FIELD OF THE TECHNOLOGY

[0001] The present disclosure is directed to the field of lithium extraction, production, and amplification. In particular, the present disclosure encompasses an isolated microorganism modified relative to wild-type and useful to extract, produce and/or amplify lithium from various substrates.

BACKGROUND

[0002] Lithium is rare non-ferrous metal, which has been widely used in a secondary battery, a special glass, a single-crystal oxide, an aircraft, a spring material, etc. Global demands for lithium have recently increased along with demand expansion of information technology devices. Demands for the lithium will further increase. Producing countries of the lithium are concentrated, and it is therefore desirable to recover the lithium in a stable manner in areas having no mineral resources for lithium.

[0003] An amount of lithium mined from the earth in consideration of economical feasibility of a lithium mineral resource is only about 4.1 million tons in the world, and a lithium resource is a rare resource expected to be depleted within the next decade.

[0004] In general, lithium is recovered by a technology of performing acid treatment after embedding inorganic compound particles such as a manganese oxide as a lithium ion molecular sieve in a polymer such as polyvinyl chloride (PVC) or putting the inorganic compound particles in a storage made of a polymer membrane to selectively exchange ions.

[0005] However, since it takes a significantly long time to adsorb the specific ion, economical feasibility and efficiency are low, and since a toxic material such as an acid should be used in a post-treatment process for recovering the ion such as an ion separation process, there are problems such as corrosion of a system, environmental contamination, and the like.

[0006] Therefore, a need in the art exists for compositions and improved methods to extract, produce and/or amplify lithium. SUMMARY

[0007] Among the various aspects of the present disclosure provide compositions comprising an isolated modified bacterial strain Glycomyces lithiumensis. In some embodiments, the bacterial strain is referred to as ECO002, which has been designated Accession number NRRL No. B-68190, deposited in accordance with the Budapest Treaty at the Agricultural Research Service Culture Collection (USDA, ARS, 1815 North University Street, Peoria, IL, 61064) on August 15, 2022.

[0008] In one aspect, the disclosure provides a method of extracting lithium comprising contacting a substrate having trace amounts of lithium with a composition comprising an isolated modified bacterial strain Glycomyces lithiumensis. In some embodiments, the bacterial strain is ECO002. In some embodiments, the composition includes one or more microbes used in industrial mining and as described herein.

[0009] In one aspect, the disclosure provides a method of extracting lithium comprising contacting an agriculture crop residue/stubble substrate having trace amounts of lithium with a composition comprising an isolated modified bacterial strain Glycomyces lithiumensis. In some embodiments, the bacterial strain is ECO002. In some embodiments, the composition includes one or more microbes used in industrial mining and as described herein.

[0010] In one aspect, the disclosure provides a method of extracting lithium comprising contacting a oil, gas, or frac water substrate having trace amounts of lithium with a composition comprising an isolated modified bacterial strain Glycomyces lithiumensis. In some embodiments, the bacterial strain is ECO002. In some embodiments, the composition includes one or more microbes used in industrial mining and as described herein.

[0011] In another aspect, the disclosure provides a method of amplifying lithium comprising contacting a substrate with a composition comprising an isolated modified bacterial strain Glycomyces lithiumensis. In some embodiments, the bacterial strain is ECO002. In some embodiments, the composition includes one or more microbes used in industrial mining.

[0012] In one aspect, the disclosure provides a method of recovering lithium by contacting a substrate with a composition comprising an isolated modified bacterial strain Glycomyces lithiumensis. In some embodiments, the bacterial strain is EC0002. In some embodiments, the composition includes one or more microbes used in industrial mining.

[0013] In some embodiments, the methods further comprising adding a fertilizer, nutrient and/or by product composition one or more times to the substrate after inoculation with the bacterial strain.

[0014] In some embodiments, the methods of the disclosure include allowing sufficient time for the bacterial strain to colonize and exponentially grow on or in the substrate. In some embodiments, the methods of the disclosure comprise using an anodic and cathodic LED having a wavelength generator set at a range of 100 Hz - 200 KHz in the liquid substrate.

[0015] In some embodiments, the methods of the disclosure comprise harvesting the lithium by separating trace lithium from the substrate.

[0016] The details of one or more embodiments of the disclosure are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several examples, and from the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

[0017] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way. It should be noted that the drawings are submitted in black and white, but the color versions as filed in the priority United States provisional application no. 63420356, filed October 28, 2022, are explicitly incorporated herein by reference,

[0018] FIG. 1 shows a schematic of the microbial transmutation of lithium by a bacterial strain of the disclosure.

[0019] FIG. 2A shows the microbial urease enzyme assay, where the left tube liquid is yellow/orange in color, and the right tube liquid is bright pink.

