MATERIALS AND METHODS FOR REMEDIATING AND MITIGATING IRON POLLUTION

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No. 63/359,248, filed July 8, 2022, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Iron ore is a mineral substance that yields metallic iron (Fe) and can comprise iron oxides such as, for example, magnetite (FC3O4) and hematite (Fe2O3). Further, iron ore is the source of primary iron for the world’s iron and steel industries and is essential for the production of steel. Iron ore is mined in over 50 countries, with seven countries comprising over three-quarters of the total world production: Australia, Brazil, China, India, Russia, Ukraine, and Canada.

The mining of iron ore is a highly energy intensive process and causes air pollution in the form of greenhouse gasses including nitrous oxide, carbon dioxide, carbon monoxide, and sulfur dioxide from diesel generators, trucks, and other equipment. Additionally, in mining environments, soil and water can become contaminated with excess levels of heavy metals, including iron. Mining iron ore can also cause pollution of water with heavy metals and acids that drain from the mines, which can continue for many years even after mining stops in a location. Furthermore, processing and dumping of ore waste products can lead to the deposition of these metals into the environment

Other causes for iron pollution can include, for example, industrial drainage, corrosion of iron or steel well casing or water pipes, excessively low pH in soil and water environments, leaching from metal scraps such as ammunition and construction waste, and toxic waste disposal.

Excessive iron exposure can cause serious disruption to ecosystems and can be health hazards 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 conditions such as liver disease, heart conditions and diabetes. Furthermore, chromic inhalation of iron oxide fumes or dust can also result in lasting respiratory illnesses, including cancer.

In plants, excessive iron absorption can cause leaf discoloration and stunted root growth, which can ultimately lead to premature death.

Heavy metal pollution can destabilize ecosystems through the bioaccumulation of metals in living organisms. All heavy metals can have toxic effects on living organisms, for example, through metabolic interference and mutagenesis. Iron pollution, in particular, can cause lasting physiological damage to exposed organisms; thus, improved methods and compositions are needed for remediating excess iron levels in heavy metal-polluted environments. BRIEF SUMMARY OF THE INVENTION

The subject invention pertains to methods and compositions for remediating and/or mitigating heavy metal pollution. More specifically, the subject invention provides compositions that, when applied to an environment in which iron pollution is occurring, or may occur, can reduce the level of free iron present in that environment. Advantageously, the compositions and methods can be useful for reducing harm to humans, animals and the environment as a result of iron pollution.

In preferred embodiments, the subject invention provides methods for remediating and/or mitigating heavy metal pollution, wherein a composition comprising an iron-capturing ingredient is introduced into an environment in which iron pollution is occurring, or may occur. The methods can be utilized in environments including, for example, soil, water, and air. Advantageously, in certain embodiments, the methods result in capture of free iron present in the environment. In one embodiment, iron pollution has been determined to be present prior to the introduction of the composition.

In one embodiment, the composition sequesters the iron from the soil, water and/or air, thus reducing iron pollution in that environment and reducing the potential for iron toxicity.

In one embodiment, the methods comprise applying the composition to a mining environment. In such environments, soil and water can become contaminated with excess levels of heavy metals, including iron. Processing and dumping of ore waste products can lead to the deposition of these metals into the environment, which can cause serious disruption to ecosystems as well as health hazards to humans and animals.

In another embodiment, the method comprises applying the composition to an environment where firearms are, or have previously been, heavily used, such as hunting grounds, firing ranges and war zones/battlefields. Soil and water in such areas can become polluted with iron and other metals that leach from residual ammunition.

In one embodiment, the composition can be applied to reagents in scrubbers utilized in pollution control. Dry scrubbers utilize dry reagents, which are contacted with exhaust streams to bind and/or remove pollutants, while wet scrubbers utilize liquid reagents to absorb or dissolve pollutants from the exhaust. Thus, the subject invention provides methods for capturing iron particulates from industrial exhaust streams in both wet and dry scrubbers, wherein the composition is contacted with the exhaust stream as part of a wet or dry scrubbing reagent.

The subject invention further provides a composition comprising one or more ingredients that capture iron. In certain embodiments, the iron-capturing ingredient is a beneficial microorganism, a growth by-product of an iron-capturing microorganism, or some other compound known to bind iron. In certain embodiments, more than one iron-capturing ingredient is included in the composition. In preferred embodiments, the beneficial microorganisms of the subject invention are non- pathogenic fungi, yeasts and/or bacteria capable of sequestering iron, either naturally or through genetic modification.

In one embodiment, the beneficial microorganism is a strain of Bacillus subtilis. In a specific embodiment, the strain is B. subtilis B4 (NRRL B-68031). The B4 strain is preferably administered in spore form but grows in biofilm form when exposed to acidic environments.

Surprisingly, B4 was found to produce one or more compounds capable of sequestering, chelating or otherwise capturing iron. In certain embodiments, the compounds are pulcherrimin and/or pulcherriminic acid. Advantageously, the microbes and/or the exopolysaccharide (EPS) of the microbes when grown in biofilm form effectively hoard freely-available iron using an iron-capturer such as pulcherrimin and/or pulcherriminic acid upon exposure to an acidic pH (e.g., less than 6.8, preferably less than 5.0).

B4 is also particularly advantageous over other traditional probiotic microorganisms due to its ability to produce digestive enzymes, including, for example, cellulases and amylases.

In certain embodiments, the composition can comprise other non-pathogenic microorganisms that are capable of producing compounds that can sequester, chelate or otherwise capture iron. The microorganism(s) can be in biofilm form, spore form, planktonic form, or any other form.

In certain embodiments, the microorganisms are also capable of producing one or more of the following: surface active agents, such as lipopeptides and/or glycolipids; bioactive compounds with antimicrobial and immune-modulating 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.

In certain embodiments, the iron-capturing ingredient of the subject composition is a crude form or purified siderophore or phytosiderophore, or other molecule with high iron affinity, for example, pulcherrimin, pulcherriminic acid, citrate, citric acid, EDTA (Ethylenediaminetetraacetic acid), ferric EDTA, DTPA (Diethylenetriaminepentaacetic acid), EDDHA (Ethylenediamine di(o- hydroxyphenylacetic acid), N,N-dihydroxy-N,N'-diisopropylhexanediamide (DPH), 2,3- dihydroxybenzoic acid, azotochelin, transferrin, enterobactin, pyoverdine, protochelin, pyochelin, bacillibactin, vibriobactin, vibrioferrin azotobactin, aminochelin, yersiniabactin, agrobactin, staphyloferrin, ferrichrome, defarasirox, deferiprone, desferrioxamine, fusarinine, chrysobactin, achromobactin, omibactin, rhodotorulic acid, lysine, glutamic acid, gluconic acid, iron oxyhydroxide minerals, ferrihydrite, magnetite, hematite, geothite, sideritehydroxamate, catecholates, salicylates, carboxylates, mugineic acid, ferulic acid, caffeic acid, and/or nicotianamine. In certain embodiments, the composition comprises an organic or inorganic acid. Preferably, the acid is present in an amount suitable for adjusting the pH of the composition or the environment to which it is applied to 6.8 or lower, preferably, 5.0 or lower, more preferably, 4.8 or lower.

Advantageously, in certain embodiments, the composition according to the subject invention can be effective at reducing the level of toxic compounds in, for example, soil, water, and air. Furthermore, the methods of the subject invention do not require complicated equipment or high energy consumption, and production of the composition can be performed on site, for example, at a mine or at an oil well.

BRIEF DESCRIPTION OF THE FIGURES

Figures 1A-1C show growth and exopolysaccharide (EPS) formation of B4 after 24 hours (A) and 48 hours (B) at acidic pH (top row, pH 4.8) and neutral pH (bottom row, pH 6.8).

Figure 2 shows purified B4 EPS with a pink hue.

Figure 3 shows result of an amylase test for B4. Agar streaked with B4 produced an orange color around the bacterial growth, indicating the breakdown of starch.

Figure 4 show results of a cellulase test for B4. Agar streaked with B4 produced a yellow zone of clearing around the bacterial growth, indicating the breakdown of cellulose.

Figures 5A-5C show B4 siderophore production and activity after 6 hours in aerobic (top plates) and anaerobic (bottom plates) environments and on different growth media. (A) shows B4 culture grown in MRS-sucrose (left side) and M23-6 (right side) media. (B) shows B4 culture grown in minimal media with Tween (left side) and minimal media without Tween (right side). (C) shows dried B4 spores grown in minimal medium.

