FIELD OF THE INVENTION

[0001 ] Provided herein are a biological sorbent reagent for extracting a metal from a solution comprising the metal, comprising one or more of a Mucoromycota fungus and a biomass produced by the Mucoromycota fungus, a biological sorbent reagent for extracting a metal from a solution comprising the metal, comprising one or more of a fungus and a biomass produced by the fungus, wherein the biomass is hydrophilic and has a point of zero charge (pHPZC) corresponding to the pH of the solution comprising the metal, and a method for extracting a metal from a solution comprising the metal, comprising contacting the solution comprising the metal with a filter comprising the biological sorbent reagent of the present disclosure.

BACKGROUND OF THE INVENTION

[0002] Waste and industrial waters are commonly charged with metals. Some of such metals are toxic (e.g., heavy metals), but others are economically valuable (e.g., critical metals). Therefore, there has been a need for efficiently and sustainably extracting the metals (especially of aluminum (Al), calcium (Ca), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), nickel (Ni), zinc (Zn), and vanadium (V)) from a solution comprising the metals so that they can be reintroduced in an industrial circuit.

STATE OF THE ART

[0003] The so called Clean TeQ Water’s CLEAN-IX® technology provides highly efficient extraction and purification of a range of metals from slurries and solutions. CLEAN-IX® is a combination of continuous ion exchange and counter- current ion exchange, where the resin and slurry/solution are continuously moved around the system, and are directly contacted in countercurrent to each other. This achieves an extraction rate of typically more than 98% of the contained metal in the solution, and provides an economical method for recovery and concentration of dilute streams.

[0004] CLEAN IX metal recovery technologies achieve very high eluate concentrations, by providing customized flowsheets, with each ion exchange process step occurring in a dedicated vessel which is optimally sized for its task.

[0005] However, this conventional system is complex and there exist needs to provide methods and tools that could be used in the efficient recovery in rapid, cost efficient and more ecological manner.

SUMMARY OF THE INVENTION

[0006] The present disclosure provides a sustainable approach based on the use of microbe-mineral interactions (i.e., immobilizing metals in a solution; a process called biosorption). The biosorption method is sustainable as it affords advantageously a lower carbon footprint compared to an abiotic method, such as Clean TeQ Water’s CLEAN-IX® technology.

[0007] In particular, the present disclosure provides a biological sorbent reagent for extracting a metal from a solution comprising the metal, comprising one or more of a Mucoromycota fungus and a prepared (or processed) biomass of the Mucoromycota fungus. In one embodiment, the Mucoromycota fungus is Mucor moelleri, especially the strain NEUM140 identified by GENBANK® ID accession number MZ374564. Others preferred examples of Mucoromycota fungus are M. hiemalis, especially the strain NEUM 144 identified by GENBANK® ID accession number OR478153 and M. saturninus, especially the strain NEUM 172 identified by GENBANK® ID accession number OR478154. In another embodiment, the biomass is produced by culturing the Mucoromycota fungus in a medium comprising glucose, dextrose or glycerol, which are carbon sources for microbial (or Mucoromycota fungus) growth. Glycerol or crude glycerol is the main byproduct of biodiesel production and may be used as carbon source for growth of Mucoromycota fungus. Here, the metal may be one or more selected from the group consisting of aluminum (Al), calcium (Ca), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), nickel (Ni), zinc (Zn), and vanadium (V).

[0008] The present disclosure also provides a biological sorbent reagent for extracting a metal from a solution comprising the metal, comprising one or more of a fungus and a prepared or processed biomass of the fungus, wherein the biomass is hydrophilic and has a point of zero charge (pH _PZ c) corresponding to the pH of the solution comprising the metal. In one embodiment, the point of zero charge (pHpzc) corresponding to the pH of the solution comprising the metal is pH 2 to 8. In another embodiment, the fungus is a Mucoromycota fungus. In another embodiment, the Mucoromycota fungus is Mucormoelleri, especially the strain NEUM140 identified by GENBANK® Accession Number MZ374564. Others preferred examples of Mucoromycota fungus are M. hiemalis, especially the strain NEUM 144 identified by GENBANK® Accession Number OR478153 and M. saturninus, especially the strain NEUM 172 identified by GENBANK® Accession Number OR47815. The biomass may be produced by culturing the fungus in a medium comprising a carbon source such as glucose, dextrose or glycerol or crude glycerol. In addition, the metal may be one or more selected from the group consisting of aluminum (Al), calcium (Ca), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), nickel (Ni), zinc (Zn), and vanadium (V).

