Patent Application
20240132992
The invention relates to an isolated Pseudomonas bacteria capable of the recovery of metals, a new species of genus Pseudomonas, termed Candidatus Pseudomonas pretiosorbens, the use thereof for metal recovery, a composition containing the new species and an isolated nucleic acid.
A number of living microorganisms, but also nonviable, inactivated cells have the ability to bind metal ions or nanoparticles. Metal binding can occur via adsorption to the cell surface or via active intracellular accumulation of metals that is often referred to as biosorption or bioadsorption. In the case of nonviable, inactivated cells—metal ion or nanoparticle binding is believed to occur exclusively via surface adsorption. The biosorption capacity as a general characteristic of biomass results from the presence of chelating groups (e.g. carboxyl-, amide-hydroxyl-, phosphate-, and thiol-groups) contributed by carbohydrates, lipids and proteins that are displayed on the cell surface. It has been described that amounts of metals of up to 50% of the cell dry weight can be accumulated by biomass (Vieira and Volesky, 2000). United States Patent 1991/5055402 describes a process for removing metal ions from aqueous solution, using a matrix prepared from metal-binding microorganisms that have been heat-inactivated at temperatures of 300-500° C. However, specific binding mechanisms by organic surface structures are obviated by this procedure.
EP 0673350 B1 describes the accumulation of metals, including some rare earth elements such as lanthanium and yttrium, by reacting phosphate ions generated by a microorganism and metals to polyphosphates. Accumulation of the metal-polyphosphates by the microorganism of the genus Acetobacter makes the metals accessible to precipitation and depletion thus enabling purification of metal-polluted water. WO 1991/003424 describes a biomining procedure for leaching of gallium and germanium from ores using an admixture of bacteria, culture medium and crushed ore. However, no process has been described to date that could be used to recover platinum group metals in significant amounts from liquid waste materials using a microorganism from which biomass can be produced in a large scale.
Karavaiko et al., Biotechnology Letters, 18 (1996) 1291-1296 describes biosorption of scandium and yttrium from solutions using biomass of Saccharomyces cerevisiae and Aspergillus terreus. MINT Technologies (WO 2018/080326 A1 and WO 2018/084723 A1) describe methods for recovering metals, especially gold, from solid feedstocks using Cupriavidus metallidurans, Bacillus subtilis and Pseudomonas putida as microorganisms. Yet, there is a need for microorganisms which are capable of preferential binding and separation of metals, particularly precious metals, specifically platinum group metals. Furthermore, there is a need for non-pathogenic robust microorganisms from which biomass can be produced in a large scale.
This object is solved by claim 1 of the present invention, which is directed to an isolated Pseudomonas bacteria capable of the recovery of metals, having at least 94% identity to SEQ ID No 2.
Surprisingly the inventors have found that the isolated Pseudomonas bacteria according to the invention has a lot of advantageous characteristics, for example
In a preferred embodiment according to the invention, the isolated Pseudomonas bacteria has at least 96% identity to SEQ ID No 2. In a more preferred embodiment according to the invention, the isolated Pseudomonas bacteria has at least 98% identity to SEQ ID No 2.
In a most preferred embodiment according to the invention, the isolated Pseudomonas bacteria is a species of genus Pseudomonas, identified as Pseudomonas sp. BR8764 termed Candidatus Pseudomonas pretiosorbens, having Accession Deposit Number DSM 33684.
As used herein the terms “species”, “strain” (with regard to Pseudomonas sp. BR8764) and “Pseudomonas pretiosorbens” can be used synonymously since the terms all relate to Candidatus Pseudomonas pretiosorbens, identified as Pseudomonas sp. BR8764 (having Accession Deposit Number DSM 33684).
Platinum group metals according to the invention are defined as Ruthenium (Ru), Rhodium (Rh), Palladium (Pd)) (light platinum metals) and Osmium (Os), Iridium (Ir), Platinum (Pt) (heavy platinum metals) and their mixtures.
Precious metals according to the invention are defined as being selected from the platinum group metals and additionally gold and silver and their mixtures.
As used herein, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof. Thus, for example, reference to “liquid material,” “the liquid material,” or “a liquid material” also includes a plurality of liquid materials; use of the term “a nucleic acid” optionally includes, as a practical matter, many copies of that nucleic acid molecule.
As used herein, the term “about” indicates that a value includes the inherent variation of error for the method being employed to determine a value, or the variation that exists among.
As used herein the term “biosorption” or “biosorption-based processes” means the recovery and concentration of metals from materials. In a preferred embodiment according to the invention, the recovery from platinum group metals and/or precious metals from materials. In a further preferred embodiment, the recovery from platinum group metals and/or precious metals from liquid materials.
BIOSORPTION: As already stated above, the isolated Pseudomonas bacteria according to the invention is capable of recovering metals, particularly precious metals, more specifically platinum group metals from liquid materials by biosorption.