[0020] FIG. 2B shows the microbial respiration assay.

[0021] FIG. 2C shows microbial phosphorus solubilization assay, where the composition in the tubes (left to right) are, respectively red; purple; pink; white on bottom and pink on top; red on bottom and very dark red on top. [0022] FIG. 2D shows microbial hydrogen sulfide assay, where the composition in the left tube is yellow and the composition in the right tube is black.

[0023] FIG. 2E shows microbial chitinase enzyme assay.

[0024] FIG. 2F shows microbial cellulase enzyme activity.

[0025] FIG. 2G shows ligninase enzyme activity.

DETAILED DESCRIPTION

[0026] The present disclosure is based, at least in part, on the discovery of a bacterium isolated from the EcoBiome Innovation and Discovery apparatus. In brief, the wild type of isolate was harvested from a spent lithium battery from Houston, TX. The battery cell and allowed to suspend in bacteria culture media for 10 days. The discovery, characterization and harvest of lithium-ion specific bacteria was commenced to specifically target and capture a lithium wild type of isolate. The Ecobiome apparatus and methods of use thereof are described in U.S. Patent Application number 16/258,1 12 and is herein incorporated by reference in its entirety. The spent lithium battery sample was loaded into the Ecobiome for microbial gradation and speciation. The prepped spent lithium battery sample was allowed to equilibrate, and microbial growth promoted. After some time, a population of microorganisms were isolated, including a newly discovered wild-type bacteria. After isolation, the wild-type bacteria was subjected to a number of extreme conditions culture conditions, such as high and low pH, high and low temperature, and high and low salinity conditions which minimally forced enzymatic change (e.g., nitrate reductase, ligninase, cellulase, chitinase, and/or urease) thereby producing a modified bacterial strain relative to wild-type. Thus, as used herein, the term “modified” refers to a bacterial strain that has been forced to change, minimally, with respect to enzymatic gain of function. See, for example, FIG. 2 and the below examples.

[0027] Modification of the originally isolated wild-type bacteria resulted in the isolation and characterization of ECO002 (Glycomyces lithiumensis). Through various characterization methods it was found that the ECO002 facilitated lithium recovery from a variety of environmental solid and liquid substrata. The isolated ECO002 demonstrated transmutase and/or amplificase activity to produce and/or concentrate lithium from starting substrates with no or trace detectable lithium concentrations. [0028] Altogether, the present disclosure provides multiple lines of evidence showing the presently disclosed bacterium and methods of using the same to extract, produce and/or amplify lithium from a variety of environmental substrates. Other aspects and iterations of the invention are described more thoroughly below.

[0029] The bacterial strain disclosed in this description has been deposited under conditions that assure that access to the cultures will be available during the pendency of this application. The bacterial strain disclosed in this description has been deposited in the Agricultural Research Service Culture Collection (USDA, ARS, 1815 North University Street, Peoria, HL, 61064). The bacterial strain deposited was designated as Glycomyces lithiumensis. The deposit was received by the NRRL on August 15, 2022 and was given an accession number by the International Depository Authority of B-68190. The deposit has been made to and received by the International Depository Authority under the provisions of the Budapest Treaty, and all restrictions upon public access to the deposit will be irrevocably removed upon the grant of a patent on this application. The deposits will be 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 the deposits does not constitute a license to practice the subject invention.

[0030] Further, 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., they will be stored with all the care necessary to keep them viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of a deposit, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the cultures. The depositor acknowledges the duty to replace the deposit(s) should the depository be unable to furnish a sample when requested due to the condition of the deposits.

[0031 ] Accordingly, one aspect of the present disclosure encompasses an isolated bacteria strain Glycomyces lithiumensis (ECO002) which is modified relative to wild-type. Another aspect of the disclosure provides a mutant or derivative of ECO002 having the ability to extract, produce and/or amplify lithium as described herein. The term “mutant or derivative” thereof includes naturally occurring and artificially induced mutants which retain their ability to extract, produce and/or amplify lithium. Production of such mutants or derivatives will be well known by those skilled in the art including transgenic expression of heterologous nucleic acid sequences and/or genomic modifications.

[0032] In another aspect, the present disclosure provides compositions comprising ECO002. The concentration of ECO002 will vary depending on the type of composition. Suitable ECO002 concentrations include but are not limited to at least about 0.5 x 10 ¹⁰ CFU/Gm, least about 1 .0 x 10 ¹⁰ CFU/Gm, least about 1.5 x 10 ¹⁰ CFU/Gm, least about 2.0 x 10 ¹⁰ CFU/Gm, least about 2.5 x 10 ¹⁰ CFU/Gm, least about 3.0 x 10 ¹⁰ CFU/Gm, least about 3.5 x 10 ¹⁰ CFU/Gm, least about 4.0 x 10 ¹⁰ CFU/Gm, least about 4.5 x 10 ¹⁰ CFU/Gm, least about 5.0 x 10 ¹⁰ CFU/Gm or greater.

