical fertilizers that pollute the environment. The end product (ammonia) of atmospheric nitrogen fixation can be enhancedCO 2 To bicarbonate and minerals, as ammonia has been reported to increase pH, thereby accelerating mineralization. Because ammonia produced by the microorganisms disclosed herein is significantly higher than other soil-dwelling microorganisms, even in the presence of an external nitrogen source, the mineralization process of the strains disclosed herein may be faster than other microorganisms that inhabit the soil.
In some embodiments of the present invention, in some embodiments, the microorganism is selected from Acetobacter, actinobacillus, bacillus alcaligenes, aminophilia, bacillus bifidus, anaerobic Bacillus, thiobacillus, anaerobic Bacillus, brevibacillus, thermoanaerobacter, caminiella, prunus, clostridium, ke Enshi, ke Kesi, acidocella, desulphurized Enterobacter, bacillus, and Bacillus Desulfosporomula, desulfocampylobacter, desulfosporula, desulfosporium, protosporium, linear, gelria, geosporobacter, cellularomyces, halophilum, haloatron, solar, japanese, leishmania, chrysosporium, lysine bacillus, mahela, metabacterium, mushroom, mortierella, and Mortierella Desulfosporomula, desulfocampylobacter, desulfosporula, protosporium, linear, gelria, geosporium, geosporobacter, bacillus, halomatron, solar bacillus, solar bacterium, leishmania, chronic Bacillus, lysine bacillus, mahela, metabacterium, mushroom, bacillus, and Bacillus, microorganisms of the genus Thermoflavum, thermovelabalum, bacillus, cladosporium and Vulcanobacillus. In some embodiments, the microorganism is a microorganism selected from the group consisting of Acetobacter, actinobacillus, bacillus, chryseobacterium, ke Kesi, sword, gluconobacter, microbacterium, and Serratia. In some embodiments, the microorganism is acetobacter. In some embodiments, the microorganism is an actinomycete. In some embodiments, the microorganism is bacillus. In some embodiments, the microorganism is a chrysobacterium. In some embodiments, the microorganism is of the genus Ke Kesi. In some embodiments, the microorganism is a genus xiphoid. In some embodiments, the microorganism is a genus glutamate. In some embodiments, the microorganism is a genus microbacterium. In some embodiments, the microorganism is pantoea. In some embodiments, the microorganism is serratia. In some embodiments, the microorganism is an endospore of any one of the microorganisms.
In some embodiments, the microorganism is selected for one or more characteristics related to its ability to interact with the plant. In some embodiments, the microorganism is selected for compatibility. In some embodiments, the microorganism is selected to ensure that predation or antagonism does not occur. In some embodiments, the microorganism is selected based on stability during storage. In some embodiments, microorganisms are selected based on rapid plant colonization and survival within the associated tissue. In some embodiments, the microorganism is selected for optimal incorporation into one or more plants. In some embodiments, the microorganism remains present throughout the plant life cycle.
In some embodiments, the microorganism incorporated into the plant is stable after incorporation. In some embodiments, the microorganism is stable for more than 30 days, more than six months, more than one year, or more than two years. In some embodiments, the microorganism is stable for more than 30 days. In some embodiments, the microorganism is stable for more than six months. In some embodiments, the microorganism is stable for more than one year. In some embodiments, the microorganism is stable for more than two years.
Definition of the definition
Whenever the term "at least", "more than" or "more than or equal to" precedes the first value in a series of two or more values, the term "at least", "more than" or "more than or equal to" applies to each of the values in the series. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.
Whenever the term "no more than", "less than" or "less than or equal to" precedes the first value in a series of two or more values, the term "no more than", "less than" or "less than or equal to" applies to each of the values in the series. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.
The use of absolute or sequential terms such as "to," "not to," "should," "must," "first," "initially," "next," "subsequently," "preceding," "following," "last" and "final" are not meant to limit the scope of the embodiments of the invention disclosed herein, but rather as examples.
As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "includes," has, "" with, "or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term" comprising.
As described herein, the term "irrigation system" may be an artificial process that applies controlled amounts of water to aid in crop production and plant growth, where it may be referred to as "watering. In some embodiments, the term "irrigation system" may include spraying foliage, in-furrow fertilizer treatment, spraying systems, humidifiers, or spraying systems.
As used herein, the phrases "at least one," "one or more," and/or "are open-ended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions "at least one of A, B and C", "at least one of A, B or C", "one or more of A, B and C", "one or more of A, B or C" and "A, B and/or C" refers to a alone, B alone, C, A and B, A and C, B and C alone, or A, B and C.
As used herein, "or" may refer to "and," "or" and/or, "and may be used exclusively and inclusively. For example, the term "a or B" may refer to "a or B", "a but not B", "B but not a", and "a and B". In some cases, the context may specify a particular meaning.
Any of the systems, methods, software, and platforms described herein are modular. Thus, terms such as "first" and "second" do not necessarily imply a priority, order of importance, or order of behavior.
When referring to an amount or numerical range, the term "about" means that the amount or numerical range referred to is an approximation within the experimental variable range (or within statistical experimental error), and may vary from, for example, 1% to 15% of the stated amount or numerical range. In an example, the term "about" refers to ±10% of the stated quantity or value.
As used herein, the term "increasing" generally refers to increasing a static significant amount. In some aspects, the term "increase" refers to an increase of at least 10% compared to a reference level, e.g., an increase of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or up to and including a 100% increase or any increase between 10% -100% compared to a reference level, standard or control. Other examples of "increasing" include increasing by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold, or more as compared to a reference level.
The term "reducing" is generally used herein to refer to reducing by a statistically significant amount. In some aspects, "reduced" refers to a reduction of at least 10% from a reference level, e.g., a reduction of at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or up to and including a 100% reduction (e.g., a level that is absent or undetectable from the reference level) or any reduction between 10% -100% from the reference level. In the context of signs or symptoms, these terms refer to a statistically significant reduction in such levels. The reduction may be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and preferably is reduced to a level that is acceptable within the normal range for an individual without the given disease.
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. The invention is not limited by the specific embodiments provided in the specification. While the invention has been described with reference to the foregoing specification, the description and illustrations of the embodiments herein are not intended to be construed in a limiting sense. Many variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it is to be understood that all aspects of the invention are not limited to the specific descriptions, configurations, or relative proportions set forth herein, depending on various conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the present invention will also cover any such alternatives, modifications, variations, or equivalents. The following claims are intended to define the scope of the invention and methods and structures within the scope of these claims and their equivalents are covered thereby.
Numbered embodiments
1. A composition comprising one or more microorganisms, wherein the one or more microorganisms are located at a gap between a coating and a cellular layer of a plant or portion thereof and are or are derived from one or more microorganisms selected to produce or promote bicarbonate, carbonate, or one or more mineral formation.
2. The composition according to any one of the preceding embodiments, wherein the plant or the part thereof is a plant root, a plant stem, a plant leaf, a plant seed, a plant fruit, a plant tuber or a plant root nodule.
3. The composition according to any one of the preceding embodiments, wherein the plant or the part thereof comprises a commercial plant or part thereof.
4. The composition according to any one of the preceding embodiments, wherein the commercial plant or part thereof is corn, wheat, rice, sorghum, barley, rye, sugarcane, millet, oat, soybean, cotton, alfalfa, bean, quinoa, lentil, peanut, sunflower, canola, cassava, oil palm, potato, sugar beet, cocoa, coffee, lettuce, tomato, pea, cabbage, fruit trees, nut trees, forests, grasslands, or turf grasses.
5. The composition according to any one of the preceding embodiments, wherein the portion thereof is a plant seed, and wherein the one or more microorganisms associated with the plant seed are disposed in the gap between the seed coat and the seed embryo of the plant seed.
6. The composition according to any one of the preceding embodiments, wherein the one or more microorganisms associated with the plant or the part thereof are arranged as a coating of the plant or the part thereof.
7. The composition according to any one of the preceding embodiments, wherein the one or more microorganisms associated with the plant or the part thereof are applied to the plant seed by an irrigation system.
8. The composition according to any of the preceding embodiments, wherein the irrigation system comprises in-furrow treatment techniques.
9. The composition according to any of the preceding embodiments, wherein the irrigation system comprises a spray technique.
10. The composition according to any of the preceding embodiments, wherein the bicarbonate sequesters carbon.
11. The composition according to any one of the preceding embodiments, wherein the carbon is gaseous carbon.
12. The composition according to any of the preceding embodiments, wherein the gaseous carbon is carbon dioxide.
13. The composition according to any one of the preceding embodiments, wherein the carbonate sequesters carbon.
14. The composition according to any one of the preceding embodiments, wherein the carbon is gaseous carbon.
15. The composition according to any of the preceding embodiments, wherein the gaseous carbon is carbon dioxide.
16. The composition according to any one of the preceding embodiments, wherein the one or more minerals sequester carbon.
17. The composition according to any one of the preceding embodiments, wherein the carbon is gaseous carbon.
18. The composition according to any of the preceding embodiments, wherein the gaseous carbon is carbon dioxide.
19. The composition according to any one of the preceding embodiments, wherein the portion thereof is a plant seed, and wherein the one or more microorganisms associated with the plant seed are disposed in a gap between a seed pericarp and a seed aleurone cell layer of the plant seed.