The term “liquid material” means solutions of metal ions, metal complexes or metal nanoparticles that are based on water, non-aqueous solvents or organic solvents or a mixture thereof. Water-based liquid materials are for example mine drainages, communal or industrial waste waters, natural waterbodies and aquifers, as well as acidic and alkaline bioleaching solutions. Liquid materials based on concentrated or diluted non-aqueous or organic solvents or mixtures thereof usually originate from chemical processes as employed in the mining, metal refining and metal recycling industries. In a preferred embodiment according to the invention, the liquid material is water-based.
Pseudomonas s. BR8764: According to a preferred embodiment of the invention, Pseudomonas sp. BR8764 is provided as new organism. This organism can be used for recovering metals, particularly precious metals, more specifically platinum group metals from liquid materials. Surprisingly, this organism is capable of biosorption of target metals also from materials that contain elevated levels of toxic metals such as lead, chromium and cadmium. This allows the conclusion that the biomass of organism is not only able of biosorption of metals or metal recovery, particularly precious metals, more specifically platinum group metals from liquid materials but the biomass has also a high tolerance against external conditions such as toxic elements and compounds, heat and extreme pH conditions.
It has been found that Pseudomonas sp. BR8764 represents a novel species of the genus Pseudomonas. Classification of Pseudomonas sp. BR8764 to a novel species of genus Pseudomonas was supported by phenotypic (API, BIOLOG) and genotypic data. Genomic data were used for further phylogenetic analysis of partial sequences of the housekeeping genes rpoB, rpoD and gyrB and calculation of average nucleotide identity (ANI) based on BLAST algorithm using scaffold genomes. As shown in
Furthermore, the physiology of Pseudomonas sp. BR8764 was found to be distinct from Pseudomonas species known so far as indicated by the API (Biomerieux API 20 NE) and BIOLOG (BIOLOG PM1, PM2A) assays.
DEPOSIT OF BIOLOGICAL MATERIAL The isolated and identified species Pseudomonas sp. BR8764 was deposited on Nov. 5, 2020, under the provisions of the Budapest Treaty in the Leibniz Institute DSMZ—German Collection of Microorganisms and Cell Cultures. It has been assigned to the Accession number DSM 33684.
In a preferred embodiment according to the invention, the isolated Pseudomonas bacteria according to the invention, preferably the new species Pseudomonas sp. BR8764, belongs to risk group 1. The term “risk group” is a classification system which is used in many countries for the classification of microorganisms. The affiliation to a risk group depends for example on the following factors
In an embodiment according to the invention the ribosomal DNA of Pseudomonas sp. BR8764 has SEQ ID No. 1.
USE: In one embodiment according to the invention, the isolated Pseudomonas bacteria having at least 94% identity to SEQ ID No. 2 is used for the recovery of metals, particularly precious metals, specifically platinum group metals from liquid material streams.
Preferably, the liquid material streams are selected from mine drainage waters, communal or industrial waste waters, process streams, for example from metal refining or recycling processes as well as supernatants from bioleaching processes.
As already stated above, the platinum group metals are defined as Ruthenium (Ru), Rhodium (Rh), Palladium (Pd)) (light platinum metals) and Osmium (Os), Iridium (Ir), Platinum (Pt) (heavy platinum metals) and their mixtures.
In a preferred embodiment according to the invention, the isolated Pseudomonas bacteria has at least 96% identity to SEQ ID No 2. In a more preferred embodiment according to the invention, the isolated Pseudomonas bacteria has at least 98% identity to SEQ ID No 2.
Furthermore preferred is the use of Pseudomonas sp. BR8764 for the recovery of metals, particularly precious metals, specifically platinum group metals from liquid material streams.
In one preferred embodiment, the Pseudomonas bacteria according to the invention can be used in the form of free biomass. The biomass can consist of living cells or inactivated cells or cross-linked cells. In another preferred embodiment, the Pseudomonas bacteria can be used as a composition that contains the cells within or on the surface of a sold carrier material or cross-linked cells as described as follows.
COMPOSITION: A further embodiment according to the invention relates to a composition comprising:
In a preferred embodiment, at least one liquid material (b) is selected from secondary resources, wherein the secondary resource is selected from waste waters from mine drainages, communal or industrial waste waters, process streams, for example from metal refining or recycling processes as well as supernatants from bioleaching processes.
As already stated above, the platinum group metals are defined as Ruthenium (Ru), Rhodium (Rh), Palladium (Pd)) (light platinum metals) and Osmium (Os), Iridium (Ir), Platinum (Pt) (heavy platinum metals) and their mixtures.