[0009] In addition, the present disclosure provides a method for extracting a metal from a solution comprising the metal, comprising contacting the solution comprising the metal with a filter comprising a biological sorbent reagent comprising one or more of a Mucoromycota fungus and a prepared or processed biomass of the Mucoromycota fungus. In one embodiment, the filter comprises a membrane comprising the biological sorbent reagent. In another embodiment, the membrane further comprises a fabric. The membrane may be prepared from the biological sorbent reagent using electrospinning. In another embodiment, the filter is a bead or a hollow spherical cavity comprising the biological sorbent reagent. In another embodiment, the filter comprises a plurality of hollow spherical cavities ranging from 0.05 - 1 mm; preferably 0.1 - 0.5 mm, wherein the biological sorbent reagent is contained within the cavity. The hollow spherical cavities may comprise polymeric (such as polypropylene) material, metallic material (Al based), non-metallic material (Si based) and I or ceramic material (silica or alumina).

[0010] The present disclosure also provides a method for extracting a metal from a solution comprising the metal, comprising contacting the solution comprising the metal with a filter, a bead or a compact column comprising the biological sorbent reagent comprising one or more of a fungus and a prepared or processed biomass of the fungus, wherein the biomass is hydrophilic and has a point of zero charge (pHpzc) corresponding to the pH of the solution comprising the metal. In one embodiment, the fungus is a Mucoromycota fungus. In one embodiment, the filter comprises a membrane comprising the biological sorbent reagent. In another embodiment, the membrane further comprises a fabric. In another embodiment, the filter comprises a porous material. In one embodiment, the filter comprises a porous polymer, such as polypropylene. In another embodiment is filter comprises a porous material such as metals, ceramics. In one embodiment, the filter is a bead or a compact column comprising the biological sorbent reagent. In another embodiment, the membrane is prepared from the biological sorbent reagent using electrospinning. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIGS. 1A, 1 B and 1 C show data relating to the amount of the residual metals and adsorption capacity.

[0012] FIGS. 2 (a-c) show the pH _pzc of Mucor moelleri (NEUM140), Mucor saturnius (NEUM144) and Mucor hiemalis (NEUM 172).

[0013] FIG.3 represents the results (mg of vanadium /kg of biomas).of Biosorption experiment

[0014] FIG. 4 represents a custom-made packed bed reactor having porous filter (sponge) comprising biological sorbent reagent (M. moelleri inoculated inside the sponge).

[0015] FIG.5 represents different removal rates (% of Metal removal with fungal biosorption according to time (minutes)) obtained during M. moelleri continuous flow experiment

DEFINITIONS

[0016] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments described herein, some preferred methods, compositions, devices, and materials are described herein. However, before the present materials and methods are described, it is to be understood that this disclosure is not limited to the particular molecules, compositions, methodologies or protocols herein described, as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the embodiments described herein.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. However, in case of conflict, the present specification, including definitions, will control. Accordingly, in the context of the embodiments described herein, the following definitions apply.

[0018] As used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise.

[0019] As used herein, the term “comprise” and linguistic variations thereof denote the presence of recited feature(s), element(s), method step(s), etc., without the exclusion of the presence of additional feature(s), element(s), method step(s), etc. Conversely, the term “consisting of’ and linguistic variations thereof, denotes the presence of recited feature(s), element(s), method step(s), etc., and excludes any unrecited feature(s), element(s), method step(s), etc., except for ordinarily-associated impurities. The phrase “consisting essentially of’ denotes the recited feature(s), element(s), method step(s), etc., and any additional feature(s), element(s), method step(s), etc., that do not materially affect the basic nature of the composition, system, or method. Many embodiments herein are described using open “comprising” language. Such embodiments encompass multiple closed “consisting of” and/or “consisting essentially of’ embodiments, which may alternatively be claimed or described using such language.

[0020] As used herein, the term “biological sorbent reagent for extracting a metal from a solution” refers to a reagent which is able to adsorb a metal from a solution including the metal and other materials, preferably as complexes.

[0021] As used herein, the terms “prepared (or processed) biomass produced by the Mucoromycota fungus”, mean a biomass obtained from a liquid culture of the Mucoromycota fungus that has been autoclaved and/or washed and dried and/or ground to obtain a powder. [0022] As used herein, the terms “Mucoromycota fungus” refer to a division or phylum within the kingdom fungi, and include a diverse group of various molds. This division includes at least Mucor moelleri, especially the strain NEUM140 identified by GenBank® Accession Number MZ374564; Mucor hiemalis, especially the strain NEUM144 identified by GenBank® Accession Number OR478153 and M. saturninus, especially the strain NEUM172 identified by GENBANK® Accession Number OR478154 .

[0023] As used herein, the term “biomass produced by the Mucoromycota fungus (or the fungus)” refers to a material produced by the Mucoromycota fungus (or the fungus) while culturing it.

[0024] As used herein, the term “hydrophilic” refers to the physicochemical property where a material, in particular a fungal surface of this invention, has an affinity for a solution where the solvent is water (H2O) represented by a water contact angle that is less than 60° or preferably less than 45°.

