Materials and methods for remediating and mitigating iron contamination

Abstract

Compositions and methods for remediating and mitigating iron contamination are provided. In a preferred embodiment, a composition comprising an iron trapping fraction is contacted with an environment subject to iron contamination to reduce the amount of iron in the environment.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 359,248, filed on July 8, 2022, the contents of which are incorporated herein by reference in their entirety. Background Art

[0003] Iron ore is a mineral that produces metallic iron (Fe) and may contain iron oxides such as, for example, magnetite (Fe3O4) and hematite (Fe2O3). In addition, iron ore is the main source of iron for the world's steel industry and is essential for steel production. More than 50 countries mine iron ore, with seven countries accounting for more than three-quarters of the world's total production: Australia, Brazil, China, India, Russia, Ukraine and Canada.

[0004] The mining of iron ore is a highly energy-intensive process and contributes to air pollution in the form of greenhouse gases, including nitrous oxide, carbon dioxide, carbon monoxide, and sulfur dioxide, produced by diesel generators, trucks, and other equipment. Additionally, in mining environments, soil and water may be contaminated with excessive amounts of heavy metals, including iron. Mining iron ore can also result in mines discharging contaminated water with heavy metals and acids. This contamination can persist for years even after mining stops in a location. In addition, handling and dumping of ore waste can result in the deposition of these metals into the environment.

[0005] Other causes of iron contamination may include, for example, industrial drainage, corrosion of iron or steel well casings or water pipes, low pH in soil and water environments, leaching of metal waste such as ammunition and construction debris, and disposal of toxic waste.

[0006] Excessive exposure to iron can wreak havoc on ecosystems and pose a health hazard to humans, plants and animals. In humans and animals, iron can accumulate in organs such as the liver, heart and pancreas. In excess, this accumulation can cause or exacerbate diseases such as liver disease, heart disease and diabetes. In addition, inhalation of iron oxide fumes or dust can also lead to persistent respiratory diseases, including cancer.

[0007] In plants, excessive iron absorption can lead to leaf discoloration and stunted root growth, ultimately leading to premature death.

[0008] Heavy metal pollution can destabilize ecosystems through bioaccumulation of metals in organisms. All heavy metals can produce toxic effects on organisms, such as through metabolic disruption and mutagenesis. Iron pollution, in particular, can cause lasting physiological damage to exposed organisms; therefore, improved methods and compositions are needed to remedy excessive iron content in heavy metal contaminated environments. Summary of the invention

[0009] The present invention relates to methods and compositions for remediating and / or mitigating heavy metal pollution. More specifically, the present invention provides compositions that, when applied to an environment where iron pollution is occurring or is likely to occur, can reduce the free iron content present in the environment. Advantageously, these compositions and methods can be used to reduce the harm of iron pollution to humans, animals and the environment.

[0010] In a preferred embodiment, the present invention provides a method for remediating and / or mitigating heavy metal pollution, wherein a composition comprising an iron trapping component is introduced into an environment where iron pollution is occurring or may occur. These methods can be used in environments including, for example, soil, water, and air. Advantageously, in certain embodiments, these methods result in the capture of free iron present in the environment. In one embodiment, the presence of iron pollution has been determined prior to the introduction of the composition.

[0011] In one embodiment, the composition sequesters iron from soil, water, and / or air, thereby reducing iron contamination in the environment and reducing the potential for iron toxicity.

[0012] In one embodiment, the method comprises applying the composition to a mining environment. In such environments, soil and water may be contaminated by excessive amounts of heavy metals, including iron. The processing and dumping of ore wastes may result in the deposition of these metals into the environment, which may cause severe damage to ecosystems and pose a health hazard to humans and animals.

[0013] In another embodiment, the method comprises applying the composition to an environment where firearms are or have been used in large quantities, such as hunting grounds, shooting ranges, and combat zones / battlefields. The soil and water in these areas may be contaminated by iron and other metals leached from residual ammunition.

[0014] In one embodiment, the composition can be applied to a reagent in a scrubber used in pollution control. Dry scrubbers utilize dry reagents in contact with the exhaust stream to bind and / or remove pollutants, while wet scrubbers utilize liquid reagents to adsorb or dissolve pollutants in the exhaust gas. Thus, the present invention provides a method for capturing iron particles from industrial exhaust streams in wet scrubbers and dry scrubbers, wherein the composition is contacted with the exhaust stream as part of the wet scrubbing reagent or the dry scrubbing reagent.

[0015] The present invention also provides a composition comprising one or more components that capture iron. In certain embodiments, the iron-capturing component is a beneficial microorganism, a growth byproduct of an iron-capturing microorganism, or some other compound known to bind iron. In certain embodiments, the composition comprises more than one iron-capturing component.

[0016] In a preferred embodiment, the beneficial microorganisms of the present invention are non-pathogenic fungi, yeasts and / or bacteria that are able to accumulate iron naturally or through genetic modification.

[0017] In one embodiment, the beneficial microorganism is a strain of Bacillus subtilis. In a specific embodiment, the strain is Bacillus subtilis B4 (NRRL B-68031). The B4 strain is preferably applied in the form of spores, but grows in the form of biofilm when exposed to an acidic environment.

[0018] Surprisingly, it was found that B4 produces one or more compounds that can gather, chelate or otherwise capture iron. In certain embodiments, the compound is pulcherrimone and / or pulcherrimone acid. Advantageously, when microorganisms and / or microorganisms' extracellular polysaccharides (EPS) grow in the form of biofilms, these microorganisms and / or microorganisms' extracellular polysaccharides (EPS) when exposed to acidic pH values ​​(e.g., less than 6.8, preferably less than 5.0), use iron-capturing agents (such as pulcherrimone and / or pulcherrimone acid) to store freely available iron.

[0019] B4 is also particularly advantageous compared to other traditional probiotic microorganisms due to its ability to produce digestive enzymes (including, for example, cellulases and amylases).

[0020] In certain embodiments, the composition may include other non-pathogenic microorganisms that are capable of producing compounds that can sequester, chelate or otherwise trap iron. The microorganisms may be in biofilm form, spore form, planktonic form, or any other form.

[0021] In certain embodiments, these microorganisms are also capable of producing one or more of the following: surfactants, such as lipopeptides and / or glycolipids; bioactive substances with antimicrobial and immunomodulatory effects; polyketides; acids; peptides; anti-inflammatory compounds; enzymes, such as amylases, cellulases, proteases and / or lipases; and sources of amino acids, vitamins and other nutrients.

[0022] In certain embodiments, the iron-trapping component of the composition of the present invention is a crude or purified siderophore or plant siderophore, or other molecules with high iron affinity, for example, pulcherrimin, pulcherriminic acid, citrate, citric acid, EDTA (ethylenediaminetetraacetic acid), iron EDTA, DTPA (diethylenetriaminepentaacetic acid), EDDHA (ethylenediamine-di-o-hydroxyphenylacetic acid), N,N-dihydroxy-N,N′-diisopropyladipamide (DPH), 2,3-dihydroxybenzoic acid, azotocin, transferrin, enterocoele bacteriocin, pyocyanin, protochelatin, chelatrocin, bacteriocin, vibriocin, vibrioferrin, azotocin, aminochelatin, yersin, agarobactin, grape agglutinin, ferrochrome, deferasirox, deferiprone, deferoxamine, fusarin, chrysin, chromatin, ornithine, rhodotorulic acid, lysine, glutamic acid, gluconic acid, ferric oxyhydroxide minerals, ferrihydrite, magnetite, hematite, goethite, siderocite, hydroxamate, catecholase, salicylate, carboxylate, mugineic acid, ferulic acid, caffeic acid and / or nicotinamide.

[0023] In certain embodiments, the composition comprises an organic acid or an inorganic acid. Preferably, the acid is present in an amount suitable for adjusting the pH of the composition or the environment in which it is used to 6.8 or lower, preferably 5.0 or lower, more preferably 4.8 or lower.

[0024] Advantageously, in certain embodiments, the composition according to the present invention can effectively reduce the content of toxic compounds in, for example, soil, water and air. In addition, the method of the present invention does not require complex equipment or high energy consumption, and the production of the composition can be carried out on site such as a mine or oil well. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figures 1A to 1C Shown are the growth and exopolysaccharide (EPS) formation of B4 after 24 h (A) and 48 h (B) at acidic pH (top row, pH 4.8) and neutral pH (bottom row, pH 6.8).

[0026] Figure 2 The purified B4 EPS is shown to be pink.

[0027] Figure 3 Results of an amylase test of B4 are shown. Agar streaked with B4 produced an orange color around bacterial growth, indicating that starch was broken down.

[0028] Figure 4 Results of the cellulase test of B4 are shown. Agar streaked with B4 produced a yellow clearing zone around bacterial growth, indicating that cellulose was broken down.

[0029] FIG. 5A to FIG. 5CThe production and activity of B4 siderophores after 6 hours in aerobic (top panel) and anaerobic (bottom panel) environments and in different growth media are shown. (A) B4 cultures grown in MRS-sucrose (left) medium and B4 cultures grown in M23-6 (right) medium are shown. (B) B4 cultures grown in minimal medium with Tween (left) and minimal medium without Tween (right) are shown. (C) Dry B4 spores grown in minimal medium are shown.

[0030] FIG. 6A to FIG. 6C The production and activity of B4 siderophores after 24 hours in aerobic (top panel) and anaerobic (bottom panel) environments and in different growth media are shown. (A) B4 cultures grown in M23-6 (left) medium and MRS-sucrose (right) medium are shown. (B) B4 cultures grown in minimal medium with Tween (left) and minimal medium without Tween (right) are shown. (C) Dry B4 spores grown in minimal medium are shown.

[0031] Figure 7 Shown are the results of iron assays of B4 (BSSL) cultures grown in different media. DETAILED DESCRIPTION

[0032] The present invention provides compositions and methods for remediating and mitigating iron pollution. More specifically, the present invention provides compositions that, when contacted with an environment containing free iron, result in a reduction in free iron in the environment. Advantageously, in some embodiments, these compositions and methods may also improve the overall health and productivity of living organisms in the environment in which these methods and compositions are used.

[0033] Selected Definitions

[0034] As used herein, "biofilm" is a complex aggregate of microorganisms (such as bacteria) in which cells adhere to each other and / or to a surface. In certain embodiments, adhesion is achieved via extracellular polysaccharide substances produced by the bacteria. Cells in a biofilm are physiologically different from planktonic cells of the same organism, which are single cells that can float or swim in a liquid culture medium.