In a preferred embodiment according to the invention, the Pseudomonas bacteria according to the invention is heat inactivated at temperatures >70° C. In another embodiment the polymer used in b) is for example an alginate, a poly vinyl alcohol-alginate, gelrite, a silicate or a sol gel matrix. In a preferred embodiment according to the invention, the isolated Pseudomonas bacteria has at least 96% identity to SEQ ID No 2. In a more preferred embodiment according to the invention, the isolated Pseudomonas bacteria has at least 98% identity to SEQ ID No 2. Preferably, the Pseudomonas bacteria is selected from Pseudomonas sp. BR8764.
Further preferred is a composition comprising
Suitable materials for solid carriers are ceramic materials such as bentonite or materials such as polyurethane foam and plaster of paris, gypsum or nylon.
In a further embodiment of the invention, the preparation of living or inactivated cells of the Pseudomonas bacteria is cross-linked with, for example, glutaraldehyde or polyethyleneimine, or oxalic acid and polyethyleneimine.
In a preferred embodiment according to the invention, the isolated Pseudomonas bacteria has at least 96% identity to SEQ ID No 2. In a more preferred embodiment according to the invention, the isolated Pseudomonas bacteria has at least 98% identity to SEQ ID No 2. Preferably, the Pseudomonas bacteria is selected from Pseudomonas sp. BR8764.
METHOD: One embodiment according to the invention relates to a method for recovering metals from liquid material with the Pseudomonas bacteria having at least 94% identity to SEQ ID No 2. In a preferred embodiment according to the invention, this method comprises or consists of the following steps:
In step (a), the metals ions, complexes or nanoparticles bind to the biomass. In step (b), the metal ions, complexes or nanoparticles are concentrated and separated from the liquid material. This step can for example be realized by standard solid-liquid separation methods, for example by centrifugation or sedimentation.
In a preferred embodiment according to the invention, the isolated Pseudomonas bacteria has at least 96% identity to SEQ ID No 2. In a more preferred embodiment according to the invention, the isolated Pseudomonas bacteria has at least 98% identity to SEQ ID No 2. Preferably, the Pseudomonas bacteria is selected from Pseudomonas sp. BR8764.
ISOLATED NUCLEIC ACID: A further embodiment according to the invention relates to an isolated nucleic acid having at least 94% identity to SEQ ID No. 2.
In a preferred embodiment according to the invention the isolated nucleic acid having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID No. 2. In another preferred embodiment according to the invention the isolated nucleic acid having 100% identity to SEQ ID No. 2. In other words, the isolated nucleic acid is SEQ ID No. 2.
As used herein, the term “identity” when used in relation to nucleic acids, describes the degree of similarity between two or more nucleotide sequences. The percentage of “sequence identity” between two sequences can be determined by comparing two optimally aligned sequences over a comparison window, such that the portion of the sequence in the comparison window may comprise additions or deletions (gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. A sequence that is identical at every position in comparison to a reference sequence is said to be identical to the reference sequence and vice-versa. An alignment of two or more sequences may be performed using any suitable computer program. For example, a widely used and accepted computer program for performing sequence alignments is CLUSTALW vl 0.6 (Thompson, et al. (1994) Nucl. Acids Res., 22:4673-4680).
SEQ ID No. 2 represents an artificial sequence. This artificial sequence was nanopore sequenced and represents the genome sequence of Pseudomonas sp. BR8764. Furthermore, the nanopore sequence was further polished with the Illumina technique. Nanopore sequencing is a third generation approach used in the sequencing of biopolymers-specifically, polynucleotides in the form of DNA or RNA. By using nanopore sequencing, a single molecule of DNA or RNA can be sequenced without the need for PCR amplification or chemical labeling of the sample. The DNA/RNA is transported through a pore which has the size of a nanometer. When it passes through, the voltage on this pore is changed. The change in the voltage is specific for each of the four nucleobases, whereby the sequence can be recognized. The nanopore itself consists of a recombinant protein that is embedded in a polymer membrane.
The environmental sample for isolation of Pseudomonas sp. BR8764 was taken in Germany in Grube Segen Gottes in Haslach on 28.022012. Therefore, there are no Nagoya Protocol restrictions for this strain. This strain was isolated according to the following procedure: A small amount of environmental sample was suspended in about 500 μl 0.9% NaCl solution and plated on TSB agar plates. Incubation temperature was 10° C. Appearing microbial colonies were transferred to LB agar plates and purified by a “clean streak” on fresh LB agar plates. Small scale cultivation was done at 28° C. in LB medium (such as Bacto Tryptone #211705 (BD) 1% (w/v), Bacto Yeast Extract #212750 (BD) 0.5% (w/v), NaCl #141659.1221 (Applichem) 0.5% (w/v).
