CROSS-REFERENCE TO RELATED APPLICATIONS

This is application claimed priority to U.S. provisional patent application 61/837,760 filed on June 21 , 2013 which is herewith enclosed in its entirety.

TECHNOLOGICAL FIELD

The present disclosure relates to homoserine lactone derivatives, particularly N-acyl homoserine lactone derivatives, for improving the recovery of a mineral element from an ore during a biomining process. BACKGROUND

While world demand for various elements, such as copper, is growing rapidly to fuel the increasing industrialization of developing countries, higher-grade elements reserves are continuously diminishing. In order to satisfy increasing demand the mining industry is therefore faced with the need to implement cost effective methods to treat lower grade ores, overburden as well as waste from existing mining operations. It is within this context of cost effective treatment of low-grade ores that bioprocessing of mineral elements has become an increasingly attractive option for the mining industry.

The biotechnological subfield of mineral bioprocessing, or biomining, is a microbial-based biotechnology that had a major impact on the global mining industry. For example, heap bioleaching of copper sulphides was initially targeted for secondary copper sulphides (mainly from chalcocite but also from covellite and bornite) and designed to recover copper oxides from acid leaching.

Chalcopyrite (CuFeS ₂ ) is the most commonly occurring copper ore in nature. Despite chalcopyrite constituting the majority of world copper reserves, it only represents a small fraction of the copper being recovered from leaching operations to date. Instead, chemical leaching of copper oxides as well as bioleaching of secondary sulphides, such as chalcocite (Cu ₂ S), constitute the great majority of hydrometallurgical processes implemented by the industry.

Conventionally, copper sulphides are usually concentrated on site using froth flotation before being shipped off to a smelter for further processing. Froth flotation processing of chalcopyrite from ore bodies with less than 0.8% copper is difficult. Since the next generation of mines are likely to be closer to 0.6% copper head grade, this projected drop is also likely to significantly reduce the competitiveness for conventional processing via froth flotation and smelting.

The application of heap bioleaching applications to chalcopyrite is impeded due to low recovery. Standard mesophilic microbial cultures for instance, that are very successful at leaching secondary sulfide minerals, only manage to oxidize low levels of chalcopyrite (30- 60%) with prolonged leach times unable to improve yields.

It would be highly desirable to be provided with to be provided with improved methods of bioleaching ores, especially lower grade ores. Preferably such methods would be compatible with existing bioleaching processes.

BRIEF SUMMARY

The present disclosure provides a bioming additive for improving the recovery of a target mineral element. The biomining additive is believed to potentiate the biomining properties of bacterial populations capable of leaching a target mineral element from an ore.

In accordance with the present disclosure, there is provided a biomining composition comprising (i) a biomining additive of formula (I) :

AHL-N-C(0)-R _r R ₂ or

wherein AHL is an homoserine lactone derivative; Ri is a lower linear or branched alkyl, cycloalkyl, a lower linear or branched alkenyl, cycloalkenyl or aryl; a C ₉ to Ci ₉ linear or branched alkyl, cycloalkyl, linear or branched alkenyl, cycloalkenyl or aryl; or a C ₉ to Ci ₉ linear or branched alkylcarbonyl, cycloalkylcarbonyl, linear or branched alkenylcarbonyl, cycloalkenylcarnonyl or arylcarbonyl; and R ₂ is absent or an halogen. It is to be understood that, when R ₂ is absent, the bond between Ri and R ₂ is also absent; and (ii) an excipient. In an embodiment, R-i is a C ₁₃ to C _ig or a C ₁₄ to C ₁₉ linear or branched alkyl, cycloalkyl, linear or branched alkenyl, cycloalkenyl or aryl. In another embodiment, is a C ₁₃ to C ₁₉ , a C ₁₄ to C ₁₉ or a Ci5 to C ₁₉ linear or branched alkyl, cycloalkyl, linear or branched alkenyl, cycloalkenyl or aryl. In yet another embodiment, Ri is a Ci ₃ or a C ₁₅ linear or branched alkyl, cycloalkyl, linear or branched alkenyl, cycloalkenyl or aryl. In some specific embodiments, Ri is a linear or branched alkyl, and in further specific embodiments, Ri is a linear alkyl. In some embodiments, Ri is an unbranched alkyl. In other embodiment, the biomining additive is of formula (II) :

AHL - N - C(O) - (CH ₂ ) _n - CH ₃ or

In the biomining additives of formula (II), n can be between 13 and 18, between 14 and 18. In another embodiment, n can be 13, 14 or 15. In some embodiments, R-i is a branched alkyl, for example can be branched with at least one lower alkyl, cycloalkyl, a lower alkenyl, cycloalkenyl or aryl or, in yet a further embodiment, R^ can be branched with at least one methyl, propyl, butyl, phenyl or, in still a further embodiment, can be branched (e.g., at the terminal carbon atom of the chain) with an halogen (bromide for example). In still another embodiment, R^ is a C ₁₃ to C ₁₉ or a C ₁₄ to C ₁₉ linear or branched alkylcarbonyl, cycloalkylcarbonyl, linear or branched alkenylcarbonyl, cycloalkenylcarnonyl or arylcarbonyl. In another embodiment, R-i is a Ci ₅ to C ₁₉ linear or branched alkylcarbonyl, cycloalkylcarbonyl, linear or branched alkenylcarbonyl, cycloalkenylcarnonyl or arylcarbonyl. In still another embodiment, R^ is a Ci _3, C ₁₄ or C ₁₅ linear or branched alkylcarbonyl, cycloalkylcarbonyl, linear or branched alkenylcarbonyl, cycloalkenylcarnonyl or arylcarbonyl. In yet another embodiment, Ri is a linear or branched alkylcarbonyl (for example a 3-oxo alkylcarbonyl). In still another embodiment, R-i is a linear alkylcarbonyl (unbranched alkylcarbonyl or branched alkylcarbonyl). For example, R^ can be branched with at least one lower alkyl, cycloalkyl, a lower alkenyl, cycloalkenyl or aryl, such as, for example at least one methyl, propyl, butyl or phenyl. In another example, Ri can be branched with at least one halogen (bromide for example). In some embodiments, Ri can be methyl, propyl, butyl or phenyl.