20. The composition according to any of the preceding embodiments, wherein the one or more microorganisms comprise one or more carbonic anhydrases.
21. The composition according to any of the preceding embodiments, wherein the one or more carbonic anhydrases comprise an alpha-carbonic anhydrase.
22. The composition according to any of the preceding embodiments, wherein the one or more carbonic anhydrases comprise a beta-type carbonic anhydrase.
23. The composition according to any of the preceding embodiments, wherein the one or more carbonic anhydrases comprise gamma-type carbonic anhydrases.
24. The composition according to any of the preceding embodiments, wherein the one or more carbonic anhydrases comprise a delta-type carbonic anhydrase.
25. The composition according to any one of the preceding embodiments, wherein the one or more carbonic anhydrases comprise a zeta-type carbonic anhydrase.
26. The composition according to any of the preceding embodiments, wherein the one or more carbonic anhydrases comprise class η carbonic anhydrase.
27. A composition according to any one of the preceding embodiments, wherein the one or more carbonic anhydrases comprise iota-type carbonic anhydrases.
28. The composition according to any one of the preceding embodiments, wherein the one or more microorganisms comprise bacteria, archaebacteria, fungi or viruses.
29. The composition according to any one of the preceding embodiments, wherein the one or more microorganisms comprise the bacteria.
30. The composition according to any one of the preceding embodiments, wherein the bacteria comprise endospore-forming bacteria.
31. The composition according to any of the preceding embodiments, wherein the bacteria comprise a bacterial strain from the genus Acetobacter, the genus actinomyces, the genus Bacillus, the genus Aminophilia, the genus Bacillus, the genus anaerobic, the genus Thiobacillus, the genus anaerobic bacillus, the genus Bacillus, the genus Brevibacterium, the genus thermal anaerobic, the genus Xeothermia, the genus Caminiella, the genus Prunella, the genus Clostridium, the genus Ke Enshi, the genus Ke Kesi, the genus Saccharomyces, the genus Enterobacter, the genus Desulfosporula, the genus Campylobacter, the genus Desulfovirginia, the genus Desulfosporium, the genus desulphus, the genus Brevibacterium, the genus Gelria, the genus Georrobacter, the genus Cellum, the genus Salmonella, the genus Halobacterium, the genus Halonella, the genus Bacillus, the genus solar bacillus, the genus Bacillus, the genus Leuconostoc, the genus Bacillus, the genus lysine, the genus Mahezia, the genus Malus, the genus Max Metacteria, mushroom, natronella, paenibacillus, oreneia, ornithine, oxalic acid bacteria, acetobacter, paenibacillus, hairyveromyces, pelospora, anaerobic Enterobacter, bacillus, flavobacterium, haibacterium, propionibacterium, salickiella, qinghai, shimadzu, paenibacillus, paenidium, paenibacillus, and so forth Acetobacter, sporobacter, bacillus, lactobacillus, banana, sporobacter, sporotalaea, enteromorpha, cotrophic, bacillus, warm, geobacillus, deep sea Bacillus, acetobacter, thermoactinomyces, thermoalactobacillus, bacillus, acetobacter, bacillus, and methods of producing a strain of Acetobacter, bacteria of the genus Thermoanaerobacter, thermobacillus, thermoflavobacterium, thermovenabaum, bacillus megaterium, bacillus, vulcanobacillus, or combinations thereof.
32. The composition according to any one of the preceding embodiments, wherein the bacteria comprise bacteria belonging to the phylum firmicutes.
33. The composition according to any one of the preceding embodiments, wherein the bacteria comprise rhizosphere bacteria.
34. The composition according to any one of the preceding embodiments, wherein the rhizosphere bacteria comprises bacillus, paenibacillus, or both.
35. The composition according to any one of the preceding embodiments, wherein the bacteria comprises bacillus amyloliquefaciens, bacillus laterosporus, bacillus licheniformis, bacillus macerans, bacillus cereus, bacillus circulans, bacillus firmus, bacillus subtilis, bacillus megaterium, bacillus coagulans, bacillus brevis, bacillus sphaericus, bacillus thuringiensis, bacillus mycoides, bacillus cucumeris, bacillus plantarum, bacillus pumilus, bacillus bailii, bacillus mucilaginosus, bacillus tertagatous, bacillus methylotrophicus, or any combination thereof.
36. The composition according to any one of the preceding embodiments, wherein the bacteria comprises bacillus subtilis S3C23, bacillus subtilis MP2, bacillus subtilis RO2C15, bacillus subtilis RO2C22, bacillus megaterium 6, bacillus megaterium S3C21, bacillus megaterium RO2C12, bacillus cucumber S3C14, bacillus plantarum 5, or any combination thereof.
37. The composition according to any one of the preceding embodiments, wherein the bacterium comprises bacillus subtilis S3C23.
38. The composition according to any one of the preceding embodiments, wherein the bacteria comprises bacillus subtilis MP2.
39. The composition according to any one of the preceding embodiments, wherein the bacteria comprise a C3C 10 strain.
40. The composition according to any one of the preceding embodiments, wherein the bacteria comprises paenibacillus polymyxa, paenibacillus persicae, paenibacillus pocheonensis, paenibacillus aceris, paenibacillus catalpa, paenibacillus wetland, paenibacillus feed, paenibacillus brazil, or any combination thereof.
41. The composition of any one of the preceding embodiments, wherein the bacteria comprises paenibacillus polymyxa RO3C16, paenibacillus persicae TY4D5, paenibacillus pocheonensis S C3, paenibacillus aceris VF2D2, catalpa ovata TY2B5, paenibacillus wetland TY2D5, paenibacillus feed PG2A8, or any combination thereof.
42. The composition according to any one of the preceding embodiments, wherein the bacteria comprise non-endospore-forming bacteria.
43. The composition according to any one of the preceding embodiments, wherein the bacteria comprise bacteria belonging to the phylum Proteus.
44. The composition according to any one of the preceding embodiments, wherein the bacteria comprises klebsiella, rhizobium, bradyrhizobium, pallidobacter, sinorhizobium, xanthobacter, methylobacterium, actinomyces, coxsackie, azotobacter, acetobacter, rhodospirillum, pseudomonas, paraburkholderia, rocentrotus, geobacillus, serratia, pantoea, swordsmalls, enterobacter, or any combination thereof.
45. The composition according to any one of the preceding embodiments, wherein the bacteria comprise bacteria belonging to the phylum actinomycetes.
46. The composition according to any one of the preceding embodiments, wherein the bacteria comprises streptomyces, ke Kesi, frank's bacteria.
47. The composition according to any one of the preceding embodiments, wherein the bacteria comprise bacteria belonging to the phylum cyanobacteria.
48. The composition according to any one of the preceding embodiments, wherein the bacteria comprises cyanobacteria.
49. The composition according to any one of the preceding embodiments, wherein the bacteria comprise bacteria belonging to the phylum green-bending bacteria.
50. The composition according to any one of the preceding embodiments, wherein the one or more microorganisms comprise one or more fungi associated with the plant or the part thereof.
51. The composition according to any one of the preceding embodiments, wherein the one or more fungi associated with the plant or the part thereof is arranged in the gap between the seed coat and the seed embryo of the plant or the part thereof.
52. The composition according to any one of the preceding embodiments, wherein the one or more fungi associated with the plant or the part thereof are arranged as a coating of the plant or the part thereof.
53. A composition according to any one of the preceding embodiments, wherein the one or more fungi associated with a plant or part thereof is applied to the plant or part thereof using in-furrow techniques.
54. The composition according to any one of the preceding embodiments, wherein the one or more fungi associated with the plant or the part thereof are applied to the plant or the part thereof using a spray technique.
55. The composition according to any one of the preceding embodiments, wherein the one or more fungi associated with the plant or the part thereof is applied to the plant or the part thereof by the irrigation system.
56. The composition according to any one of the preceding embodiments, wherein the one or more fungi associated with the plant or the part thereof is arranged in the gap between the seed pericarp and the seed aleurone cell layer of the plant or the part thereof.
57. The composition according to any one of the preceding embodiments, wherein the one or more fungi comprises arbuscular mycorrhizal fungi.
58. The composition according to any one of the preceding embodiments, wherein the one or more fungi comprises ectomycorrhizal fungi.
59. The composition according to any one of the preceding embodiments, wherein the one or more fungi comprises a fungus from the genus trichoderma.
60. The composition according to any one of the preceding embodiments, wherein the one or more fungi comprises a fungus from the genus penicillium.
61. The composition according to any one of the preceding embodiments, wherein the one or more minerals comprise calcite, aragonite, dolomite, limestone or any combination thereof.
62. The composition according to any one of the preceding embodiments, wherein the one or more minerals comprise CaCO 3 、MgCO 3 、CaMg(CO 3 ) 2 Or any combination thereof.
63. The composition according to any one of the preceding embodiments, wherein said promoting production of said one or more minerals comprises production of ammonia and a resulting increase in pH in a medium in which plants derived from said plants or said parts thereof are grown.