[0033] A composition comprising ECO002 according to the present disclosure may comprise one or more additional components, including but not limited to, biosolvents ethyl lactate, ATP, ADP, pyrophosphate, soy based solvents, chemical solvents, green solvents, range of organic acids, lactic acid, malic acid, ascorbic acid, alkanes, alkenes, alkynes, saturates, aromatics, resinoids, asphaltenes, light, mid chain and heavy chain hydrocarbons, sodium nitrate, sodium nitrite, ethanol, sulfur, sulfate, sulfite, nitrogen, chemical surfactants (ionic, anionic, cationic, zwitterionic surfactants), polymers (low, mid, heavy chains), biosurfactants, glycolipids, rhamnolipids (J1 and J2), glycerin, propylene glycol, carbon sugars, dextrose, galactose, sucrose, fructose, complex carbohydrates, starch, cellulose, lignin, keratin, proteins and amino acids, fertilizer NPK (e.g., organic and inorganic fertilizers), manures, composts, green waste, sludge material, humic and fulvic acids, coal ash and coal derived waste, alumina cytokinins and seaweed extracts.

[0034] A composition comprising ECO002 according to the present disclosure may comprise a water source for microbial culturing or final product carrier. Non-limiting examples include deionized water, distilled water, filtered water, well water, tap water, fresh water, sea water, brackish water, mineralized water, carbonated water, saline water, ionically charged water, ionized water, and hydrogen water. Thus, according to the present disclosure compositions comprising microorganisms of the disclosure for use within the methods of the disclosure may comprise a water source for microbial culturing or final product carrier. Non-limiting examples include deionized water, distilled water, filtered water, well water, tap water, fresh water, sea water, brackish water, mineralized water, carbonated water, saline water, ionically charged water, ionized water, and hydrogen water. The aqueous solution may contain sufficient nutrients to support microbial growth. The useful nutrients are both inorganic and organic compounds commonly used to grow and nourish microbes. Inorganic nutrients include nitric acid, ammonium nitrate, ammonium chloride, ammonium sulfate, sodium nitrate, sulfur, sodium sulfide, sodium chloride, sodium bicarbonate, sodium phosphate, potassium phosphate, sulfuric acid, nitric acid, cyanide, uranium, mercury, lead, lithium, sodium metabisulfite, ammonium nitrate, fertilizers, gluconic acid, phosphogypsum, ferric chloride, calcium chloride, and ammonium phosphate. Organic nutrients include microbial biomass, glucose, dextrose, sodium acetate, amino acids, and purines. Vitamins that can be included in the nutrient solution include pyridoxine, pyridoxamine-HCI, riboflavin, thiamine, niacin, pantothenic acid, p-aminobenzoic acid, folic acid, and biotin. Small amounts of trace elements such as iron, copper, molybdenum and zinc can also be provided in the nutrient solution. Useful nutrients can also be mineral ores used for recovery of metals.

[0035] A composition comprising ECO002 according to the present disclosure may be formulated as a soil mixture, liquid, sludge or slurry substrate.

[0036] In some embodiments, a composition comprising ECO002 according to the present disclosure may comprise sulfuric acid, nitric acid, cyanide, uranium, mercury, lead, lithium, sodium metabisulfite, ammonium nitrate, fertilizers, gluconic acid, or phosphogypsum.

[0037] In one aspect, an ECO002 composition of the present disclosure may comprise Glycomyces lithiumensis in a water-soluble powder form within a container, for example, a large glass ampule or easy use vessel. In some embodiments, the kit also contains the selective Lithium Drive unit to be used with the microbe Glycomyces lithiumensis. All instructions for use will be included for the culture, extraction, and amplification of Lithium.

[0038] Another aspect of the present disclosure is a method to extract, produce and/or amplify lithium comprising culturing ECO002 along with one or more suitable microbe or plurality of suitable microbes. Non-limiting examples of suitable microbes include acidophilic archaea such as Sulfolobus metallicus and Metallosphaera sedula', mesophilic bacteria of the genera Acidithiobacillus or Leptospirillum ferrooxidans', Pyrococcus furiosus', thermoacidophilic archaeon Sulfolobus (Metallosphaera sedula); and Pyrobaculum islandicum. These microorganisms are basically 10, belonging to Bacteria: Acidiphilium sp., Leptospirillum sp., Sulfobacillus sp., Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans; and Archaea: Acidianus sp., Ferroplasma sp., Metallosphaera sp., Sulfolobus sp. and Thermoplasma sp.