Figures 6A-6C show B4 siderophore production and activity after 24 hours in aerobic (top plates) and anaerobic (bottom plates) environments and on different growth media. (A) shows B4 culture grown in M23-6 (left side)and MRS-sucrose (right side) media. (B) shows B4 culture grown in minimal media with Tween (left side) and minimal media without Tween (right side). (C) shows dried B4 spores grown in minimal medium.

Figure 7 shows results of an iron assay for B4 (BSSL) cultures grown in different media.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention provides compositions and methods for remediating and mitigating iron pollution. More specifically, the subject invention provides compositions that, when contacted to an environment that contains free iron, lead to a reduction of free iron in that environment. Advantageously, in some embodiments, the compositions and methods can also improve the overall health and productivity of living organisms in the environment in which the methods and compositions are employed.

Selected Definitions

As used herein, a “biofilm” is a complex aggregate of microorganisms, such as bacteria, wherein the cells adhere to each other and/or to a surface. In certain embodiments, adherence is achieved via an exopolysaccharide substance produced by the bacteria. The cells in biofilms are physiologically distinct from planktonic cells of the same organism, which are single cells that can float or swim in liquid medium.

As used herein “preventing” or “prevention” of a disease, condition or disorder means delaying, inhibiting, suppressing, forestalling, and/or minimizing the onset or progression of a particular sign or symptom thereof. Prevention can include, but does not require, indefinite, absolute or complete prevention throughout a subject’s lifetime, meaning the sign or symptom may still develop at a later time. Prevention can include reducing the severity of the onset of such a disease, condition or disorder, and/or inhibiting the progression of the condition or disorder to a more severe condition or disorder.

As used herein, “treating” or “treatment” of a disease, condition or disorder means the eradicating, improving, reducing, ameliorating or reversing of at least one sign or symptom of the disease, condition or disorder (e.g., an infection). Treatment can include, but does not require, a complete cure of the disease, condition or disorder, meaning treatment can also include partial eradication, improvement, reduction, amelioration or reversal.

As used herein, an “isolated” or “purified” nucleic acid molecule, polynucleotide, polypeptide, protein, organic compound such as a small molecule (e.g., those described below), or other compound is substantially free of other compounds, such as cellular material, with which it is associated in nature. For example, a purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) is free of the genes or sequences that flank it in its naturally-occurring state. A purified or isolated polypeptide is free of the 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, the isolated strain may exist as, for example, a biologically pure culture, or as spores (or other forms of the strain) in association with a carrier.

In certain embodiments, purified compounds are at least 60% by weight the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. For example, a purified compound is one that is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of the desired compound by weight. Purity is measured by any appropriate standard method, for example, by column chromatography, thin layer chromatography, or high-performance liquid chromatography (HPLC) analysis. As used herein, “ionophores” are carboxylic polyether non-therapeutic antibiotics that disrupt the ion concentration gradient (Ca2+, K+, H+, Na+) across microorganisms, which causes them to enter a futile ion cycle. The disruption of the ion concentration prevents the microorganism from maintaining normal metabolism and causes the microorganism to expend extra energy. Ionophores function by selecting against or affecting the metabolism of gram-positive bacteria, such as methanogens, and protozoa.

As used herein, “siderophores” are compounds produced by different organisms for the purpose of scavenging iron from the surrounding environment. Siderophores are typically small, low molecular weight compounds with high affinity for ferric iron (Fe ³⁺ ), forming strong ferric chelate complexes that can, in some instances be taken up by the organisms. As used herein, “phytosiderophores” are siderophores produced by plants.

A “metabolite” refers to any substance produced by metabolism (e.g., a growth by-product) or a substance necessary for taking part in a particular metabolic process. A metabolite can be an organic compound that is a starting material, an intermediate in, or an end product of metabolism. Examples of metabolites can include, but are not limited to, enzymes, toxins, acids, solvents, alcohols, proteins, carbohydrates, vitamins, minerals, microelements, amino acids, polymers, polyketides, and surfactants.

As used herein, a “methanogen” is a microorganism that produces methane gas as a by-product of metabolism. Methanogens are archaea that can be found in the digestive systems and metabolic waste of ruminant animals and non-ruminant animals (e.g., pigs, poultry and horses). Examples of methanogens include, but are not limited to, Methanobacterium spp. (e.g., M. formicicum), Methanobrevibacler spp. (e.g., M. ruminantium), Methanococcus spp. (e.g., M. paripaludis), Melhanoculleus spp. (e.g., M. bourgensis), Methanoforens spp. (e.g., M. stordalenmirensis), Methanofollis liminatans, Methanogenium wolfei, Methanomicrobium spp. (e.g., M. mobile), Methanopyrus kandleri, 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.

As used herein, “ore” refers to a naturally occurring solid material from which a valuable substance, mineral and/or metal can be profitably extracted. Ores are often mined from ore deposits, which comprise ore minerals containing the valuable substance. “Gangue” minerals are minerals that occur in the deposit but do not contain the valuable substance. Examples of ore deposits include hydrothermal deposits, magmatic deposits, laterite deposits, volcanogenic deposits, metamorphically reworked deposits, carbonatite-alkaline igneous related deposits, placer ore deposits, residual ore deposits, sedimentary deposits, sedimentary hydrothermal deposits and astrobleme-related deposits. Ores, as defined herein, however, can also include ore concentrates or tailings, copper, or even other sources of metal or valuable minerals, including but not limited to, jewelry, electronic scraps, and other scrap materials.

As used herein, “industrial wastewater” refers to the water that is produced as the result of an industrial activity, including, for example, petroleum refining, chemical production, mining, oil and gas extraction, power production, iron and steel production, battery production, paper making, smelting, textile production, and municipal wastewater treatment.

As used herein “produced water” refers to naturally occurring water that is removed from ground during the extraction of oil and gas, as geological formations that contain oil and gas also contain water.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 1, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, “nested sub-ranges” that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 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.

As used herein, “reduction” means a negative alteration and “increase” means a positive alteration, wherein the positive or negative alteration 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%.

The transitional term “comprising,” which is synonymous with “including,” or “containing,” is inclusive or open-ended and does not exclude additional, un-recited elements or method steps. By 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 a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. Use of the term “comprising” contemplates other embodiments that “consist” or “consist essentially of’ the recited component(s).

Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a,” “and” and “the” are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of 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 embodiments or portions thereof.

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

Compositions

The subject invention provides a composition for use according to the subject methods, wherein the composition comprises one or more ingredients that capture iron. In certain embodiments, the iron- capturing ingredient is a beneficial microorganism, a growth by-product of a microorganism, or some other compound known to bind iron. In certain embodiments, more than one iron-capturing ingredient is included in the composition.

The total iron-capturing ingredients) in the composition preferably comprise from 0.0001% to 100% of the composition by weight or by volume, or from 0.001 to 95%, from 0.01 to 90%, from 0.1% to 85%, from 0.5 to 80%, from 0.75 to 75%, from 1.0 to 70%, from 1.25 to 65%, from 1.5 to 60%, from 1 .75 to 55%, from 2.0 to 50%, or from 5.0 to 25% by weight or by volume.

In certain embodiments, the composition is a “microbe-based composition,” meaning a composition that comprises components that were produced as the result of the growth of microorganisms or other cell cultures. Thus, the microbe-based composition may comprise the microbes themselves and/or by-products of microbial growth. The microbes may be in a vegetative state, in spore form, in mycelial form, in any other form of microbial propagule, or a mixture of these. The microbes may be planktonic or in a biofilm form, or a mixture of both. The by-products of growth may be, for example, metabolites, cell membrane components, expressed proteins, and/or other cellular components. The microbes may be intact or lysed. The cells may be totally absent, or present at, for example, a concentration of at least 1 x 10 ⁴ , 1 x 10 ⁵ , 1 x 10 ⁶ , 1 x 10 ⁷ , 1 x 10 ⁸ , 1 x 10 ⁹ , 1 x 10 ¹⁰ , 1 x 10 ¹¹ , 1 x 10 ¹² , 1 x 10 ¹³ or more CFU per milliliter or CFU/g of the composition.

Advantageously, in preferred embodiments, the subject compositions can sequester free iron in environments that are, or may be, polluted with heavy metals. Thus, in some embodiments, the composition can be used for remediating and/or mitigating iron pollution in an environment, which can also enhance the health of soil, water, air and living organisms.