64. The composition according to any one of the preceding embodiments, wherein the part thereof is a plant seed, and wherein the one or more microorganisms are not naturally present in the gap between the seed pericarp and the seed aleurone cell layer of the plant or the part thereof.
65. The composition according to any one of the preceding embodiments, wherein the plant or the part thereof is a monocot or dicot.
66. A method of promoting mineralization, the method comprising:
a. cultivating a plant or part thereof, one or more microorganisms associated with said plant or said part thereof,
wherein the one or more microorganisms are or are derived from microorganisms selected to produce or promote bicarbonate, carbonate, or one or more mineral formation.
67. The method according to any one of the preceding embodiments, wherein the plant or the part thereof is a commercial plant, plant root, plant stem, plant leaf, plant seed, plant fruit, plant tuber or plant root nodule.
68. The method according to any one of the preceding embodiments, wherein the one or more microorganisms associated with the plant or part thereof are disposed on the plant root or rhizosphere of the plant or part thereof.
69. The method according to any one of the preceding embodiments, wherein the one or more microorganisms associated with the plant or the part thereof are disposed on the plant root or the rhizosphere of the plant or the part thereof by an irrigation system.
70. The method according to any of the preceding embodiments, wherein the irrigation system comprises in-furrow treatment techniques.
71. The method according to any of the preceding embodiments, wherein the irrigation system comprises spray technology.
72. The method according to any one of the preceding embodiments, wherein the plant or part thereof originates from a seedling, which is integrated with the microorganism by the irrigation system to stimulate the plant or part thereof to produce the one or more minerals.
73. The method according to any one of the preceding embodiments, wherein the bicarbonate sequesters carbon.
74. The method according to any one of the preceding embodiments, wherein the carbon is gaseous carbon.
75. The method according to any one of the preceding embodiments, wherein the gaseous carbon is carbon dioxide.
76. The method according to any one of the preceding embodiments, wherein the carbonate sequesters carbon.
77. The method according to any one of the preceding embodiments, wherein the carbon is gaseous carbon.
78. The method according to any one of the preceding embodiments, wherein the gaseous carbon is carbon dioxide.
79. The method according to any one of the preceding embodiments, wherein the one or more minerals sequester carbon.
80. The method according to any one of the preceding embodiments, wherein the carbon is gaseous carbon.
81. The method according to any one of the preceding embodiments, wherein the gaseous carbon is carbon dioxide.
82. The method according to any one of the preceding embodiments, wherein the one or more microorganisms comprise bacteria, archaebacteria, fungi, or viruses.
83. The method according to any one of the preceding embodiments, wherein the one or more microorganisms comprise the bacteria.
84. The method according to any one of the preceding embodiments, wherein the bacteria comprise endospore-forming bacteria.
85. The method according to any one of the preceding embodiments, wherein the bacteria comprise rhizosphere bacteria.
86. The method according to any one of the preceding embodiments, wherein the rhizosphere bacteria comprises bacillus, paenibacillus, or both.
87. The method according to any one of the preceding embodiments, wherein the bacteria comprises bacillus amyloliquefaciens, bacillus laterosporus, bacillus licheniformis, bacillus macerans, bacillus cereus, bacillus circulans, bacillus firmus, bacillus subtilis, bacillus sphaericus, bacillus megaterium, bacillus coagulans, bacillus brevis, bacillus thuringiensis, bacillus mycoides, bacillus cucumeris, bacillus plantarum, bacillus pumilus, bacillus bailii, bacillus mucilaginosus, bacillus tertavatus, bacillus methylotrophicus, or any combination thereof.
88. The method according to any one of the preceding embodiments, wherein the bacteria comprises bacillus subtilis S3C23, bacillus subtilis MP2, bacillus subtilis RO2C15, bacillus subtilis RO2C22, bacillus megaterium 6, bacillus megaterium S3C21, bacillus megaterium RO2C12, bacillus cucumber S3C14, bacillus plantarum 5, or any combination thereof.
89. The method according to any one of the preceding embodiments, wherein the bacterium comprises bacillus subtilis S3C23.
90. The method according to any one of the preceding embodiments, wherein the bacteria comprise bacillus subtilis MP2.
91. The method according to any one of the preceding embodiments, wherein the bacteria comprise a C3C 10 strain.
92. The method according to any one of the preceding embodiments, wherein the one or more microorganisms comprise one or more fungi associated with the plant or the part thereof.
93. The method according to any one of the preceding embodiments, wherein the one or more fungi associated with the plant or the part thereof is arranged on the plant root or the rhizosphere of the plant or the part thereof by the irrigation system.
94. The method according to any one of the preceding embodiments, wherein the one or more fungi comprises arbuscular mycorrhizal fungi.
95. The method according to any one of the preceding embodiments, wherein the one or more fungi comprises ectomycorrhizal fungi.
96. The method according to any one of the preceding embodiments, wherein the one or more fungi comprises a fungus from the genus trichoderma.
97. The method according to any one of the preceding embodiments, wherein the one or more fungi comprises a fungus from the genus penicillium.
98. The method according to any one of the preceding embodiments, wherein the one or more microorganisms produce the formation of one or more carbonic anhydrases.
99. The method according to any one of the preceding embodiments, wherein the one or more microorganisms produce the formation of one or more carbonic anhydrases belonging to class a.
100. The method according to any one of the preceding embodiments, wherein the one or more microorganisms produce the formation of one or more carbonic anhydrases belonging to class β.
101. The method according to any one of the preceding embodiments, wherein the one or more microorganisms produce the formation of one or more carbonic anhydrases belonging to the class γ.
102. The method according to any one of the preceding embodiments, wherein the one or more microorganisms produce the formation of one or more carbonic anhydrases belonging to class delta.
103. The method according to any one of the preceding embodiments, wherein the one or more microorganisms produce the formation of one or more carbonic anhydrases belonging to the zeta class.
104. The method according to any one of the preceding embodiments, wherein the one or more microorganisms produce the formation of one or more carbonic anhydrases belonging to class η.
105. The process according to any one of the preceding embodiments, wherein the one or more microorganisms produce the formation of one or more carbonic anhydrases belonging to the iota class.
106. The method according to any one of the preceding embodiments, wherein the one or more minerals comprise calcite, aragonite, dolomite, limestone or any combination thereof.
107. The method according to any one of the preceding embodiments, wherein the promoting production of the one or more minerals comprises production of ammonia and a resulting increase in pH in a medium in which the plant or the part thereof is grown.
108. The method according to any one of the preceding embodiments, wherein the one or more microorganisms are not naturally present on the one or more roots.
109. The method according to any one of the preceding embodiments, wherein the plant or the part thereof is a monocot or dicot.
110. The method according to any one of the preceding embodiments, wherein the plant or the part thereof comprises a commercial plant or part thereof.
111. The method according to any one of the preceding embodiments, wherein the commercial plant or part thereof comprises a group consisting essentially of: corn, wheat, rice, sorghum, barley, rye, sugarcane, millet, oat, soybean, cotton, alfalfa, bean, quinoa, lentils, peanut, sunflower, canola, cassava, oil palm, potato, sugar beet, cocoa, coffee, lettuce, tomato, peas, cabbage, fruit trees, nut trees, forests, grasslands, or turf grasses.
112. A composition comprising one or more microorganisms, wherein the one or more microorganisms are or are derived from microorganisms selected to produce or promote bicarbonate, carbonate, or one or more mineral formations.
113. The composition according to any one of the preceding embodiments, wherein the plant or the part thereof is a plant root, a plant stem, a plant leaf, a plant seed, a plant fruit, a plant tuber or a plant root nodule.
114. The composition according to any one of the preceding embodiments, wherein the plant or the part thereof comprises a commercial plant or part thereof.
115. The composition according to any one of the preceding embodiments, wherein the commercial plant or part thereof is corn, wheat, rice, sorghum, barley, rye, sugarcane, millet, oat, soybean, cotton, alfalfa, bean, quinoa, lentil, peanut, sunflower, canola, cassava, oil palm, potato, sugar beet, cocoa, coffee, lettuce, tomato, pea, cabbage, fruit trees, nut trees, forests, grasslands, or turf grasses.
116. The composition according to any of the preceding embodiments, wherein the bicarbonate sequesters carbon.
117. The composition according to any one of the preceding embodiments, wherein the carbon is gaseous carbon.
118. The composition according to any of the preceding embodiments, wherein the gaseous carbon is carbon dioxide.
119. The composition according to any one of the preceding embodiments, wherein the carbonate sequesters carbon.
120. The composition according to any one of the preceding embodiments, wherein the carbon is gaseous carbon.
121. The composition according to any of the preceding embodiments, wherein the gaseous carbon is carbon dioxide.
122. The composition according to any one of the preceding embodiments, wherein the one or more minerals sequester carbon.
123. The composition according to any one of the preceding embodiments, wherein the carbon is gaseous carbon.
124. The composition according to any of the preceding embodiments, wherein the gaseous carbon is carbon dioxide.
125. The composition according to any one of the preceding embodiments, wherein the one or more microorganisms comprise bacteria, archaebacteria, fungi or viruses.