[0035] As used herein, "preventing" or "prevention" a disease, condition, or disorder refers to delaying, arresting, inhibiting, preventing, and / or minimizing the onset or progression of specific signs or symptoms thereof. Prevention may include, but does not require, indefinite, absolute, or complete prevention during the lifetime of a subject, meaning that signs or symptoms may still develop later. Prevention may include reducing the severity of the onset of such a disease, condition, or disorder, and / or inhibiting the progression of a condition or disorder to a more severe condition or disorder.

[0036] As used herein, "treating" or "treatment" of a disease, condition, or disorder refers to eradication, improvement, reduction, alleviation, or reversal of at least one sign or symptom of a disease, condition, or disorder (e.g., infection). Treatment may include but does not require complete cure of the disease, condition, or disorder, meaning that treatment may also include partial eradication, improvement, reduction, alleviation, or reversal.

[0037] As used herein, an "isolated" or "purified" nucleic acid molecule, polynucleotide, polypeptide, protein, organic compound such as a small molecule (such as those described below), or other compound is substantially free of other compounds associated with it in nature (e.g., microporous materials). For example, a purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) is free of flanking genes or sequences in its naturally occurring state. A purified or isolated polypeptide is free of amino acids or sequences that flank it in its naturally occurring state. A purified or isolated microbial strain is removed from the environment in which it exists in nature. Thus, an isolated strain may exist, for example, as a biologically pure culture or as a spore (or other form of strain) associated with a vector.

[0038] In certain embodiments, the purified compound is at least 60% by weight of the compound of interest. Preferably, the preparation is at least 75% by weight of the compound of interest, more preferably at least 90%, and most preferably at least 99%. For example, the purified compound is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99% or 100% (w / w) by weight of the desired compound. Purity is measured by any appropriate standard method such as column chromatography, thin layer chromatography or high performance liquid chromatography (HPLC) analytical methods.

[0039] As used herein, "ionophores" are carboxylated polyether non-therapeutic antibiotics, wherein the carboxylated polyether non-therapeutic antibiotics disrupt the ion concentration gradient (Ca2+, K+, H+, Na+) of microorganisms, thereby causing them to enter an ineffective ion cycle. The disruption of ion concentrations makes it impossible for microorganisms to maintain normal metabolism and causes microorganisms to consume additional energy. The functions of ionophores include: selecting against Gram-positive bacteria (such as methanogens) and protists or affecting the metabolism of Gram-positive bacteria and protists.

[0040] As used herein, "siderophores" are compounds produced by various organisms to remove iron from the surrounding environment. Siderophores are generally small, low molecular weight compounds that have the ability to bind to ferric iron (Fe 3+ ) with a higher affinity, thereby forming strong iron chelate complexes that can be taken up by organisms under certain circumstances. As used herein, "plant siderophores" are siderophores produced by plants.

[0041] "Metabolite" refers to any substance produced by metabolism (e.g., a growth byproduct) or a substance necessary to participate in a specific metabolic process. A metabolite can be an organic compound that is a starting material, intermediate, or end product of metabolism. Examples of metabolites can include, but are not limited to, enzymes, toxins, acids, solvents, alcohols, proteins, carbohydrates, vitamins, minerals, trace elements, amino acids, polymers, polyketides, and surfactants.

[0042] As used herein, "methanogens" are microorganisms that produce methane gas as a byproduct of metabolism. Methanogens are archaea that can be found in the digestive systems and metabolic wastes of ruminants and non-ruminants (e.g., pigs, poultry, and horses, among others). Examples of methanogens include, but are not limited to, Methanobacterium spp. (e.g., M. formicicum), Methanobrevibacter spp. (e.g., M. ruminantium), Methanococcus spp. (e.g., M. paripaludis), Methanoculleus spp. (e.g., M. bourgensis), Methanoforens spp. (e.g., M. stordalenmirensis), Methanofollis liminatans, Methanogenium wolfei, Methanomicrobium spp. (e.g., M. mobile), Methanomicrobium candlerii, and Methanomicrobium spp. spp., Methanoregula boonei, Methanosaeta spp. (e.g., M. concilii, M. thermophile), Methanosarcina spp. (e.g., M. barkeri, M. mazeii), Methanosphaera stadtmanae, Methanospirillium hungatei, Methanothermobacter spp., and / or Methanothrix sochngenii.

[0043] As used herein, "ore" refers to a naturally occurring solid material from which valuable substances, minerals and / or metals can be extracted. Ores are typically mined from a deposit consisting of ore minerals containing valuable substances. "Gange" minerals refer to minerals present in a deposit but without valuable substances. Examples of deposits include hydrothermal deposits, magmatic deposits, laterite deposits, volcanogenic deposits, metamorphic altered deposits, carbonate alkaline igneous rock-related deposits, placer deposits, residual ore deposits, sedimentary deposits, sedimentary hydrothermal deposits, and crater-related deposits. However, ores as defined herein may also include concentrates or tailings, copper, or even other sources of metals or valuable minerals, including but not limited to jewelry, electronic waste, and other waste materials.

[0044] As used herein, "industrial wastewater" refers to water generated by the following industrial activities: including, for example, oil refining, chemical production, mining, oil and gas extraction, power generation, steel production, battery production, paper making, smelting, textile production and municipal wastewater treatment.

[0045] As used herein, "produced water" refers to naturally occurring water that is removed from the ground during the extraction of oil and gas, because formations containing oil and gas gases also contain water.

[0046] The scope provided herein is understood as a shorthand expression of all numerical values ​​in the scope. For example, the scope of 1 to 50 is understood to include any number, combination of numbers or the sub-range of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 and all decimal values ​​(such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 and 1.9) between the above integers. About sub-range, " nested sub-range " extending from any end point of scope is particularly considered. For example, nested sub-ranges of the exemplary range 1 to 50 may include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0047] As used herein, "decrease" refers to a negative change and "increase" refers to a positive change, wherein the positive or negative change is at least 0.25%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

[0048] The transitional term "comprising," which is synonymous with "including" or "containing," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. In contrast, the transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of" limits the scope of the claim to the specified materials or steps "and those that do not materially affect the basic, novel characteristic." Use of the term "comprising" refers to other embodiments consisting of "consisting of" or "consisting essentially of" the referenced components.

[0049] Unless otherwise specified or obvious from the context, the term "or" as used herein should be understood to be inclusive. Unless otherwise specified or obvious from the context, as used herein, the terms "a", "and" and "the" are understood to be singular or plural.

[0050] Unless otherwise specified or apparent from the context, as used herein, the term "about" should be understood to be within the normal tolerance range in the art, such as within 2 standard deviations of the mean. "About" can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the specified value.

[0051] The recitation of a list of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of the listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0052] All references cited herein are hereby incorporated by reference in their entirety.

[0053] Composition

[0054] The present invention provides a composition for use according to the method of the present invention, wherein the composition comprises one or more components that capture iron. In certain embodiments, the iron-capturing component is a beneficial microorganism, a growth byproduct of a microorganism, or some other compound known to bind iron. In certain embodiments, the composition comprises more than one iron-capturing component.

[0055] The total amount of iron-trapping components in the composition preferably comprises 0.0001% to 100% of the composition, or 0.001% to 95%, 0.01% to 90%, 0.1% to 85%, 0.5% to 80%, 0.75% to 75%, 1.0% to 70%, 1.25% to 65%, 1.5% to 60%, 1.75% to 55%, 2.0% to 50% or 5.0% to 25% of the composition by weight or by volume.

[0056] In certain embodiments, the composition is a "microbial-based composition," which means that the composition contains components produced by the growth of a microorganism or other cell culture. Thus, a microbial-based composition may include the microorganism itself and / or microbial growth byproducts. The microorganism may be in an asexual state, in spore form, in mycelial form, in any other form of microbial propagule, or a mixture thereof. The microorganism may be in the form of a planktonic microorganism or a biofilm, or a mixture of the two. The byproducts of growth may be, for example, metabolites, cell membrane components, expressed proteins, and / or other cell components. The microorganism may be a complete microorganism or a lysed microorganism. The cells may be completely absent, or present in an amount of, for example, at least 1×10 per milliliter of composition or at least 1×10 per CFU / g of composition. 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 or more CFU concentrations.

[0057] Advantageously, in preferred embodiments, the compositions of the present invention can collect free iron in environments that are or may be contaminated by heavy metals. Thus, in some embodiments, the compositions can be used to remediate and / or mitigate iron pollution in the environment, which can also enhance the health of soil, water, air, and organisms.

[0058] In a preferred embodiment, the beneficial microorganisms of the composition of the present invention are non-pathogenic fungi, yeasts and / or bacteria that can collect iron naturally or by genetic modification. The beneficial microorganisms can be in active form, inactive form and / or dormant form. In a preferred embodiment, the microorganism is a microorganism described as "generally recognized as safe" by the relevant regulatory agency or GRAS.

[0059] In certain embodiments, these microorganisms are also capable of producing one or more of the following: surfactants, such as lipopeptides and / or glycolipids; bioactive substances with antimicrobial and immunomodulatory effects; polyketides; acids; peptides; anti-inflammatory compounds; enzymes, such as amylases, cellulases, proteases and / or lipases; and sources of amino acids, vitamins and other nutrients.

[0060] The microorganism of the present invention can be a natural microorganism or a genetically modified microorganism. For example, the microorganism can be transformed with a specific gene to show a specific characteristic. The microorganism can be a mutant of a desired strain. As used herein, "mutant" refers to a strain, genetic variant or subtype of a standard bacterium, wherein the mutant has one or more genetic variations (e.g., point mutation, missense mutation, nonsense mutation, deletion, duplication, frameshift mutation or amplification mutation) compared to the standard bacterium. The process of preparing mutants is well known in the field of microbiology. For example, UV mutagenesis and nitrosoguanidine are widely used for this purpose.

[0061] In some embodiments, beneficial microorganisms are selected based on natural or acquired resistance to certain antibiotics applied to an environment containing iron-trapping pathogens, e.g., to control pathogenic and / or harmful microorganisms in a living subject or elsewhere in the environment.