[0025] As used herein, the term “point of zero charge (pHpzc) corresponding to the pH of the solution comprising the metal” refers to the pH at which the net charge of total particle surface (i.e. absorbent’s surface) is equal to zero.

[0026] As used herein, the term “electrospinning” refers to a method to produce ultrafine (in nanometers) fibers by charging and ejecting a polymer melt or solution through a spinneret under a high-voltage electric field and to solidify or coagulate it to form a filament.

[0027] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

DETAILED DESCRIPTION OF THE INVENTION

The biological sorbent reagent for extracting a metal from a solution comprising the metal

[0028] The present disclosure provides a biological sorbent reagent for extracting a metal from a solution comprising the metal, comprising one or more of a Mucoromycota fungus and a prepared (or processed) biomass produced by the Mucoromycota fungus. Here, the metal may be extracted (or recovered) from the solution by being adsorbed to the biological sorbent reagent. In one embodiment, the Mucoromycota fungus may be Mucor moelleri. Mucor moelleri has been isolated at the laboratory of Microbiology of the University of Neuchatel from a soil sample taken at the Creux-du-Van (Neuchatel, Switzerland), and has been deposited to GenBank®, the National Center for Biotechnology Information genetic sequence database at 8600 Rockville Pike, Bethesda, MD 20894, United States of America on July 2 ^nd 2021 under the accession number of MZ374564.

[0029] In another embodiment, the biomass is produced by culturing the Mucoromycota fungus in a medium comprising glycerol. In one embodiment, the prepared (or processed) biomass is produced by autoclaving and/or washing a liquid culture of the Mucoromycota fungus, and drying and/or grinding it to obtain a powder.

[0030] In another embodiment, the biomass may be pre-treated (preconditioned) with acidic and alkaline solutions or not pre-treated with these acidic and/or alkaline solutions. Furthermore, a rehydrating step of the biomass is possible. However, in the claimed method this rehydrating step is not present. Here, the metal may be one or more selected from the group consisting of aluminum (Al), calcium (Ca), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), nickel (Ni), zinc (Zn), and vanadium (V).

[0031] The present disclosure also provides a biological sorbent reagent for extracting a metal from a solution comprising the metal, comprising one or more of a fungus and a prepared or processed biomass of the fungus, wherein the biomass is hydrophilic and has a point of zero charge (pH _PZ c) corresponding to the pH of the solution comprising the metal. Here, the point of zero charge (pHpzc) corresponding to the pH of the solution comprising the metal may be pH 2 to 8. In one embodiment, the concentration of the metal in the solution is approximately between 2 and 5 mg/L. In one embodiment, the fungus is a Mucoromycota fungus. In another embodiment, the Mucoromycota fungus is Mucor moelleri (NEUM 140); preferably the strain corresponding to the GENBANK® accession number MZ374564. Others preferred examples of Mucoromycota fungus are M. hiemalis (NEUM 144) , preferably the strain corresponding to the GENBANK® accession number OR478153 and M. saturninus (NEUM172 preferably the strain corresponding to the GENBANK® accession number OR478154. The biomass may be produced by culturing the fungus in a medium comprising glycerol. In one embodiment, the biomass is produced by autoclaving and/or washing a liquid culture of the Mucoromycota fungus, and drying and/or grinding it to obtain a powder. [0032] In addition, the metal may be one or more selected from the group consisting of aluminum (Al), calcium (Ca), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), nickel (Ni), zinc (Zn), and vanadium (V).

