Patent Application
20150267276
The foregoing applications, and all documents cited therein or during their prosecution (“appln cited documents”) and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.
The object of the invention is the plasmid pSheB, particularity a plasmid which may comprise a fragment of pSheB which may comprise the arr module or functional derivatives thereof, and strains which may comprise such a plasmid, particularity Shewanella sp. O23S strain, which are capable of removing arsenic from mineral resources, raw materials industry waste and soils, in particular, post-mining (e.g. soils) under anaerobic conditions, by dissolution of minerals and reduction of arsenates to arsenites. The object of the invention is also the method and the use of such bacterial strains or a composition which may comprise them, for the selective removal of arsenic from mineral resources, raw materials industry waste or the soil.
Arsenic is an element that very often co-occurs in copper minerals and constitutes their specific impurity. In pyrometallurgical processes of roasting and smelting of copper concentrates, volatile arsenic compounds are released into the atmosphere, which, due to the toxicity of these compounds, constitutes a major threat to the environment. During the smelting process, most of the arsenic is removed as a volatile compound As4O6 at concentrations up to 0.5 mg/l, while only 0.04%-0.06% is removed in a solid, stable form with slags [Piret, 1999]. Apart from the volatile arsenic compounds, high concentrations of arsenic are also found in dusts. For this reason, it is very important from both an economic and an environmental point of view, to develop an effective method of controlled removal of arsenic form copper deposits and the products of their processing.
In order for the removal of arsenic from copper minerals to bring the expected economic and environmental benefits, this process must be conducted at the early stages of the copper deposits processing, such as flotation. The traditional copper flotation systems are insufficient and inadequate for separation and division of sulfide minerals containing arsenic (e.g. enargite Cu3AsS4 or tennantite (Cu,Fe)12As4S13) from copper sulfides not containing arsenic, present in the ores. An aid to the conventional flotation methods are the methods proposed in recent years. One of the methods relates to the selective oxidation of sulfides, based on the electrochemical properties of the separated compounds [Fornasiero et al., 2001]. Another method of selective flotation utilizes the differences in the flotation pulp potentials [Guo and Yen, 2005]. Using the separation method based on the difference in the pulp's potentials, minerals containing arsenic, e.g. enargite (Cu3AsS4), can be separated from copper sulfides not containing arsenic. As a result of these processes, two fractions of concentrates are produced: (i) with a low arsenic content, and (ii) with a high arsenic content. The former can be used in pyrometallurgical processes, whereas the latter fraction of concentrates, containing copper minerals contaminated with arsenic, still requires adequate treatment [Senior et al., 2006].
One of the ways that can help to solve the problem of removal of arsenic from copper minerals, is the application of biohydrometallurgical methods, using microorganisms to recover metals from minerals and deposits. The use of microorganisms for the extraction of copper or gold from their ores and concentrates, is a well-known process, and is often described in the literature [Xia, L. et al., 2010; Xia, L. et al., 2009; Olson et al., 2003; Rawlings and Johnson, 2007]. Most of the biohydrometallurgical processes are based on the processes of oxidation of minerals, and lead to (i) an increase in the accessibility to chemical solvents (biooxidation) or (ii) their direct dissolution (bioleaching) [Rawlings and Johnson, 2007]. Unfortunately, these methods are non-specific, because they are based on the oxidation of sulfur and/or iron from minerals, and are associated with the release of all the metals associated with this type of minerals. A further limitation of the traditional biohydrometallurgical methods is the use of acidophilic bacteria in leaching from neutral or slightly alkaline deposits, which is often inefficient, and sometimes even impossible, due to the need for the use of considerable amounts of sulfuric acid to acidify the deposits. In the literature microorganisms are described, mainly chemolithoautotrophic, sulfur oxidizing bacteria, belonging to the genus: Thiobacillus, Halothiobacillus, Thiomonas, and iron oxidizing bacteria, such as: Galionella feruginea or Leptothrix ochracea, Thiothrix and Beggiatoa, which can be used in bioleaching processes at neutral pH, but are very difficult to cultivate and are still poorly understood. Furthermore, bioleaching with the use of these microorganisms is time-consuming and these processes are carried out as long as several months [Sklodowska and Madakowska, 2007]. A confirmation of the lack of suitable microorganisms, capable of recovering metals under neutral or slightly alkaline conditions is the current situation in the mining market. Currently, there are no known and commercially available bitechnological methods of removing precious metals, occurring in the form of sulphides, under the conditions of neutral or slightly alkaline deposits.