[0062] In one embodiment, the composition includes about 1×10 6 About 1×10 13 , about 1×10 7 About 1×10 12 , about 1×10 8 About 1×10 11 , or about 1×10 9 About 1×10 10 CFU / g of various types of microorganisms.

[0063] In one embodiment, the composition includes a total of about 0.001% to 100%, about 1% to 90%, or about 10% to 75% total microorganisms by volume.

[0064] In certain embodiments, the composition comprises growth byproducts of microorganisms, but does not comprise live microorganisms. For example, in certain embodiments, pathogenic microorganisms are only used to produce growth byproducts of the composition according to the present invention, rather than being directly applied to the environment.

[0065] The microorganisms may include yeast, bacteria and / or fungi, such as Acaulospora, Acidithiobacillus spp. (e.g., A.ferooxidans, A.albertensis, A.caldus, A.cuprithermicus, A.ferrianus, A.ferridurans, A.ferriphilus, A.ferrivorans, A.ferrooxidans, A.sulfuriphilus, and A.thiooxidans), Acremonium chrysogenum, Agrobacterium (e.g., A. radiobacter), Aspergillus, Aureobasidium (e.g., A. pullulans), Azospirillum (e.g., A. brasiliensis), Azotobacter (A. vinelandii, A. chroococcum), Bacillus (e.g., B. amyloliquefaciens, B. coagulans, B. firmus, B. laterosporus), orus, B. licheniformis, B. megaterium, B. mucilaginosus, B. subtilis), Blakeslea, Candida (e.g., C. albicans, C. apicola, C. batistae, C. bombicola, C. floricola, C. kuoi, C. riodocensis, C. nodaensis, C. stellate), Cryptococcus, Debaryomyces (e.g., D. hansenii, D.hansenii), Dipodascopsism, Entomophthora, Escherichia coli, Frateuria (e.g., F. aurantia), Hanseniaspora (e.g., H. uvarum), Hansenula, Issatchenkia, Kluyveromyces (e.g., K. phaffii), Lentinula spp. (e.g., L. edodes), Legionella pneumophila, Lipomyces, Magnetospirillummagneticum, Magnetococcus marinus, methanogens, Metschnikowia sp. (M. pulcherrimia), Meyerozyma (e.g., M. guilliermondii, M. caribbica), Monascus purpureus, Mortierella, Mucor (e.g., M. piriformis), Neisseria meningitidis, Pantoea (e.g., P. agglomerans, P. allii), Penicillium, Phythium, Phycomyces, Pichia (e.g., P. anomala, P. guilliermondii, P. occidentalis, P. kudriavzevii), Pleurotus erythrocytes, otus (e.g., P. ostreatus, P. ostreatus, P. sajorcaju, P. cystidiosus, P. cornucopiae, P. pulmonarius, P. tuberregium, P. citrinopileatus, and P. flabellatus), Pseudomonas (e.g., P. chlororaphis, P. aeruginosa, P.aeruginosa, Pseudomonas koreensis), Pseudozyma (e.g., P. aphidis, P. antarctica), Rhizobium radiobacter, Rhizopus, Rhodospirillum (e.g., R. rubrum), Rhodotorula (e.g., R. bogoriensis), Saccharomyces (e.g., Saccharomyces cerevisiae, S. boulardii, S. torula), Sphingomonas (e.g., S. pauc imobilis), Starmerella (e.g., S. bombicola), Streptomyces, Torulopsis, Thraustochytrium, Trichoderma (e.g., T. reesei, T. harzianum, T. viridae), Ustilago (e.g., U. maydis), Vibrio cholerae. cholerae, Wickerhamiella (e.g., W. domericqiae), Wickerhamomyces (e.g., W. anomalus), Williopsis (e.g., W. mrakii), Zygosaccharomyces (e.g., Z. bailii), and others (including those listed as pathogens elsewhere in this disclosure).

[0066] If present, the fungus may be in the form of living or non-living cells, mycelium, spores and / or fruiting bodies. The fruiting bodies (if present) may be, for example, chopped and / or mixed into granular and / or powdered form.

[0067] If present, the yeast may be in the form of live or inactivated cells or spores, and may also be in the form of dry cells and / or dormant cells (eg, yeast hydrolysate).

[0068] If present, the bacteria may be in the form of vegetative or planktonic cells, biofilms, spores, and / or dried cell or spore masses.

[0069] In some embodiments, dried microorganisms (eg, spores) may be mixed with fillers known in the art, such as, for example, microcrystalline cellulose (MCC).

[0070] In one embodiment, the composition comprises one or more Bacillus bacteria and / or growth byproducts thereof. In certain embodiments, the Bacillus is Bacillus amyloliquefaciens, Bacillus subtilis, Spore lactic acid bacteria and / or Bacillus licheniformis.

[0071] In one embodiment, the composition comprises Bacillus amyloliquefaciens NRRL B-67928 "B.amy" and / or its growth byproducts. Cultures of the Bacillus amyloliquefaciens "B.amy" microorganism have been deposited at the Northern Regional Research Laboratory (NRRL) Culture Collection of the Agricultural Research Service at 1815 N. University St., Peoria, IL, USA. The deposit has been assigned the accession number NRRL B-67928 and was deposited on February 26, 2020.

[0072] In one embodiment, the composition comprises a strain of Bacillus subtilis and / or its growth byproducts. In a specific embodiment, the strain is Bacillus subtilis B4 (NRRL B-68031). Cultures of the B4 microorganism have been deposited at the Northern Regional Research Laboratory (NRRL) Culture Collection of the Agricultural Research Service at 1815 North University St., Peoria, IL, USA. The deposit has been assigned a deposit number, NRRL B-68031, and was deposited on May 6, 2021.

[0073] B4 is a Gram-positive spore-forming strain of Bacillus subtilis capable of anaerobic growth (obligate anaerobe). The B4 strain is preferably administered as a spore, but germinates in an acidic environment where it can grow as a biofilm. Surprisingly, it was found that B4, when grown as a biofilm, produces one or more compounds that can gather, chelate or otherwise trap iron. In certain embodiments, the compound is pulcherrimone and / or pulcherrimone acid.

[0074] Advantageously, the microorganisms and / or exopolysaccharides (EPS) of the biofilm efficiently store freely available iron using an iron-capturing agent such as pulcherrimidine and / or pulcherrimidine acid when subjected to low pH values ​​(e.g., less than 6.8, less than 5.0, or less than 4.8).

[0075] B4 is also particularly advantageous because it is able to produce relatively large amounts of the lipopeptide surfactin (eg, higher than wild-type B. subtilis), as well as digestive enzymes including, for example, cellulases and amylases.

[0076] The proprietary cultures described herein have been deposited under conditions that ensure that they are available during the pendency of this patent application to persons whom the Director of the Patent and Trademark Office deems entitled to access them pursuant to 37 CFR 1.14 and 35 U.S.C. 122. Deposits of the present invention may be made available in accordance with the requirements of foreign patent laws in countries in which the counterpart of the present application or its progeny has been filed. It should be understood, however, that the provision of a deposit does not constitute a derogation from patent rights granted by governmental action, but rather constitutes a license to practice the present invention.

[0077] Furthermore, each of the deposited cultures of the present invention is maintained and made available to the public, in accordance with the provisions of the Budapest Treaty on the Deposit of Microorganisms, by all necessary means to preserve its viability and protect it from contamination, for a period of at least five years after the last request for samples of the deposit and, in any case, for at least 30 (thirty) years after the date of deposit or for the enforceability of any patent that may issue disclosing the culture. The depositor acknowledges that it is responsible for replacing the deposit if the depositor is unable to provide samples upon request due to the state of the deposit. Upon the grant of a patent disclosing a deposit of cultures of the present invention, all restrictions on making the culture available to the public are irrevocably lifted.

[0078] In certain embodiments, the composition may include other microorganisms that are capable of naturally producing or genetically engineering pulcherrimin and / or pulcherriminic acid, or other compounds that are capable of sequestering, chelating or otherwise trapping iron. In specific embodiments, the microorganism is capable of growing as a biofilm.

[0079] In certain embodiments, the microorganism is a naturally occurring microorganism or a transgenic microorganism capable of regulating genes involved in iron capture and transport, for example, HFE, GDF15, TWSG1, ERFE, matrix enzyme 2, TF, TFR1, TFR2, HAMP, and HJV.

[0080] In certain embodiments, the composition may comprise a crude or purified siderophore or plant siderophore, or other molecules with high iron affinity, for example, pulcherrimin, pulcherriminic acid, citrate, citric acid, EDTA (ethylenediaminetetraacetic acid), iron EDTA, DTPA (diethylenetriaminepentaacetic acid), EDDHA (ethylenediamine-di-o-hydroxyphenylacetic acid), N,N-dihydroxy-N,N′-diisopropyladipamide (DPH), 2,3-dihydroxybenzoic acid, azotocin, transferrin, enterobactin, purulent azotocin, protochelatin, chelatin, bacteriocin, vibriocin, vibrioferrin, azotocin, aminochelatin, yersin, agarobactin, grape agglutinin, ferrochrome, deferasirox, deferiprone, deferoxamine, fusarin, chrysin, achromatin, ornithine, rhodotorulic acid, lysine, glutamic acid, gluconic acid, ferric oxyhydroxide minerals, ferrihydrite, magnetite, hematite, goethite, siderocite, hydroxamate, catecholase, salicylate, carboxylate, mugineic acid, ferulic acid, caffeic acid and / or nicotinamide.

[0081] The composition may also include other microbial growth byproducts. The microbial growth byproducts may be produced by the microorganisms of the composition, and / or they may be produced separately, for example, by the microorganisms listed herein and added to the composition.

[0082] In certain embodiments, the composition may include residual substrates from the culture process and / or purified or unpurified growth byproducts, such as biosurfactants, killer factors, enzymes, polyketides, and / or other metabolites. The microorganism may be active or inactive, although in preferred embodiments, if the microorganism is considered a pathogen, the microorganism is inactivated and / or removed from the composition.

[0083] In one embodiment, the growth by-products are purified from the substratum producing the by-products. In addition, in one embodiment, the growth by-products are used in a crude form. The crude form may include, for example, a liquid supernatant generated by the microorganisms that culture the relevant growth by-products (comprising residual cells and / or nutrients).

[0084] Growth byproducts may include metabolites or other biochemicals produced as a result of cell growth, including, for example, amino acids, peptides, polyketides, antibiotics, proteins, enzymes, biosurfactants, solvents, vitamins, and / or other metabolites.