126. The composition according to any one of the preceding embodiments, wherein the one or more microorganisms comprise the bacteria.
127. The composition according to any one of the preceding embodiments, wherein the bacteria comprise endospore-forming bacteria.
128. The composition according to any one of the preceding embodiments, wherein the bacteria comprise rhizosphere bacteria.
129. The composition according to any one of the preceding embodiments, wherein the rhizosphere bacteria comprises bacillus, paenibacillus, or both.
130. The composition according to any one of the preceding embodiments, wherein the bacteria comprises bacillus amyloliquefaciens, bacillus laterosporus, bacillus licheniformis, bacillus macerans, bacillus cereus, bacillus circulans, bacillus firmus, bacillus subtilis, bacillus sphaericus, bacillus megaterium, bacillus coagulans, bacillus brevis, bacillus thuringiensis, bacillus mycoides, bacillus cucumeris, bacillus plantarum, bacillus pumilus, bacillus bailii, bacillus mucilaginosus, bacillus tertagatous, bacillus methylotrophicus, or any combination thereof.
131. The composition according to any one of the preceding embodiments, wherein the bacteria comprises bacillus subtilis S3C23, bacillus subtilis MP2, bacillus subtilis RO2C15, bacillus subtilis RO2C22, bacillus megaterium 6, bacillus megaterium S3C21, bacillus megaterium RO2C12, bacillus cucumber S3C14, bacillus plantarum 5, or any combination thereof.
132. The composition according to any one of the preceding embodiments, wherein the bacterium comprises bacillus subtilis S3C23.
133. The composition according to any one of the preceding embodiments, wherein the bacteria comprises bacillus subtilis MP2.
134. The composition according to any one of the preceding embodiments, wherein the bacteria comprise a C3C 10 strain.
135. The composition according to any one of the preceding embodiments, wherein the one or more microorganisms comprise one or more fungi associated with the plant or the part thereof.
136. The composition according to any one of the preceding embodiments, wherein the one or more fungi associated with the plant or the part thereof is arranged on the plant root or the rhizosphere of the plant or the part thereof by the irrigation system.
137. The composition according to any one of the preceding embodiments, wherein the one or more fungi comprises arbuscular mycorrhizal fungi.
138. The composition according to any one of the preceding embodiments, wherein the one or more fungi comprises ectomycorrhizal fungi.
139. The composition according to any one of the preceding embodiments, wherein the one or more fungi comprises a fungus from the genus trichoderma.
140. The composition according to any one of the preceding embodiments, wherein the one or more fungi comprises a fungus from the genus penicillium.
141. The composition according to any one of the preceding embodiments, wherein the one or more minerals comprise calcite, aragonite, dolomite, limestone or any combination thereof.
142. The composition according to any one of the preceding embodiments, wherein said promoting production of said one or more minerals comprises production of ammonia and a resulting increase in pH in a medium in which said plant or said part thereof is grown.
143. The composition according to any of the preceding embodiments, wherein the one or more microorganisms comprise one or more carbonic anhydrases.
144. The composition according to any of the preceding embodiments, wherein the one or more carbonic anhydrases comprise an alpha-carbonic anhydrase.
145. The composition according to any of the preceding embodiments, wherein the one or more carbonic anhydrases comprise a beta-type carbonic anhydrase.
146. The composition according to any of the preceding embodiments, wherein the one or more carbonic anhydrases comprise gamma-type carbonic anhydrases.
147. The composition according to any of the preceding embodiments, wherein the one or more carbonic anhydrases comprise a delta-type carbonic anhydrase.
148. The composition according to any one of the preceding embodiments, wherein the one or more carbonic anhydrases comprise a zeta-type carbonic anhydrase.
149. The composition according to any of the preceding embodiments, wherein the one or more carbonic anhydrases comprise class η carbonic anhydrase.
150. A composition according to any one of the preceding embodiments, wherein the one or more carbonic anhydrases comprise iota-type carbonic anhydrases.
Examples
The methods and compositions of the present disclosure are designed to microbially sequester carbon by forming bicarbonate and one or more minerals in association with the plant or plant seed.
Definition of microbial preparation: to achieve early adaptation, the present disclosure employs seed treatment compositions comprising synthetic consortia or individual isolated bacterial strains or endospores in suspension medium. In general, plant culture compositions and methods include diverse and environmentally-friendly plant-associated bacteria, the fines Bacteria belong to a variety of bacterial genera, distributed among different taxonomies within the phylum Proteobacteria (α -, β -, γ -and δ -Proteobacteria), the phylum Thick-walled, the phylum Bacteroides and the phylum Actinomyces. The present inventors have isolated and characterized rhizobacteria belonging to various genera, which generally contain plant-related microorganisms, which within these large taxonomic groups can be applied to seeds using the methods of the present disclosure in order to effectively colonize the roots and ultimately sequester CO 2 . The composition comprises one, two, three or several different bacterial strains cultured separately and mixed for Microprime TM And (5) seed treatment.
Example 1 seed treatment to increase non-endospore forming bacteria, endospore forming bacteria and/or bacterial endospore loading: microprime TM Techniques.
Although CO 2 Immobilized microorganisms are present in the soil, but CO is due to the following reasons 2 The seal is invalid. Many microorganisms cannot utilize CO 2 As its energy source. CO is generated by many organisms 2 The roots discharged are not close enough and their number is always low, as other microorganisms kill them. Microprime TM Seed treatment at CO 2 The release site delivers the microorganism directly and promotes 100% efficient colonization on the root.
Microprime TM Seed treatment is a stable microbial seed treatment process by which plant beneficial bacteria and/or synthetic consortia of microorganisms and/or secretions thereof and/or individualised biomolecules thereof as endospores or vegetative cells are enclosed inside the seed by an industrial scale process. The process takes into account process costs, time, stability over time (for plant embryos and inoculants), multi-soil compatibility, stability under different environmental conditions, and compatibility with conventional distribution chains of agricultural inputs. The method involves controlled, economical and rapid imbibition of seeds in an aqueous solution of an osmotically active liquid medium supplemented with specific amounts of, in addition to surfactants that enhance the intra-seed permeability and/or a set of nutrients that enhance the colonization of microorganisms within the seed and/or supplemental agents that enhance bacterial endospores formationThe beneficial microorganism or synthetic consortium of microorganisms and/or its secretions and/or its personalized biomolecules. Through Microprime TM The seed technology ensures long-term survival of the biological agent, genetic modulation of the embryo and extended shelf life of the treated seed. Finally, the method does not require a seed drying process, which makes it an economically unfeasible process, only $0.20 per acre, possibly even more reduced.
Microprime using endospores TM Seed treatment has 100% efficiency. This means that 100% of the treated seeds were effectively loaded with the desired endospores within 30 seconds, and that the average order of magnitude of bacterial cells per seed was 10 5 (Table 2). This high loading efficiency of microbial cells into seeds is Microprime TM Unique characteristics of seed treatment. This ensures that the desired bacteria are effectively delivered to the field. The colonization of plant roots by the loaded bacteria is 100%, which means that 100% of the treated seeds/plants are effectively colonized by the initially loaded bacteria. When seeds were sown, the root colonization process of the plants was immediately started, and was released from the dormant state and started to germinate (table 2, fig. 5 to fig. 7).
Microprime use in monocot and dicot seeds TM Seed treatment, the efficiency and effectiveness were the same (table 2). Briefly, microprime was performed using endospores of bacillus subtilis strain S3C23 TM Seed treatment is applied to monocot (corn and rice) and dicot (soybean) seeds. The percentage of root colonization plants and the percentage of seedling emergence were measured 14 days after sowing. For seed loading, the data represent the average of three pools, each pool having five seeds. For root colonisation, the data represent the average of 10 plants per treatment. For the percentage of seedlings that appeared, the data represent the average of 36 corn plants per treatment and 72 soybean and rice plants per treatment.
Microprime, as required by traditional seed industry and agricultural practices TM Seed treatment exhibited high compatibility with commercial seeds because embryo and bacterial stability was ensured over time (fig. 8). Described more specifically in WO2020214843Microprime TM Seed treatment process, which is incorporated herein by reference in its entirety.
Table 2: examples of bacterial Loading into seeds
Table 3: microprime applied to monocotyledonous plants and dicotyledonous plants TM Summary of seed treatments
Example 2: biological systems plant-microorganism effect on carbon dioxide removal.
The carbonless world is a global initiative and many internationally known companies (such as microsoft, amazon and google) promise to achieve carbon balance or no carbon for 2040 years. These companies create a high carbon footprint and in order to become carbon balanced, in the near future, these companies will never be able to effectively capture CO 2 To purchase carbon credits. The focus of this technology is not only on reducing carbon emissions, but also on converting it into agriculturally useful products or bio-durable products. Effective CO will be ensured using the compositions and methods disclosed herein 2 Sequestration, which would generate carbon credits for farmers and at the same time slow down climate change. Because the overall cost of the compositions and methods disclosed herein is significantly lower than any prior art in the marketplace, their adoption should be rapid, which would allow for the generation of large amounts of carbon credits to provide them to companies that are increasingly in need of carbon footprint reduction.