In preferred embodiments, the beneficial microorganisms of the subject compositions are non- pathogenic fungi, yeasts and/or bacteria capable of sequestering iron, either naturally or through genetic modification. The beneficial microorganisms may be in an active, inactive and/or dormant form. In preferred embodiments, the microorganism is one that is characterized as “generally regarded as safe,” or GRAS, by the appropriate regulatory agency. In certain embodiments, the microorganisms are also capable of producing one or more of the following: surface active agents, such as lipopeptides and/or glycolipids; bioactive compounds with antimicrobial and immune-modulating effects; polyketides; acids; peptides; anti-inflammatoiy compounds; enzymes, such as amylases, cellulases, proteases and/or lipases; and sources of amino acids, vitamins, and other nutrients.

The microorganisms of the subject invention may be natural, or genetically modified microorganisms. For example, the microorganisms may be transformed with specific genes to exhibit specific characteristics. The microorganisms may also be mutants of a desired strain. As used herein, “mutant” means a strain, genetic variant, or subtype of a reference microorganism, wherein the mutant has one or more genetic variations (e.g., a point mutation, missense mutation, nonsense mutation, deletion, duplication, frameshift mutation or repeat expansion) as compared to the reference microorganism. Procedures for making mutants are well known in the microbiological art. For example, UV mutagenesis and nitrosoguanidine are used extensively toward this end.

In some embodiments, the beneficial microorganisms are selected based on a natural or acquired resistance to certain antibiotics administered to an environment comprising an iron-capturing pathogen to, for example, control pathogenic and/or deleterious microbes in a living subject or elsewhere in an environment.

In one specific embodiment, the composition comprises about 1 x 10 ⁶ to about 1 x 10 ¹³ , about 1 x 10 ⁷ to about 1 x 10 ¹² , about 1 x 10 ⁸ to about 1 x 10", or about 1 x 10 ⁹ to about 1 x 10'° CFU/g of each species of microorganism present in the composition.

In one embodiment, the composition comprises about 0.001 to 100% microorganisms total by volume, about 1 to 90%, or about 10 to 75%.

In certain embodiments, the composition comprises a growth by-product of a microorganism but no living microorganism. For example, in certain embodiments, a pathogenic microorganism is utilized only in the production of growth by-products for producing a composition according to the subject invention as opposed to direct administration to an environment.

The microorganisms can include yeasts, bacteria and/or fungi, including, for example, Acaulospora, Acidithiobacillus spp. (e.g., A. ferooxidans, A. albertensis, A. caldus, A. cuprithermicus, A. ferrianus, A. ferridurans, A. ferriphilus, A. ferrivorans, A. ferrooxidans, A. sulfur iphilus, 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, B. licheniformis, B. megaterium, B. mucilaginosus, B. subtilis), Blakeslea, Candida (e.g., C. albicans, C. apicola, C. hatistae, C. bombicola, C. floricola, C. kuoi, C. riodocensis, C. nodaensis, C. stellate), Cryptococcus, Debaryomyces (e.g., 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, Magnetospirillum magneticum, 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 (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. koreensis), Pseudozyma (e.g., P. aphidis, P. antarctica), Rhizobium radiobacter, Rhizopus, Rhodospirillum (e.g., R. rubrum), Rhodotorula (e.g., R. bogoriensis), Saccharomyces (e.g., 5. cerevisiae, S. boulardii, S. torula), Sphingomonas (e.g., .S' paucimobilis), Starmerella (e.g., S. bombicola), Streptomyces, Torulopsis, Thraustochytrium, Trichoderma (e.g., T. reesei, T. harzianum, T. viridae), Ustilago (e.g., U maydis), Vibrio cholerae, Wicker hamiella (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).

If present, fungi can be in the form of live or inactive cells, mycelia, spores and/or fruiting bodies. The fruiting bodies, if present, can be, for example, chopped and/or blended into granules and/or a powder form.

If present, yeasts can be in the form of live or inactive cells or spores, as well as in the form of dried and/or dormant cells (e.g., a yeast hydrolysate).

If present, bacteria can be in the form of vegetative or planktonic cells, biofilms, spores, and/or a dried cell or spore mass.

In some embodiments, dried microbes, e.g., spores, can be mixed with fillers known in the art, such as e.g., microcrystalline cellulose (MCC).

In one embodiment, the composition comprises one or more Bacillus spp. bacteria and/or growth by-products thereof. In certain embodiments, the Bacillus spp. are B. amyloliquefaciens, B. subtilis, B. coagulans and/or B. licheniformis .

In one embodiment, the composition comprises B. amyloliquefaciens NRRL B-67928 “B. amy” and/or a growth by-product thereof. A culture of the B. amyloliquefaciens “B. amy” microbe has been deposited with the Agricultural Research Service Northern Regional Research Laboratory (NRRL) Culture Collection, 1815 N. University St., Peoria, IL, USA. The deposit has been assigned accession number NRRL B-67928 by the depository and was deposited on February 26, 2020.

In one embodiment, the composition comprises a strain of Bacillus subtilis and/or a growth byproduct thereof. In a specific embodiment, the strain is B. subtilis B4 (NRRL B-68031). A culture of the B4 microbe has been deposited with the Agricultural Research Service Northern Regional Research Laboratory (NRRL) Culture Collection, 1815 N. University St., Peoria, IL, USA. The deposit has been assigned accession number NRRL B-68031 by the depository and was deposited on May 06, 2021.

B4 is a Gram-positive spore-forming strain of B. subtilis that is capable of anaerobic growth (obligate anaerobe). The B4 strain is preferably administered in spore form but germinates in acidic environments, wherein it can grow in biofilm form. Surprisingly, B4 was found to produce one or more compounds capable of sequestering, chelating or otherwise capturing iron when grown in biofilm form. In certain embodiments, the compounds are pulcherrimin and/or pulcherriminic acid.

Advantageously, the microbes and/or the exopolysaccharide (EPS) of the biofilm effectively hoard freely-available iron using an iron-capturer such as, e.g., pulcherrimin and/or pulcherriminic acid, while traveling through low pH (e.g., less than 6.8, less than 5.0, or less than 4.8).

B4 is also particularly advantageous due to its ability to produce increased amounts of the lipopeptide surfactin (e.g., greater than wild type B. subtilis), as well as digestive enzymes, including, for example, cellulases and amylases.

The proprietary cultures described herein have been deposited under conditions that assure that access to the cultures will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR § 1.14 and 35 U.S.C § 122. The deposit is available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

Further, each of the subject culture deposits will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., it will be stored with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposit, and in any case, for a period of at least 30 (thirty) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the culture. The depositor acknowledges the duty to replace the deposit should the depository be unable to furnish a sample when requested, due to the condition of the deposit. All restrictions on the availability to the public of the subject culture deposit will be irrevocably removed upon the granting of a patent disclosing it.

In certain embodiments, the composition can comprise other microorganisms that are capable, either naturally or by genetic modification, of producing pulcherrimin and/or pulcherriminic acid, or other compounds capable of sequestering, chelating or otherwise capturing iron. In specific embodiments, the microbes are capable of growing as a biofilm. In certain embodiments, the microorganism is a naturally-occurring or genetically-modified microorganism capable of regulating genes involved in iron capture and transport, e.g., HFE, GDF15, TWSG1, ERFE, Matriptase 2, TF, TFR1 , TFR2, HAMP and HJV.

In certain embodiments, the composition can comprise a crude form or purified siderophore or phytosiderophore, or other molecule with high iron affinity, for example, pulcherrimin, pulcherriminic acid, citrate, citric acid, EDTA (Ethylenediaminetetraacetic acid), ferric EDTA, DTPA (Diethylenetriaminepentaacetic acid), EDDHA (Ethylenediamine di(o-hydroxyphenylacetic acid), N,N-dihydroxy-N,N'-diisopropylhexanediamide (DPH), 2,3-dihydroxybenzoic acid, azotochelin, transferrin, enterobactin, pyoverdine, protochelin, pyochelin, bacillibactin, vibriobactin, vibrioferrin azotobactin, aminochelin yersiniabactin, agrobactin, staphyloferrin, ferrichrome, defarasirox, deferiprone, desferrioxamine, fusarinine, chrysobactin, achromobactin, omibactin, rhodotorulic acid, lysine, glutamic acid, gluconic acid, iron oxyhydroxide minerals, ferrihydrite, magnetite, hematite, geothite, sideritehydroxamate, catecholates, salicylates, carboxylates, mugineic acid, ferulic acid, caffeic acid, and/or nicotianamine.