In accordance with the present disclosure, there is provided a method for improving the recovery of an element during a biomining process. Broadly, the method comprises contacting an ore comprising the element, a bacterial population capable of exhibiting a biomining activity and the biomining additive described herein under conditions suitable for leaching the element from the ore into a leaching solution and/or for oxidizing the ore in the leaching solution. In an embodiment, the bacterial population comprises Acidithiobacillus sp. (Acidithiobacillus ferrooxidans for example). In still another embodiment, the leaching solution is an acidic solution (a sulfuric acid solution for example). In some embodiments, the element is copper and/or the ore is chalcopyrite. In another embodiment, the biomining process is a bioleaching process (a heap leaching process for example). In a further embodiment, the element is gold and/or the ore is pyrite or arsenopyrite. In still a further embodiment, the biomining process is a biooxidation process (performed in a continuous stirred thank reactor for example). In some embodiments, the method further comprises, prior to the contacting step: combining the biomining additive with the bacterial population to provide an unprimed bacterial population; culturing the unprimed bacterial population under conditions to obtain a primed bacterial population; and adding the primed bacterial population to the ore.

In the present disclosure, the term "alkyl" represents an optionally linear or branched carbon moiety having at least 1 carbon atoms. In some embodiments, the alkyl comprises between 2 and 20, between 10 and 20, between 14 and 18 carbon atoms. In some alternative embodiments, the number of carbons atoms in the alkyl is 16. The term "lower alkyl" specifically refers to a linear or branched moiety having 1 to 6 or preferably 1 to 3 carbon atoms. The term "alkyl" includes the subclasses primary, secondary and tertiary alkyls, such as, but not limited to, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, isohexyl or neohexyl.

The terms "alkenyl" and "alkynyl" represent optionally substituted linear or branched hydrocarbon moiety which has one or more double bonds or triple bonds in the chain. The number of carbon atoms in an alkenyl is at least 2. In some embodiments, it can comprises between 2 and 20, between 10 and 20, between 14 and 18 carbon atoms. In some alternative embodiments, the number of carbons atoms in the alkelyl is 16. The term includes the subclasses of primary and secondary alkynyl. Examples of alkenyl and alkynyl groups include, but are not limited to, allyl, vinyl, acetylenyl, ethylenyl, propenyl, isopropenyl, butenyl, isobutenyl, hexenyl, butadienyl, pentenyl, pentadienyl, hexenyl, hexadienyl, hexatrienyl, heptenyl, heptadienyl, heptatrienyl, octenyl, octadienyl, octatrienyl, octatetraenyl, propynyl, butynyl, pentynyl and hexynyl. The term "aryl" represents an optionally substituted carbocyciic moiety containing at least one benzenoid-type ring (i.e., may be monocyclic or polycyclic). Examples include but are not limited to phenyl, tolyl, dimethylphenyl, aminophenyl, anilinyl, naphthyl, anthryl, phenanthryl, indanyl, tetralinyl or biphenyl. Preferably, the aryl comprises 6 to 10 or more preferably 6 carbon atoms. In some embodiments, the aryl is located on the terminal carbon.

The term "carbonyl" is intended to indicate a radical of the formula -C(0)-R', wherein R' represents an alkyl, a cycloalkyl, an alkenyl, a cycloalkenyl or an aryl as indicated above. The number of carbon atoms in the carbonyl is at least 2. In some embodiments, it can comprises between 2 and 20, between 10 and 20, between 14 and 18 carbon atoms. In some alternative embodiments, the number of carbons atoms in the carbonyl is 16.

The term "halogen atom" or "halogen" refers specifically to a fluorine atom (or a fluoride), chlorine atom (or a chloride), bromine atom (or a brominde) or iodine atom (or an iodide). Preferably, the halogen is a bromide. The term "haloalkyl" is intended to indicate an alkyl group as defined above substituted with one or more halogen atoms. In one embodiment, the halogen substitution is made at the terminal carbon atom of the chain moiety of the AHL derivative.