The method for extracting a metal from a solution comprising the metal

[0033] The present disclosure further provides a method for extracting a metal from a solution comprising the metal, comprising contacting the solution comprising the metal with a filter, a bead or a compact column comprising a biological sorbent reagent comprising one or more of a Mucoromycota fungus and a prepared (or processed) biomass of the Mucoromycota fungus. Here, the metal may be extracted (or recovered) from the solution by being adsorbed to the biological sorbent reagent. Contacting the solution with a filter may include any methods for exposing the biological sorbent reagent to the solution comprising the metal so that the metal may be adsorbed to the biological sorbent reagent. In one embodiment, this may be done by flowing the solution comprising the metal through the filter, upon beads or through the compact column which comprises the biological sorbent reagent. In one embodiment, a circular flow may be used to expose the biological sorbent reagent to the solution comprising the metal. The circular flow, however, ensures that no desorption of metal takes place. In another embodiment, a parallelized flow or a combination of filters may be used to expose the biological sorbent reagent to the solution comprising the metal. Here, the filter may be a membrane filter which is made of, or includes the biological sorbent reagent, or where the biological sorbent reagent is immobilized. The membrane may further comprise a fabric. Here, the fabric may be a waste material that acts as a physical substrate for a microbial growth but does not contain any bioavailable/bioaccessible carbon for growth. In one embodiment, the membrane is prepared from the biological sorbent reagent using electrospinning. In addition, the filter may also be a bead or a compact column comprising the biological sorbent reagent. Here, the bead, or the compact column for instance, may include the biological sorbent reagent upon one or more of its surface(s). In one embodiment, the bead and the compact column may be hydrophilic and/or porous. The porous compact column may comprise polymeric materials such as a sponge comprising polypropylene. In another embodiment, the Mucoromycota fungus is Mucormoelleri, especially the strain NEUM140 identified by GENBANK® accession number MZ374564. Others preferred examples of Mucoromycota fungus are M. hiemalis (NEUM 144) , preferably the strain corresponding to the GENBANK® accession number OR478153 and M. saturninus. (NEUM 172), preferably the strain corresponding to the GENBANK® accession number OR478154. The biomass may be produced by culturing the fungus in a medium comprising glucose or glycerol or crude glycerol. In one embodiment, the biomass is produced by autoclaving and/or washing a liquid culture of the Mucoromycota fungus, and drying and/or grinding it to obtain a powder. In another embodiment, the biomass may be pretreated with acidic and alkaline solutions or not. In addition, the metal may be one or more selected from the group consisting of aluminum (Al), calcium (Ca), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), nickel (Ni), zinc (Zn), and vanadium (V).

[0034] The present disclosure also provides a method for extracting a metal from a solution comprising the metal, comprising contacting the solution comprising the metal with a filter, beads or a compacts column comprising the biological sorbent reagent comprising one or more of a fungus and a prepared (or processed) biomass of the fungus, wherein the biomass is hydrophilic and has a point of zero charge (pHPZC) corresponding to the pH of the solution comprising the metal. Here, the metal may be extracted (or recovered) from the solution by being absorbed to the biological sorbent reagent. Contacting the solution with a filter; beads or a compact column may include any methods for exposing the biological sorbent reagent to the solution comprising the metal so that the metal may be adsorbed to the biological sorbent reagent. In one embodiment, this may be done by flowing the solution comprising the metal through the filter; beads or compact column, which comprises the biological sorbent reagent. In one embodiment, a circular flow may be used to expose the biological sorbent reagent to the solution comprising the metal. Here, the filter may be a membrane filter which is made of, or includes the biological sorbent reagent, or where the biological sorbent reagent is immobilized. The membrane may further comprise a fabric. Here, the fabric may be a waste material that acts as a physical substrate for a microbial growth but does not contain any bioavailable/bioaccessible carbon for growth. In one embodiment, the membrane is prepared from the biological sorbent reagent using electrospinning. In addition, the filter may also be a bead or a compact column comprising the biological sorbent reagent. Here, the bead or the compact columns, for instance, may include the biological sorbent reagent upon one or more of its surface(s). In one embodiment, the bead or the compact column may be hydrophilic and/or porous. The porous compact column may comprise polymeric materials such as a sponge comprising polypropylene. Here, the point of zero charge (pHPZC) corresponding to the pH of the solution comprising the metal may be pH 2 to 8, preferably 2 to 5. In one embodiment, the concentration of the metal in the solution is approximately between 2 and 5 mg/L. In one embodiment, the fungus is a Mucoromycota fungus. In another embodiment, the Mucoromycota fungus is Mucor moelleri (NEUM 140), preferably the strain having the GENBANK® accession number MZ374564. Others preferred examples of Mucoromycota fungus are M. hiemalis (NEUM 144) , preferably the strain corresponding to the GENBANK® accession number OR478153 and M. saturninus. (NEUM 172), preferably the strain corresponding to the GENBANK® accession number OR478154. The biomass may be produced by culturing the fungus in a medium comprising glucose or glycerol. In one embodiment, the biomass is produced by autoclaving and/or washing a liquid culture of the Mucoromycota fungus, and drying and/or grinding it to obtain a powder. In addition, the metal may be one or more selected from the group consisting of aluminum (Al), calcium (Ca), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), nickel (Ni), zinc (Zn), and vanadium (V).The present invention will be described in details in the following examples and in reference to the enclosed drawings, both being presented as preferred, but not limiting preferred embodiments of the present invention

EXAMPLES

1. Confirmation of of the bioloqical sorbent to metals

Biomass synthesis /production

[0035] The fungal biomass production is described on Figure 1 The flowsheet applies for all Mucor strains used into this project (NEUM140 corresponding to M. moelleri which is the most used strain on project experiments, NEUM144 corresponding to M. saturninus and NEUM172 corresponding to M. hiemalis).