An alternative to the oxidation processes are selective bioreduction processes, in which the selected elements, associated with the metabolic activity of microorganisms, are released. Although there are several examples of application of the bioreduction processes, methods for removing arsenic from copper minerals using microbial reduction are unknown. Many strains of bacteria that dissimilatory reduce arsenates have been identified, but the use of most of them is limited to the transformation of soluble arsenic compounds [Newman et al., 1998] or secondary arsenic minerals, resulting from iron compounds [Zobrist et al, 2000]. Strains capable of removing arsenic from copper concentrates and flotation tailings have also been described [Mantur et al., 2011], but the dissimilatory arsenate reduction process, carried out by the aforementioned strains, is not fully balanced and part of the arsenic may be removed out of the cells in the form of volatile, toxic arsenic compounds (unpublished data). Apart from that, the strains described by Mantur et al., 2011, simultaneously release copper and arsenic from minerals, thus lowering the value of the obtained copper concentrate.
Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.
In the tight of the described state of the art, the aim of the present invention is to overcome the indicated inconveniences and to provide a plasmid which may comprise genetic information ensuring the capability of dissimilatory arsenate reduction and selective removal of arsenic, particularity from copper deposits. The aim of the invention is to provide novel bacterial strains which may comprise such a plasmid, compositions which may comprise them and uses thereof, and the methods for the selective removal of arsenic using such strains. Furthermore, it is desirable for such microorganisms, possessing the capability of arsenate reduction, not to produce volatile, and, at the same time, toxic arsenic compounds. The Shewanella sp. O23S strain, that has been isolated from microbial mats from a gold mine in Zloty Stok, possesses such properties [Drewniak, 20091]. The Shewanella sp. O23S strain has been deposited on 24 Jul. 2012 in the IAFB Collection of Industrial Microorganisms Institute of Agricultural and Food Biotechnology in Warsaw, Poland under the deposit number KKP 2045p. This strain is capable of anaerobic growth using arsenates as the final electron acceptor and can mobilize arsenic from rocks from the gold mine in Zloty Stok [Drewniak et al., 2010]. It was unexpectedly found, that these properties are ensured by the plasmid pSheB, isolated from Shewanella sp. O23S, the sequence of which has been shown in SEQ ID NO: 1, particularly its region which may comprise the fragment from 63978 to 72599, which encodes, among others, dissimilatory arsenate reductase, determining these properties.
Accordingly, it is an object of the invention not to encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method, It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product. It may be advantageous in the practice of the invention to be in compliance with Art. 53(c) EPC and Rule 28(b) and (c) EPC. Nothing herein is to be construed as a promise. 100111 It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.
These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.
The Shewanella sp. O23S strain has been deposited on 24 Jul. 2012 in the IAFB Collection of Industrial Microorganisms Institute of Agricultural and Food Biotechnology in Warsaw, Poland under the deposit number KKP 2045p.
The Deposits with IAFB Collection of industrial Microorganisms institute of Agricultural and Food Biotechnology in Warsaw, Poland, under deposit accession number KKP 2045p were made pursuant to the terms of the Budapest Treaty. Upon issuance of a patent, all restrictions upon the deposit will be removed, and the deposit is intended to meet the requirements of 37 CFR §§ 1.801-1.809. The deposit will be irrevocably and without restriction or condition released to the public upon the issuance of a patent. The deposit will be maintained in the depository for a period of 30 years, or 5 years after the last request, or for the effective life of the patent, whichever is longer, and will be replaced if necessary during that period.
The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings.
For a better understanding of the invention, it has been illustrated in the examples of embodiments and in the accompanying figures, in which:
Bacterial strains which may comprise a plasmid including the fragment from 63978 to 72599 of SEQ ID NO: 1, plasmid pSheB or their functional derivatives, particularity the Shewanella sp. O23S strain (deposited as KKP 2045p), are capable of dissimilatory arsenate reduction and selective removal of arsenic, particularly from mineral resources, raw materials industry waste and from the soil, particularly preferable is that they are capable of dissimilatory arsenate reduction and selective removal of arsenic from copper deposits, preferably under neutral or slightly alkaline conditions. The bacterial strains which may comprise the plasmid which may comprise the fragment from 63978 to 72599 of SEQ ID NO: 1, pSheB or their functional derivatives, particularity the Shewanella sp. 023S strain (deposited as KKP 2045p) do not produce toxic, volatile arsenic compounds and stably persist in the environment.