The terms "optionally substituted", "optionally substituent" or "substituent" represents, at each occurrence, and independently, one or more halogen, amino, amidino, amido, azido, cyano, guanido, hydroxyl, nitro, nitroso, urea, a cycloether, OS(0) ₂ R _A (wherein R _A is selected from Ci_ ₆ alkyl, C ₆ -i ₀ aryl or 3-10 membered heterocycle), OS(0) ₂ OR _B (wherein R _B is selected from H, Ci_ ₆ alkyl, C ₆ -io aryl or 3-10 membered heterocycle), S(0) ₂ OR _c (wherein R _c is selected from H, Ci_ ₆ alkyl, C ₆ -io aryl and 3-10 membered heterocycle), S(0) _O _ ₂ RD (wherein R _D is selected from H, alkyl, C ₆ -i ₀ aryl or 3-10 membered heterocycle), OP(0)OR _E OR _F , P(0)OR _E OR _F (wherein R _E and R _F are each independently selected from H or C-i- ₆ alkyl, alkyl, C ₆ _ ₁₀ aryl, C _1-6 alkyl, C ₆ _ ₁₀ aryl, C _1-6 alkoxy, C _6-10 aryl, C _1-6 alkyloxy, C ₆ _ ₁₀ aryloxy, 3-10 membered heterocycle), C(0)R _G (wherein R _G is selected from H, C _1-6 alkyl, C _6- io aryl, C _6-10 aryl, C _1-6 alkyl or 3-10 membered heterocycle), C(0)OR _H (wherein R _H is selected from H, C _1-6 alkyl, C ₆ . ₁₀ aryl, C ₆ . ₁₀ aryl, C _1-6 alkyl or 3-10 membered heterocycle), NR|C(0)Rj (wherein R| is H or C _1-6 alkyl and Rj is selected from H, C _1-6 alkyl, C _6- io aryl, C ₆ . ₁₀ aryl, C _1-6 alkyl or 3-10 membered heterocycle, or R| and Rj are taken together with the atoms to which they are attached to form a 3 to 10 membered heterocycle) or S0 ₂ NR _K R|_ (wherein R _K and R _L are each independently selected from H, C _1-6 alkyl, C _6- io aryl, C _3-10 heterocycle or C ₆ . ₁₀ aryl, C _1-6 alkyl). In another embodiment, the terms "optionally substituted", "optionally substituent" or "substituent" preferably represent halogen, Ci _-6 alkyl, C ₂ -6 alkenyl, C ₂ -6 alkynyl, Ci _-6 alkoxy, C ₂ -6 alkenyloxy, C ₂ -6 alkynyloxy, -NR RN, -C(0)NR _M RN, -NR _M COR _n , carboxy, azido, cyano, hydroxyl, nitro, nitroso, -OR _M , -SR _M , -S(O) ₀ - ₂ RM, -C(0)R _M , -C(0)OR _m and -S0 ₂ NR _M R _N ; wherein R _M and R _N are each independently H, Ci_ _s alkyl, C ₂ _ ₆ alkenyl or C ₂ _ ₆ alkynyl.

In still another embodiment, the term "optionally substituted", "optionally substituent" or "substituent" preferably represent halogen, Ci_s alkyl, C ₂ .6 alkenyl, Ci _-6 alkoxy, cycloether, - NR _M RN, -C(0)NR _M R _N , -NRMCORN, carboxy, hydroxyl, nitro, -SR _M , -S(O) ₀ - ₂ RM, -C(0)R _M , - C(0)OR _M and -S0 ₂ NR R _N ; wherein R _M and R _N are each independently H, or C _1-6 alkyl. The term "independently" refers to a substituent that can be the same or a different definition for each item.

The term "hydrocarbon" is intended to indicate a compound containing only hydrogen and carbon atoms, it may contain one or more double and/or triple carbon-carbon bonds, and it may comprise cyclic moieties in combination with branched or linear moieties. Said hydrocarbon preferably comprises 1-20, e.g. 1-18, e.g. 1-12 carbon atoms. The term includes alkyl, alkenyl, alkynyl and aryl, as indicated above.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus generally described the nature of the disclosure, reference will now be made to the accompanying drawings, showing by way of illustration, a preferred embodiment thereof, and in which:

Figure 1 illustrates the results obtained with different AHL derivatives on copper leaching using the rhodamine assay. Results are shown for the AHL-derivatives in function of the amount of copper leached after 16 days (g/L). For each AHL derivative, results are provided in function of a combination of ore and bacteria (upper column for each AHL derivative tested, white bars), a combination of ore, bacteria and a blank (middle column for each AHL tested, dark gray bars) or a combination of ore, bacteria and the AHL derivative (lower column for each AHL derivative tested, light grey bars). Results are shown as the compilation of at least three technical replicates. Error bars refer to standard deviations.

Figure 2 illustrates the rolling bottle tests results obtained in the presence or absence of a C16-AHL derivative. Representative results are shown as the concentration of copper in solution (mg/L) in function of leach time (hours) for chalcopyrite incubated in the presence (regular line) or absence (bold line) of the C16-AHL derivative. Figure 3 illustrates the substrate utilization preferences of A. ferrooxidans cultured in the presence or absence of different concentrations of C16-AHL. (A) Results of iron oxidation (measured as g/L) are provided for A. ferrooxidans cultured for three days in the presence of various concentration (in μΜ) of the C16-AHL additive in the presence of iron and sulfur substrates. From left to right, results are provided for the following concentrations of the C16-AHL additive: 0 μΜ (DMSO), 1 μΜ, 2.5 μΜ, 5 μΜ, 7.5 μΜ, 10 μΜ and 15 μΜ. (Β) Results of iron oxidation (measured as g/L) are provided for A. ferrooxidans cultured for three days in the presence of various concentration (in μΜ) of the C16-AHL additive in the presence of iron substrates but in the absence of alternative sulfur substrates. From left to right, results are provided for the following concentrations of the C16-AHL additive: 0 μΜ (DMSO), 1 μΜ, 2.5 μΜ, 5 μΜ, 7.5 μΜ, 10 μΜ and 15 μΜ.