[0036] First, Mucor asexual spores are transferred to an agitated and baffled reactor together with the cultivation medium. The cultivation medium is composed by two main ingredients (Potato starch and D(+)-glucose - or dextrose). The proportion of potato infusion powder and glucose used are preferably 4 g/L and 20 g/L, respectively. Glycerol can replace glucose on the cultivation medium. The mixture of spores and cultivation medium is kept under room temperature (22-25°C) and 120 rpm agitation. Total residence time is between 4 to 8 days, preferably 7 days. After the cultivation step is finished, the resulting biomass is transferred to an autoclave step where the mixture is heated at 121 °C for 20 minutes.

[0037] The slurry obtained from the autoclave step is then transferred to a filtration unit (preferably a disk or pressure filter) where solid-liquid separation followed by a washing step will be performed. The washing is performed using bi-distilled water, wherein the ratio of slurry to water is preferably 1 :5. After the washing step, the remaining solid material is transferred to a drying step. Drying is performed according to common heating techniques to achieve less than 1 % moisture. Temperature for drying step is 60°C and total residence time is between 4 hours to 48 hours, preferably 24 hours. The dried biomass is then submitted to a griding step, where the material is pulverized into powder. The grinding step is performed using an ultra-turrax® homogenizer. The biomass can then be used for metals adsorption steps.

[0038] According to a preferred embodiment, the biomass for the biological sorbent reagent according to the invention was obtained from liquid cultures of Mucor moelleri (Mucoromycota fungus) in potato-dextrose broth (20g.1’1 D- glucose, 4 g.l’ ¹ potato starch) which have been autoclaved, lyophilized, and grinded into a thin powder. Batch experiments were held in 250 ml Erlenmeyer flasks at room temperature, on a rotary shaker (120 rpm) for 30 min with 100 mg of prepared biomass. The biomass was separated from the solution through a 0.1 - 0.5 pm (preferably between 0.2 and 0.25 microns) cellulose membrane, such as nitrocellulose or cellulose acetate, using a vacuum pump.

Contact angle measurement

[0039] Water contact angle was used to evaluate the hydrophobicity of Mucor mycelium. For this, asexual spores were inoculated in 20 ml of malt broth (12 g/L) placed in 50 ml Schott bottles. Cultures were incubated at room temperature and under constant agitation on a rotary shaker (120 rpm) until the presence of biomass visible to the naked-eye. Fungi used in this study were: Aspergillus niger, Mucor moelleri, Mucor hiemalis, Mucor saturninus, Beauveria bassiana, B. caledonica, B. brongniartii, Penicillium commune, P. brevicompactum and P. spinulosum.

[0040] The resulting biomass, consisting of mycelial pellets, was recovered through a sieve, transferred into saline water (0.9% NaCI), and homogenized using an ultra-turrax®. The obtained suspension was centrifuged at 5000rpm for 5 minutes and the supernatant discarded. The pellet was washed with 5 ml saline water and centrifuged as described previously. Finally, biomass was resuspended in 5 ml saline water and deposited on the surface of a nitrocellulose filter (pore size 0.45pm, diameter of the filter 0 22mm) by vacuum filtration.

[0041] Dry biomass was also assessed. For this, biomass obtained from a liquid culture was sieved, autoclaved at 121 °C for 20 minutes, and dried for 48 hours at 60°C. After this, biomass was crushed with a mortar and pestle into a powder. Then powders are placed on a tape for contact angle measurements according to the technique known in the art.

[0042] This biomass was then used to measure the contact angle of 30 pL water droplets at their surface using a goniometer connected to a camera. The contact angles as measured for Mucor mycelium were lower than 60°. pH of Point Zero(pHpzc)

[0043] The aim of this experiment is to measure the pH value at which biomass reaches a point of zero charge, in other words a pH value when there is a perfect balance between negative and positive charges at the surface of the biomass. Favorable biosorption of anions and cations occurs at pH values smaller and greater than pHpzc, respectively. Maximum adsorption of vanadium ions in a solution at pH 4.5 was obtained using state-of-the-art technique as described in Saudi journal of biological sciences 25.8 (2018): pages 1664-1669.

[0044] Solutions having a pH of 2, 3, 4, 5, 6 and 8 were obtained using 25 ml of 0.1 M NaCI with addition of NaOH or HCI until the required pH are attained. 0.05 g of prepared fungal biomass is added to the individual solutions and placed for 24h on a rotary shaker at 120 rpm. Initial and final pH are measured to calculate the pHpzc. Similar to the measurement of the contact angle, a number of fungus species were tested including those belonging to the group Mucor. Two of the Mucor species shows pH _pzc at about 4.5 (4.62 for Mucor moelleri (NEUM140) and 4.66 for Mucor saturnius (NEUM144). Figure 2 (a-c) shows the obtained pH _pzc of the tested strains Mucor moelleri (NEUM140), Mucor saturnius (NEUM144) and Mucor hiemalis (NEUM 172).