The invention therefore relates to the isolated plasmid which may comprise the fragment of the nucleotide sequence from 63978 to 72599 of the plasmid pSheB, having the sequence shown in SEQ ID NO: 1 or its functional derivative.
The invention also relates to the plasmid pSheB shown in SEQ ID NO: 1 or its functional derivative.
The term “functional derivative of the plasmid” or “functional derivative of the sequence” may comprise plasmids/sequences having a nucleotide sequence coding for open reading frames, which encode products which may comprise an amino-acid or a nucleotide sequence identical or highly homologous to the sequences coded by the indicated sequences, wherein the coding sequences or other sequences of the plasmid/sequence have been modified e.g. by substitution, replacement, deletion or insertion, such that it does not essentially alter the activity of the products of these open reading frames, and enables the maintenance of functional features carried by such plasmid/sequence. The indicated sequence will therefore be the region which may comprise the fragment from 63978 to 72599 of SEQ ID NO: 1, equally preferably it will be the sequence of the plasmid pSheB shown in SEQ :ID NO: 1. A highly homologous sequence means that the sequence is homologous, preferably identical in at least 70%, preferably 80%, more preferably 90%, most preferably, in at least 95%. The term “functional derivative of the plasmid” means, therefore, plasmids having a nucleotide sequence coding for open reading frames, encoding products which may comprise an amino-acid or a nucleotide sequence identical or highly homologous to the sequences coded by the fragment from 63978 to 72599 of SEQ ID NO: 1 and/or to the sequence of the plasmid pSheB shown in SEQ ID NO: 1, wherein the coding sequences or other plasmid sequences have been modified e.g. by substitution, replacement, deletion or insertion, such that it does not essentially alter the activity of the products of these open reading frames, and enables the maintenance of functional features carried by such a plasmid.
The essence of the present invention is thus based on an unexpected finding, that it is possible to use a strain containing the plasmid which may comprise the nucleotide fragment from 63978 to 72599 of SEQ ID NO: 1, pSheB shown in SEQ ID NO: 1 or their functional derivatives, particularity the Shewanella sp. O23S strain, deposited as KKP 2045p, for the selective removal of arsenic from mineral resources, raw materials industry: waste and soils, preferably under neutral or slightly alkaline conditions. It was unexpectedly found, that the strain which may comprise the plasmid pSheB, shown in SEQ ID NO: 1, particularly the Shewanella sp. O23S strain, is: capable of (i) growth in mineral media containing bituminous black shales, flotation tailings, and post-mining soils containing arsenic, (ii) selective release of arsenic from copper minerals containing arsenic, (iii) tolerating the toxic effects of heavy metals released as a result of dissolution of minerals, is (iv) lacking the ability to produce volatile arsenic compounds, is (v) lacking the ability to mobilize copper from mineral resources. The present invention, therefore, also relates to the Shewanella sp. O23S strain, which may comprise the plasmid pSheB, deposited under the number KKP2045p in the IAFB Collection of Industrial Microorganisms Institute of Agricultural and Food Biotechnology in Warsaw.
The invention also relates to a composition which may comprise the isolated plasmid according to the invention and/or a bacterial strain according to the invention or a combination thereof.
The invention also relates to use of a bacterial strain according to the invention, a composition according to the invention, for the selective removal of arsenic from mineral resources, raw materials industry waste or the soil.
Particularly preferred is the use of the bacterial strain, Shewanella sp. O23S, which may comprise the natural plasmid pSheB, carrying: (i) all the genes necessary for dissimilatory arsenate reduction, (ii) arsenite and arsenate resistance genes, and (iii) genes coding for the replication-stabilization system for selective removal of arsenic from mineral resources, raw materials industry waste and post-mining soils. The complete sequence of the plasmid pSheB of Shewanella sp. O23S has been shown in SEQ ID NO: 1.
The presented solutions according to the invention enable the removal of arsenic from mineral resources, raw materials waste, and post-mining soils, preferably under neutral or slightly alkaline conditions, preferably at a pH in the range of about 6 to about 8, using a strain which may comprise the plasmid pSheB or its derivative, more preferably Shewanella sp. O23S, without the need for acidification of the “environment” and without the risk of releasing toxic, volatile arsenic compounds. By the invention, it is possible to selectively remove arsenic without the undesirable release of the target metals, e.g. copper or gold.