Figure 4 illustrates copper concentration in the leach liquor of bottle roll containing chalcopyrite incubated 14 days with either the C12-AHL, the C14-AHL or the C16-AHL additive in the presence of the native consortia. Results are shown as copper concentration (in ppm) in function of the additive used: control (DMSO), C12 (C12-AHL), C14 (C14-AHL) and C16 (C16-AHL). Error bars refers to standard deviations.

Figure 5 characterizes a chalcopyrite leached after 5 months in 50 kg columns using either a control (DMSO) or a C14-AHL additive (+AHL). (A) Results are shown as the copper concentration in the leach liquor (g/L) in function of additive: control (dark grey bar) or C14- AHL (light grey bar). (B) Results are shown as the Fe ²⁺ /Fe ³⁺ ratio in the leach liquor in function of additive: control (dark grey bar) or C14-AHL (light grey bar). (C) Results are shown as the reduction potential (units) in the leach liquor (g/L) in function of additive: control (dark grey bar) or C14-AHL (light grey bar). (D) Results are shown as the total iron in the leach liquor (g/L) in function of additive: control (dark grey bar) or C14-AHL (light grey bar).

DETAILED DESCRIPTION

In accordance with the present disclosure, there is provided a biomining additive for improving the recovery of a mineral element from an ore. The biomining additive is an homoserine lactone derivative which promotes the intrinsic biomining activity of a bacterial population.

As it is known in the art, the formation of biofilms (aggregations of bacteria colonizing surfaces via extracellular oligosaccharides) is known to influence the rate and yield of the bioleaching of mineral elements from ores. The formation of biofilms is known to be controlled by quorum sensing (QS) molecules, which are low molecular weight compounds synthesized and sensed by bacteria that trigger physiological and morphological changes. It is however unknown whether biofilms of leaching bacteria act to inhibit further leaching (for example, forming a passivating layer) or act to enhance leaching (by creating a chemical microenvironment that increases local leaching rates) (Gonzalez et al., 2013). As explained in Oliviera-Nappa et al, "[t]he determination of the actual chalcopyrite passivation mechanism remains a problem difficult to ascertain experimentally given the complexity and interconnectivity of the physical, chemical, and biological processes" (Oliviera-Nappa et al., 2010). Consequently, and as it will be presented below, there is no consensus in the art between the promotion of biofilm formation and the bioleaching of mineral elements from ores.

The present disclosure thus provide chemically synthesized homoserine lactone derivative as a biomining additive to improve the recovery of a mineral element from an ore. Advantages associated with some of the derivatives include, but are not limited, to addition to a heap via irrigation at low concentrations (e.g., sub-micromolar), low toxicity and biodegradability.

Biomining additive

The biomining additives provided herewith have the ability to promote biomining activities of a bacterial population. The present disclosure provides a biomining additive having the following formula (I) :

ln the biomining additive of formula (I), Ri can be a lower linear or branched, optionally substituted alkyl, cycloalkyl, a lower linear or branched, optionally substituted, alkenyl, cycloalkenyl or aryl. R< _\ is preferably a lower linear, optionally substituted, alkyl, cycloalkyl, a lower linear alkenyl, cycloalkenyl or aryl. In an embodiment, R ₁ can be a methyl, propyl, butyl or phenyl.

In the biomining additive of formula (I), R- can be a C _g to C ₁₉ linear or branched, optionally substituted, alkyl, cycloalkyl, linear or branched, optionally substituted alkenyl, cycloalkenyl or aryl. In an embodiment, the number of carbons in the alkyl, cycloalkyl, alkenyl, cycloalkenyl or aryl and preferably has between 13 to 19, 14 to 19, 15 to 19 total carbon atoms. In an embodiment, R-i can have 13 or 15 total carbon atoms.

In still yet another embodiment, in the biomining additive of formula (I), R-i can be a C ₉ to C ₁₉ linear or branched, optionally substituted, alkylcarbonyl, cycloalkylcarbonyl, linear or branched, optionally substituted, alkenylcarbonyl, cycloalkenylcarnonyl or arylcarbonyl. In an embodiment, the number of carbons in the alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, cycloalkenylcarnonyl or arylcarbonyl can have preferably between 13 to 19, 14 to 19, 15 to 19 total carbon atoms. In an embodiment, can have 13 or 15 total carbon atoms. In the biomining additives of formula (I), R ₂ can be absent or be an halogen. When R ₂ is absent, it is understood that the bond between R-i and R ₂ is also absent.

In an embodiment of the biomining additive of formula (I), is a linear or branched, optionally substituted alkyl. In an advantageous embodiment, Ri is a linear and unbranched alkyl. An exemplary compound in which Ri is a linear and unbranched alkyl is the compound of formula (II) :

wherein n is between 8 to 18, preferably 13 to 18, more preferably 14 to 18 and even more preferably 13, 14 or 15.