The composition can also comprise other microbial growth by-products. The microbial growth by-product can be produced by the microorganisms of the composition, and/or they can be produced separately, e.g., by a microorganism listed herein, and added to the composition.

In certain embodiments, the composition can comprise substrate leftover from cultivation, and/or purified or unpurified growth by-products, such as biosurfactants, killer toxins, enzymes, polyketides, and/or other metabolites. The microbes can be live or inactive, although, in preferred embodiments, if the microbe is considered a pathogen, the microbe is inactivated and/or removed from the composition.

In one embodiment, the growth by-product has been purified from the cultivation medium in which it was produced. Alternatively, in one embodiment, the growth by-product is utilized in crude form. The crude form can comprise, for example, a liquid supernatant resulting from cultivation of a microbe that produces the growth by-product of interest, including residual cells and/or nutrients.

The growth by-products can 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.

Additional Components

Further components can be added to the composition, for example, carriers, other microbebased compositions, biosurfactants, enzymes, catalysts, solvents, buffers, emulsifying agents, lubricants, solubility controlling agents, preservatives, stabilizers, ultra-violet light resistant agents, viscosity modifiers, preservatives, tracking agents, biocides, surfactants, pH adjusting agents, essential oils, botanical extracts, cross-linking agents, chelators, fatty acids, alcohols, reducing agents, syndetics, dyes, colorants, fragrances, antimicrobial compounds, antibiotics, foaming agents, foam reducers, polymers, thickeners, chelators, and other ingredients specific for an intended use.

In certain specific embodiments, the composition comprises a chelating agent including, but are not limited to, ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), a phosphonate, succimer (DMSA), diethylenetriaminepentaacetate (DTPA), A-acetylcysteine, n- hydroxyethylethylenediaminetriacetic acid (HEDTA), organic acids with more than one coordination group (e.g., rubeanic acid), STPP (sodiumtripolyphosphate, Na5P3O10), trisodium phosphate (TSP), water, carbohydrates, organic acids with more than one coordination group (e.g., citric acid), lipids, steroids, amino acids or related compounds (e.g., glutathione), peptides, phosphates, nucleotides, tetrapyrrols, ferrioxamines, ionophores, orphenolics, sodium citrate, sodium gluconate, ethylenediamine disuccinic acid (EDDS), iminodisuccinic acid (IDS), L-glutamic acid diacetic Acid (GLDA), GLDA-Na4, methyl glycindiacetic acid (MGDA), polyaspartic acid (PASA), hemoglobin, chlorophyll, lipophilic P-diketone, and (14,16)-hentriacontanedione, 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), trimethyl glycine (TMG), Tiron, or any combination thereof.

In certain embodiments, the composition comprises a germination enhancer for enhancing germination of spore-form microorganisms used in the microbe-based composition. In specific embodiments, the germination enhancers are amino acids, such as, for example, L-alanine and/or L- leucine. In one embodiment, the germination enhancer is manganese.

In certain embodiments, the composition comprises an organic acid selected from, 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 acids, succinic acid, carbonic acid, glutaric acid, decanoic acid, and ascorbic acid. In some embodiments, the composition comprises an inorganic acid selected from, 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 of the composition or the environment to which it is applied to 6.8 or lower, preferably, 5.0 or lower, more preferably, 4.8 or lower.

In one embodiment, the composition comprises one or more fatty acids. The fatty acids can be produced by the microorganisms of the composition, and/or produced separately and included as an additional component. In certain preferred embodiments, the fatty acid is a saturated long-chain fatty acid, having a carbon backbone of 14-20 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, a saturated long-chain fatty acid can inhibit methanogenesis and/or increase cell membrane permeability of methanogens.

In certain embodiments, the composition comprises one or more enzymes that help digest 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 an additional component. Non-limiting examples of digestive enzymes include amylases, maltases, lactases, lipases, proteases, sucrases and cellulases.

In some embodiments, the composition can comprise additional components known to reduce methane production from methanogens, such as, for example, nitrates (e.g., calcium nitrate, ammonium nitrate, sodium nitrate, potassium nitrate, and magnesium nitrate); seaweed (e.g., Asparagopsis laxiformis and/or Asparagopsis armata); kelp; nitrooxypropanols (e.g., 3 -nitrooxypropanol and/or ethyl-3-nitrooxypropanol); anthraquinones; ionophores (e.g., monensin and/or lasalocid); polyphenols (e.g., saponins, tannins); Yucca schidigera extract (steroidal saponin-producing plant species); Quillaja saponaria extract (triterpenoid saponin-producing plant species); organosulfurs (e.g., garlic extract); flavonoids (e.g., quercetin, rutin, kaempferol, naringin, and anthocyanidins; bioflavonoids from green citrus fruits, rose hips and black currants); carboxylic acid; and/or terpenes (e.g., d-limonene, pinene and citrus extracts).

In one embodiment, the composition can comprise one or more biosurfactants. Biosurfactants are a structurally diverse group of surface-active substances produced by microorganisms, which are biodegradable and can be efficiently produced using selected organisms on renewable substrates. All biosurfactants are amphiphiles. They consist of two parts: a polar (hydrophilic) moiety and non-polar (hydrophobic) group. The common lipophilic moiety of a biosurfactant molecule is the hydrocarbon chain of a fatty acid, whereas the hydrophilic part is formed by ester or alcohol groups of neutral lipids, by a carboxylate group of fatty acids or amino acids (or peptides), an organic acid in the case of flavolipids, or, in the case of glycolipids, by a carbohydrate.

Due to their amphiphilic structure, biosurfactants increase the surface area of hydrophobic water-insoluble substances, increase the water bioavailability of such substances, and change the properties of bacterial cell surfaces. Biosurfactants accumulate at interfaces, thus reducing interfacial tension and leading to the formation of aggregated micellar structures in solution. Safe, effective microbial biosurfactants reduce the surface and interfacial tensions between the molecules of liquids, solids, and gases. The ability of biosurfactants to form pores and destabilize biological membranes permits their use as antibacterial, antifungal, and hemolytic agents.

Advantageously, in certain embodiments, biosurfactants can help disrupt and/or penetrate biofilms for increased effectiveness of antibacterial compounds. Biosurfactants according to the subject invention can include, for example, glycolipids, lipopeptides, flavolipids, phospholipids, fatty acid esters, and high molecular weight polymers such as lipoproteins, lipopolysaccharide-protein complexes, and polysaccharide-protein-fatty acid complexes.

In one embodiment, the biosurfactant is a glycolipid. Glycolipids can include, for example, sophorolipids, rhamnolipids, cellobiose lipids, mannosylerythritol lipids and trehalose lipids. In one embodiment, the biosurfactant is a lipopeptide. Lipopeptides can include, for example, surfactin, iturin, arthrofactin, viscosin, fengycin, and lichenysin. In certain embodiments, a mixture of biosurfactants is used.

In one embodiment, the biosurfactant has been purified from the fermentation medium in which it was produced. Alternatively, in one embodiment, the biosurfactant is utilized in crude form comprising fermentation broth resulting from cultivation of a biosurfactant-producing microbe. This crude form biosurfactant solution can comprise from about 0.001% to 99%, from about 25% to about 75%, from about 30% to about 70%, from about 35% to about 65%, from about 40% to about 60%, from about 45% to about 55%, or about 50% pure biosurfactant, along with residual cells and/or nutrients.

In one embodiment, the composition comprises a saponin at 1 to 10 ml/L, or 2 to 6 ml/L of ruminal fluid. Saponins are natural surfactants that are found in many plants and that exhibit similar characteristics to microbial biosurfactants, for example, self-association and interaction with biological membranes. There are three basic categories of saponins, including triterpenoid saponins, steroidal saponins, and steroidal glycoalkaloids.

Some well-known triterpenoid saponin-accumulating plant families include the Leguminosae, Amaranthaceae, Apiciceae. Caryophyllaceae, Aquifoliaceae, Araliaceae, Cucurbitaceae, Berberidaceae, Chenopodiaceae, Myrsinaceae and Zygophyllaceae, among many others. Quillaja and legumes such as soybeans, beans and peas are a rich source of triterpenoid saponins. The steroidal saponins are typically found in members of the Agavaceae, Alliaceae, Asparagaceae, Dioscoreaceae, Liliaceae, Amaryllidaceae, Bromeliaceae, Palmae and Scrophulariaceae families and accumulate in abundance in crop plants such as yam, alliums, asparagus, fenugreek, yucca, and ginseng. The steroidal glycoalkaloids are commonly found in members of the Solanaceae family including tomato, potato, aubergines and capsicum.