Carbon Dioxide Removal (CDR) is a climate engineering in which CO 2 Removed from the atmosphere and stored for a long period of time. CDR methods include forestation, agricultural practices to sequester carbon in the soil, bioenergy with carbon capture and storage, enhanced weathering (enhanced weathering), marine fertilization, and direct air capture in combination with storage.
The 2019 consensus report of the national academy of sciences, engineering and medical sciences concludes that it is possible to remove and sequester carbon dioxide as much as 10Gt per year using existing CDR methods on a scale that can be safely and economically deployed. This will offset greenhouse gas emissions by about one fifth of the production rate.
For carbon sequestration, three conditions are required: storage, space and low cost power. Current technologies for capturing carbon from air, such as Direct Air Capture (DAC), require a large space for their operation and a large amount of energy to extract CO from ambient air 2 The theoretical minimum energy required is about 250 kWh/ton CO 2 。
On the other hand, by using existing crop parks, fruit trees and forests to support food production for the global population, and also using wood as commodity for several industries, the advantages in terms of installed capacity (installed capacity) and low cost power are unparalleled.
In more detail, with respect to capturing CO 2 FAO prediction, global arable land use will continue to be extended from 15.8 hectares (3.9 x 10) of 2014 9 Acre) to 16.6 hundred million hectares (4.1×10) at 2050 9 Acre) and the total area of forests is 40.6 hundred million hectares, the installed capacity already exists and must be used.
Regarding low energy power, the use of microorganisms (such as bacteria) that interact tightly with plant roots is CO 2 The most cost-effective solution for the sequestration plant. This is because plants provide a rich mixture of carbon and energy sources through their root secretions, which can be used by soil bacteria to proliferate, and on the other hand, plants benefit in a variety of ways through colonization by these bacteria. Such biological system plants-bacteria thrive without additional power input.
Finally, by using plant-related carbonic anhydrase expressing microorganisms, CO2 can be efficiently sequestered by producing bicarbonate and ultimately carbonate minerals in the soil.
Example 3: methods for measuring CA activity and/or bicarbonate and mineral formation.
On a laboratory scale, e.g. firstSelected microorganisms were cultured in CA-producing medium as described previously (Zhuang et al, 2018). CA activity units were measured at different time points using the CA activity measurement method described in Zhuang et al, 2018. Alternatively, relative transcript abundance can be measured to see which CA has the highest activity, as many microorganisms encode more than one CA in their genome. In addition, the production of bicarbonate and carbonate ions, ammonium (NH 4 + ) Concentration, growth curve and pH change.
Biological and non-biological carbonate minerals (MgCO) were tested using the method described in Han et al 2020 3 And CaCO (CaCO) 3 ) Is formed by the steps of (a). The precipitate in the form of carbonate minerals was characterized using Scanning Electron Microscopy (SEM). The mineralization purposes and the effectiveness of the different cations were tested and the stability was analyzed. X-ray diffractometer (XRD) analysis was performed to characterize the morphology of the minerals (Zhuang et al, 2018). To further demonstrate the biological origin of these minerals, stable carbon isotope values can be considered. Although mineralization occurs on the bacterial surface, mineralization to some extent may also be internal to the bacterial cell. The detection of intracellular mineral formation was performed by the method described in Han et al 2020.
Testing selected mineralized CO with enhancement in a greenhouse 2 Microorganisms capable of being used in the greenhouse, microprime TM Seed technology loads these microorganisms into seeds of major crops (such as soybean, corn, etc.), the Microprime TM Seed technology ensures efficient and guaranteed colonization of the root by microorganisms when the root emerges. Allowing the plants to grow under standard conditions, after a given time, the plants will be root-extracted and the bacteria will be extracted, followed by counting and mineralizing the CO on their surface 2 . In addition, soil samples closer to the roots were analyzed to quantify total carbon, bicarbonate, carbonate, and minerals, while data was compared to control soil (without Microprime TM Seed/plant of (a). As previously mentioned, stable C isotopes may be used 13 C]To resolve CO 2 Immobilization, respiration of the soil microflora and soil respiration. Finally, selection in large soybean and corn field testingTo measure CO 2 Fixation, mineral production and its influence on productivity.
Example 4: strains that enhance CA activity and/or formation are engineered.
To fully utilize the microorganisms disclosed herein to immobilize CO via CA 2 Genetic engineering will be employed to increase its expression, stability, activity and secretion. According to Han et al, 2019, the concentration of bacillus subtilis CA increases from 0h up to about 30h to 17.14U/L, followed by a slight decrease from 31h to 350 h. In bacillus subtilis, it has been demonstrated that mRNA is continuously produced even if the bacteria are not growing. This suggests that mRNA stability is enhanced, and thus it is strategically significant to increase its production. Although CA production may be tightly regulated, genetic strategies will be applied to relieve such regulation, including but not limited to making expression constitutive. For this approach, several high expressing housekeeping gene promoters or strongly expressed constitutive promoter genes, such as P, will be tested by substituting the natural promoter of CA liaG 、P lepA 、P veg 、P gsiB 、P43、P trnQ 、P lial (bacitracin-inducible) and P xylA (xylose inducible). Increased CA transcript abundance may maintain the highest level of CA. The strength of the promoters was tested using a relative transcript abundance study via qRT-PCR, and CA proteins were labeled to assess the corresponding increases in transcript and protein levels.
Many microorganisms can exocytosis CA, which catalyzes CO under alkaline conditions 2 Hydration to form HCO 3 - . Extracellular secretion or targeting of CA to the periplasmic space facilitates efficient CO sequestration by mineral carbonation 2 . Whole cells as CA catalysts have been shown to be important for mineralization even at slightly acidic or near neutral pH conditions. This is particularly critical for mineralization in the soil, as the pH of the soil may be slightly acidic or neutral and may not reach pH 9.0 (ideal pH for mineralization). Jo et al, 2013 targets CA (ngCA) of Neisseria gonorrhoeae (Neisseria gonorrhoeae) to the cytoplasmic and periplasmic space of E.coli (E.coli) and concludes that ngCA is in E.coli periplasmThe expression of (C) greatly accelerates CaCO 3 The rate of formation. Microorganisms were tested for their ability to transport CA to the periplasmic space or their extracellular secretion. Various methods can be used to alter the targeting of CA in a strain. These strategies involve the addition of periplasmic signal sequences or extracellular secretion signals to the 5' end of CA. The twin arginine transport (Tat) pathway may be used to extracellular transport fully folded proteins. For extracellular secretion of CA, the actual Tat signal (TorA leader sequence) will be fused to the 5' end of CA. Alternatively, a general secretory pathway (Sec) that initiates translocator protein upon protein synthesis may be used, and a true Sec signal such as PelB may be used. These new strains were tested for their ability to target CA in specific compartments of bacterial cells, followed by the Microprime-passing tests as described above TM Is a microorganism of the genus (A).
The soil environment is dynamic and may contain CA inhibitors, to test this, the activity of CA will be tested in the presence of sample soil. To increase its fitness and to evolve the CA enzyme, directed evolution methods may be used. The method is used for improving the thermal stability and alkali resistance of CA of common Vibrio desulphurisation (Desulfovibrio vulgaris). Alvizo et al, 2014 used directed evolution to increase temperature resistance to 107 ℃ in the presence of a 4.2M alkaline amine solvent at pH >10.0, and this increase was a 4,000,000 fold improvement over the native enzyme.
Example 5: concentration of bicarbonate and calcium carbonate in soil-2020 season:
corn was grown in the western U.S. field with Microprime bacillus subtilis S3C23 treated seed and untreated seed (control). IN the middle of the growing season, 9 samples were taken from three different fields (Paxton, IL), milford, IL and Wolcott, indiana) at a time treatment. Soil samples were analyzed for calcium carbonate and bicarbonate content. The results are shown in table 4. An average of 3.19 tons of CO compared to the soil of the untreated plant (control plant) 2 Acres are sealed in soil containing bacillus subtilis strain S3C 23. This value reflects what was detected up to the V11 maize stage (45 days after sowing, 110 days total)Bicarbonate and calcium carbonate concentrations.
Table 4: soil analysis corn 2020 season results.
Example 6: concentration of bicarbonate and calcium carbonate in soil-2021 season:
corn was grown in the western U.S. field with Microprime bacillus subtilis S3C23 treated seed and untreated seed (control). IN the middle of the growing season, 9 samples were taken from four different fields (milford, indiana, renssel (IN), beaver Damn (wife, WI), and Monticello, IL) at a time. Soil samples were analyzed for calcium carbonate and bicarbonate content. The results are shown in table 5. On average 8.18 tons of CO compared to the soil of the untreated plant (control plant) 2 Acres are sealed in soil containing bacillus subtilis strain S3C 23. This value reflects the concentrations of bicarbonate and calcium carbonate detected up to the V11 corn stage (45 days after sowing, 110 days total).
Table 5: soil analysis corn 2021 season results.