[0039] In a preferred embodiment, the methods include contacting a substrate with the bacterium strain ECO002 or a mutant or derivative thereof. Without wishing to be bound by theory, the mode of action of producing precious lithium is due to the ECO002’s innate ability to extract, produce and/or amplify lithium from the substrate. Isolated ECO002 is found to express various proteins and biochemicals which are modulated by the base concentrations of lithium in its surrounding environment. Additionally, the Drive apparatus is used in conjunction with the microbe to accelerate the recovery, extraction and amplification of the metal.

[0040] For purposes of this disclosure, the term “mineral” or “mineral ore” means a composition that comprises precious metal values. Thus, a mineral may be a mined mineral, ancient seabed deposit, ancient lakebed deposit, black sands, an ore concentrate, metal bearing sea water, and waste products, such as mining tails, industrial waste water, oil well brine, coal tars, oil shales, tar sands, salt flats and oil sands. Useful minerals contain trace amounts of precious metals. Trace amount means the detection limit or below detection limits of conventional assay procedures such as fire assay, AAS (atomic adsorption spectroscopy), ICP-MS (inductive coupled plasma-mass spectrometer), ICP-AES (atomic emission spectroscopy) and other spectroscopic instrumentation commonly used in analytical laboratories. Some spectroscopic methods can detect as little as 1 ppt (part per trillion) to 0.1 ppb (part per billion).

[0041] As used herein, the term “rare earth metals” or “RE” may refer to lithium (Li), scandium (Sc), yttrium (Y), lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), Selenium (Se) and/or lutetium (Lu). As used herein, the term “precious earth metals” may refer to gold, silver, aluminum, rhenium, indium, platinum, gallium, germanium, ruthenium, rhodium, beryllium, palladium, osmium, iridium, tellurium, bismuth, platinum palladium, titanium, zinc, and zirconium.

[0042] In some embodiments, the methods of extracting, producing and/or amplifying precious metals and/or rare earth metals generally comprise farming the precious metals and/or rare earth metals including the steps of inoculating the bacterial strains disclosed herein on solid substrates or geological substrates. A geologic substrate is a surface (or volume) of sediment or rock where physical, chemical, and biological processes occur, such as the movement and deposition of sediment, the formation of bedforms, and the attachment, burrowing, feeding, reproduction, and sheltering of organisms. Non limiting examples of a geological substrate useful for the present disclosure include sandstone, limestone, shale, coal, chalk deposit formations, refractory rock ore (e.g., single, double and triple refractory rock ore). Additional solid substrates include, but are not limited to a sample collected from any terrestrial, aquatic or marine source such as soil, biofilms, sediments (e.g. coral or other marine sediments, aquifer sediments and the like), native metal rocks, sludge residue, aluminum, bauxite (red mud), spodumene, lithium mine substrates, salt flats, lithium batteries, electronic waste, phospho gypsum, fertilizer mines, and agriculture residue including corn, soybean, rice, cotton stubble and all other crop residue. In some embodiments, the solid substrate is disinfected prior to inoculation of the bacterial strains disclosed herein. Disinfection techniques include but are not limited to steam, autoclave, oven, microwave, biocide and fungicide solutions. Additional substrates include but are not limited to animal manures, bauxite, base metals, calcium phosphate, calcium silicate, clays and silicates, aluminum oxide, diatomaceous earth, diammonium phosphate, erionite and zeolites, feldspar, flint, food wastes, granite, graphite, gypsum, humic and fulvic acids, marble, mica, molten rock and lava, monoammonium phosphate, potash, pumice, silica, slate, seaweed, talc and recycled electronics and commercial devices.

[0043] In some embodiments, ECO002 may be applied to any solid substrate in a rock, powder, granulated or broken form for improved precious metals, platinum metals and rare earth metal leaching and extraction (in situ and/or ex situ applications). In some embodiments, solid substrates are used in traditional farming, specialty farming, potted and greenhouse farming, hydroponics and aeroponics techniques. In some embodiments, the solid substrate includes old or recycled electronic components or batteries.

[0044] In one embodiment, the biological process using microbes according to the disclosure is conducted in commercially available bioreactor consisting of a reactor having an agitation means. The agitation means can be mechanical stirring with a flat bladed impeller, percolation column, or air agitated pachuca reactor. The bioreactor can have air intake means, sterilization means, harvesting means, heating and/or cooling means, temperature controller means, pH controller means, filtration means and pressure controller means. All these features of bioreactors are known and commercially available in the biotechnology industry.