In one embodiment, the subject composition can comprise one or more additional substances and/or nutrients to supplement the needs of the beneficial microorganism of the composition and/or to supplement the needs of soil, water or plants in the environment to which it is applied. These can include, for example, sources of amino acids (including essential amino acids), peptides, proteins, vitamins, microelements, 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 produce and/or provide these substances.

In certain embodiments, the composition can further comprise one or more carriers and/or excipients suitable for delivery of the composition to an environment, including soil, water or air.

Carriers and/or excipients according the subject invention can include any and all solvents, diluents, buffers (such as, e.g., neutral buffered saline, phosphate buffered saline, or optionally Tris- HC1, acetate or phosphate buffers), oil-in-water or water-in-oil emulsions, aqueous compositions with or without inclusion of organic co-solvents, solubilizers (such as, e.g., Tween 80, Polysorbate 80), colloids, dispersion media, vehicles, fillers (e.g., MCC), chelating agents (such as, e.g., EDTA or glutathione), amino acids (such as, e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavorings, aromatizers, thickeners, coatings, preservatives (such as, e.g., Thimerosal, benzyl alcohol), antioxidants (such as, e.g., ascorbic acid, sodium metabisulfite), tonicity controlling agents, absorption delaying agents, adjuvants, bulking agents (such as, e.g., lactose, mannitol) and the like. In certain embodiments, the composition comprises a filler, such as microcrystalline cellulose (MCC).

In certain embodiments, the composition can comprise glucose (e.g., in the form of molasses), glycerol and/or glycerin, as, or in addition to, an osmoticum substance, to promote osmotic pressure during storage and transport of a dry product.

The compositions can be formulated into preparations in, for example, solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, pressed pellets, powders, granules, gels, solutions, steaks/spikes (for soil), drops, aerosols, suspensions, concentrates, and other preparations as suitable for a particular application, liquid, dust, granules, microgranules, pellets, wettable powder, flowable powder, emulsions, microcapsules, oils, or aerosols.

To improve or stabilize the effects of the composition, it can be blended with suitable adjuvants and then used as such or after dilution, if necessary.

Exemplary Embodiments

In some embodiments, the composition of the subject invention comprises:

A) one or more microorganisms (yeasts, fungi and/or bacteria) capable of capturing iron and/or producing an iron-capturing growth by-product, wherein preferably at least one of the one or more microorganisms is a Bacillus sp., and wherein even more preferably, the Bacillus sp. is B. subtilis NRRL B-68031 or B. amyloliquefaciens NRRL B-67928;

B) an iron-capturing substance selected from pulcherrimin, pulcherriminic acid, citrate, citric acid, EDTA (Ethylenediaminetetraacetic acid), ferric EDTA, DTPA (Diethylenetriaminepentaacetic acid), EDDHA (Ethylenediamine di(o-hydroxyphenylacetic acid), N,N-dihydroxy-N,N'-diisopropylhexanediamide (DPH), 2,3-dihydroxybenzoic acid, azotochelin ferrichrome, defarasirox, deferiprone, desferrioxamine, fusarinine, chrysobactin, achromobactin, omibactin, rhodotorulic acid, lysine, glutamic acid, gluconic acid, iron oxyhydroxide minerals, ferrihydrite, magnetite, hematite, geothite, siderite, transferrin, enterobactin, bacillibactin, vibriobactin, azotobactin, aminochelin, pyoverdine, yersiniabactin, agrobactin, staphyloferrin, hydroxamate, catecholate, salicylate, carboxylate, mugineic acid, ferulic acid, caffeic acid, enterobactin, pyoverdine, protochelin, pyochelin, vibrioferrin and/or nicotianamine;

C) a carrier/excipient including solvents, diluents, buffers (such as, e.g., neutral buffered saline, phosphate buffered saline, or optionally Tris-HCl, acetate or phosphate buffers), oil-in-water or water-in-oil emulsions, aqueous compositions with or without inclusion of organic co-solvents, solubilizers (such as, e.g., Tween 80, Polysorbate 80), colloids, dispersion media, vehicles and/or fillers (e.g., MCC);

D) one or more acids selected from, 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 acids, succinic acid, carbonic acid, glutaric acid, decanoic 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 the environment to which it is applied to 6.8 or lower, preferably, 5.0 or lower, more preferably, 4.8 or lower; and/or

E) a biosurfactant selected from sophorolipids, rhamnolipids, cellobiose lipids, mannosylerythritol lipids, trehalose lipids, surfactin, iturin, arthrofactin, viscosin, fengycin, and lichenysin.

In some embodiments, the composition comprises each of components A-E. In some embodiments, the composition comprises any combination of A-E, or any one of A-E individually.

In some embodiments, the composition comprises spore, vegetative and/or biofilm-form B. subtilis NRRL B-68031 or B. amyloliquefaciens NRRL B-67928; and pulcherrimin and/or pulcherriminic acid.

In some embodiments, the composition comprises spore, vegetative and/or biofilm-form B. subtilis NRRL B-68031 or B. amyloliquefaciens NRRL B-67928; and a carrier/excipient.

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

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

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

Production of Microorganisms and/or Microbial Growth By-Products

The subject invention utilizes methods for cultivation of microorganisms and production of microbial metabolites and/or other by-products of microbial growth. The subject invention further utilizes cultivation processes that are suitable for cultivation of microorganisms and production of microbial metabolites on a desired scale. These cultivation processes include, but are not limited to, submerged cultivation/fermentation, solid state fermentation (SSF), and modifications, hybrids and/or combinations thereof.

As used herein “fermentation” refers to cultivation or growth of cells under controlled conditions. The growth could be aerobic or anaerobic. In preferred embodiments, the microorganisms are grown using SSF and/or modified versions thereof.

In one embodiment, the subject invention provides materials and methods for the production of biomass (e.g., viable cellular material), extracellular metabolites, residual nutrients and/or intracellular components.

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

In a further embodiment, the vessel may also be able to monitor the growth of microorganisms inside the vessel (e.g., measurement of cell number and growth phases). Alternatively, a daily sample may be taken from the vessel and subjected to enumeration by techniques known in the art, such as dilution plating technique.

In one embodiment, the method includes supplementing the cultivation 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 may be used independently or in a combination of two or more.

The method can provide oxygenation to the growing culture. One embodiment utilizes slow motion of air to remove low-oxygen containing air and introduce oxygenated air. In the case of submerged fermentation, the oxygenated air may be ambient air supplemented daily through mechanisms including impellers for mechanical agitation of liquid, and air spargers for supplying bubbles of gas to liquid for dissolution of oxygen into the liquid.

The method can further comprise supplementing the cultivation with a carbon source. The carbon source is typically a carbohydrate, such as glucose, sucrose, lactose, fructose, trehalose, mannose, mannitol, and/or maltose; organic acids such as acetic acid, fumaric acid, citric acid, propionic acid, malic acid, malonic acid, and/or pyruvic acid; alcohols such as ethanol, propanol, butanol, pentanol, hexanol, isobutanol, and/or glycerol; fats and oils such as soybean oil, canola oil, rice bran oil, olive oil, com oil, sesame oil, and/or linseed oil; etc. These carbon sources may be used independently or in a combination of two or more.

In one embodiment, growth factors and trace nutrients for microorganisms are included in the medium. This is particularly preferred when growing microbes that are incapable of producing all of the vitamins they require. Inorganic nutrients, including trace elements such as iron, zinc, copper, manganese, molybdenum and/or cobalt may also be included in the medium. Furthermore, sources of vitamins, essential amino acids, and microelements can be included, for example, in the form of flours or meals, such as com flour, or in the form of extracts, such as yeast extract, potato extract, beef extract, soybean extract, banana peel extract, and the like, or in purified forms. Amino acids such as, for example, those useful for biosynthesis of proteins, can also be included.

In one embodiment, inorganic salts may also be included. Usable inorganic salts can 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 independently or in a combination of two or more.

In one embodiment, one or more biostimulants may also be included, meaning substances that enhance the rate of growth of a microorganism. Biostimulants may be species-specific or may enhance the rate of growth of a variety of species.

In some embodiments, the method for cultivation may further comprise adding an antimicrobial in the medium before, and/or during the cultivation process.