[0085] Additional components

[0086] Other components may be added to the composition, such as carriers, other microbial-based compositions, biosurfactants, enzymes, catalysts, solvents, buffers, emulsifiers, lubricants, solubility regulators, preservatives, stabilizers, UV inhibitors, viscosity regulators, preservatives, tracers, antimicrobial agents, surfactants, pH adjusters, essential oils, plant extracts, cross-linking agents, chelating agents, fatty acids, alcohols, reducing agents, synthetic agents, dyes, colorants, fragrances, antimicrobial compounds, antibiotics, foaming agents, foam reducers, polymers, thickeners, chelates and other components specific to the intended use.

[0087] In certain embodiments, the composition may include a chelating agent, which may be, but is not limited to, ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), phosphonates, succinic acid (DMSA), diethylenetriaminepentaacetic acid (DTPA), N-acetylcysteine, N-hydroxyethylethylenediaminetriacetic acid (HEDTA), organic acids with more than one coordinating group (e.g., erythromycin), STPP (sodium tripolyphosphate, Na5P3O 10) , trisodium phosphate (TSP), water, carbohydrates, organic acids with more than one coordinating group (e.g., citric acid), lipids, steroids, amino acids or related compounds (e.g., glutathione), peptides, phosphates, nucleotides, tetrapyrroles, ferroamines, ionophores, or phenols, sodium citrate, sodium gluconate, ethylenediamine disuccinic acid (EDDS), iminodisuccinic acid (IDS), L-glutamic acid diacetic acid (GLDA), GLDA Na4, methylglycine diacetic acid (MGDA), polyaspartic acid (PASA), hemoglobin, chlorophyll, lipophilic β-diketone and (14,16)-heteronedione, ethylenediamine-N,N'-diglutaric acid (EDDG), ethylenediamine-N,N'-dimalonic acid (EDDM), 3-hydroxy-2,2-iminodisuccinic acid (HIDS), 2-hydroxyethyliminodiacetic acid (HEIDA), pyridine-2,6-dicarboxylic acid (PDA), trimethylglycine (TMG), titanium reagent, or any combination thereof.

[0088] In certain embodiments, the composition includes a germination enhancer for enhancing germination of spore-shaped microorganisms in a microbial-based composition. In a specific embodiment, the germination enhancer is an amino acid, such as, for example, L-alanine and / or L-leucine. In one embodiment, the germination enhancer is manganese.

[0089] In certain embodiments, the composition comprises an organic acid selected from the following: for example, acetic acid, citric acid, formic acid, lactic acid, malic acid, oxalic acid, tartaric acid, uric acid, propionic acid, butyric acid, sorbic acid, fumaric acid, benzoic acid, hydrofluoric acid, caproic acid, salicylic acid, gluconic acid, pyruvic acid, adipic acid, trichloroacetic acid, glycolic acid, cinnamic acid, carboxylic acid, succinic acid, carbonic acid, glutaric acid, capric acid and ascorbic acid. In some embodiments, the composition comprises an inorganic acid selected from the following: for example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, boric acid, perchloric acid, hydrofluoric acid, hydrobromic acid and sulfonic acid. Preferably, the acid is present in an amount suitable for adjusting the pH value of the composition or the environment in which it is used to 6.8 or lower, preferably 5.0 or lower, more preferably 4.8 or lower.

[0090] In one embodiment, the composition includes one or more fatty acids. The fatty acids can be produced by the microorganisms of the composition, and / or produced separately and included as additional components. In certain preferred embodiments, the fatty acids are saturated long-chain fatty acids having a carbon backbone of 14 to 20 carbons, such as, for example, myristic acid, palmitic acid, or stearic acid. In some embodiments, a combination of two or more saturated long-chain fatty acids is included in the composition. In some embodiments, saturated long-chain fatty acids can inhibit methane production and / or increase cell membrane permeability of methanogens.

[0091] In certain embodiments, the composition comprises one or more enzymes that help break down food sources into smaller units, such as volatile fatty acids (e.g., propionate, acetate, butyrate), glucose, and amino acids. These enzymes can be produced by the microorganisms of the composition, and / or produced separately and included as additional components. Non-limiting examples of digestive enzymes include amylase, maltase, lactase, lipase, protease, sucrase, and cellulase.

[0092] In some embodiments, the composition may include additional components that are known to reduce methane production in methanogens, such as, for example, nitrates (e.g., calcium nitrate, ammonium nitrate, sodium nitrate, potassium nitrate, and magnesium nitrate); seaweed (e.g., Asparagopsis taxiformis and / or Asparagopsis armata, etc.); giant kelp; nitrooxypropanols (e.g., 3-nitrooxypropanol and / or ethyl 3-nitrooxypropanol, etc.); anthraquinones; ionophores (e.g., monensin and / or lasalocid, etc.); polyphenols (e.g., saponins, tannins, etc.); Yuccaschidigera extracts (e.g., steroidal saponin-producing plant species, etc.); Quillaja saponaria extract (a triterpenoid saponin-producing plant species); organosulfurs (e.g., garlic extract, etc.); flavonoids (e.g., quercetin, rutin, kaempferol, naringin, and anthocyanins, etc.); bioflavonoids from green citrus fruits, rose hips, and / or red currants; carboxylic acids; and / or terpenes (e.g., d-limonene, pinene, and citrus extract, etc.).

[0093] In one embodiment, the composition may include one or more biosurfactants. Biosurfactants are a group of structurally diverse surface-active substances produced by microorganisms that are biodegradable and can be efficiently produced by selected organisms on renewable substrates. All biosurfactants are amphiphilic molecules. Amphiphilic molecules consist of two parts: a polar (hydrophilic) group and a non-polar (hydrophobic) group. The lipophilic portion of the biosurfactant molecule is usually a hydrocarbon chain of a fatty acid, while the hydrophilic portion is formed by an ester or alcohol group of a neutral lipid, a carboxylate group of a fatty acid or an amino acid (or peptide), an organic acid in the case of flavonoids, or a carbohydrate in the case of glycolipids.

[0094] Biosurfactants, due to their amphiphilic structure, increase the surface area of ​​hydrophobic insoluble substances, improve the water bioavailability of these substances, and change the properties of bacterial cell surfaces. Biosurfactants accumulate at interfaces, thereby reducing interfacial tension, resulting in the formation of aggregated micellar structures in solution. Safe and effective microbial biosurfactants reduce surface tension and interfacial tension between liquid, solid, and gas molecules. The ability of biosurfactants to form pores and destabilize biofilms allows biosurfactants to be used as antibacterial, antifungal, and hemolytic agents.

[0095] Advantageously, in certain embodiments, biosurfactants can help disrupt and / or penetrate biofilms to increase the effectiveness of antimicrobial compounds.

[0096] The biosurfactant according to the present invention may include, for example, glycolipids, lipopeptides, flavonoids, phospholipids, fatty acid esters, and high molecular weight polymers (such as lipoproteins, lipopolysaccharide-protein complexes, and polysaccharide-protein-fatty acid complexes, etc.).

[0097] In one embodiment, the biosurfactant is a glycolipid. Glycolipids may include, for example, sophorolipids, rhamnolipids, cellobiose lipids, mannoerythritol lipids, and trehalose lipids. In one embodiment, the biosurfactant is a lipopeptide. Lipopeptides may include, for example, surfactin, iturin, sphingomyelin, mucin, fengycin, and lichenin. In certain embodiments, a mixture of biosurfactants is used.

[0098] In one embodiment, the biosurfactant has been purified from a fermentation medium in which the biosurfactant was produced. Additionally, in one embodiment, the biosurfactant is used in a crude form (including a fermentation broth from a culture of a microorganism that produces the biosurfactant). The crude biosurfactant solution may include about 0.001% to 99%, about 25% to about 75%, about 30% to about 70%, about 35% to about 65%, about 40% to about 60%, about 45% to about 55%, or about 50% pure biosurfactant, as well as residual cells and / or nutrients.

[0099] In one embodiment, the composition includes 1 ml / L to 10 ml / L of saponin or 2 ml / L to 6 ml / L of rumen fluid. Saponin is a natural surfactant present in many plants and exhibits properties similar to microbial surfactants (e.g., self-binding and interacting with biofilms). Saponin has three basic categories: triterpenoid saponins, steroidal saponins, and steroidal glycoalkaloids.

[0100] Some well-known triterpenoid saponin plant families include Leguminosae, Amaranthaceae, Apiaceae, Caryophyllaceae, Aquifoliaceae, Araliaceae, Cucurbitaceae, Berberidaceae, Chenopodiaceae, Myrsinaceae, and Zygophyllaceae, etc. Quillaja sapodilla and legumes (such as soybeans, beans, and peas) are rich sources of triterpenoid saponins. Steroidal saponins are commonly found in members of the following families: Agavaceae, Alliaceae, Asparagaceae, Dioscoreaceae, Liliaceae, Amaryllidaceae, Bromeliaceae, Palmae, and Scrophulariaceae, and are abundantly accumulated in crop plants such as Dioscorea, Allium, Asparagus, Fenugreek, Yucca, and Ginseng. Steroidal glycoalkaloids are commonly found in plants of the Solanaceae family such as tomatoes, potatoes, eggplants, and peppers.

[0101] In one embodiment, the composition of the present invention may include one or more additional substances and / or nutrients to supplement the needs of beneficial microorganisms in the composition and / or to supplement the needs of soil, water or plants in its application environment. These may include, for example, sources of amino acids (including essential amino acids), peptides, proteins, vitamins, trace elements, fats, fatty acids, lipids, carbohydrates, sterols, enzymes and minerals (such as calcium, magnesium, phosphorus, potassium, sodium, chlorine, sulfur, chromium, cobalt, copper, iodine, iron, manganese, molybdenum, nickel, selenium and zinc). In some embodiments, the microorganisms of the composition generate and / or provide these substances.

[0102] In certain embodiments, the composition may further comprise one or more carriers and / or excipients suitable for delivering the composition to the environment, including soil, water, or air.