[0045] The biological process using microbes according to the disclosure can also be done by heap leaching techniques. In heap bio leaching techniques, a large body of mineral ore is treated with mutant microbes in nutrient solution in large contaminant ponds with no agitation and/or only occasional agitation. Generally, the contact time for heap type bio treatment is substantially longer than the agitated bioreactors and range from 10 days to 100 days.

[0046] As used herein, the term “inoculating” refers to the act of introducing a microorganism or a plurality of microorganisms (e.g. ECO002) into a substrate where it will be metabolically active and/or propagate. In preferred embodiments, the step of inoculating is performed using aseptic technique. In some embodiments, the bacterial strain is inoculated at a concentration of about 1 .0 x 10 ³ CFU/gm, about 1 .0 x 10 ⁴ CFU/gm, about 1 .0 x 10 ⁴ CFU/gm, about 1 .0 x 10 ⁶ CFU/gm, about 1 .0 x 10 ⁷ CFU/gm, about 1 .0 x 10 ⁸ CFU/gm, about 1 .0 x 10 ⁹ CFU/gm, about 1 .0 x 10 ¹⁰ CFU/gm, about 1 .0 x 10 ¹¹ CFU/gm, about 1 .0 x 10 ¹² CFU/gm, about 1 .0 x 10 ¹³ CFU/gm, about 1 .0 x 10 ¹⁴ CFU/gm, about 1 .0 x 10 ¹⁵ CFU/gm, about 1 .0 x 10 ¹⁶ CFU/gm, or about 1 .0 x 10 ¹⁷ CFU/gm. In a preferred embodiment, the bacterial strain is inoculated at a concentration from about 1 .0 x 10 ⁶ CFU/gm to about 1 .0 x 10 ¹² CFU/gm. The inoculating step can occur one or more times during the duration of extracting, producing and/or amplifying lithium from the substrate.

[0047] Inoculation of the substrate can occur by any means know to the skilled artisan which provides the microbe to the substrate in a sufficient amount. In some embodiments, after inoculation additional substrate is added to increase to surface area of the substrate which is in contact with the bacterial strain. In one aspect, additional substrate is added to create a 4-6 inch depth over the initial inoculation depth.

[0048] After inoculation the substrate is optionally irrigated and/or fertilized one or more times to stimulate colonization and exponential growth throughout of the bacterial strain throughout the solid substrate. Exemplary fertilizers include a low NPK plus micronutrient fertilizer, solutions comprising a complex or simple sugar, a seaweed or cytokine and a vitamin blend. In addition, specialty nutrients and by-products can be added to the inoculated solid substrate one or more times to establish new, increased and rigorous colonization by the bacterial strain.

[0049] In addition, the solid substrate may be covered to maintain a stable temperature or allow for an increase in the solid substrate temperature, for example using a poly covering for consistent temperature control and to control microbial contaminants from colonizing. In some embodiments, the inoculated solid substrate is maintained at a temperature between about 20°C to about 60°C including any range therein. In a preferred embodiment, the inoculated solid substrate is maintained at a temperature between about 29°C to about 50°C.

[0050] After the bacterial strain has had sufficient time to colonize and biochemically process the substrate (non-limiting example 2-10 weeks) the substrate can be tested and processed for lithium production. The testing and/or processing steps include harvesting the solid substrate which has been colonized by the bacterial strain, generating a slurry by adding a solution to the substrate, and centrifugation at a minimum of 8,000 RPM to concentrate the precipitate which contains the de novo precious metals and/or rare earth metals. These steps may optionally include a bacterial lysis step to release any metals within the bacterial strains intracellular matrix.

[0051] In a still another aspect, the methods of extracting, producing and/or amplifying lithium metals generally comprise inoculating the bacterial strains disclosed herein in liquid substrates. Suitable liquid substrates include but are not limited to balanced salt and nutrient solutions, broths, environmental samples collected from any aquatic or marine source, waste waters, sludge waters, saltwater, freshwater, irrigation systems, ponds, lakes, rivers, estuaries, produced water, brine water and frac water. In some embodiments, the liquid substrate is disinfected prior to inoculation of the bacterial strains disclosed herein. Disinfection techniques include but are not limited to steam, autoclave, oven, microwave, biocide and fungicide solutions. In a preferred embodiment, the disinfection step will reduce microbial colony and propagule concentrations to below or at about 5.0 x 10 ⁵ CFU/ml.

[0052] Inoculation of the liquid substrate can occur by any means known to the skilled artisan at concentration described above for the solid substrate. The inoculating step can occur one or more times during the duration of extracting, producing and/or amplifying lithium from the liquid substrate. After inoculation the liquid substrate is preferably agitated during the extraction, production and/or amplification of the lithium. In an exemplary embodiment, agitation of the liquid substrate occurs using an air pump for aerobic respiration.