In certain embodiments, an antibiotic can be added to a culture at low concentrations to produce microbes that are resistant to the antibiotic. The microbes that survive exposure to the antibiotic are selected and iteratively re-cultivated in the presence of progressively higher concentrations of the antibiotic to obtain a culture that is resistant to the antibiotic. This can be performed in a laboratory setting or industrial scale using methods known in the microbiological arts. In certain embodiments, the amount of antibiotic in the culture begins at, for example, 0.0001 ppm and increases by about 0.001 to 0.1 ppm each iteration until the concentration in the culture is equal to, or about equal to, the dosage that would typically be applied to a iron-capturing pathogen.

The pH of the mixture should be suitable for the microorganism of interest. Buffers, and pH regulators, such as carbonates and phosphates, may be used to stabilize pH near a preferred value. When metal ions are present in high concentrations, use of a chelating agent in the medium may be necessary.

The microbes can be grown in planktonic form or as biofilm. In the case of biofilm, the vessel may have within it a substrate upon which the microbes can be grown in a biofilm state. The system may also have, for example, the capacity to apply stimuli (such as shear stress) that encourages and/or improves the biofilm growth characteristics.

In one embodiment, the method for cultivation of microorganisms is carried out at about 5° to about 100° C, preferably, 15 to 60° C, more preferably, 25 to 50° C. In a further embodiment, the cultivation may be carried out continuously at a constant temperature. In another embodiment, the cultivation may be subject to changing temperatures.

In one embodiment, the equipment used in the method and cultivation process is sterile. The cultivation equipment such as the reactor/vessel may be separated from, but connected to, a sterilizing unit, e.g., an autoclave. The cultivation equipment may also have a sterilizing unit that sterilizes in situ before starting the inoculation. Air can be sterilized by methods know in the art. For example, the ambient air can pass through at least one filter before being introduced into the vessel. In other embodiments, the medium may be pasteurized or, optionally, no heat at all added, where the use of low water activity and low pH may be exploited to control undesirable bacterial growth.

In one embodiment, the subject invention further provides a method for producing microbial metabolites such as, for example, biosurfactants, enzymes, proteins, ethanol, lactic acid, beta-glucan, peptides, metabolic intermediates, polyunsaturated fatty acid, and lipids, by cultivating a microbe strain of the subject invention under conditions appropriate for growth and metabolite production; and, optionally, purifying the metabolite. The metabolite content produced by the method can be, for example, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

The biomass content of the fermentation medium may be, for example, from 5 g/1 to 180 g/1 or more, or from 10 g/1 to 150 g/1. The cell concentration may be, for example, at least 1 x 10 ⁹ , 1 x 10 ¹⁰ , 1 x 10 ¹¹ , 1 x 10 ¹² or 1 x 10 ¹³ cells per gram of final product. The microbial growth by-product produced by microorganisms of interest may be retained in the microorganisms or secreted into the growth medium. The medium may contain compounds that stabilize the activity of microbial growth by-product.

The method and equipment for cultivation of microorganisms and production of the microbial by-products can be performed in a batch, a quasi-continuous process, or a continuous process.

In one embodiment, all of the microbial cultivation composition is removed upon the completion of the cultivation (e.g., upon, for example, achieving a desired cell density, or density of a specified metabolite). In this batch procedure, an entirely new batch is initiated upon harvesting of the first batch.

In another embodiment, only a portion of the fermentation product is removed at any one time. In this embodiment, biomass with viable cells, spores, conidia, hyphae and/or mycelia remains in the vessel as an inoculant for a new cultivation batch. The composition that is removed can be a cell-free medium or contain cells, spores, or other reproductive propagules, and/or a combination of thereof. In this manner, a quasi-continuous system is created.

Advantageously, the method does not require complicated equipment or high energy consumption. The microorganisms of interest can be cultivated at small or large scale on site and utilized, even being still-mixed with their media.

Preparation of Microbe-based Products

A “microbe-based product,” is a product to be applied in practice to achieve a desired result. The microbe-based product can be simply a microbe-based composition harvested from a microbe cultivation process. Alternatively, a microbe-based product may comprise further ingredients that have been added. These additional ingredients can include, for example, stabilizers, buffers, carriers (e.g., water or salt solutions), added nutrients to support further microbial growth, non-nutrient growth enhancers and/or agents that facilitate tracking of the microbes and/or the composition in the environment to which it is applied. The microbe-based product may also comprise mixtures of microbebased compositions. The microbe-based product may also comprise one or more components of a microbe-based composition that have been processed in some way such as, but not limited to, filtering, centrifugation, lysing, drying, purification and the like.

One microbe-based product of the subject invention is simply the fermentation medium containing a microorganism and/or the microbial metabolites produced by the microorganism and/or any residual nutrients. The product of fermentation may be used directly without extraction or purification. If desired, extraction and purification can be easily achieved using standard extraction and/or purification methods or techniques described in the literature. The microorganisms in the microbe-based product may be in an active or inactive form. Furthermore, the microorganisms may be removed from the composition, and the residual culture utilized. The microbe-based products may be used without further stabilization, preservation, and storage. Advantageously, direct usage of these microbe-based products preserves a high viability of the microorganisms, reduces the possibility of contamination from foreign agents and undesirable microorganisms, and maintains the activity of the by-products of microbial growth.

The microbes and/or medium (e.g., broth or solid substrate) resulting from the microbial growth can be removed from the growth vessel and transferred via, for example, piping for immediate use.

In one embodiment, the microbe-based product is simply the growth by-products of the microorganism. For example, biosurfactants produced by a microorganism can be collected from a submerged fermentation vessel in crude form, comprising, for example about 50% pure biosurfactant in liquid broth.

In other embodiments, the microbe-based product (microbes, medium, or microbes and medium) can be placed in containers of appropriate size, taking into consideration, for example, the intended use, the contemplated method of application, the size of the fermentation vessel, and any mode of transportation from microbe growth facility to the location of use. Thus, the containers into which the microbe-based composition is placed may be, for example, from 1 gallon to 1,000 gallons or more. In other embodiments the containers are 2 gallons, 5 gallons, 25 gallons, or larger.

Upon harvesting, for example, the yeast fermentation product, from the growth vessels, further components can be added as the harvested product is placed into containers and/or piped (or otherwise transported for use). The additives can be, for example, buffers, carriers, other microbe-based compositions produced at the same or different facility, viscosity modifiers, preservatives, nutrients for microbe growth, tracking agents, solvents, biocides, other microbes and other ingredients specific for an intended use.

Other suitable additives, which may be contained in the formulations according to the invention, include substances that are customarily used for such preparations. Examples of such additives include surfactants, emulsifying agents, lubricants, buffering agents, solubility controlling agents, pH adjusting agents, preservatives, stabilizers and ultra-violet light resistant agents.

In one embodiment, the product may further comprise buffering agents including organic and amino acids or their salts. Suitable buffers include citrate, gluconate, tartarate, malate, acetate, lactate, oxalate, aspartate, malonate, glucoheptonate, pyruvate, galactarate, glucarate, tartronate, glutamate, glycine, lysine, glutamine, methionine, cysteine, arginine and a mixture thereof. Phosphoric and phosphorous acids or their salts may also be used. Synthetic buffers are suitable to be used but it is preferable to use natural buffers such as organic and amino acids or their salts listed above. In a further embodiment, pH adjusting agents include potassium hydroxide, ammonium hydroxide, potassium carbonate or bicarbonate, hydrochloric acid, nitric acid, sulfuric acid or a mixture.

In one embodiment, additional components such as an aqueous preparation of a salt, such as sodium bicarbonate or carbonate, sodium sulfate, sodium phosphate, or sodium biphosphate, can be included in the formulation.

Advantageously, in accordance with the subject invention, the microbe-based product may comprise broth in which the microbes were grown. The product may be, for example, at least, by weight, 1%, 5%, 10%, 25%, 50%, 75%, or 100% broth. The amount of biomass in the product, by weight, may be, for example, anywhere from 0% to 100% inclusive of all percentages therebetween.

Optionally, the product can 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 live cells are present in the product, the product is stored at a cool temperature such as, for example, less than 20° C, 15° C, 10° C, or 5° C. On the other hand, a biosurfactant composition can typically be stored at ambient temperatures.

Methods for Remediating and/or Mitigating Iron Pollution

In preferred embodiments, the subject invention provides methods for remediating and/or mitigating heavy metal pollution, wherein a composition comprising a metal chelator is introduced into an environment in which metal pollution is occurring, or may occur. The methods can be utilized in environments including, for example, soil, water, air, or waste (e.g., municipal or industrial waste).