First of all the affiliation of strain Pseudomonas sp. BR8764 to the genus Pseudomonas was shown by performing 165 rRNA gene analysis of partial 165 rRNA gene sequences (
To analyze physiological traits of Pseudomonas sp. BR8764 the standardized system for identification of non-fastidious, non-enteric Gram-negative rods that is API 20 NE (BIOMtRIEUX) and carbon utilization tests based on BIOLOG PM1 and PM2A plates (BIOLOG) were used according to the manufacturer's instructions. Results were compared with selected results from literature of physiological tests of relevant strains. Relevant strains were chosen based on above phylogenetic sequence analysis results (
These data further underline that Pseudomonas sp. BR8764 can be classified as a novel species of the genus Pseudomonas termed Candidatus Pseudomonas pretiosorbens.
The isolated and identified strain has been submitted on Nov. 5, 2020 to the Leibniz Institute DSMZ—German Collection of Microorganisms and Cell Cultures. It has the accession number DSM 33684.
First of all, Pseudomonas sp. BR8764 was cultured on LB agar plate for two days at 28° C. Then cells from LB agar plate were suspended in 500 μl LB-medium. 50 μl were transferred to 10 mL of Riesenberg medium (see below) with 5 g glycerol per l. After over night incubation in a shaker, 417 μl of preculture were used for inoculation of 50 ml of the same medium to a final OD of 0.05. The optical density of the culture was 32 after 7.67 hours. For generation of working cell bank cryo-culture, 900 μl of suspended culture were filled in a tube, mixed with 900 μl of 30% glycerol solution and frozen at −80° C. One preculture of 50 mL Riesenberg medium in 500 mL Erlenmeyer flask was inoculated with one working cell culture. After over night incubation in a shaker bioreactor culture was inoculated using preculture to get start a OD of OD 0.1. Pseudomonas sp. BR8764 was cultured in 2 L bioreactor (Sartorius, Biostat B) filled with about 1.8 L medium for 16 hours in batch fermentation. Riesenberg medium was prepared with 26 mM KH2PO4 (AppliChem, #A3620), 30 mM (NH4)2HPO4 (AppliChem (#A2291), 9 mM C6H8O7*H2O (AppliChem (#A4212). Then, after autoclaving 5 mM MgSO4*7H2O (AppliChem, #A4101), 1 ml trace element solution (50 mM FeSO4*7H2O, 10 mM MnSO4*1H2O, 10 mM ZnSO4*7H2O, 2 mM CoSO4*7H2O, 2 mM CuSO4*5H2O, 2 mM NiSO4*6H2O, 2 mM Na2MoO4*2H2O, 2 mM Na2SeO3, 2 mM H3BO3) and 15 g glycerol per liter (Sigma-Aldrich, W252506-25KG-K) was added. pH was adjusted to 6.8 using 5M NaOH-solution. After harvesting of biomass and washing with 0.9% NaCl-solution the pellet was frozen at −80° C. and lyophilized, referred to as dry matter (DM).
To generate test solutions commercially acquired ICP-standard solutions from ESI-Elemental Scientific (www.icpms.de) were diluted to about 100 ppm in either 10 [%] HCl for test solution 1 or 7.5 [%] HNO3 for test solution 2. Following standards were used: PdCl2 (10% v/v HCl, 1000 ppm), CGPD1-125ML; Pt (10% v/v HCl, 1000 ppm), CGPT1-125ML; RhCl3 (15% v/v HCl, 1000 ppm) CGRH1-125ML; IrCl3 (10% v/v HCl, 1000 ppm) CGIR1-125ML; Al (3% v/v HNO3, 1000 ppm) CGAL1-125ML; Fe (2% v/v HNO3, 1000 ppm) CGFE1-125ML To analyse elementary composition in test solutions ion-coupled plasma mass spectrometry (ICP-MS) was applied. Elementary composition of test solution 1 and 2 was as shown in Table 1.
Experimental set-up for biosorption experiments was as described as follows. Dry matter (DM) of biomass prepared as described above was weight into 2 mL-test tubes. Then 1.5 mL test solution were added and incubated at room temperature for 3 hours by mixing using an overhead shaker. Afterwards the samples were centrifuged for 10 min at 13300 rpm. Supernatant was taken to analyze elementary composition by ICP-MS. Metal binding to biomass was calculated by subtracting ICP-MS-element value of supernatant from value in test solution. Recovery was expressed as [%] bound as shown in Table 2.
As shown in Table 2 using dry matter of Pseudomonas sp. BR8764 recovery of palladium above 90% was shown in acid HCl- or HNO3-leachates. Recovery of platinum was observed up to around 80%. In HCl-acid leachate recovery of rhodium or iridium up to around 30% was shown. In HNO3-acid leachate recovery or rhodium or iridium was even higher. Recovery of up to around 40% was determined. However, some aluminium or iron was also recovered, but preferential biosorption of Pd was observed.
| Number | Date | Country | Kind |
|---|---|---|---|
| 21159994.9 | Mar 2021 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/054554 | 2/23/2022 | WO |