[0103] Carriers and / or excipients according to the present invention may include any and all solvents, diluents, buffers (such as, for example, neutral buffered saline, phosphate buffered saline or optionally Tris-HCl, acetate or phosphate buffer), oil-in-water or water-in-oil emulsions, aqueous compositions with or without organic co-solvents, solubilizers (such as, for example, Tween 80, polysorbate 80), colloids, dispersion media, carriers, fillers (e.g., MCC), chelating agents (such as, for example, EDTA or glutathione), amino acids (such as, for example, glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, fragrances, thickeners, coatings, preservatives (such as, for example, thimerosal, benzyl alcohol), antioxidants (such as, for example, ascorbic acid, sodium metabisulfite), tonicity control agents, absorption delaying agents, adjuvants, bulking agents (such as, for example, lactose, mannitol), etc. In certain embodiments, the composition comprises a filler, such as microcrystalline cellulose (MCC).

[0104] In certain embodiments, the composition may include glucose (eg, in the form of molasses), glycerol, and / or glycerol as an osmotic pressure substance or as a supplement to an osmotic pressure substance to increase the osmotic pressure during storage and transportation of the dry product.

[0105] The composition can be formulated into, for example, solid, semi-solid, liquid or gaseous forms such as tablets, capsules, powder tablets, powders, granules, gels, solutions, steaks / spikes (for soil), drops, aerosols, suspensions, concentrates and other formulations suitable for a particular application. Liquids, dusts, granules, microgranules, granules, wettable powders, flowable powders, emulsions, microcapsules, oils or aerosols.

[0106] In order to enhance or stabilize the effect of the composition, it may be mixed with a suitable adjuvant and then used directly or after dilution (if necessary).

[0107] Exemplary embodiments

[0108] In some embodiments, the compositions of the present invention comprise the following:

[0109] A) one or more microorganisms (yeast, fungi and / or bacteria) capable of capturing iron and / or producing iron capturing growth byproducts, wherein preferably at least one of the one or more microorganisms is of the genus Bacillus, and wherein even more preferably the Bacillus is Bacillus subtilis NRRL B-68031 or Bacillus amyloliquefaciens NRRL B-67928;

[0110] B) Iron-capturing substances selected from the group consisting of pulcherrimone, pulcherrimone acid, citrate, citric acid, EDTA (ethylenediaminetetraacetic acid), iron EDTA, DTPA (diethylenetriaminepentaacetic acid), EDDHA (ethylenediamine-di-o-hydroxyphenylacetic acid), N,N-dihydroxy-N,N′-diisopropylhexanediamine (DPH), 2,3-dihydroxybenzoic acid, azotocin, ferrochrome, deferasirox, deferiprone, deferoxamine, fusarin, chrysin, achromatin, ornithine , rhodotorulin, lysine, glutamic acid, gluconic acid, iron oxyhydroxide minerals, ferrihydrite, magnetite, hematite, goethite, siderite, transferrin, enterobactin, bacteriocin, vibriocin, azotocin, aminochelatin, pyocyanin, yersinic acid, agarobactin, grape agglutinin, hydroxamic acid, catecholase, salicylate, carboxylate, mugineic acid, ferulic acid, caffeic acid, enterobactin, pyocyanin, protochelatin, chelatin, vibrioferrin and / or nicotinamide.

[0111] C) carriers / excipients, which include solvents, diluents, buffers (such as, for example, neutral buffered saline, phosphate buffered saline or optionally Tris-HCl, acetate or phosphate buffer), oil-in-water or water-in-oil emulsions, aqueous compositions with or without organic co-solvents, solubilizers (such as, for example, Tween 80, polysorbate 80), colloids, dispersion media, carriers and / or fillers (e.g., MCC);

[0112] D) one or more acids selected from the group consisting of, for example, acetic acid, citric acid, formic acid, lactic acid, malic acid, oxalic acid, tartaric acid, uric acid, propionic acid, butyric acid, sorbic acid, fumaric acid, benzoic acid, hydrofluoric acid, caproic acid, salicylic acid, gluconic acid, pyruvic acid, adipic acid, trichloroacetic acid, glycolic acid, cinnamic acid, carboxylic acid, succinic acid, carbonic acid, glutaric acid, capric acid, ascorbic acid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, boric acid, perchloric acid, hydrofluoric acid, hydrobromic acid and sulfonic acid in an amount suitable for adjusting the pH of the composition or its application environment to 6.8 or less, preferably 5.0 or less, more preferably 4.8 or less; and / or

[0113] E) A biosurfactant selected from the group consisting of sophorolipids, rhamnolipids, cellobiose lipids, mannoerythritol lipids, trehalose lipids, surfactins, iturin, sphingolipids, mucins, fengycins and lichenin.

[0114] In some embodiments, the composition comprises each of Component A to Component E. In some embodiments, the composition comprises any combination of Component A to Component E, or comprises any one of Component A to Component E alone.

[0115] In some embodiments, the composition comprises Bacillus subtilis NRRL B-68031 or Bacillus amyloliquefaciens NRRL B-67928 in spore form, plant form, and / or biofilm form; and pulcherrimin and / or pulcherriminic acid.

[0116] In some embodiments, the composition comprises Bacillus subtilis NRRL B-68031 or Bacillus amyloliquefaciens NRRL B-67928 in spore form, plant form, and / or biofilm form; and a carrier / excipient.

[0117] In some embodiments, the composition comprises Bacillus subtilis NRRL B-68031 or Bacillus amyloliquefaciens NRRL B-67928 in spore form, plant form and / or biofilm form; and one or more acids listed in point D).

[0118] In some embodiments, the composition comprises Bacillus subtilis NRRL B-68031 or Bacillus amyloliquefaciens NRRL B-67928 in spore form, plant form, and / or biofilm form; pulcherrimin and / or pulcherriminic acid; and a carrier / excipient.

[0119] In some embodiments, the composition comprises Bacillus subtilis NRRL B-68031 or Bacillus amyloliquefaciens NRRL B-67928 in spore form, plant form and / or biofilm form; pulcherrimin and / or pulcherriminic acid; a carrier; and one or more acids listed in point D).

[0120] In some embodiments, the composition comprises Bacillus subtilis NRRL B-68031 or Bacillus amyloliquefaciens NRRL B-67928 in spore form, plant form, and / or biofilm form; pulcherrimin and / or pulcherriminic acid; a carrier; and a biosurfactant.

[0121] Production of microorganisms and / or microbial growth by-products

[0122] The present invention utilizes methods for the cultivation of microorganisms and the generation of microbial metabolites and / or other byproducts of microbial growth. The present invention further utilizes culture methods suitable for cultivating microorganisms and generating microbial metabolites at a desired scale. These culture processes include, but are not limited to, liquid culture / fermentation, solid state fermentation (SSF) and modifications, hybrids and / or combinations thereof.

[0123] As used herein, "fermentation" refers to the cultivation or growth of cells under controlled conditions. Growth can be aerobic or anaerobic. In a preferred embodiment, the microorganisms are grown using SSF and / or a modified form thereof.

[0124] In one embodiment, the invention provides materials and methods for generating biomass (eg, living cellular material), extracellular metabolites, residual nutrients, and / or intracellular components.

[0125] The microbial growth vessel used according to the present invention can be any fermenter or culture reactor for industrial use. In one embodiment, the vessel can have functional controls / sensors or can be connected to functional controls / sensors to measure important factors in the culture process, such as pH, oxygen, pressure, temperature, humidity, microbial density and / or metabolite concentration, etc.

[0126] In another embodiment, the vessel can also monitor the growth of microorganisms in the vessel (e.g., measuring cell number and growth stage). Additionally, daily samples can be taken from the vessel and counted by techniques known in the art (such as dilution plate techniques).

[0127] In one embodiment, the method comprises supplementing the culture with a nitrogen source. The nitrogen source can be, for example, potassium nitrate, ammonium nitrate, ammonium sulfate, ammonium phosphate, ammonia, urea, and / or ammonium chloride. These nitrogen sources can be used alone or in combination of two or more.

[0128] This method can provide oxygenation for the growing culture. One embodiment utilizes the slow movement of air to remove air with low oxygen content and introduce oxygenated air. In the case of submerged fermentation, the oxygenated air can be ambient air supplemented daily by mechanical means including an impeller for mechanically agitating the liquid and an air distributor for supplying gas bubbles to the liquid to dissolve oxygen into the liquid.

[0129] The method may further include supplementing the culture with a carbon source. The carbon source is typically a carbohydrate such as glucose, sucrose, lactose, fructose, trehalose, mannose, mannitol and / or maltose; an organic acid such as acetic acid, fumaric acid, citric acid, propionic acid, malic acid, malonic acid and / or pyruvic acid; an alcohol such as ethanol, propanol, butanol, amyl alcohol, hexanol, isobutanol and / or glycerol; and a fat and oil such as soybean oil, rapeseed oil, rice bran oil, olive oil, corn oil, sesame oil and / or linseed oil. These carbon sources may be used alone or in combination of two or more.

[0130] In one embodiment, the growth factors and micronutrients of microorganisms are included in the culture medium. When the growing microorganisms cannot generate all the vitamins they need, this is particularly preferred. Inorganic nutrients may also be included in the culture medium, which include trace elements such as iron, zinc, copper, manganese, molybdenum and / or cobalt. In addition, the source of vitamins, essential amino acids and trace elements may be included in the culture medium in the form of fine powder or meal (for example, corn flour) or in the form of extracts (such as yeast extract, potato extract, beef extract, soybean extract, banana peel extract, etc.) or in purified form. Amino acids (such as amino acids that can be used for protein biosynthesis) may also be included.

[0131] In one embodiment, the culture medium may also include an inorganic salt. Available inorganic salts may be potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, magnesium chloride, iron sulfate, iron chloride, manganese sulfate, manganese chloride, zinc sulfate, lead chloride, copper sulfate, calcium chloride, sodium chloride, calcium carbonate and / or sodium carbonate. These inorganic salts may be used alone or in combination of two or more.

[0132] In one embodiment, one or more biostimulants, meaning substances that enhance the growth rate of microorganisms, may also be included. Biostimulants may be species specific and may enhance the growth rate of various species.

[0133] In some embodiments, the method for culturing may further include adding an antimicrobial agent to the culture medium before and / or during the culturing process.