In certain specific embodiments, the methods are used for remediating and/or mitigating iron pollution. The composition can sequester free iron from the soil, water and/or air, thus reducing iron pollution in that environment and reducing the potential for iron toxicity to humans, animals and plants.

In some embodiments, the method comprises applying an iron-capturing ingredient according to the subject invention alongside an acid, wherein the acid modulates the pH of the composition or the environment to 6.8 or lower, preferably 5.0 or lower, more preferably 4.8 or lower. The acid can be an organic acid selected from, 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 acids, succinic acid, carbonic acid, glutaric acid, decanoic acid, and ascorbic acid. The acid can also be an inorganic acid selected from, for example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, boric acid, perchloric acid, hydrofluoric acid, hydrobromic acid, and sulfonic acid.

As used herein, “applying” a composition or product, or “treating” an environment refers to contacting a composition or product with a target or site such that the composition or product can have an effect on that target or site. The effect can be due to, for example, microbial growth and/or the action of a metabolite, enzyme, biosurfactant or other growth by-product.

If present in the composition, the applied microbes can be either live (or viable) or inactive at the time of application. In some embodiments, the microbes are in the form of yeast extract and/or another microbial hydrolysate.

Microbial growth by-products can be applied in addition to the growth by-products produced by the microorganism(s) of the composition, or they can be applied on their own without the microorgan ism(s).

The methods can further comprise adding materials to enhance microbe growth during application (e.g., adding nutrients and/or prebiotics). Thus, live microorganisms can grow in situ and produce the active compounds onsite. Consequently, a high concentration of microorganisms and their growth by-products can be achieved easily and continuously in an environment. t In some embodiments, the method comprises applying an entire microbial culture, comprising inactivated cells in submerged or solid-state fermentation medium. Advantageously, this reduces the amount of waste products produced during production of the subject compositions while increasing efficiency of production by removing the steps of extraction and/or purification of microbial metabolites. Furthermore, inclusion of inactive cells and residual fermentation medium provides rich sources of organic and inorganic nutrients that are essential for supporting soil and/or plant health.

In one embodiment the composition is dispersed in an environment while being supported on a carrier. The carrier can be made of materials that can retain microorganisms thereon relatively mildly and thus allow easy release of microorganisms thus proliferated. The carrier is preferably inexpensive and in some embodiments can act as a nutrient source for the microorganisms thus applied, particularly a nutrient source that can be gradually released. Biodegradable carrier materials include comhusk, sugar industiy waste, or any agricultural waste. The water content of the carrier typically varies from 1% to 99% by weight, preferably from 5% to 90% by weight, more preferably from 10% to 85% by weight.

Substances that enhance the growth of microorganisms and the production of microbial growth by-products of interest may also be added to the composition and/or the treatment site. These substances include, but not limited to, oil, glycerol, sugar, or other nutrients. For example, a carbon substrate that supports the growth of the microorganism may be added to the composition or the targeted areas.

In certain embodiments, the methods of the subject invention result in at least a 25% decrease in the amount of free heavy metal particles in an environment, preferably at least a 50% decrease, after one treatment. In certain embodiments, the environment can be treated multiple times to further decrease the amount of metal pollution. In certain embodiments, the time period in which the composition can be contacted with the polluted, or potentially polluted, environment ranges from about 1 second to about 1 year, about 1 minute to about 1 year, about 1 minute to about 6 months, about 1 minute to about 1 month, about 1 minute to about 1 week, about 1 minute to about 48 hours, about 30 minutes to 40 hours, or preferably about 12 hours to 24 hours.

In certain embodiments, the methods comprise applying the composition to the environment for the period of time until the amount of pollutants, particularly iron, in the environment is determined to be satisfactory or, which can be readily determined by one skilled in the art. The amount of iron or other metal pollutants that may be considered acceptable and/or safe depends on the context. For example, water that is made available to humans, animals or plants might require less iron content than water that is to be utilized down a fracking well as fracking fluid.

In certain embodiments, the method comprising applying a composition of the subject invention to soil. The methods can be utilized in, for example, agricultural fields, pastures, orchards, prairies, plots, and/or forests. The methods can also be utilized in areas containing soil that is significantly uninhabitable by plant life, for example, soils that have been over-cultivated and/or where crop rotation has not been implemented or has been insufficient to retain the soil’s fertility; soils that have been polluted by over-treatment with pesticides, fertilizers and/or herbicides; soils with high salinity; soils that have been polluted by dumping, metal scrap accumulation (e.g., in junkyards or scrapyards), or chemical or hydrocarbon spills (e.g., SuperFund site, oil and gas recovery sites); soils that have been polluted by ammunition (e.g., hunting grounds, firing ranges and warzones); and/or soils in areas damaged by natural or anthropogenic causes, including fire, flooding, pest infestation, construction and development (e.g. commercial, residential or urban building), digging, oil and gas recovery, mining, logging, steel production, livestock rearing, and other causes.

In certain embodiments, the polluted soil has an iron level above 300 ppm. In preferred embodiments, the method reduces the iron level in the soil to 200 ppm or less, more preferably to 150 ppm or less, even more preferably to between 50 and 100 ppm.

In some embodiments application to soil is performed by spreading a composition of the present invention onto the soil surface. This may be performed using a standard spreader or sprayer device. In some embodiments, a single spreading step may complete the application process, wherein all of the components are included in a single formulation. In other embodiments, which use two- or multiplepart formulations, multiple spreading steps may be used.

In one embodiment, the composition may be rubbed, brushed, or worked into the soil using a mechanical action, for example, by tilling. In still further embodiments, the application of a composition may be subsequently followed by application of a liquid, such as water. The water may be applied as a spray, using standard methods known to one of ordinary skill in the art. Other liquid wetting agents and wetting formulations may also be used.

In certain embodiments, the compositions are applied to the soil surface without mechanical incorporation. The beneficial effect of the soil application can be activated by rainfall, sprinkler, flood, or drip irrigation, and subsequently dispersed throughout the soil. In an exemplary embodiment, the compositions can be efficiently applied via a center pivot irrigation system or with a spray over the seed furrow.

In some embodiments, the compositions, either in a dry or in liquid formulation, are applied as a seed treatment or to the surface of a plant or plant part (e.g., to the surface of a plant’s leaves or roots).

Application of the subject compositions can be performed either alone or in combination with application of other compounds for enhancing soil health and/or plant health. For example, commercial and/or natural fertilizers, pesticides, herbicides and/or other soil amendments can be applied alongside the compositions.

Advantageously, the methods can help enhance agricultural yields, even in depleted or damaged soils; restore depleted greenspaces, such as pastures, forests, wetlands and prairies; and restore uncultivatable land so that it can be used for farming, reforestation and/or natural regrowth of plant ecosystems. Additionally, through improved agricultural practices, the methods can help reduce pollution caused by emissions of greenhouse gases.

In certain embodiments, enhancing soil health comprises improving one or more qualities of soil. This can comprise, for example, removing and/or reducing pollutants in the soil, improving the nutrient content and nutrient availability of the soil, improving drainage and/or moisture retention properties of the soil, improving the salinity of the soil, improving the soil microbiome diversity, and/or controlling a soil-borne pest. Other improvements can include adding bulk and/or structure to soils that have been eroded by wind and/or water, as well as preventing and/or delaying erosion of soil by wind and/or water.

In certain embodiments, the methods comprise a step of characterizing the soil type and/or soil health status prior to treating the soil according to the subject methods. Accordingly, the method can also comprise tailoring the composition in order to meet a specific soil type and/or soil health need. Methods of characterizing soils are known in the agronomic arts.

In some embodiments, the methods are used for restoring soil health, wherein the soil being treated was once healthy, but deteriorated over some period of time. The restoration may bring the soil back to its previous state of health and/or an enhanced state of health.

In certain embodiments, the method results in removal and/or reduction of pollutants from soil, including remediation of soils contaminated with iron. In some embodiments, the pollutants are degraded directly by the applied microorganisms of the composition. In some embodiments, the growth by-products of the microorganisms, e.g., iron-capturing compounds and/or biosurfactants, facilitate the sequestration and/or degradation of pollutants, and can chelate and form a complex with ionic and nonionic metals to release them from the soil. Soil pollutants include, for example, residual fertilizers, pesticides, herbicides, fungicides, hydrocarbons, chemicals (e.g., dry cleaning treatments, urban and industrial wastes), benzene, toluene, ethylbenzene, xylene, and heavy metals. In preferred embodiments, the soil pollutant is iron.