In the western U.S. field, soybeans were planted with Microprime bacillus subtilis MP1, bacillus subtilis MP2, and sabia adhesion S3C10 treated seed and untreated seed (control). In the middle of the growing season, eachSecondary treatments 9 samples were taken from one field (Flanagan, IL). Soil samples were analyzed for calcium carbonate and bicarbonate content. The results are shown in table 6. On average 8.63 tons of CO compared to the soil of the untreated plant (control plant) 2 Acres are sealed in soil containing Andes Microprime microbial treatments. This value reflects the concentrations of bicarbonate and calcium carbonate detected up to 45 days after sowing.
The data presented in tables 5 and 6 indicate that 2020 data as shown in Table 4 is not merely coincidental, andes Microprime TM Treatment and Andes-specific microorganisms are treating large amounts of CO 2 The sequestration into the soil is essentially effective.
Table 6: soil analysis soybean 2021 season results.
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. The invention is not limited by the specific embodiments provided in the specification. While the invention has been described with reference to the foregoing specification, the description and illustrations of the embodiments herein are not intended to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it is to be understood that all aspects of the invention are not limited to the specific descriptions, configurations, or relative proportions set forth herein, depending on various conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the present invention will also cover any such alternatives, modifications, variations, or equivalents. The following claims are intended to define the scope of the invention and methods and structures within the scope of these claims and their equivalents are covered thereby.

Claims (167)

1. A composition comprising a plant seed and one or more microorganisms associated with the plant seed, wherein the one or more microorganisms are or are derived from one or more microorganisms selected to produce or promote bicarbonate, carbonate, or one or more mineral formations.
2. The composition of claim 1, wherein the plant seed is a commercial plant seed, a fruit tree seed, a nut tree seed, a shrub seed, a bulb seed, a grassland seed, a lawn seed, or any combination thereof.
3. The composition of claim 1, wherein the one or more microorganisms associated with the plant seed are disposed in a gap between a seed coat and a seed embryo of the plant seed.
4. The composition of claim 1, wherein the one or more microorganisms associated with the plant seed are disposed as a coating of the plant seed.
5. The composition of claim 1, wherein the one or more microorganisms associated with the plant seed are applied to the plant seed by an irrigation system.
6. The composition of claim 5, wherein the irrigation system comprises in-furrow treatment techniques.
7. The composition of claim 5, wherein the irrigation system comprises spray technology.
8. The composition of any of the preceding claims, wherein the bicarbonate sequesters carbon.
9. The composition of claim 8, wherein the carbon is gaseous carbon.
10. The composition of claim 9, wherein the gaseous carbon is carbon dioxide.
11. The composition of any of the preceding claims, wherein the carbonate sequesters carbon.
12. The composition of claim 11, wherein the carbon is gaseous carbon.
13. The composition of claim 12, wherein the gaseous carbon is carbon dioxide.
14. The composition of any one of the preceding claims, wherein the one or more minerals sequester carbon.
15. The composition of claim 14, wherein the carbon is gaseous carbon.
16. The composition of claim 15, wherein the gaseous carbon is carbon dioxide.
17. The composition of any one of the preceding claims, wherein the one or more microorganisms associated with the plant seed are disposed in a gap between a seed pericarp and a seed aleurone cell layer of the plant seed.
18. The composition of any one of the preceding claims, wherein the one or more microorganisms comprise one or more carbonic anhydrases.
19. The composition of claim 18, wherein the one or more carbonic anhydrases comprise carbonic anhydrases that belong to class a.
20. The composition of claim 18, wherein the one or more carbonic anhydrases comprise carbonic anhydrases that belong to class β.
21. The composition of claim 18, wherein the one or more carbonic anhydrases comprise carbonic anhydrases belonging to the class gamma.
22. The composition of claim 18, wherein the one or more carbonic anhydrases comprise carbonic anhydrases that belong to class delta.
23. The composition of claim 18, wherein the one or more carbonic anhydrases comprise carbonic anhydrases belonging to the zeta class.
24. The composition of claim 18, wherein the one or more carbonic anhydrases comprise carbonic anhydrases that belong to class η.
25. The composition of claim 18, wherein the one or more carbonic anhydrases comprise carbonic anhydrases belonging to the iota class.
26. The composition of any one of the preceding claims, wherein the one or more microorganisms comprise bacteria, archaea, fungi, or viruses.
27. The composition of claim 26, wherein the one or more microorganisms comprise the bacteria.
28. The composition of claim 27, wherein the bacteria comprise endospore-forming bacteria.
29. The composition according to claim 27, wherein the bacterium comprises a strain derived from Acetobacter, actinomyces, bacillus, alkaleidosporium, aminophilia, bacillus bifidus, anaerobic Bacillus, thiobacillus, anaerobic Bacillus, brevibacterium, thermoanaerobacter, caminiella, prunus, clostridium, ke Enshi, ke Kesi, tree-derived spore bacillus, bacillus, and a nucleic acid sequence derived from Bacillus, such as Bacillus, and Bacillus desulphurized enterobacteria, desulfosporidium, desulphurized campylobacter, desulfosporidium, desulphurized agaricus, geotrichum, bacillus gracillus, bacillus camptotheca, halonatronum, solar bacillus, mesophilus, rice bacillus, chroogomphatus, lysine bacillus, mahela, metaacter, metacter desulphurized enterobacteria, desulfospormus, desulphurized campylobacter, desulfospormus, desulphurized Sheng Bacillus, desulphurized Bacillus, protomyces, linear Bacillus, gelria, geobacillus Geosporobacter, bacillus, halomatron, solar, japanese, leuconostoc, chromobacterium, lysine, mahela, metacter, metatinum, and Metatinum, bacteria of the genera Thermoanaerobomonas, thermobacillus, thermoflavus, thermovelabaum, bacillus, cladosporium, vulcanobacillus, or combinations thereof.
30. The composition of claim 27, wherein the bacteria comprise bacteria belonging to the phylum firmicutes.
31. The composition of claim 27, wherein the bacteria comprise rhizosphere bacteria.
32. The composition of claim 31, wherein the rhizosphere bacteria comprises bacillus, paenibacillus, or both.
33. The composition of claim 27, wherein the bacteria comprises bacillus amyloliquefaciens, bacillus laterosporus, bacillus licheniformis, bacillus macerans, bacillus cereus, bacillus circulans, bacillus firmus, bacillus subtilis, bacillus megaterium, bacillus coagulans, bacillus brevis, bacillus sphaericus, bacillus thuringiensis, bacillus mycoides, bacillus cucumeris, bacillus plantarum, bacillus pumilus, bacillus bailii, bacillus mucilaginosus, bacillus tertiarydorus, bacillus methylotrophicus, or any combination thereof.
34. The composition of claim 27, wherein the bacteria comprises bacillus subtilis S3C23, bacillus subtilis MP2, bacillus subtilis RO2C15, bacillus subtilis RO2C22, bacillus megaterium 6, bacillus megaterium S3C21, bacillus megaterium RO2C12, bacillus cucumber S3C14, bacillus plantarum 5, or any combination thereof.
35. The composition of claim 27, wherein the bacteria comprises bacillus subtilis S3C23.
36. The composition of claim 27, wherein the bacteria comprises bacillus subtilis MP2.
37. The composition of claim 27, wherein the bacteria comprises a C3C 10 bacterium.
38. The composition of claim 27, wherein the bacteria comprises paenibacillus polymyxa, paenibacillus persicae, paenibacillus pocheonensis, paenibacillus aceris, paenibacillus catalpa, paenibacillus wetland, paenibacillus feed, paenibacillus brazil, or any combination thereof.
39. The composition of claim 27, wherein the bacteria comprise paenibacillus polymyxa RO3C16, paenibacillus persicae TY4D5, paenibacillus pocheonensis S C3, paenibacillus aceris VF2D2, paenibacillus catalpa TY2B5, paenibacillus wetland TY2D5, paenibacillus feed PG2A8, or any combination thereof.
40. The composition of claim 27, wherein the bacteria comprise non-endospore forming bacteria.
41. The composition of claim 27, wherein the bacteria comprise bacteria belonging to the phylum proteus.
42. The composition of claim 27, wherein the bacteria comprises klebsiella, rhizobium, bradyrhizobium, pallium, sinorhizobium, xanthobacter, methylobacterium, actinomyces, kosakochia, azotobacter, acetobacter, rhodospirillum, pseudomonas, paraburkholderia, ralstonia, geobacillus, serratia, pantoea, rapier, enterobacter, or any combination thereof.
43. The composition of claim 27, wherein the bacteria comprise bacteria belonging to the phylum actinomycetes.
44. The composition of claim 27, wherein the bacteria comprises streptomyces, ke Kesi, frank's bacteria.
45. The composition of claim 27, wherein the bacteria comprise bacteria belonging to the phylum cyanobacteria.
46. The composition of claim 27, wherein the bacteria comprises cyanobacteria.
47. The composition of claim 27, wherein the bacteria comprise bacteria belonging to the phylum green-curved.
48. The composition of claim 26, wherein the one or more microorganisms comprise one or more fungi associated with the plant seed.
49. The composition according to claim 48, wherein said one or more fungi associated with said plant seed is disposed in the interstice between the seed coat and the seed embryo of said plant seed.
50. The composition according to claim 48, wherein said one or more fungi associated with said plant seed is arranged as a coating of said plant seed.