[0134] In certain embodiments, antibiotics can be added to the culture at low concentrations to generate microorganisms that are resistant to antibiotics. Microorganisms that survive contact with the antibiotics are selected and repeatedly re-cultured with increasing antibiotic concentrations to obtain cultures that are resistant to antibiotics. This can be done in a laboratory setting or on an industrial scale using methods known in the art of microbiology. In certain embodiments, for example, the amount of antibiotic in the culture starts at 0.0001 ppm and increases by about 0.001 ppm to 0.1 ppm each iteration until the concentration in the culture is equal to or approximately equal to the dose typically applied to iron-trapped pathogens.

[0135] The pH of the mixture should be appropriate for the microorganism of interest. Buffers and pH adjusters (such as carbonates and phosphates) can be used to stabilize the pH near the preferred value. When metal ions are present in high concentrations, it may be necessary to use chelating agents in the culture medium.

[0136] The microorganisms can grow in a planktonic form or in a biofilm. If a biofilm is present, the vessel can contain a substrate on which the microorganisms can grow in a biofilm state. The system can also have the ability to apply stimuli (such as shear stress) that encourage and / or improve the growth characteristics of the biofilm.

[0137] In one embodiment, the culture method of the microorganism is carried out at about 5° to about 100° C., preferably 15° C. to 60° C., more preferably 25° C. to 50° C. In another embodiment, the culture can be carried out continuously at a constant temperature. In another embodiment, the culture may be subjected to temperature changes.

[0138] In one embodiment, the equipment used in the method and the culture process is a sterile device. Culture equipment (such as a reactor / vessel) can be separated from a sterilizing device (for example, an autoclave), but is connected thereto. Culture equipment can also have a sterilizing device that sterilizes on the spot before starting inoculation. Air can be sterilized by methods known in the art. For example, ambient air can pass through at least one filter before being introduced into the vessel. In other embodiments, the culture medium can be pasteurized, or alternatively not heated at all, but can utilize low water activity and low pH value to control the growth of undesirable bacteria.

[0139] In one embodiment, the present invention further provides a method for producing microbial metabolites by culturing the microbial strain of the present invention under conditions suitable for growth and metabolite production, and optionally purifying the metabolites, such as, for example, biosurfactants, enzymes, proteins, ethanol, lactic acid, β-glucans, peptides, metabolic intermediates, polyunsaturated fatty acids and lipids. The metabolite content generated by the method can be, for example, at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.

[0140] The biomass content of the fermentation medium may be, for example, 5 g / l to 180 g / l or more, or 10 g / l to 150 g / l, etc. The cell concentration may be, for example, at least 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 or 1×10 13 cells.

[0141] Microbial growth byproducts produced by the microorganism of interest can be retained in the microorganism or secreted into the growth medium. The medium can contain compounds that stabilize the activity of the microbial growth byproducts.

[0142] The methods and apparatus for culturing microorganisms and producing microbial byproducts can be performed in a batch process, a quasi-continuous process, or a continuous process.

[0143] In one embodiment, all of the microbial culture composition is removed when the culture is complete (eg, when a desired cell density or density of a particular metabolite is reached). In this batch process, a completely new batch is started when the first batch is harvested.

[0144] In another embodiment, only a portion of the fermentation product is removed at any one time. In this embodiment, the biomass with living cells, spores, conidia, hyphae and / or mycelium remains in the vessel as an inoculant for a new culture batch. The removed composition can be a cell-free culture medium or contain cells, spores or other reproductive propagules and / or combinations thereof. A quasi-continuous system is created in this way.

[0145] Advantageously, the method does not require complex equipment or high energy consumption. The microorganisms of interest can be cultured and used on-site on a small or large scale, even while still mixed with their culture medium.

[0146] Preparation of microbial-based products

[0147] "Microbial-based products" are products that are applied in practice to achieve a desired effect. Microbial-based products can be simply microbial-based compositions obtained from a microbial culture process. In addition, microbial-based products may include further ingredients that have been added. These additional ingredients may include, for example, stabilizers, buffers, carriers (e.g., water or saline), nutrients that support further microbial growth addition, non-nutrient growth enhancers and / or agents that help track microorganisms and / or compositions in their application environment. Microbial-based products may also include mixtures based on microbial compositions. Microbial-based products may also include one or more components based on microbial compositions that have been treated in some manner, such as but not limited to filtration, centrifugation, lysis, drying, purification, etc.

[0148] A microbial-based product of the present invention is simply a fermentation medium containing the microorganism and / or microbial metabolites and / or any residual nutrients produced by the microorganism. The fermented product can be used directly without extraction or purification. If necessary, extraction and purification can be easily achieved using standard extraction and / or purification methods or techniques described in the literature.

[0149] The microorganisms in the microbial products can be in active or inactivated form. In addition, the microorganisms and the residual culture used can be removed from the composition. The microbial products can be used without further stabilization, preservation and storage. Advantageously, the direct use of these microbial products maintains the high vitality of the microorganisms, reduces the possibility of contamination by foreign agents and undesirable microorganisms, and maintains the activity of the by-products of microbial growth.

[0150] The microorganisms and / or culture medium (eg, fermentation broth or solid matrix) produced by the growth of the microorganisms can be removed from the growth vessel and transferred via tubing or the like for immediate use.

[0151] In one embodiment, the microbial-based product is the only microbial growth byproduct.For example, a biosurfactant produced by a microorganism can be collected from a liquid fermentation tank in a crude form that includes about 50% pure biosurfactant in the liquid fermentation broth.

[0152] In other embodiments, the microbial-based product (microorganism, culture medium, or microorganism and culture medium) can be placed in a container of appropriate size, taking into account, for example, the intended use, the intended method of use, the size of the fermenter, and any mode of transportation from the microbial growth facility to the point of use. Thus, the container in which the microbial-based composition is placed can be, for example, 1 gallon to 1,000 gallons or larger. In other embodiments, the container is 2 gallons, 5 gallons, 25 gallons or larger.

[0153] For example, when the yeast fermentation product is harvested from the growth vessel, when the obtained product is placed in a container and / or piped (or otherwise transported for use), other components may be added. Additives may be, for example, buffers, carriers, other microbial-based compositions produced in the same or different facilities, viscosity modifiers, preservatives, nutrients for microbial growth, tracers, solvents, antimicrobial agents, other microorganisms, and other ingredients specific to the intended use.

[0154] Other suitable additives that may be included in the formulations according to the invention include substances commonly used in such preparations. Examples of such additives include surfactants, emulsifiers, lubricants, buffers, solubility regulators, pH regulators, preservatives, stabilizers and UV inhibitors.

[0155] In one embodiment, the product may also include a buffer comprising an organic acid and an amino acid or its salt. Suitable buffers include citrate, gluconate, tartrate, malate, acetate, lactate, oxalate, aspartate, malonate, glucoheptonate, pyruvate, galactate, gluconate, propanol glycolate, glutamate, glycine, lysine, glutamine, methionine, cysteine, arginine, and mixtures thereof. Phosphoric acid and phosphorous acid, or their salts may also be used. Synthetic buffers may be used, but preferably natural buffers may be used, such as the organic acids and amino acids listed above, or their salts.

[0156] In another embodiment, the pH adjuster comprises potassium hydroxide, ammonium hydroxide, potassium carbonate or bicarbonate, hydrochloric acid, nitric acid, sulfuric acid, or a mixture.

[0157] In one embodiment, additional components may be included in the formulation, such as an aqueous solution formulation of a salt (such as sodium bicarbonate or carbonate, sodium sulfate, sodium phosphate, sodium hydrogen phosphate).

[0158] Advantageously, according to the present invention, microbial-based products may include fermentation broth in which the microorganisms are grown. The product may be, for example, at least 1%, 5%, 10%, 25%, 50%, 75% or 100% fermentation broth by weight. The biomass in the product may be, for example, 0% to 100% (including all percentages between 0% and 100%) by weight.

[0159] Alternatively, the product may be stored prior to use. The storage time is preferably short. Thus, the storage time may be less than 60 days, 45 days, 30 days, 20 days, 15 days, 10 days, 7 days, 5 days, 3 days, 2 days, 1 day or 12 hours. In a preferred embodiment, if living cells are present in the product, the product is stored at a cool temperature, such as, for example, below 20° C., 15° C., 10° C. or 5° C. On the other hand, the biosurfactant composition can generally be stored at ambient temperature.

[0160] Methods for remediation and / or mitigation of iron pollution

[0161] In a preferred embodiment, the invention provides methods for remediating and / or mitigating heavy metal pollution, wherein a composition comprising a metal chelator is introduced into an environment where metal pollution is occurring or may occur. These methods can be used in environments including, for example, soil, water, air or waste (e.g., municipal waste or industrial waste).

[0162] In certain embodiments, these methods are used to remediate and / or mitigate iron pollution. The compositions can concentrate free iron from soil, water, and / or air, thereby reducing iron pollution in the environment and reducing iron toxicity to humans, animals, and plants.

[0163] In some embodiments, the method comprises applying the iron trapping component according to the present invention together with an acid, wherein the acid adjusts the pH of the composition or environment to 6.8 or less, preferably 5.0 or less, and more preferably 4.8 or less. The acid may be an organic acid selected from the following items: for example, acetic acid, citric acid, formic acid, lactic acid, malic acid, oxalic acid, tartaric acid, uric acid, propionic acid, butyric acid, sorbic acid, fumaric acid, benzoic acid, hydrofluoric acid, caproic acid, salicylic acid, gluconic acid, pyruvic acid, adipic acid, trichloroacetic acid, glycolic acid, cinnamic acid, carboxylic acid, succinic acid, carbonic acid, glutaric acid, capric acid, and ascorbic acid. The acid may also be an inorganic acid selected from the following items: for example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, boric acid, perchloric acid, hydrofluoric acid, hydrobromic acid, and sulfonic acid.

[0164] As used herein, "applying" a composition or product, or "treating" an environment means contacting a composition or product with a target or site so that the composition or product can have an effect on the target or site. The effect can be caused, for example, by the growth of microorganisms and / or the action of metabolites, enzymes, biosurfactants or other growth byproducts.

[0165] If present in the composition, the applied microorganism may be alive (or viable) or inactive when applied.In some embodiments, the microorganism is in the form of a yeast extract and / or another microbial hydrolysate.

[0166] The microbial growth by-products may be used in addition to the microbial generated growth by-products of the composition or may be used alone without the microorganisms.

[0167] The method may also include adding materials to promote microbial growth (e.g., adding nutrients and / or prebiotics) during the application process. Thus, live microorganisms can grow in situ and produce active compounds in situ. Thus, high concentrations of microorganisms and their growth byproducts can be easily and continuously achieved in the environment.