[0053] After inoculation the liquid substrate is optionally specialty nutrients and by-products can be added to the inoculated solid substrate one or more times to establish new, increased and rigorous colonization by the bacterial strain, for example by adding solutions comprising a complex or simple sugar, a seaweed or cytokinin and a vitamin blend. In addition, for accelerated reactions establish an anodic and cathodic LED using a wavelength generator set at a range of 100 Hz - 200 KHz.

[0054] After the bacterial strain has had sufficient time to colonization and biochemically process the liquid substrate (non-limiting example 12-72 hours) the liquid substrate can be tested and processed for lithium production. The testing and/or processing steps include collecting the liquid substrate which has been colonized by the bacterial strain, generating a slurry by adding a solution to the solid substrate, and centrifugation of the liquid solution through an in line and continuous centrifuge at a minimum of 8,000 RPM to concentrate the precipitation. These steps may optionally include a bacterial lysis step to release any metals within the bacterial strains intracellular matrix.

[0055] In each of the above embodiments, a bioreactor, fermenter, reaction vessel can be used in the disclosed methods. Moreover, the present disclosure contemplates the use of the disclosed microbes for bioleaching and heap leaching and therefore the use of leach pits are contemplated within the methods as well.

[0056] Bio treatment temperature ranges from 15 degrees centigrade to 50 degrees centigrade, preferably from 20 degrees to 30 degrees centigrade. pH can be acidic (pH 1 to 3) or basic (pH 9 to 12), although slightly acidic (pH 4) to slightly basic (pH 8) pH ranges are preferred. The most preferred pH ranges are the neutral range of from pH 6.5 to pH 7.5.

[0057] In accordance with the methods of the present disclosure, pressure is not critical and can be at atmospheric, below atmospheric, and/or above atmospheric. The biological transmutation process can be conducted in aerobic or anaerobic conditions. The biological transmutation process can be conducted in the presence of nitrogen, carbon dioxide, and oxygen in the atmosphere. Oxygen can be provided chemically, for example, with hydrogen peroxide, or as a gas from pressurized vessels.

[0058] Microbe concentration is not critical. At low microbe concentration, the contact duration is generally longer to allow the microbe to grow and multiply. However, microbe concentration should not exceed the maximum microbe concentration that the nutrient solution can sustain. Contact time can vary from a few hours to several weeks and depends in part on the type and mesh size of the mineral ore digested. Contact time ranges can be from 1 day to 30 days, more preferably from 1 day to 10 days.

[0059] The biological process using microbes according to the disclosure can be conducted in aerobic or anaerobic conditions. However, preferably conducted in the presence of oxygen, nitrogen and carbon dioxide in the atmosphere. Oxygen can also be provided chemically, for example, with hydrogen peroxide, or as a gas from pressurized vessels.

[0060] Nutrients can also be provided during the biological transmutation process to support growth of the mutant microbes. Nutrients can be inorganic, including nitric acid, sulfur, ammonium nitrate, ammonium chloride, ammonium sulfate, sodium nitrate, sodium chloride, sodium bicarbonate, sodium phosphate, potassium nitrate, potassium phosphate, ferric chloride, calcium chloride, and ammonium phosphate, and organic, including glucose, dextrose, sodium acetate, amino acids, and purines. Vitamins that can be included in the nutrient solution include pyridoxine, pyridoxamine-HCI, riboflavin, thiamine, niacin, pantothenic acid, p- aminobenzoic acid, folic acid, and biotin. Small amounts of traces elements such as iron, copper, molybdenum and zinc can also be provided in the nutrient solution. [0061] In a still another aspect, the biological transmutation process using microbes according to the disclosure occurs in liquid substrates. Suitable liquid substrates include but are not limited to balanced salt and nutrient solutions, broths, environmental samples collected from any aquatic or marine source, waste waters, sludge waters, saltwater, freshwater, irrigation systems, ponds, lakes, rivers, and estuaries. In some embodiments, the liquid substrate is disinfected prior to inoculation of the bacterial strains disclosed herein.

[0062] Inoculation of the liquid substrate (in situ and/or ex situ applications) can occur by any means known to the skilled artisan at concentration described above for the solid substrate. The inoculating step can occur one or more times during the duration of extracting, producing and/or amplifying precious metals and/or rare earth metals from the liquid substrate. After inoculation the liquid substrate is preferably agitated during the extraction.