The invention therefore relates to the method for selective removal of arsenic from mineral resources, raw materials industry waste, or the soil, in which the dissimilatory arsenate reduction step is carried out using a bacterial strain according to the invention and/or a composition according to the invention. Preferably, the dissimilatory arsenate reduction step is carried out under neutral or slightly alkaline conditions. Preferably, the mineral resources are copper deposits.
The invention also relates to the method for selective removal of arsenic from various mineral resources, raw materials industry, or soils, in which the removal of arsenic is carried out by dissimilatory arsenate reduction, using a bacterial strain according tote invention, preferably which may comprise the plasmid pSheB having the sequence shown in SEQ ID NO: 1 or its functional derivative, more preferably the Shewanella sp. O23S strain, deposited as KKP2045p, which method may comprise the following steps:
It is preferable when step b) is followed by step c) of selective removal of arsenic, released from the solutions obtained is step b). Such removal of arsenic from the solutions (culture fluids), will preferably be carried out using the already developed methods, e.g. by flotation, preferably by selective precipitation of arsenites with sulfides, as a consequence of which a stable, water-insoluble compound, arsenic sulfide As2S3 is formed [Robins, 1985].
In the preferred method, step a) is carried out by: (i) shredding and fractionation of mineral resources, wastes and soils, preferably to the fraction of 125-250 μm (having the average particle size), due to the highest performance obtainable in this range of particle sizes. Equally preferred is (ii) the preparation of an appropriate culture medium R1-R2 with additives of suitable substrates, among which are: sodium lactate as the source of carbon and energy, yeast extract as an additional source of carbon and vitamins. Tuovinen's mineral salts (Tuovinen slats (Tuovinen and Kelly, 1974)) as the source of microelements. Furthermore, in the preferred method, the medium does not contain NO3− and Fe3+.
It is equally preferred if in step b) the culture is carried out under conditions of an appropriate, anaerobic atmosphere, which is obtained by conducting the culture with flushing of the medium with a mixture of gases N2:CO2, preferably in a ratio 4:1. The flushing of the medium with the mixture of gases N2:CO2, preferably in a ratio 4:1, can be equally preferred already in step a) of the method of dissimilatory arsenate reduction.
In the preferred method, in order to obtain the highest performance for the Shewanella sp. O23S strain, step b) is carried out at a temperature in the range from 15° C. to 30° C., preferably about 22° C. with shaking (160 rpm), for at least 21 days. It is preferred if the density of the culture at the beginning of the process is at least about 106 cells/ml. In the preferred method, the inoculum of the Shewanella sp. O23S strain is flushed several times with saline solution or is passaged several times to R1-R2 medium, enriched with sodium arsenate.
Publications cited in the description, and the references given therein, are in their entirety incorporated herein as references.
The following examples are presented merely to illustrate the invention and to clarify its various aspects, but are not intended to be limitative, and should not be equated with all its scope, which is defined in the appended claims.
In the following examples, unless it was otherwise indicated, standard materials and methods described in Sambrooket al., 2001. Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, New York. were used, or the manufacturers' instructions for specific materials and methods were followed.
Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined in the appended claims.
The present invention will be further illustrated in the following Examples which are given fur illustration purposes only and are not intended to limit the invention in any way.
Characteristics of the Plasmid pSheB and Determination of its Complete Sequence
Plasmid pSheB, of the size of 81 kbp, was isolated from the Shewanella sp. O23S strain. In order to sequence the plasmid, plasmid pSheB was isolated from 200 ml of overnight culture of Shewanella sp. O23S by alkaline lysis method. Plasmid pSheB was sequenced by pyrosequencing method, using “shotgun” strategy on the GS FIX Titanium (454) sequencer (in the Oligo Pl. centre). For the construction of the DNA library, approx. 5 μg of pSheB DNA was used and reagent kits provided by the manufacturer were applied ((GS FLX Titanium Library Preparation Kit, Roche). The constructed library was sequenced and assembled using the software from the Newbler de novo assembler package (Roche). The obtained sequences were then assembled into contigs using Seqman software from Lasergene package (DNAStar). Annotation of the plasmid (identification of the open reading frames and determination of their potential functions) were performed using Artemis program and BLAST programs (from the NCBI database). The complete sequence of the plasmid has been shown in the SEQ ID NO: 1. Sequencing of the plasmid pSheB showed that it is a DNA particle of the size 81 591 by and the GC-content of 44.04%. It comprises 87 open reading frames (ORF), which constitute 89.6% of the sequence of the plasmid. Table 1, below, features a detailed description of the identified ORFs within SEQ ID NO: 1.