Alternatively, Ri is a branched, optionally substituted, alkyl. In such embodiment, the alkyl can be branched at one or more carbon atoms. Exemplary branching moieties include, but are not limited to a lower alkyl (such as, for example, methyl, propyl, butyl or phenyl), cycloalkyl, a lower alkenyl, cycloalkenyl, aryl and/or halogen (a bromide for example). In instances where more than one branching moieties are present, it is contemplated that the same or different branching moieties be used. In an embodiment, the branching is observed on last carbon of the chain (e.g., terminal carbon). In some additional embodiments, the branching is observed only on the last carbon of the chain (e.g., terminal carbon). In an embodiment of the biomining additive of formula (I), Ri is a linear or branched, optionally substituted, alkylcarbonyl. In an advantageous embodiment, Ri is a linear and unbranched alkylcarbonyl. In one non-limiting example of an linear and unbranched alkylcarbonyl, the biomining additive can be a 3-oxo alkylcarbonyl having the following formula (III) :

wherein n is between 6 to 16, preferably 11 to 16, more preferably 12 to 16, even more preferably 10 or 12.

Alternatively, the biomining additive of formula (I), (II) or (III) is a branched, optionally substituted, alkylcarbonyl. In such embodiment, the alkylcarbonyl can be branched at one or more carbon atoms. Exemplary branching moieties include, but are not limited to a lower alkyl (such as, for example, methyl, propyl, butyl or phenyl), cycloalkyl, a lower alkenyl, cycloalkenyl, aryl and/or halogen (a bromide for example). In instances where more than one branching moieties are present, it is contemplated that the same or different branching moieties be used. In an embodiment, the branching is observed on last carbon of the chain (e.g., terminal carbon). In some additional embodiments, the branching is observed only on the last carbon of the chain (e.g., terminal carbon).

Since the biomining additive is going to be admixed in an acidic leaching solution, it is preferable that the biomining additive be considered soluble or substantially soluble in the leaching solution used in the biomining operations. In some embodiments, the acidic leaching solution has a pH between about 1.0 and about 4.0 and, consequently, the biomining additive must be soluble or substantially soluble in the leaching solution. In an optional embodiment, the leaching solution is an inorganic acidic leaching solution such as, for example, a sulfuric acid solution. In an embodiment, the biomining additive has a solubility in the leaching solution of at least 0.001 mg/mL. In other embodiments, the biomining additive has a solubility in the leaching solution of at least 1 mg/mL, 5 mg/mL, 10 mg/mL, 20 mg/mL or 30 mg/mL. In some specific embodiments, the solubility of the biomining additive in the leaching solution is between 20 and 30 mg/mL. In some embodiments, the biomining additive presented herein are based on unnatural chemically-synthesized acyl homoserine lactones to supplement a biomining process.

In some embodiments, the biomining additives defined herein may include a chiral center which gives rise to enantiomers. Such additives may thus exist in the form of two different optical isomers, that is (+) or (-) enantiomers. All such enantiomers and mixtures thereof, including racemic or other ratio mixtures of individual enantiomers, are included within the scope of the disclosure. The single enantiomer can be obtained by methods well known to those of ordinary skill in the art, such as chiral HPLC, enzymatic resolution and chiral auxiliary derivatization. It will also be appreciated that, in some embodiment, the biomining additives can contain more than one chiral centre. The compounds of the present disclosure may thus exist in the form of different diastereomers. All such diastereomers and mixtures thereof are included within the scope of the disclosure. The single diastereomer can be obtained by methods well known in the art, such as HPLC, crystalisation and chromatography. There is also provided salts of the biomining additives described herein. For example, conventional salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, perchloric and the like, as well as salts prepared from organic acids such as formic, acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxy-benzoic, fumaric, toluenesulfonic, methanesulfonic, benzenesulphonic, naphthalene-2-sulphonic, ethane disulfonic, oxalic, isethionic, trifluoroacetic and the like.

Depending on the intended use, a single species of the biomining additive can be used during the biomining operations. Alternatively, more than one species of the biomining additive can be used during the biomining operations. These plurality of species of biomining additive can be added simultaneously or subsequently, depending on the intended use. As such, the present disclosure also provides a composition comprising a biomining additive (or a combination of biomining additives) in the presence of an excipient (DMSO for example). In addition, the present disclosure also provides a kit comprising at least two doses of the (same or different) biomining additive.

In some embodiments, the biomining additive can be formulated in a biomining composition. For example, the biomining additive can be combined with a biomining acceptable excipient (e.g., an excipient amenable to biomining processes), such as, for example, DMSO. In some further embodiments, the biomining additive can be admixed with at least one bacterial medium component or even, in some additional embodiments, with a biomining bacterial population. The biomining composition can be provided as a liquid or solid (e.g. lyophilized).

Improved heap leaching method with bioleaching additive Biomining is broadly divided into of bioleaching and biooxidation. Bioleaching refers to the solubilisation of the mineral element from ores using a microbial population (also referred to as a microbial consortia). For example, copper is currently being bioleached from copper- containing ores such as chalcocite and covellite. On the other hand, biooxidation refers to the dissolution of the ore (containing the mineral element) for facilitation subsequent extraction steps. For example, gold-containing ores are currently being biooxidized by a microbial population prior to the cyanidation extraction of gold. Biomining can be applied to various types of reactors such as those used in dump, heap, in situ or continuous stirred tank reactor (CSTR) leaching.

In the present disclosure, the biomining additive can be used for improving the recovery of a mineral element from an ore. When compared to a biomining process which does not use the biomining additive described herein, this improvement is observed by a reduction in the amount of time to obtain a determined level of a leached element or oxidized element containing-ore and/or an increase of the level of the leached element or oxidized element containing-ore.