[0063] After inoculation the liquid substrate is optionally specialty nutrients and by-products can be added to the inoculated solid substrate one or more times to establish new, increased and rigorous colonization by the bacterial strain, for example by adding solutions comprising a complex or simple sugar, a seaweed or cytokinin and a vitamin blend. In addition, for accelerated reactions establish an anodic and cathodic LED using a wavelength generator set at a range of 100 Hz - 200 KHz.

[0064] In each of the above embodiments, a bioreactor, fermenter, reaction vessel can be used in the disclosed methods. Moreover, the present disclosure contemplates the use of the disclosed microbes for bioleaching and heap leaching and therefore the use of leach pits are contemplated within the methods as well.

[0065] After the biological transmutation process, the recovery of metal produced from the starting material and microbial solution can be performed by conventional metallurgical methods such as smelting, leaching, electrolysis, resins and other methods known to those skilled in art of metallurgy. In another embodiment, the precious metals in the microbes or biomass of dead microbes can be recovered by methods described for recovery of precious metals from mineral ore. [0066] Fire assaying and cupellation are described by C. W. Ammen, Recovery and Refining of Precious Metals, second edition 1993, Chapter 12, pp 302- 329.

[0067] In an exemplary embodiment, a premeasured amount of crop residue is harvested and added to a slurry blend at a range of 25 - 45% pulp density. The bacterium Glycomyces lithiumensis is added to residue slurry along with bio nutrients and allowed to culture for 96 - 120 hours. During this time, the microbial activity of Lithium liberation and recovery occurs resulting in an appreciable extraction and recovery activity whereby the bacteria possess a significant amount of Lithium in their intracellular bodies.

[0068] Also provided are kits. Such kits can include an agent or composition described herein and, in certain embodiments, instructions for use. Such kits can facilitate performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use. Components include but are not limited to ECO002 compositions and formulations for use or stability, as described herein. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components.

[0069] Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline or sterile each of which has been packaged under a neutral non-reacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules, and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, and the like.

[0070] In certain embodiments, kits can be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic-readable medium, such as a floppy disc, mini-CD-ROM, CD- ROM, DVD-ROM, Zip disc, videotape, audio tape, and the like. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit.

[0071] Compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see, e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001 ) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005) Protein Expr Purif. 41 (1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley- VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

General Techniques

[0072] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R. I. Freshney, ed. 1987); Introuction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds. 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991 ); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed. 1985); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds.(1985»; Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984»; Animal Cell Culture (R.l. Freshney, ed. (1986» ; Immobilized Cells and Enzymes (IRL Press, (1986» ; and B. Perbal, A practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.).

[0073] So that the present disclosure may be more readily understood, certain terms are first defined. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention pertain. Many methods and materials similar, modified, or equivalent to those described herein can be used in the practice of the embodiments of the present invention without undue experimentation, the preferred materials and methods are described herein. In describing and claiming the embodiments of the present invention, the following terminology will be used in accordance with the definitions set out below.

[0074] Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. As an illustration, a numerical range of “about 2 to about 50” should be interpreted to include not only the explicitly recited values of 2 to 50, but also include all individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 2, 2.4, 3, 3.7, 4, 5.5, 10, 10.1 , 14, 15, 15.98, 20, 20.13, 23, 25.06, 30, 35.1 , 38.0, 40, 44, 44.6, 45, 48, and sub-ranges such as from 1 -3, from 2-4, from 5-10, from 5-20, from 5-25, from 5-30, from 5-35, from 5- 40, from 5-50, from 2-10, from 2-20, from 2-30, from 2-40, from 2-50, etc. This same principle applies to ranges reciting only one numerical value as a minimum or a maximum. Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.

[0075] The term “about,” as used herein, refers to variation of in the numerical quantity that can occur, for example, through typical measuring techniques and equipment, with respect to any quantifiable variable, including, but not limited to, mass, volume, time, distance, and amount. Further, given solid and liquid handling procedures used in the real world, there is certain inadvertent error and variation that is likely through differences in the manufacture, source, or purity of the ingredients used to make the compositions or carry out the methods and the like. The term “about” also encompasses these variations, which can be up to ± 5%, but can also be ± 4%, 3%, 2%, 1 %, etc. Whether or not modified by the term “about,” the claims include equivalents to the quantities.