In some embodiments, the polluted soil is combined with nonhazardous organic amendments such as manure or agricultural wastes. The presence of these organic materials supports the development of a rich microbial population and elevated temperature characteristics of composting. Thus, the rate of bioremediation can be increased.

In certain embodiments, the methods can also help improve soil microbiome diversity by promoting colonization of the soil and plant roots growing therein with beneficial soil microorganisms. Growth of nutrient-fixing microbes, such as rhizobium and/or mycorrhizae, can be promoted, as well as other endogenous and applied microbes, thereby increasing the number of different species within the soil microbiome.

In certain embodiments, the method comprising applying a composition of the subject invention to water. The methods can be utilized in, for example, municipal and industrial wastewater, produced water, and natural or man-made waterways. The water can be present in, for example, mining environments, oil and gas fields, dumping sites, SuperFund sites, industrial waste sites, landfills, agricultural fields, metalworking sites, construction sites, hunting grounds, firing ranges, and war zones/battlefields.

In certain embodiments, the polluted water has an iron level of 25 mg/L or more. In preferred embodiments, the method reduces the level of iron 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, and even more preferably to 0.3 mg/L or less.

The method preferably comprises contacting a composition according to the subject invention with the water for a period of time to yield a mixture comprising treated water; and, optionally, separating the composition and/or pollutant from the water.

The method can be carried out using, for example, a column, a filter, a membrane, a solvent, electrolysis, or using any other laboratory or industrial sized reactor. The method can also be carried out in situ, for example, in the case of natural or man-made waterways.

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

In certain embodiments, the source of the water is produced water. Produced water is naturally occurring water that is removed from the ground during the extraction of oil and gas, as geological formations that contain oil and gas also contain water. In certain embodiments, the source of the water is groundwater or precipitation including, for example, at industrial sites such as mines, quarries, and oil and gas wells.

In certain embodiments, the source of the water is the water that is pumped or otherwise applied into the ground or geologic formation, during, for example, hydraulic fracturing.

In certain embodiments, the source of the water is industrial or municipal wastewater, which is being treated for reintroduction and use by communities.

The compositions can be applied to liquids or vessels that contain liquids that reside at a range of temperatures and aquatic environments, such as, for example, a stream, river, waterway, ocean, sea, lake, pond, runoff area, containment ponds, piping, filter, press, membrane, column, screen, cone, dewaterer, classifier, scraper, hydrocyclone, agitator, drum, disk, or industrial wastewater treatment/holding tank. In certain embodiments, the composition can be added to the vessels that contain liquids before the liquid composition is added to said vessel.

In one embodiment, the composition can be applied to reagents in scrubbers utilized in pollution control. Dry scrubbers utilize dry reagents, which are contacted with exhaust streams to bind and/or remove pollutants, while wet scrubbers utilize liquid reagents to absorb or dissolve pollutants from the exhaust. Thus, the subject invention provides methods for capturing iron particulates from industrial exhaust streams in both wet and dry scrubbers, wherein the composition is contacted with the exhaust stream as part of a wet or dry scrubbing reagent.

In some instances, this can also be useful for capturing iron oxide fumes emitted in metalworking and welding environments.

Advantageously, the methods of the present invention can be used for reducing metal pollution in the environment, particularly iron pollution. Thus, in some embodiments, the methods can also be useful for reducing the occurrence or, and/or preventing, a disease or condition caused by over-exposure to iron, i.e., iron toxicity. Such conditions include, for example, myocardial siderosis, liver damage, liver cirrhosis, pancreatic islet cell damage, diabetes, hypothyroidism, hypogonadism, conjunctivitis, choroiditis, and retinitis.

In certain embodiments, the composition can also be administered to a human, animal or plant that is suffering from iron toxicity in order to treat and/or prevent a condition caused by over-exposure to iron and/or iron toxicity.

EXAMPLES

A greater understanding of the present invention and of its many advantages may be had from the following examples, given by way of illustration. The following examples are illustrative of some of the methods, applications, embodiments and variants of the present invention. They are not to be considered as limiting the invention. Numerous changes and modifications can be made with respect to the invention.

EXAMPLE 1 - PH GROWTH TESTING OF B4 STRAIN

The B4 strain was spread on Tryptic Soy Agar (TSA) at a neutral (6.8) and acidic (4.8) pH to look for differences in growth, with the goal of determining how it would behave in the various pH environments within a cow’s digestive system.

Growth at neutral pH (6.8) was faster within 24 hours compared to the acidic plates, but at 48 hours, the growth on pH 4.8 agar was equal or greater. FIGS. 1A-1B. Additionally, a significant amount of exopolysaccharide (EPS) was produced when grown on an acidic agar. FIG. 1C. This is a result of environmental stress.

The dried B4 spores were also added in sterile PBS adjusted to pH 2.8 and left for 24 hours. The same was then plated on neutral (6.8) TSA plates. A lawn of growth was present, but no EPS was produced (similar to pH 6.8 plates from FIG. 1). This shows that dried spores exposed to an overall harsh environment were still intact and viable. Overall, the pH of the growing media/environment influences EPS production.

EXAMPLE 2 - EPS

B4 was grown in a liquid medium at pH 4.8 specifically for the production of EPS. The assumed EPS was isolated out of the culture and purified.

FTIR analysis confirmed the purified sample to be an EPS and HPLC analysis confirmed the existence of a large peak denoting a sugar oligomer. A C=O bond was also observed via UV absorption.

Additionally, and surprisingly, when the culture was processed for EPS extraction, there was a purple stripe present in the cell pellet. When the EPS purification was completed, the sample was a pink color, suggesting the presence of pulcherrimin or pulcherriminic acid. FIG. 2.

EXAMPLE 3 - ENZYME ASSAY TESTING - AMYLASE

Amylase is an enzyme that hydrolyzes the glycosidic bonds in starch molecules by converting complex carbohydrates to simple sugars. Agar, a starch, was inoculated with B4 and incubated for growth. FIG. 3. After running the plate assay, an orange color around the bacterial growth was observed, indicating the breakdown of starch. This is a positive amylase test.

EXAMPLE 4 - ENZYME ASSAY TESTING - CELLULASE

Cellulases are enzymes that convert cellulose to glucose. B4 was tested for cellulase activity using carboxymethylcellulose agar (CMCA) media plates. B4 was grown in tryptic soy broth with and without cellobiose added. After 48 hours of growth, the liquid culture was streaked onto CMCA plates. After inoculation, and once growth was present on the agar, an iodine solution was introduced to the plates. A yellow zone of clearing around the bacterial growth indicates the breakdown of cellulose and the presence of cellulase enzymes. FIG. 4.

EXAMPLE 5 - DETECTION OF SIDEROPHORES

Chrome azurol S (CAS) assay was used for the detection of siderophores from B4 cultures and dried B4 spores. The tests were run in aerobic and anaerobic environments on different growth media: MRS-sucrose, M23-6, minimal media with Tween, and minimal media without Tween. The plates were observed at 6 hours (FIGS. 5A-5C) and 24 hours (FIGS. 6A-6C).

Siderophores scavenge iron from an Fe-CAS-hexadecyltrimethylammonium bromide complex, and the release of the CAS dye results in a color change from blue to orange. Any observable color change on CAS agar plates indicates a qualitative detection of siderophores.

All B4 media cultures tested were positive for siderophore production and activity. MRS- sucrose (the richest media) produced the strongest activity. There were no differences in siderophore activity between aerobic and anaerobic conditions with the four media cultures.

Dried B4 spores produced less siderophore activity compared to the B4 cultures, which may due to spore dormancy. The dried spores performed better in aerobic conditions.

EXAMPLE 6 - IRON ACTIVITY ASSAY

To understand how B4 interacts with iron, and to determine how iron activity differs between culture media, an iron assay kit (Sigma-Aldrich) was used to determine the concentration of ferrous (Fe ²⁺ ), ferric (Fe ³⁺ ) and total iron present in different B4 cultures.

Iron is released from the sample by the addition of an acidic buffer. Released iron is reacted with chromagen, resulting in a colorimetric (593nm) product that is proportional to the iron present.

The results in FIG. 7 correlate with the siderophore results reported in FIGS. 5-6, meaning higher siderophore activity correlates with lower total iron levels. For example, MRS-sucrose media is the richest media with the highest siderophore activity and the lowest total iron levels. Additionally, the minimal media used is designed for increased production of pulcherrimin production, which is a ferric chelate. Greater levels of pulcherrimin in the culture should increase ferric and total iron concentrations.