Adsorption Kinetics (determination of minimum contact time / extraction)

[0045] To determine the adsorption kinetics, the inventors have prepared several batches of Mucor biomass which have been stored at 4°C until further use. Solutions containing vanadium ions (sodium orthovanadate (NasVC ) or vanadium (III) chloride or (VCI3)) at 1 ppm or 3.5 ppm at a pH set at 4.5 were prepared using bi-distilled water to avoid any other metal presence. These two vanadium ion containing solutions are also compared with actual processed water that contains 3.5 ppm of vanadium ions. Said otherwise, the processed water from an industrial chemical synthesis has an initial concentration of vanadium of 3.5mg/L and a pH of 4.5.

Biosorption experiments

[0046] To test the effect of time on the adsorption of vanadium, 0.1 g of prepared biomass was added to 25 ml of a vanadium solution and shaken at 120 rpm at room temperature for 30, 180, 360 and 480 minutes. Control was shaken at the same conditions for 120 minutes.

[0047] After contact between biomass and vanadium solutions for the given time, samples were vacuumed through 47mm cellulose membranes to separate the biomass from the liquid phase. Metal contents of both fractions were analyzed with an ICP-OES. Liquid phase samples were previously acidified with HNO3 and biomass samples were first mineralized to dissolve the organic material. The analyses results are shown in Fig 3.

[0048] The adsorption capacity of the biological sorbent (M. moelleri biomass as described above) reagent obtained was evaluated with vanadium containing solutions: 1) processed water from an industrial chemical synthesis that has an initial concentration of vanadium of 3.5mg/L and a pH of 4.5; and 2) a solution of NasVC dissolved in bi-distilled water (referred to as V solution in FIGS. 1 A, 1 B and 1 C), with a concentration of vanadium of 3.5mg/L and a pH of 4.5.

[0049] As shown in FIGS. 1A, 1 B and 1 C, where “Control” stands for the experiment run without biomass and “Fungus” with prepared M. moelleri biomass, the biological sorbent reagent (Fungus) removed vanadium from both the processed water and the NasVO4 solution (FIG 1A), corresponding to approximately 75-78% of vanadium in solution (FIG. 1 B). Adsorption capacity (mg/g) is similar to other biological sorbents described in the scientific literature to-date (FIG. 1 C).

[0050] After contact with the vanadium solution (1 ppm), prepared biomass of M. moelleri adsorbed Vanadium ions. Vanadium in the solution decreased significantly after 30 minutes contact with the biomass. Conversely, Vanadium ion concentration increased rapidly in the solid biomass, and it reaches a plateau between 20 mg and 25 mg of vanadium for 1 kg of biomass after 180 minutes as shown in Figure 3c. After this, no significant change is found in the vanadium concentration in either the liquid or the biomass fractions after 3 hours.

[0051] The adsorption capacity (Q mg/g) and rate (R %) of M. moelleri were defined using the following equations (1) and (2):

[0052] where Q (mg/g) is the adsorption capacity, R (%) is the adsorption rate, V is the volume of the solution (L), M is the mass of the biosorbent (g), Co is the V concentration before adsorption (mg/L) and C is the V concentration after adsorption (mg/L). Adsorption capacity is defined as the amount of adsorbate (vanadium or other metals ions) taken up by the adsorbent per unit mass (per g of M. moelleri) of the adsorbent. In other words, the adsorption capacity is the amount of metals (mg) that M. moelleri (g) can adsorb.

[0053] Metal concentrations that increase in the solution is illustrated by the biomass releasing metal ions in the solution, therefore biomass desorbs metals. On the contrary, metals ions decreasing the solution is synonym for metal adsorption by biomass. A positive adsorption capacity (Q > 0) represents an adsorption phenomenon; a negative adsorption capacity (Q < 0) represents a desorption phenomenon. Adsorption capacities and rates were adapted in the biomass values by transforming it into its inverse.

[0054] Vanadium has been adsorbed from the solution by the solid biomass and after 3 hours between 50 - 60% of the vanadium ions in the solution have been adsorbed at the surface of prepared biomass of Mucor moelleri. The decrease of vanadium concentration in the solution is in accordance with the increase of vanadium in the prepared biomass.

[0055] The adsorption capacity of M. moelleri is promising: with only 0.1g of dried biomass, more than 10% of its weight can be uptaken in 30 minutes. As the rates show a rapid increase tending to 60% of vanadium removal, longer exposition between biomass and solution might increase to 70% or more.

(1) A hydrophilic membrane filter comprising the biological sorbent reagent

[0056] The hydrophilic membrane filter (hydrophilic mycelial mats of Mucor moelleri) is prepared by the following steps.