Consequently, the present disclosure provides a method for improving the recovery of a mineral element in a biomining process based on the addition of the biomining additive described herein. Broadly, the method comprises contacting an ore suspected of having the mineral element, a bacterial population having a biomining activity and the biomining additive described herein. The steps of the methods should be conducted in conditions (temperature, pH, oxygen/carbon oxide levels, contaminant levels, etc.) suitable to allow leaching of the target mineral element from the ore or oxidation of the element containing- target ore.

Heap leaching can be used in the biomining operations described herein. In heap leaching procedures, the ore is crushed, heaped on a leach pad (considered as the heap leach reactor) and then contacted (either via irrigation or dripping) with a leach solution to dissolve (or leach) the target mineral element. The leach solution percolates through the heap and leaches the target element. In some heap operations, a rotary drum (which agglomerates the crushed ores for obtaining more uniform particle distribution) can be added to improve the leaching process. The leach cycle usually takes between one to twenty-four months, depending on the type of ore and elements it contains. The leach solution containing the dissolved elements can be collected and optionally treated to recover the target mineral elements.

Continuous stirred tank reactor (CSTR) leaching can also be used in the biomining operations described herein. In CSTR leaching procedures, the ore is crushed and placed in a stirred tank reactor and then contacted with a leach solution to dissolve the target mineral. The stirred tank reactor provides a stirring motion to facilitate the dissolution of the target mineral element. The leach solution containing the dissolved elements can be collected and optionally treated to recover the target mineral element. In some embodiment, the leaching process can be conducted over the course of 5 to 40 days or even more, depending on the ore properties and the leaching conditions used.

The biomining additive can be combined with any type of ores amenable to biomining. Since the biomining additive improves the recovery of the target mineral element, it can be advantageously used with lower grade ores (for example those containing less than 0.8% (w/w) of the target mineral element) or refractory ores. In some embodiments, the biomining additive can be used in ores containing as little as 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1 %, 0.01 %, 0.001 % or 0.0001 % (w/w) of the target mineral element.

In some embodiments, the biomining methods described herein can be performed at temperature between 20°C and 60°C, between 20°C and 55°C, between 20°C and 50°C, between 20°C and 45°C, between 20°C and 40°C, between 20°C and 35°C, between 20°C and 30°C, between 20°C and 25°C, between 25°C and 45°C, between 25°C and 40°C, between 25°C and 35°C, between 25°C and 30°C, between 30°C and 60°C, between 30°C and 55°C, between 30°C and 50°C, between 30°C and 45°C, between 30°C and 40°C, between 30°C and 35°C, between 35°C and 60°C, between 35°C and 55°C, between 35°C and 50°C, between 35°C and 45°C, between 35°C and 40°C or at about 20°C, about 25°C , about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C or about 60°C. Consequently, the bacterial population must exhibit biomining activity at those temperatures (e.g. be considered mesophilic or thermophilic bacteria).

In an additional embodiment, the biomining methods described herein can be performed in acidic environments. For example, biomining can be conducted in a leaching solution having a pH below about 4.0, below about 3.0 or even below about 2.0. In some situations, biomining will be conducted in a leaching solution having a pH between about 1 .0 and about 4.0, between about 1.0 and about 3.0, between about 1.0 and about 2.0, between about 2.0 and about 4.0, between about 2.0 and about 3.0 or between about 3.0 and about 4.0. In some embodiments, it is even contemplated that biomining will be conducted in an environment having a fluctuating pH (for example an environment which becomes more acidic as the biomining process occurs). Consequently, the selected bacterial population must exhibit biomining activity in such conditions (e.g. be considered acidophilic bacteria).

The biomining conditions used in the methods presented herein should allow the bacterial population to exhibit its biomining activity, e.g. its ability to oxidize ferrous ions, sulfur, and/or, in embodiments where copper is leaching from chalcopyrite, solubilize copper from chalcopyrite. The microbial population used in the biomining operations described herein must have the ability to oxidize ferrous iron and sulfur in order to promote the leaching of the mineral element or the oxidation of the element containing-ore. Such biomining activity does not need to be constitutive in the bacterial population, it can be elicited prior to or during culture with the ore. Preferably, the microbial population used in the biomining operations described herein are not considered to be pathogenic to animals or humans since they do not tolerate the presence organic substances. For example, the microbial population can include bacteria from the Acidithiobacillus sp. (e.g. Acidithiobacillus ferrooxidans and/or Acidithiobacillus thiooxidans) and/or from the Leptospirillum sp. (e.g. Leptospirillum ferrooxidans). The biomoning operations described herein can be conducted with a single bacterial species/genus or a combination of bacterial species/genus.

In some embodiments, the in situ microbial population (e.g. the microbial population endogenously associated with the ore) can be used in the biomining methods described herein and no additional microbial population is introduced in the reactor during the bioleaching/biooxidation process. In such embodiment, the biomining additive, usually dissolved in the leaching solution, is placed in contact with the microbial population so as to promote the bioleaching/biooxidation process. In some embodiment, a single dose of the biomining additive is introduced during the biomining operations (usually during the initial step of the biomining operations).