[0168] In some embodiments, the method comprises applying the whole microbial culture including inactivated cells in a submerged or solid-state fermentation medium. Advantageously, this reduces the amount of waste products generated during the production of the composition of the invention while increasing production efficiency by eliminating extraction and / or purification steps of microbial metabolites. In addition, the inclusion of inactive cells and residual fermentation medium provides a rich source of organic and inorganic nutrients that are essential for supporting soil and / or plant health.

[0169] In one embodiment, the composition is dispersed in the environment and supported on a carrier. The carrier can be made of a material that can relatively gently retain the microorganism, thereby allowing the microorganisms proliferated thereby to be easily released. The carrier is preferably cheap, and in some embodiments can serve as a nutrient source for the microorganisms thus applied, particularly a nutrient source that can be gradually released. Biodegradable carrier materials include corn husks, sugar industry waste or any agricultural waste. The water content of the carrier is, by weight, typically 1% to 99%, preferably 5% to 90%, more preferably 10% to 85%.

[0170] The material that strengthens the growth of microorganisms and the generation of interested microorganism growth byproducts can also be added to the composition and / or the treatment position. These materials include but are not limited to oil, glycerol, sugar or other nutrients. For example, the carbon substrate that supports microorganism growth can be added to the composition or the target area.

[0171] In certain embodiments, the method of the present invention results in a reduction of at least 25%, preferably at least 50%, in the amount of free heavy metal particles in the environment after one treatment. In certain embodiments, the environment may be treated multiple times to further reduce the amount of metal contamination.

[0172] In certain embodiments, the period of time that the composition can be in contact with a contaminated or potentially contaminated environment is from about 1 second to about 1 year, from about 1 minute to about 1 year, from about 1 minute to about 6 months, from about 1 minute to about 1 month, from about 1 minute to about 1 week, from about 1 minute to about 48 hours, from about 30 minutes to 40 hours, or preferably from about 12 hours to 24 hours.

[0173] In certain embodiments, the method includes applying the composition to the environment for a period of time until the amount of pollutants (particularly iron) in the environment is determined to be a satisfactory amount, or an amount that can be easily determined by a person skilled in the art. The amount of iron or other metal pollutants that is acceptable and / or safe depends on the specific circumstances. For example, water available to humans, animals, or plants may require less iron content than water and fracturing fluids used in fracturing wells.

[0174] In certain embodiments, the method includes applying the composition of the present invention to soil. These methods can be used, for example, in farmland, pasture, orchard, grassland, plot and / or forest. These methods can also be used in areas containing soil that is obviously unsuitable for plant growth, such as over-cultivated soil and / or soil that does not implement crop rotation or is insufficient to maintain soil fertility; soil contaminated by excessive treatment with pesticides, fertilizers and / or herbicides; high salinity soil; soil contaminated by dumping, metal waste accumulation (such as garbage dumps or scrap yards) or chemical or hydrocarbon leaks (such as Superfund sites, oil and gas recovery sites); soil contaminated by ammunition (such as hunting grounds, shooting ranges and war zones); and / or soil in areas damaged by natural or man-made causes (such as commercial, residential or urban buildings), excavation, oil and gas extraction, mining, logging, steel production, livestock breeding and other causes including fire, floods, insect pests, construction and development, etc.

[0175] In certain embodiments, the iron content of the contaminated soil is above 300 ppm. In preferred embodiments, the method reduces the iron content in the soil to 200 ppm or less, more preferably to 150 ppm or less, even more preferably to 50 ppm to 100 ppm.

[0176] In some embodiments, the composition of the present invention is applied to the soil by spreading it onto the soil surface. This can be done using a standard spreader or sprayer device. In some embodiments, a single spreading step can complete the application process, wherein all components are included in a single formulation. In other embodiments using a two-part or multi-part formulation, multiple spreading steps can be used.

[0177] In one embodiment, mechanical action may be used, such as rubbing, brushing or working the composition into the soil by tilling. In another embodiment, after application of the composition, a liquid (such as water) may be subsequently applied. Water may be applied in the form of a spray using standard methods known to those of ordinary skill in the art. Other liquid wetting agents and wetting formulations may also be used.

[0178] In certain embodiments, the composition is applied to the soil surface without mechanical bonding. The beneficial effects of soil application can be activated by rainfall, sprinkler irrigation, flooding or drip irrigation and then dispersed throughout the soil. In an exemplary embodiment, the composition can be effectively applied via a center pivot irrigation system or by spraying on the seed furrow.

[0179] In some embodiments, the composition is applied as a seed treatment in the form of a dry or liquid formulation or applied to the surface of a plant or plant part (eg, applied to the surface of a plant leaf or root).

[0180] The application of the compositions of the present invention can be carried out alone or in combination with the application of other compounds to enhance soil health and / or plant health. For example, commercial and / or natural fertilizers, pesticides, herbicides and / or other soil conditioners can be applied with the compositions.

[0181] Advantageously, these methods can help increase agricultural yields even in poor or damaged soils; restore depleted green areas such as pastures, forests, wetlands, and grasslands; and restore non-cultivable land to make it available for agriculture, reforestation, and / or natural regeneration of plant ecosystems. Additionally, through improved agricultural practices, these methods can help reduce pollution caused by greenhouse gas emissions.

[0182] In certain embodiments, enhancing soil health includes improving one or more properties of the soil. This may include, for example, removing and / or reducing pollutants in the soil, increasing the nutrient content and nutrient availability of the soil, improving the drainage and / or moisture retention properties of the soil, increasing soil salinity, increasing soil microbiome diversity, and / or controlling soil-borne pests and diseases. Other improvements may include adding volume and / or structure to soil that is eroded by wind and / or water, and preventing and / or slowing wind and / or water erosion of the soil.

[0183] In certain embodiments, the method includes a step of characterizing the soil type and / or soil health prior to treating the soil according to the methods according to the invention. Thus, the method may also include tailoring the composition to meet specific soil type and / or soil health requirements. Soil characterization methods are known in the agronomic art.

[0184] In some embodiments, the methods are used to restore soil health, where the soil being treated was once healthy but has deteriorated over a period of time. Remediation can restore the soil to a previous healthy state and / or an enhanced healthy state.

[0185] In certain embodiments, the methods result in the removal and / or reduction of contaminants from soil, including remediation of soil contaminated with iron. In some embodiments, the contaminants are directly degraded by the microorganisms employed in the composition.

[0186] In some embodiments, microbial growth byproducts (e.g., iron trapping compounds and / or biosurfactants) contribute to the collection and / or degradation of pollutants and can chelate and form complexes with ionic and non-ionic metals to release them from the soil. Soil pollutants include, for example, residual fertilizers, pesticides, herbicides, fungicides, hydrocarbons, chemicals (e.g., dry cleaning treatments, municipal and industrial wastes), benzene, toluene, ethylbenzene, xylene, and heavy metals. In a preferred embodiment, the soil pollutant is iron.

[0187] In some embodiments, contaminated soil is combined with non-hazardous organic additives (such as manure or agricultural waste). The presence of these organic materials supports the development of rich microbial populations and the high temperature characteristics of composting. Therefore, the rate of bioremediation can be increased.

[0188] In certain embodiments, these methods can also help improve the diversity of the soil microbiome by promoting colonization of beneficial soil microorganisms in the soil and the roots of plants growing therein. The growth of nutrient-fixing microorganisms (such as rhizobia and / or mycorrhizae) and other endogenous and applied microorganisms can be promoted, thereby increasing the number of different species in the soil microbiome.

[0189] In certain embodiments, the method comprises applying the composition of the present invention to water. These methods can be used, for example, for municipal and industrial wastewater, produced water, and natural or man-made waterways. For example, water can be present in mining environments, oil and gas fields, dumps, Superfund sites, industrial waste sites, landfills, farmlands, metal processing sites, construction sites, hunting grounds, shooting ranges, and war zones / battlefields.

[0190] In certain embodiments, the iron content of the contaminated water is 25 mg / L or more. In a preferred embodiment, the method reduces the iron content in the water to 10 mg / L or less, preferably to 1 mg / L or less, more preferably to 0.5 mg / L or less, even more preferably to 0.3 mg / L or less.

[0191] The method preferably comprises contacting a composition according to the invention with water for a period of time to produce a mixture comprising treated water; and optionally separating the composition and / or contaminants from the water.

[0192] The method can be performed using, for example, columns, filters, membranes, solvents, electrolysis or using any other laboratory scale reactor or industrial scale reactor. The method can also be performed in situ (eg, in the context of a natural or artificial watercourse).

[0193] In certain embodiments, the mixture of the composition and water can be pumped or otherwise moved through a membrane, column, resin, adsorbent, or filter.

[0194] In certain embodiments, the water source is produced water. Produced water is naturally occurring water that is removed from the ground during the extraction of oil and gas because the formations containing the oil and gas also contain water.

[0195] In certain embodiments, the water source is groundwater or precipitation, including, for example, at industrial sites such as mines, quarries, and oil and gas wells.

[0196] In certain embodiments, the water source is water that is pumped or otherwise applied to a subterranean or geological formation, such as during hydraulic fracturing.

[0197] In certain embodiments, the water source is industrial or municipal wastewater that is being treated for reintroduction and use by the community.

[0198] These compositions can be applied to liquids or containers present in a range of temperatures and aquatic environments, such as, for example, streams, rivers, waterways, oceans, seas, lakes, ponds, runoff areas, containment pools, pipes, filters, filter presses, membranes, columns, sieves, cones, dehydrators, classifiers, scrapers, hydrocyclones, agitators, drums, pans, or industrial wastewater treatment tanks / industrial wastewater storage tanks. In certain embodiments, the composition can be added to a container containing a liquid before the liquid composition is added to the container.

[0199] In one embodiment, the composition can be applied to a reagent in a scrubber used in pollution control. Dry scrubbers utilize dry reagents in contact with the exhaust stream to bind and / or remove pollutants, while wet scrubbers utilize liquid reagents to adsorb or dissolve pollutants in the exhaust gas. Thus, the present invention provides a method for capturing iron particles from industrial exhaust streams in wet scrubbers and dry scrubbers, wherein the composition is contacted with the exhaust stream as part of the wet scrubbing reagent or the dry scrubbing reagent.