[0076] When introducing elements of the present disclosure or the preferred aspects(s) thereof, the articles “a,” “an,” “the,” and “said” are intended to mean that there are one or more of the elements. The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0077] In this disclosure, “comprises,” “comprising,” “containing,” and “having” and the like can have the meaning ascribed to them in U.S. Patent Law and can mean “includes,” “including,” and the like, and are generally interpreted to be open ended terms. The terms “consisting of” or “consists of” are closed terms, and include only the components, structures, steps, or the like specifically listed in conjunction with such terms, as well as that which is in accordance with U.S. Patent law. “Consisting essentially of” or “consists essentially of” have the meaning generally ascribed to them by U.S. Patent law. In particular, such terms are generally closed terms, with the exception of allowing inclusion of additional items, materials, components, steps, or elements, that do not materially affect the basic and novel characteristics or function of the item(s) used in connection therewith. For example, trace elements present in a composition, but not affecting the composition’s nature or characteristics would be permissible if present under the “consisting essentially of” language, even though not expressly recited in a list of items following such terminology. In this specification when using an open ended term, like “comprising” or “including,” it is understood that direct support should be afforded also to “consisting essentially of” language as well as “consisting of” language as if stated explicitly and vice versa.

[0078] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

[0079] As various changes could be made in the above-described materials and methods without departing from the scope of the invention, it is intended that all matter contained in the above description and in the examples given below, shall be interpreted as illustrative and not in a limiting sense.

EXAMPLES

[0080] The following examples are included to demonstrate various embodiments of the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Example 1 : Novel Microorganism Isolation and Classification

[0081 ] It has been estimated that only 2% of all microbial isolates can be cultured in a lab. Therefore, the microbes that can be grown in the laboratory represent only a small fraction of the total diversity that exists in nature. At all levels of microbial phylogeny, uncultured clades that do not grow on standard media are playing critical roles in cycling carbon, nitrogen, and other elements, synthesizing novel by-products, and impacting the surrounding organisms and environment. The ability to culture difficult to culture or previously uncultured microbial strains provides a wealth of information about their role in the environment, ecology, and nutrient cycling. But perhaps even more importantly, screening of novel isolates will reveal novel products that can have profound effects for the discovery of novel drugs, improve agricultural techniques and products, and in the production of rare and precious metals. To solve this problem the Applicant developed a novel apparatus (Ecobiome Discovery apparatus) allowing for the controlled growth, isolation and characterization of microorganisms including those that are difficult to culture or are uncultivable at the present time.

[0082] In an effort to isolate and characterize novel microbes, an experiment utilizing the Ecobiome Discovery apparatus was used. The use of the EcoBiome Discovery apparatus towards the isolation and characterization of ECO002 was utilized as an application to demonstrate its ability to respond towards external stimulation pulsed throughout the EcoBiome apparatus. The apparatus is a microbiological tool that allows for novel microbial discovery based on an aggregation of phenotypic and genotypic expression.

[0083] After isolation of the microbe population comprising the wildtype parent isolate, challenge tests were performed, which included tests specific for enriching and driving transmutase and amplificase expression. These test resulted in the isolation and characterization of ECO002 (Glycomyces lithiumensis). Table 1 - Urease Enzyme Activity

- No enzymatic activity; + Enzymatic Activity; ++ Strong Enzymatic Activity

Table 2 - Bacterial Final Electron Acceptor Metabolic Activity

Table 3 - Phosphorus Solubilization Activity

Table 4 - H ₂ S Activity (Post 2- Weeks) Table 5 - Chitinase Enzyme Activity

Table 6 - Cellulase Enzyme Activity

Table 7 - Ligninase Enzyme Activity

[0084] EC0002 is a gram-negative bacterium, non-spore former that is capable of metabolizing simple and complex polymers as well as metals through heterotrophic and chemoheterotrophic biochemical pathways. ECO002 is a facultative anaerobe and is tolerant of low and high temperatures, e.g., ranging between about 5°C to about 46°C. In addition, ECO002 is tolerant of a pH range from about 2 to about 9. The microorganism reaches exponential growth with high agitation (>360 rpm) and oxygenation (DO > 90%) within 6-8 hours.

Example 2: EcoBiome Lithium Extracting Microbes

[0085] An ion selective Lithium probe was used to conduct experimental testing of different substrates and feedstocks containing Lithium sources. Each substrate material was pre-weighed and added to the EcoBiome Discovery apparatus with water and the ECO002 microbe. All reactions were given a residence time of 2 weeks at 33°C without agitation. All readings were made in triplicate using an ion selective probe (Lithium specific).

Table 1 : Lithium Source Materials

Testing by Ion Selective Probe in solution

Table 2: Agriculture Source Materials

Corn Residue 0 m 3 8 m

Testing by Ion Selective Probe in solution Table 3: Energy/Oil & Gas Source Materials

Testing by Ion Selective Probe in solution

EQUIVALENTS

[0182] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure. [0183] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0184] The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0185] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0186] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0187] As used herein, the term “isolated” in the context of an isolated bacterial strain, is one which is altered or removed from the natural state through human intervention.