In alternative or complementary embodiments, an exogenous microbial population is used in the biomining methods described herein. For example, the exogenous microbial population can be added to an ore having an inadequate number or genus/species of bacteria to promote leaching of the element or oxidation of the ore. The exogenously-supplied microbial population can be added once during the biomining process. Alternatively, when it is determined that the bacterial population in the biomining reactor is too low to further promote the leaching of the element or the oxidation of the ore, a further microbial population can be added to the reactor. The exogenous microbial population can be mixed with the biomining additive when introduced in the bioreactor containing the ore. In one advantageous embodiment, the exogenous microbial population is first primed with the biomining additive prior to its introduction in the bioreactor. In order to do so, the microbial population is cultured with the biomining additive under conditions to promote/elicit their biomining activity. Such priming step can optionally include culturing the bacterial population with a source of elemental sulfur (for example, providing elemental sulfur as the sole substrate to the bacterial culture). Once the bacterial population has been primed, it can be added to the bioreactor containing the ore. It is contemplated that a further dose of the biomining additive be added to the bioreactor upon the introduction of the primed bacterial population in the bioreactor.

Since the addition of elemental sulfur is known to increase the biomining activity of the bacterial population, in some embodiments, the biomining operations described herein can also optionally include adding a source of elemental sulfur to the bioreactor.

In some alternate embodiments, a plurality of doses of the biomining additive is introduced at different time intervals during the biomining operations. For example, the leaching/oxidation levels can be monitored during the biomining operations and the biomining additive can be added when it is determined that the leaching of the element or the oxidation of the ore has reached a plateau or needs to be further promoted.

In the biomining operations described herein, the ore, the bacterial population and the biomining additive are contacted with a solution favoring the leaching of the element or the oxidation of the ore. The solution (referred to herein as the leaching solution) is used to absorb or store the leached element or the oxidized ore. It can be further treated (for example being submitted to a cyanidation reaction) to obtain or further purify the target element. One advantageous leaching solution is an inorganic leaching solution, such as, for example, a sulfuric acid solution, a nitric acid solution or an hydrochloric acid solution.

The biomining operations described herein are not limited to any specific ore and can be applied to recover various mineral elements such as copper, zinc, lead, arsenic, antimony, nickel, molybdenum, gold, silver, uranium and cobalt. Ores amenable to the biomining operations described herein include, but are not limited to, copper-containing ores (e.g., chalcopyrite (CuFeS ₂ ), chalcocite (Cu ₂ S), covellite (CuS), malachite (Cu ₂ C0 ₃ (OH) ₂ )), zinc- containing ores (e.g., sphalerite (ZnS)), lead-containing ores, arsenic-containing ores, antimony-containing ores, nickel-containing ores (e.g., pentlandite (Fe, Ni) ₉ S ₈ ), molybdenum-containing ores (e.g., molybdenite (MoS ₂ )), gold-containing ores (e.g. arsenopyrite), silver-containing ores (e.g., ergentite (Ag ₂ S)), uranium-containing ore (e.g., uraninite (U0 ₂ )) and cobalt-containing ores (e.g., cobaltite ((Co, Fe)AsS)). The biomining operations described herein can be advantageously used with refractory ores or lower grade ores.

The biomining methods described herein can be advantageously used to recover copper from various ores. As indicated above, it is known in the art that mineral copper is not efficiently bioleached from chalcopyrite. As shown below, the biomining additive described herein does improve the recovery of copper from chalcopyrite. Heap leaching is advantageously used to recover copper from its ore (especially chalcopyrite).

The biomining methods described herein can also be advantageously used to recover ores from various ores (such as pyrite or arsenopyrite). Continuous stirred tank reactor (CSTR) leaching is advantageously used to recover gold from its ore (especially pyrite and arsenopyrite).

As it is known in the art, gold cannot be leached directly in a leaching solution. The gold containing ore is oxidized (e.g. biooxidized in biomining operations) during the leaching process and then submitted to cyanidation to recover the mineral element. In biomining operations, the bacterial population oxidizes the sulfide minerals, thus liberating the occluded gold for subsequent recovery via cyanidation. The biooxidation process is preferably conducted in an acidic environment (e.g. having a pH between about 1.0 and about 4.0 as indicated above) and at temperature of between about 30°C to about 45°C. To ensure optimal bacterial biomining activity, a steady supply of oxygen and carbon can also be provided. The bacterially mediated oxidation of iron sulfide minerals produces iron(lll) sulfate and sulfuric acid, and in the case of arsenopyrite, arsenic acid is also produced.

The present disclosure will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.

EXAMPLE I - SYNTHESIS OF HOMOSERINE LACTONES DERIVATIVES

The synthesis of N-acyl L-homoserine lactones (AHLs) is achieved using standard amide bond formation conditions and reagents (Synthetic scheme 1 ). The L-homoserine lactone ring was made on a multigram scale (20 mmol scale, 28 g, 78% yield) from the amino acid L-methionine cyclized with bromoacetic acid and hydrobromic acid (Persson et al., 2005). Amide bond formation of the homoserine lactone ring and the appropriate n-alkyl acyl chloride (n = even numbers 4 to 16) is conducted under Schotten-Baumann biphasic condtions of chlorinated solvent dichloromethane and aqueous sodium carbonate solution (Hodgkinson et al., 201 1 ). Synihetic scheme ί - General synthesis of AHL derivatives

The desired AHLs were isolated in yields greater than 85% for alky! chains 6 to 16, with the highly water-sofuble n = 4 chain lower at 59%.

EXAMPLE II - SCREENING OF HOMOSERINE LACTONE DERIVATIVES FOR EFFECT

ON COPPER SOLUBILIZATION

Some of the AHLs synthesized in Example I were tested for their ability to so!ubilize copper from pure chalcopyrite. The screening was performed using a rhodamine dye that selectively produces a coiorimetric output in response to copper ions as described elsewhere (Huoet aL 2010). The rhodamine dye was used in a 96-weil microtiter plate format to measure copper recovery under a variety of AHLs and concentrations. The results are shown in Figure 1. It is worth noting that an AHL with a long unsubstituted acyl tail (C16-AHL) displays enhanced copper recovery. These results also highlights that C16-AHL, which has been previously shown incapable of increasing biofilm formation on pyrite (e.g., Ruiz et al., 2008), can effectively be used to increase bioleaching of copper from chalcopyrite.