(i) Preparation of a viscous basic liquid medium (VBLM): 30 g/L starch, 80 g/L molasses, 10 g/l neopeptone, and 2 g/l yeast extract.

(ii) Prepare tissue or fabric pieces in glass Petri dishes and spray with VBLM until it is fully impregnated.

(iii) Leave them to dry under a laminar flow hood and autoclave

(iv) Inoculate with an asexual spore suspension containing 103 spores/mL (trying to disperse the spores homogeneously at the tissue surface).

(v) Incubate at RT, without light.

(2) A membrane filter comprising the biological sorbent reagent in electrospunned powder form

[0057] The membrane filter comprising the biological sorbent reagent in electrospunned powder form (electrospunned powders of dried and dead Mucor moelleri biomass) is prepared as follows.

[0058] Polycaprolactone (PCL) (Mw = 80,000 g mol-1), glacial acetic acid (AA, 99%), formic acid (FA, > 95%), and tetraethylammonium bromide (TEAB, 98%) are purchased from Sigma-Aldrich (Switzerland). PUR ® (Elastollan C95A55, Mw 85,790 g mol-1) is obtained from BASF (Germany) and dimethylformamide (DMF, > 99.8%) is from VWR (France). Spinning solutions of 15% w/v PCL are prepared by dissolving PCL in a mixture of AA/FA (3:1), while for polyurethane (PUR) 14.5%w/v spinning solutions are prepared by dissolving PUR in pure DMF solvent. Additionally, 0.01 % v/v TEAB are added into the PUR solutions to increase its electrical conductivity. For preparation of melanin- blended membranes (PCL/Mel and PUR/Mel), fungal melanin powder is added into the solvent and dispersed with a Branson Ultrasonics™ Sonifier 250/450 at 83.3 W and 30% amplitude for 20 min. The ultrasonic treatment alternated 2s of stop and go under ice cooling step. The polymers (PCL and PUR) are added afterwards and kept shaking for 24h to obtain homogeneous solutions. Fiber membranes are generated on the pilot scale needless Nanospider electrospinning instrument (NS 1WS500U, Elmarco, Czech Republic). In this setup, the solution reservoir continuously moves along a wire source electrode and deposits a thin film of solution on it. Applying a high electrical field forms multiple Taylor cones along the wire; electrospinning jets are pulled towards a counter wire electrode and fibers are deposited onto a paper substrate placed before the counter electrode.

(3) A fabric membrane filter comprising the biological sorbent reagent

[0059] The fabric membrane filter comprising the biological sorbent reagent is prepared as follows.

[0060] Waste fabrics are cut into discs of 47mm diameter. Fabrics are autoclaved to ensure sterility. Fabrics are then placed on 6cm Petri dishes containing potato dextrose agar (4g/L Potato extract, 20g/L Glucose, 15g/L agar- agar). Using a Pasteur pipette, pieces of Mucor moelleri cultures are cut (approximately 5-7mm diameter) and placed on the fabric disc. These are let until whole dish is colonized, especially the fabric. The fabric is gently removed and place into a glass Petri dish before being autoclaved. This inoculated fabric will be used as a filter in a column. A water pump will flow the processed water into a column with one, two or three layers until a maximum vanadium is removed from the water.

(4) A bead comprising the biological sorbent reagent

[0061] A bead comprising the biological sorbent reagent is prepared by cultivating Mucor moelleri under constant agitation to trigger pelletization of the biomass, whereby fungal growth occurs in 3-dimensions allowing to obtain millimeter-sized and round pellets consisting of the mycelial network.

(5) A porous polypropylene sponge filter comprising the biological sorbent reagent

[0062] The polypropylene sponge filter comprising the biological sorbent reagent is prepared as follows:

[0063] Polypropylene sponges are cut into cubes of 10mm x 10 mm x 10mm. Sponges are autoclaved to ensure sterility. Sponges are then placed on 6cm Petri dishes containing potato dextrose agar (4g/L Potato extract, 20g/L Glucose, 15g/L agar-agar) and spores with a spore concentration of 1000 spore per mL. Using a pipette, 1 mL of spore suspension is dripped on to each sponge. These are let until whole dish is colonized, especially the sponge. This process takes around 4 weeks. The sponge is gently removed and place into a glass Petri dish before being autoclaved. This inoculated sponge will be used as a filter in a column (Figure 4). A water pump will flow the processed water into a column with one, two or three layers until a maximum vanadium is removed from the water.