51. The composition according to claim 48, wherein said one or more fungi associated with said plant seed is applied to said plant seed using in-furrow techniques.
52. The composition according to claim 48, wherein said one or more fungi associated with said plant seed is applied to said plant seed using spray techniques.
53. The composition according to claim 48, wherein said one or more fungi associated with said plant seed is applied to said plant seed by irrigation.
54. The composition according to claim 48, wherein said one or more fungi associated with said plant seed is disposed in a gap between a seed pericarp and a seed aleurone cell layer of said plant seed.
55. The composition according to claim 48, wherein said one or more fungi comprises arbuscular mycorrhizal fungi.
56. The composition of claim 48, wherein the one or more fungi comprise ectomycorrhizal fungi.
57. The composition according to claim 48, wherein said one or more fungi comprises a fungus from the genus trichoderma.
58. The composition according to claim 48, wherein said one or more fungi comprises a fungus from the genus penicillium.
59. The composition of any one of the preceding claims, wherein the one or more minerals comprise calcite, aragonite, dolomite, limestone, or any combination thereof.
60. The composition of any of the preceding claims, wherein the one or more minerals comprise CaCO 3 、MgCO 3 、CaMg(CO 3 ) 2 Or any combination thereof.
61. The composition of any one of the preceding claims, wherein the promoting production of the one or more minerals comprises production of ammonia and an increase in pH in a medium resulting therefrom in which a plant derived from the plant seed is grown.
62. The composition of any one of the preceding claims, wherein the one or more microorganisms are not naturally present in the gap between the seed pericarp and the seed aleurone cell layer of the plant seed.
63. The composition of any one of the preceding claims, wherein the plant seed is a monocot seed or a dicot seed.
64. The composition of claim 2, wherein the commercial plant seed is corn seed, wheat seed, rice seed, sorghum seed, barley seed, rye seed, sugarcane seed, millet seed, oat seed, soybean seed, cotton seed, alfalfa seed, bean seed, quinoa seed, lentil seed, peanut seed, sunflower seed, canola seed, cassava seed, oil palm seed, potato seed, sugar beet seed, cocoa seed, coffee beans, lettuce seed, tomato seed, pea seed, or cabbage seed.
65. A composition comprising a plant or part thereof and one or more microorganisms associated with the plant or part thereof, wherein the one or more microorganisms are or are derived from one or more microorganisms selected to produce or promote bicarbonate, carbonate, or one or more mineral formations.
66. The composition of claim 65, wherein said plant or said portion thereof is a plant root, a plant stem, a plant leaf, a plant seed, a plant fruit, a plant tuber, or a plant nodule.
67. The composition of any one of the preceding claims, wherein the plant or the portion thereof comprises a commercial plant or portion thereof.
68. The composition of claim 67, wherein the commercial plant or part thereof is corn, wheat, rice, sorghum, barley, rye, sugarcane, millet, oat, soybean, cotton, alfalfa, bean, quinoa, lentils, peanut, sunflower, canola, cassava, oil palm, potato, sugar beet, cocoa, coffee, lettuce, tomato, pea, cabbage, fruit trees, nut trees, forests, grasslands, or turf grasses.
69. The composition of claim 66, wherein said portion thereof is a plant seed, and wherein said one or more microorganisms associated with said plant seed are disposed in a gap between a seed coat and a seed embryo of said plant seed.
70. The composition of claim 65, wherein the one or more microorganisms associated with the plant or the portion thereof are disposed as a coating of the plant or the portion thereof.
71. The composition of claim 65, wherein said one or more microorganisms associated with said plant or said portion thereof are applied to said plant seed by an irrigation system.
72. The composition of claim 71, wherein the irrigation system comprises in-furrow treatment techniques.
73. The composition according to claim 71, wherein the irrigation system comprises spray technology.
74. The composition of any of the preceding claims, wherein the bicarbonate sequesters carbon.
75. The composition of claim 74, wherein the carbon is gaseous carbon.
76. The composition of claim 75, wherein said gaseous carbon is carbon dioxide.
77. The composition of any of the preceding claims, wherein the carbonate sequesters carbon.
78. The composition of claim 77, wherein said carbon is gaseous carbon.
79. The composition of claim 78, wherein the gaseous carbon is carbon dioxide.
80. The composition of any one of the preceding claims, wherein the one or more minerals sequester carbon.
81. The composition of claim 80, wherein the carbon is gaseous carbon.
82. The composition of claim 81, wherein the gaseous carbon is carbon dioxide.
83. The composition of claim 66, wherein said portion thereof is a plant seed, and wherein said one or more microorganisms associated with said plant seed are disposed in a gap between a seed pericarp and a seed aleurone cell layer of said plant seed.
84. The composition of any one of the preceding claims, wherein the one or more microorganisms comprise one or more carbonic anhydrases.
85. The composition of claim 84, wherein the one or more carbonic anhydrases comprise carbonic anhydrases that belong to class a.
86. The composition of claim 84, wherein the one or more carbonic anhydrases comprise carbonic anhydrases that belong to class β.
87. The composition of claim 84, wherein the one or more carbonic anhydrases comprise carbonic anhydrases belonging to the class gamma.
88. The composition of claim 84, wherein the one or more carbonic anhydrases comprise carbonic anhydrases that belong to class delta.
89. The composition of claim 84, wherein the one or more carbonic anhydrases comprise carbonic anhydrases that belong to the zeta class.
90. The composition of claim 84, wherein the one or more carbonic anhydrases comprise carbonic anhydrases that belong to class η.
91. The composition according to claim 84, wherein said one or more carbonic anhydrases comprise carbonic anhydrases belonging to the iota class.
92. The composition of any one of the preceding claims, wherein the one or more microorganisms comprise bacteria, archaea, fungi, or viruses.
93. The composition of claim 92, wherein the one or more microorganisms comprise the bacteria.
94. The composition of claim 93, wherein the bacteria comprise endospore-forming bacteria.
95. The composition of claim 93 which, wherein the bacterium comprises a strain derived from Acetobacter, actinomyces, bacillus, alkaleidosporium, aminophilia, bacillus bifidus, anaerobic Bacillus, thiobacillus, anaerobic Bacillus, brevibacterium, thermoanaerobacter, caminiella, prunus, clostridium, ke Enshi, ke Kesi, acidocella, bacillus, and methods of producing the same desulphurized enterobacteria, desulfosporidium, desulphurized campylobacter, desulfosporidium, desulphurized agaricus, geotrichum, bacillus gracillus, bacillus camptotheca, halonatronum, solar bacillus, mesophilus, rice bacillus, chroogomphatus, lysine bacillus, mahela, metaacter, metacter desulphurized enterobacteria, desulfospormus, desulphurized campylobacter, desulfospormus, desulphurized Sheng Bacillus, desulphurized Bacillus, protomyces, linear Bacillus, gelria, geobacillus Geosporobacter, bacillus, halomatron, solar, japanese, leuconostoc, chromobacterium, lysine, mahela, metacter, metatinum, and Metatinum, bacteria of the genera Thermoanaerobomonas, thermobacillus, thermoflavus, thermovelabaum, bacillus, cladosporium, vulcanobacillus, or combinations thereof.
96. The composition of claim 93, wherein the bacteria comprise bacteria belonging to the phylum firmicutes.
97. The composition of claim 93, wherein the bacteria comprise rhizosphere bacteria.
98. The composition of claim 97, wherein the rhizosphere bacteria comprises bacillus, paenibacillus, or both.
99. The composition of claim 93, wherein the bacteria comprises bacillus amyloliquefaciens, bacillus laterosporus, bacillus licheniformis, bacillus macerans, bacillus cereus, bacillus circulans, bacillus firmus, bacillus subtilis, bacillus megaterium, bacillus coagulans, bacillus brevis, bacillus sphaericus, bacillus thuringiensis, bacillus mycoides, bacillus cucumber, bacillus plantarum, bacillus pumilus, bacillus bailii, bacillus mucilaginosus, bacillus tertageus, bacillus methylotrophicus, or any combination thereof.
100. The composition of claim 93, wherein the bacteria comprises bacillus subtilis S3C23, bacillus subtilis MP2, bacillus subtilis RO2C15, bacillus subtilis RO2C22, bacillus megaterium 6, bacillus megaterium S3C21, bacillus megaterium RO2C12, bacillus cucumber S3C14, bacillus plantarum 5, or any combination thereof.
101. The composition of claim 93, wherein the bacteria comprises bacillus subtilis S3C23.
102. The composition of claim 93, wherein the bacteria comprises bacillus subtilis MP2.
103. The composition of claim 93, wherein the bacteria comprises a C3C 10 bacterium of the genus r.
104. The composition of claim 93, wherein the bacteria comprises paenibacillus polymyxa, paenibacillus persicae, paenibacillus pocheonensis, paenibacillus aceris, paenibacillus catalpa, paenibacillus wetland, paenibacillus feed, paenibacillus brazil, or any combination thereof.
105. The composition of claim 93, wherein the bacteria comprise paenibacillus polymyxa RO3C16, paenibacillus persicae TY4D5, paenibacillus pocheonensis S C3, paenibacillus aceris VF2D2, paenibacillus catalpa TY2B5, paenibacillus wetland TY2D5, paenibacillus feed PG2A8, or any combination thereof.