[0200] In some cases this can also be used to capture iron oxide fumes emitted in metalworking and welding environments.

[0201] Advantageously, the method of the present invention can be used to reduce metal pollution in the environment, particularly iron pollution. Therefore, in some embodiments, these methods can also be used to reduce or and / or prevent diseases or conditions (i.e., iron toxicity) caused by excessive exposure to iron. For example, these diseases include myocardial siderosis, liver damage, cirrhosis, islet cell damage, diabetes, hypothyroidism, hypogonadism, conjunctivitis, choroiditis, and retinitis.

[0202] In certain embodiments, the compositions may also be administered to humans, animals, or plants suffering from iron toxicity to treat and / or prevent diseases caused by overexposure to iron and / or iron toxicity.

[0203] Example

[0204] The present invention and its many advantages can be better understood by the following examples given by way of illustration. The following examples illustrate some methods, applications, embodiments and variations of the present invention. None of them should be considered as limitations of the present invention. For the present invention, a large number of changes and modifications may be made.

[0205] Example 1 - pH growth test of B4 strain

[0206] The B4 strain was spread on tryptic soy agar (TSA) at neutral (6.8) and acidic (4.8) pH levels to look for growth differences, in an effort to determine how it performs in the various pH environments found within the dairy cow digestive system.

[0207] Growth was faster on neutral pH (6.8) compared to acidic plates within 24 hours, but after 48 hours, growth on pH 4.8 agar was equal or faster. Figure 1A to Figure 1B Additionally, when grown on acid agar, it produces large amounts of exopolysaccharides (EPS). Figure 1C . This is the result of environmental pressure.

[0208] Dried B4 spores were also added to sterile PBS adjusted to pH 2.8 and left for 24 hours. They were then plated on neutral (6.8) TSA plates. A lawn of growth appeared, but no EPS was produced (similar to the pH 6.8 plates in Figure 1). This shows that the dried spores were still intact after being exposed to an overall harsh environment. In general, the pH of the growth medium / environment affects the production of EPS.

[0209] Example 2 - EPS

[0210] B4 was grown in liquid medium at pH 4.8 to specifically produce EPS. Putative EPS was isolated from the culture and purified.

[0211] FTIR analysis confirmed that the purified sample was EPS, and HPLC analysis confirmed the presence of a large peak, indicative of sugar oligomers. C=O bonds were also observed via UV absorption.

[0212] Additionally, surprisingly, when the culture was processed for EPS extraction, purple streaks appeared in the cell pellet. When EPS purification was complete, the sample was pink, indicating the presence of pulcherrimin or pulcherriminic acid. Figure 2 .

[0213] Example 3 - Enzyme Assay Test - Amylase

[0214] Amylase is an enzyme that hydrolyzes the glycosidic bonds in starch molecules by converting complex carbohydrates into monosaccharides. Agar (starch) is inoculated with B4 and cultured for growth. Figure 3 After running the plate assay, an orange color is observed around the bacterial growth, indicating starch breakdown. This is a positive amylase test.

[0215] Example 4 - Enzyme Assay Test - Cellulase

[0216] Cellulases are enzymes that convert cellulose into glucose. Carboxymethylcellulose agar (CMCA) plates were used to test the cellulase activity of B4.

[0217] B4 was grown in tryptone soy broth with and without cellobiose. After 48 hours of growth, the liquid culture was streaked onto CMCA plates. After inoculation, once growth appeared on the agar, an iodine solution was introduced to the plates. The yellow transparent area around the bacterial growth indicated the decomposition of cellulose and the presence of cellulase. Figure 4 .

[0218] Example 5 - Detection of siderophores

[0219] The chrome azurol S (CAS) assay was used to detect siderophores from B4 cultures and dried B4 spores. These tests were performed in aerobic and anaerobic environments in different growth media: MRS-sucrose, M23-6, minimal medium with Tween, and minimal medium without Tween. FIG. 5A to FIG. 5C ) and 24 hours ( FIG. 6A to FIG. 6C )Observe multiple plates.

[0220] Siderophores scavenge iron from the Fe-CAS-cetyltrimethylammonium chloride complex and release the CAS dye resulting in a color change from blue to orange. Any observable color change on the CAS agar plate indicates qualitative detection of siderophores.

[0221] All B4 medium cultures tested showed positive responses for siderophore production and activity. MRS-sucrose (the richest medium) produced the strongest activity. There were no differences in siderophore activity between the four medium cultures under aerobic and anaerobic conditions.

[0222] The siderophore activity produced by dried B4 spores was lower compared to that of B4 cultures, which may be due to the dormancy of the spores. The dried spores performed better under aerobic conditions.

[0223] Example 6 - Iron activity determination

[0224] To understand how B4 interacts with iron and to determine differences in iron activity between cultures, an iron assay kit (Sigma-Aldrich) was used to measure the amount of ferrous iron (Fe) present in different B4 cultures. 2+ ) concentration, iron (Fe 3+ ) concentration and total iron concentration.

[0225] Iron is released from the sample by the addition of an acidic buffer. The released iron reacts with the chromogen to produce a colorimetric (593 nm) product proportional to the iron present.

[0226] Figure 7 The results in Figures 5 and 6 correlate with the siderophore results reported in Figures 5 to 6, meaning that higher siderophore activity correlates with lower total iron content. For example, MRS-sucrose medium is the richest medium with the highest siderophore activity and the lowest total iron content. Additionally, the minimal medium used is intended to increase the production of pulchermin, which is chelated iron. Higher levels of pulchermin in the culture increase both the iron concentration and the total iron concentration.

Claims

1. A method for reducing and / or mitigating iron pollution in an environment, wherein the method comprises applying a composition comprising one or more iron-capturing components to an environment contaminated by iron.

2. The method of claim 1, further comprising applying an acid to the environment to achieve a pH of 6.8 or less.

3. The method according to claim 1, wherein the environment is soil, and wherein the soil is contaminated with an iron content higher than 200 ppm.

4. The method of claim 3, wherein the composition is applied to the soil via an irrigation system or via a sprayer applicator.

5. The method of claim 1, wherein the environment is water, and wherein the water is contaminated with an iron content higher than 10 mg / L.

6. The method of claim 1, wherein the iron-trapping component is a non-pathogenic microorganism that is capable of trapping iron or produces a growth byproduct that is capable of trapping iron. The method according to claim 6 , wherein the microorganism is a bacterium of the genus Bacillus.

8. The method of claim 7, wherein the microorganism is Bacillus subtilis B4 NRRL B-68031 or Bacillus amyloliquefaciens NRRL B-67928.

9. The method of claim 7, wherein the microorganism is applied in the form of spores but grows in the form of a biofilm when in contact with an acidic environment having a pH of 6.8 or less.

10. The method according to claim 1, wherein the iron capture component is selected from the following: pulcherrimin, pulcherriminic acid, citrate, citric acid, EDTA ethylenediaminetetraacetic acid, iron EDTA, DTPA diethylenetriaminepentaacetic acid, EDDHA ethylenediamine-di-o-hydroxyphenylacetic acid, N,N-dihydroxy-N,N′-diisopropyladipamide DPH, 2,3-dihydroxybenzoic acid, nitrogen carrying bacteria, transferrin, enterobactin, pyocyanin, protochelatin, ferritin, Bacteriocins, vibriocins, vibrioferrin, azotocin, aminochelatins, yersin, agarobactin, grape agglutinin, ferrochrome, deferasirox, deferiprone, deferoxamine, fusarin, chrysin, chromobactin, ornithine, rhodotorulic acid, lysine, glutamic acid, gluconic acid, iron oxyhydroxide minerals, ferrihydrite, magnetite, hematite, goethite, siderite hydroxamate, catecholase, salicylate, carboxylate, mugineic acid, ferulic acid, caffeic acid and / or nicotinamide.

11. The method of claim 1 , wherein the composition further comprises one or more microbial growth byproducts selected from the group consisting of biosurfactants, enzymes, organic acids, fatty acids, amino acids, proteins, peptides, alcohols, polyketides, natural antibiotics, aldehydes, amines, sterols, and vitamins.

12. A composition for reducing and / or mitigating iron pollution in the environment, the composition comprising one or more iron capturing components and optionally a carrier.

13. The composition of claim 12, further comprising an amount of an acid to adjust the pH of the composition to 6.8 or less.

14. The composition of claim 12, wherein the iron-trapping component is a non-pathogenic microorganism that is capable of trapping iron or produces a growth byproduct that is capable of trapping iron.

15. The composition according to claim 14, wherein the microorganism is a bacterium of the genus Bacillus.

16. The composition of claim 15, wherein the microorganism is Bacillus subtilis B4 NRRL B-68031 or Bacillus amyloliquefaciens NRRL B-67928.

17. The composition of claim 14, wherein the microorganism is in spore form or biofilm form.

18. The composition of claim 14, wherein the iron-trapping component is selected from the group consisting of pulcherrimin, pulcherriminic acid, citrate, citric acid, EDTA ethylenediaminetetraacetic acid, iron EDTA, DTPA diethylenetriaminepentaacetic acid, EDDHA ethylenediamine-di-o-hydroxyphenylacetic acid, N,N-dihydroxy-N,N′-diisopropyladipamide DPH, 2,3-dihydroxybenzoic acid, nitrogen-carrying bacteria, transferrin, enterobactin, pyocyanin, protochelatin The invention can be used to treat schizolin, chelatin, chelatin, bacteriocin, vibriocin, vibrioferrin, azotocin, aminochelatin, yersin, ferrochrome, deferasirox, deferiprone, deferoxamine, fusarin, chrysin, chromatin, ornithine, rhodotorulic acid, lysine, glutamic acid, gluconic acid, ferric oxyhydroxide minerals, ferrihydrite, magnetite, hematite, goethite, siderite, hydroxamate, catecholase, salicylate, carboxylate, mugineic acid, ferulic acid, caffeic acid and / or nicotinamide.

19. The composition of claim 14, further comprising a germination enhancer, wherein the germination enhancer is L-alanine, L-leucine, or manganese.

20. The composition of claim 14, further comprising one or more microbial growth byproducts selected from the group consisting of biosurfactants, enzymes, organic acids, fatty acids, amino acids, proteins, peptides, alcohols, polyketides, natural antibiotics, aldehydes, amines, sterols, and vitamins.

21. The composition of claim 14, further comprising a nutrient or a prebiotic.

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