Example 3: Metal selectivity of Mucor moelleri

[0064] To test the metal selectivity of prepared biomass of M. moelleri, 3.5 ppm solutions at pH of 4.5 in bidistilled water of the following metal ions were prepared: Al, Ca, Cr, Mg, Mn, Fe, Co, Ni, Cu, Zn, using Aluminium sulfate hexadecahydrate, Calcium chloride dihydrate, Potassium chromate, Magnesium chloride hexahydrate, Manganese(ll) sulphate monohydrate, Iron (III) chloride, Cobalt (II) sulfate heptahydrate, Nickel (II) Sulfate hexahydrate, Cupric chloride dihydrate, Zinc Sulfate heptahydrate, respectively. [0065] Previous adsorption conditions were 30 of min contact time, 100 mg of biomass in 25 mL processed water, 25 °C, 120 rpm. From these products, 100 ml of 0.1 M stock solution wwere prepared. Then, a dilution was done to obtain 200 ml of a 3.5 ppm metal concentration solution. For each metal, three control (without biomass) and three treatment (with biomass) solutions were prepared. 100 mg of prepared biomass were added to three “biomass” Erlenmeyer 200ml.

25ml of the prepared metal solution was added to all six Erlenmeyers (200ml) and installed on a rotary shaker at mentioned conditions. After contact time, solutions were filtered through a 0.45pm filter to recuperate the biomass. 10 ml of the solution was used for ICP-OES analysis. Biomass was placed at 60°C to dry and was analyzed using ICP-OES analysis. The result of the analysis is shown in Table 1.

[0066] Table 1 : Mean adsorption capacity (Q), removal of metals (R) from solutions with biomass. [0067] The selectivity coefficient for is defined as: where, index “s” represents the concentration of the ion on the biomass (mg/g) and aq represents the concentration of the ion in solution (mg/l).

[0068] The selectivity coefficient can determine the affinity of the adsorbant (biomass) for ion B over ion A (vanadium ion). As a result, for K>1 , the resin prefers ion B and for K< 1 , it prefers ion A. In the case of K equal to 1 , the ion exchanger has no preference for ion A or ion B. Therefore, the strain M. moelleri is selective for Cu>»Zn»V>AI>Ni>Co>Mn>Cr> Mg, Fe, Ca.

Example 4 :Adsorption using other Mucor species.

[0069] The aim of this study was to test two additional Mucor strains: M. hiemalis (NEUM144) and M. saturninus (NEUM172). The solutions tested in this study were the processed water from Belenos and artificial V solutions at 3.5 ppm and 100 ppm. Fungal biomasses were initially grown, autoclaved, freeze- dried and grinded into a thin powder. Then, 100 mg of the fungal powder was added to a 150 ml Erlenmeyer flask with 25 ml of a corresponding solution. Flasks were stirred for 30 min at 120 rpm before being filtered through a 0.22pm cellulose filter.

[0070] The sorbent was placed in 15 ml Falcon tubes and placed at 60°C until completely dry. The filtrated solutions were separated into two 15 ml Falcon tubes: one with 10 ml of the solution as a back-up and a second with 4 ml of the solution. The later was diluted with 8 ml of 2% HNO3. All samples were stored in a cold room until treated.

[0071] Both Mucor hiemalis (NEUM144) and Mucor saturninus (NEUM172) demonstrated the capabilities of adsorbing vanadium on all systems tested.. M. moelleri on both systems tested (3.5ppm and 100 ppm vanadium) presented the best performance, having the extraction of 78.5% and 71.6% respectively. M. hiemalis (NEUM144) presented a similar adsorption behavior when exposed to a vanadium solution containing 3.5 ppm. M. hiemalis (NEUM144) and M. saturninus (NEUM172)had a similar and worse adsorption performance when exposed to a vanadium solution containing 100 ppm.

Example 5:Continuous flow experiment using M. moelleri

[0072] A continuous flow experiment is conducted in a reactor using porous polypropylene sponge filter comprising the biological sorbent reagent which is able to immobilize the biomass for biosorption process to adsorb Al, Ca, Cr, Mg, Mn, Fe, Co, Ni, Cu, Zn, V metal ions. The flow conditions are as mentioned in Table 2.

[0073] Table 2 Continuous reactor parameter

[0074] A custom-made packed bed reactor having porous filter comprising biological sorbent reagent is shown in Figure 4.

[0075] M. moelleri strain was inoculated inside the sponge and biomass obtained is used as attachment media for continuous reactor.

[0076] The top and the bottom sponges present in the reactor are denser and used to hold the inoculated sponge in place, when flow is introduced.

[0077] In this system, input is the multi-metal wastewater and output is the treated water

[0078] As seen in Figure 5, different removal rates (% of Metal removal with fungal biosorption according to time (in minutes)) were obtained during the experiment: between 60 and 80% for most of metal ions (Al, Cr, Mg, Mn, Fe, Co, Ni, Cu, Zn), between 40% to 60% of calcium ions, and ca. 75% of vanadium ions.

The results indicates that sponges work well as an immobilization agent in continuous reactor since it could provide a constant removal rate for all metal. This result is similar to a batch experiment.