106. The composition of claim 93, wherein the bacteria comprise non-endospore forming bacteria.
107. The composition of claim 93, wherein the bacteria comprise bacteria belonging to the phylum proteus.
108. The composition of claim 93, wherein the bacteria comprises klebsiella, rhizobium, bradyrhizobium, pallium, sinorhizobium, xanthobacter, methylobacterium, actinomyces, kosakochia, azotobacter, acetobacter, rhodospirillum, pseudomonas, paraburkholderia, rocentrum, geobacillus, serratia, pantoea, rapier, enterobacter, or any combination thereof.
109. The composition of claim 93, wherein the bacteria comprise bacteria belonging to the phylum actinomycetes.
110. The composition of claim 93, wherein the bacteria comprises streptomyces, ke Kesi, frank's bacteria.
111. The composition of claim 93, wherein the bacteria comprise bacteria belonging to the phylum cyanobacteria.
112. The composition of claim 93, wherein the bacteria comprises a cyanobacterium.
113. The composition of claim 93, wherein the bacteria comprise bacteria belonging to the phylum green-curved.
114. The composition of claim 92, wherein said one or more microorganisms comprise one or more fungi associated with said plant or said portion thereof.
115. The composition of claim 114, wherein the one or more fungi associated with the plant or the part thereof is disposed in a gap between the seed coat and the seed embryo of the plant or the part thereof.
116. The composition of claim 114, wherein the one or more fungi associated with the plant or the part thereof are disposed as a coating of the plant or the part thereof.
117. The composition of claim 114, wherein the one or more fungi associated with a plant or part thereof are applied to the plant or part thereof using in-furrow techniques.
118. The composition of claim 114, wherein the one or more fungi associated with the plant or the part thereof are applied to the plant or the part thereof using a spray technique.
119. The composition of claim 114, wherein the one or more fungi associated with the plant or the part thereof are applied to the plant or the part thereof by the irrigation system.
120. The composition of claim 114, wherein the one or more fungi associated with the plant or the part thereof is disposed in a gap between a seed pericarp and a seed aleurone cell layer of the plant or the part thereof.
121. The composition of claim 114, wherein the one or more fungi comprise arbuscular mycorrhizal fungi.
122. The composition of claim 114, wherein the one or more fungi comprise ectomycorrhizal fungi.
123. The composition of claim 114, wherein the one or more fungi comprises a fungus from the genus trichoderma.
124. The composition of claim 114, wherein the one or more fungi comprises a fungus from the genus penicillium.
125. The composition of any one of the preceding claims, wherein the one or more minerals comprise calcite, aragonite, dolomite, limestone, or any combination thereof.
126. The composition of any of the preceding claims, wherein the one or more minerals comprise CaCO 3 、MgCO 3 、CaMg(CO 3 ) 2 Or any combination thereof.
127. The composition of any one of the preceding claims, wherein the promoting production of the one or more minerals comprises production of ammonia and an increase in pH in a medium resulting therefrom, in which medium plants derived from the plant or the part thereof are grown.
128. The composition of any one of the preceding claims, wherein the portion thereof is a plant seed, and wherein the one or more microorganisms are not naturally present in the gap between the seed pericarp and the seed aleurone cell layer of the plant or the portion thereof.
129. The composition of any one of the preceding claims, wherein the plant or the part thereof is a monocot or dicot.
130. A method of sequestering carbon, the method comprising:
a. cultivating a plant or part thereof and one or more microorganisms associated with said plant or said part thereof;
wherein the one or more microorganisms are or are derived from microorganisms selected to produce or promote the formation of one or more carbonate minerals to sequester carbon.
131. The method of any one of the preceding claims, wherein the plant or the portion thereof is a plant root, a plant stem, a plant leaf, a plant seed, a plant fruit, a plant tuber, or a plant nodule.
132. The method of any one of the preceding claims, wherein the plant or the portion thereof comprises a commercial plant or portion thereof.
133. The method of claim 132, wherein the commercial plant or portion thereof comprises a group consisting essentially of: corn, wheat, rice, sorghum, barley, rye, sugarcane, millet, oat, soybean, cotton, alfalfa, bean, quinoa, lentils, peanut, sunflower, canola, cassava, oil palm, potato, sugar beet, cocoa, coffee, lettuce, tomato, peas, cabbage, fruit trees, nut trees, forests, grasslands, or turf grasses.
134. The method of claim 130, wherein the one or more carbonate minerals comprise one or more gaseous carbonates.
135. The method of claim 134, wherein the one or more gaseous carbonates comprises carbon monoxide, methane, or carbon dioxide.
136. The method of claim 134, wherein the one or more gaseous carbonating species is the carbon dioxide.
137. The method of any one of the preceding claims, wherein the one or more microorganisms associated with the plant are disposed on the plant root or rhizosphere of the plant or the portion thereof.
138. The method of any one of the preceding claims, wherein the one or more microorganisms associated with the plant are disposed on the plant root or the rhizosphere of the plant or the portion thereof by an irrigation system.
139. The method of claim 138, wherein the irrigation system comprises in-furrow treatment techniques.
140. The method of claim 138, wherein the irrigation system comprises spray technology.
141. The method of any one of the preceding claims, wherein the plant or part thereof originates from a seedling that is integrated with the microorganism by the irrigation system to stimulate the plant or part thereof to produce the one or more minerals.
142. The method of any one of the preceding claims, wherein the one or more microorganisms comprise bacteria, archaebacteria, fungi, or viruses.
143. The method of claim 142, wherein the one or more microorganisms comprise the bacteria.
144. The method of claim 143, wherein the bacteria comprise endospore-forming bacteria.
145. The method of claim 143, wherein the bacteria comprise rhizosphere bacteria.
146. The method of claim 145, wherein the rhizosphere bacteria comprises bacillus, paenibacillus, or both.
147. The method of claim 143, wherein the bacteria comprises bacillus amyloliquefaciens, bacillus laterosporus, bacillus licheniformis, bacillus macerans, bacillus cereus, bacillus circulans, bacillus firmus, bacillus subtilis, bacillus megaterium, bacillus coagulans, bacillus brevis, bacillus sphaericus, bacillus thuringiensis, bacillus mycoides, bacillus cucumber, bacillus plantarum, bacillus pumilus, bacillus bailii, bacillus mucilaginosus, bacillus terteus, bacillus methylotrophicus, or any combination thereof.
148. The method of claim 143, wherein the bacteria comprises bacillus subtilis S3C23, bacillus subtilis MP2, bacillus subtilis RO2C15, bacillus subtilis RO2C22, bacillus megaterium 6, bacillus megaterium S3C21, bacillus megaterium RO2C12, bacillus cucumber S3C14, bacillus plantarum 5, or any combination thereof.
149. The method of claim 143, wherein the bacteria comprises bacillus subtilis S3C23.
150. The method of claim 143, wherein the bacteria comprises bacillus subtilis MP2.
151. The method of claim 143, wherein the bacteria comprises a C3C 10 strain of r.
152. The method of claim 142, wherein the one or more microorganisms comprise one or more fungi associated with the plant or the portion thereof.
153. The method of claim 152, wherein the one or more fungi associated with the plant or the portion thereof are disposed on the plant root or the rhizosphere of the plant or the portion thereof by the irrigation system.
154. The method of claim 152, wherein the one or more fungi comprise arbuscular mycorrhizal fungi.
155. The method of claim 152, wherein the one or more fungi comprise ectomycorrhizal fungi.
156. The method of claim 152, wherein the one or more fungi comprises a fungus from the genus trichoderma.
157. The method of claim 152, wherein the one or more fungi comprises a fungus from the genus penicillium.
158. The method of any one of the preceding claims, wherein the one or more microorganisms are not naturally present on the one or more roots.
159. The method of any one of the preceding claims, wherein the plant or the part thereof is a monocot or dicot.
160. The method of any one of the preceding claims, wherein the one or more microorganisms produce the formation of one or more carbonic anhydrases.
161. The method of any one of the preceding claims, wherein the one or more microorganisms produce the formation of one or more carbonic anhydrases belonging to the alpha class.
162. The method of any one of the preceding claims, wherein the one or more microorganisms produce the formation of one or more carbonic anhydrases belonging to class β.
163. The method of any one of the preceding claims, wherein the one or more microorganisms produce the formation of one or more carbonic anhydrases belonging to class γ.
164. The method of any one of the preceding claims, wherein the one or more microorganisms produce the formation of one or more carbonic anhydrases belonging to class delta.
165. The method of any one of the preceding claims, wherein the one or more microorganisms produce the formation of one or more carbonic anhydrases belonging to the zeta class.
166. The method of any one of the preceding claims, wherein the one or more microorganisms produce the formation of one or more carbonic anhydrases belonging to class η.
167. The process according to any one of the preceding claims, wherein said one or more microorganisms produce the formation of one or more carbonic anhydrases belonging to the iota class.
CN202180084753.3A 2020-10-21 2021-10-21 Composition and method for producing bicarbonate and minerals Pending CN116648143A (en)

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