EXAMPLE III - 2 KG BOTTLE ROLL TESTS

Bottle roil tests invofved 5 L bottles with approximately 2 kg of crushed chalcopyrite mixed in sulfuric acid. The chalcopyrite was supplemented with either DMSO solvent (as a control) or an AHL derivative dissolved in DMSO. Stock solution was made 24/48 hours before AHL addition to the bottles. Stock solutions where made at a concentration of 50 mM AHL in DMSO (311.5 mg AHL dissolved in 20 mL DMSO).

250 pL of the 50 mM stock solution was added to bottles to reach a final concentration of 5 μΜ. For negative control bottles, a final DMSO concentration of 0.01% v/v DMSO was used. No additional bacteria is inoculated in the bottles, e.g. the native consortia is the only bacteria present. Bottles were then agitated. Samples were retrieved every 48 hours and soluble copper was measured by inductively coupled plasma mass spectrometry (ICP-MS). Tests were performed ay 60°C in duplicate. Results are shown in Figure 2 for the C16-AHL additive. After 120 days, it is observed that the presence of C16-AHL enhances copper leaching.

EXAMPLE IV - INFLUENCE OF AHL DERIVATIVES ON SUBSTRATE UTILIZATION

The effect of AHL derivative treatment on substrate utilization in Acidothiobacillus ferrooxidans cell cultures was determined. All wells were inoculated with 30 pL (3%) of A. ferrooxidans cells at semi-exponential growth phase on iron. Inoculation stock had been prepared by 25% previous inoculum into 20 g/L ferrous sulphate 9K media pH 2.3 five days earlier and cultivated during shaking at 30 degrees Celsius.

Cells were inoculated into two different media. For iron media, filter sterilized tap H ₂ 0 was supplemented with 9K salts and 20 g/L ferrous iron sulphate, pH 2.5 (total volume per well: 970 pL).

For sulphur media filter, sterilized tap H ₂ 0 was supplemented with 9K salts, 20 g/L ferrous iron sulphate and 2.5 g/L 4-thionate, pH 2.5 (total volume per well: 970 pL).

As shown in Figure 3A, high concentrations of the C16-AHL resulted in lower iron oxidation in the presence of a sulphur species is present. In the absence of sulphur, as shown in Figure 3B, the amount of iron oxidized is similar across all C16-AHL concentrations. Taken together, these results indicated that the C16-AHL may modulate the substrate utilization preference of the bacteria, which may impact the redox poise of the leaching environment.

EXAMPLE V - BOTTLE ROLL TESTS Bottle roll tests involved crushed chalcopyrite mixed in sulfuric acid. The chalcopyrite was supplemented with either DMSO solvent (as a control) or an AHL derivative dissolved in DMSO. Stock solution was made 24/48 hours before AHL addition to the bottles. Stock solutions where made at a concentration of 50 mM AHL in DMSO (311.5 mg AHL dissolved in 20 mL DMSO). 250 μΙ_ of the 50 mM stock solution was added to bottles to reach a final concentration of 5 μΜ. For negative control bottles, a final DMSO concentration of 0.01% v/v DMSO was used. No additional bacteria is inoculated in the bottles, e.g. the native consortia is the only bacteria present. Bottles were then agitated. Samples were retrieved every 48 hours and soluble copper was measured by inductively coupled plasma mass spectrometry (ICP-MS). Tests were performed at 60°C in duplicate.

Results are shown in Figure 4 for the C12-AHL, C14-AHL and C16-AHL. After 14 days, the AHL derivative enhance copper leaching in samples in which no additional bacteria has been added (e.g., only the native consortia is present). The use of C14-AHL derivative increased the concentration of solubilized copper by 30% in the leach liquor, when compared to the control.

EXAMPLE VI - INFLUENCE OF AHL DERIVATIVES ON Fe LEACHING FROM

CHALCOPYRITE

Test columns containing approximately 50 kg of crushed chalcopyrite mixed in sulfuric acid were set up. The chalcopyrite was supplemented with either DMSO solvent (as a control) or a C14-AHL derivative dissolved in DMSO. Stock solution was made 24/48 hours before AHL addition to the bottles. Stock solutions where made at a concentration of 50 mM C14-AHL in DMSO (311.5 mg AHL dissolved in 20 mL DMSO). The stock solution was added to columns to reach a final concentration of 5 μΜ. For negative control bottles, a final DMSO concentration of 0.01 % v/v DMSO was used. No additional bacteria is inoculated in the bottles, e.g. the native consortia is the only bacteria present. Tests were performed at room temperature in duplicate.

After 5 months, as shown in Figure 5, the use of a C14-AHL derivative (when compared to the DMSO control) increased copper leaching (Fig. 5A), decreased the F ²⁺ /F ³⁺ ratio in the leach liquor (Fig 5B), did not modulate the reduction ratio of the leach liquor (Fig. 5C) and reduced the total iron content (Fig. 5D).

While the invention has been described in connection with specific embodiments thereof, it will be understood that the scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole. REFERENCES

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