Shewanella baltica OS 195
Shewanella baltica BA175
Shewanella baltica BA175
Shewanella baltica OS195
Shewanella baltica BA175
Shewanella baltica OS185
Shewanella baltica OS195
Shewanella baltica BA175
Shewanella baltica OS185
Shewanella baltica OS195
Shewanella baltica OS195
Shewanella baltica OS195
Shewanella baltica OS625
Shewanella baltica OS625
Scheffersomyces stipitis
Shewanella baltica OS185
Shewanella baltica OS195
Shewanella baltica OS223
Shewanella baltica OS223
Shewanella baltica OS625
Shewanella baltica OS195
Shewanella baltica OS 185
Shewanella baltica OS223
Shewanella baltica OS195
Shewanella baltica BA175
Shewanella baltica OS625
Shewanella baltica OS223
Shewanella baltica BA175
Shewanella baltica OS195
Shewanella baltica OS678
Shewanella baltica OS223
Shewanella baltica OS223
Shewanella baltica OS195
Shewanella baltica BA175
Shewanella baltica OS223
Shewanella baltica OS195
Shewanella baltica OS195
Staphylococcus aureus
Shewanella baltica OS195
Megasphaera
micronuciformis F0359
Shewanella oneidensis MR-1
Shewanella oneidensis MR-1
Shewanella baltica OS223
Shewanella baltica BA175
Shewanella baltica BA175
Bacillus subtilis subsp.
subtilis str. 168
Methylophaga thiooxydans
Methylovorus
glucosetrophus SIP3-4
Pseudoalteramonas arctica
Enterobacter cloacae SCF1
Shewanella baltica OS185
Vibrio furnissii CIP 102972
Marinomonas sp. MWYL1
Shewanella sp. ANA-3
Shewanella sp. ANA-3
Shewanella sp. ANA-3
Shewanella sp. ANA-3
Shewanella sp. ANA-3
Shewanella sp. ANA-3
Shewanella sp. ANA-3
Shewanella sp. W3-18-1
Shewanella sp. W3-18-1
Shewanella sp. W3-18-1
Shewanella sp. ANA-3
Shewanella sp. ANA-3
Shewanella putrefaciens
Shewanella putrefaciens
Shewanella sp. ANA-3
Shewanella sp. ANA-3
Shewanella sp. ANA-3
Shewanella sp. ANA-3
Shewanella sp. ANA-3
Shewanella putrefaciens
Shewanella putrefaciens
Shewanella sp. W3-18-1
Shewanella putrefaciens
Shewanella putrefaciens
Streptococcus pneumoniae
Shewanella baltica BA175
Shewanella baltica BA175
Coprococcus comes ATCC
Aeromonas hydrophila
Shewanella baltica OS117
Shewanella baltica BA175
Construction of the Plasmid-Less Strain and Functional Analysis of the Plasmid pSheB
In order to show, that the plasmid pSheB and the gene module coding for potential proteins located within it, are involved in the resistance to arsenic and dissimilatory arsenates reduction, a plasmid-less derivative of the Shewanella sp. O23S strain was constructed and its functional analysis was carried out. As the stress factor stimulating the mechanisms of plasmid removal from the cells of the host, ethidium bromide (EtBr) solution at a final concentration of 5 μM was used.
Overnight culture of the wild-type Shewanella sp. O23S strain, carried out in LB medium supplemented with 5 mM sodium arsenate was passaged to LB medium with 5 μM EtBr. The optical density (OD) of the culture at the beginning of the experiment was OD=0.1, and the culture was carried out for 24 hours at 22° C. with shaking (160 rpm). After 24 hours of incubation, culture dilutions 10−4,−6,−8,−10 were prepared, respectively, and 100 μl of each of them were plated on LB medium solidified with agar. Subsequently, 96 colonies were randomly selected and passaged by replica plating to:
The cultures in solid media were incubated for 72 hours, whereas the cultures in the liquid medium were carried out in 200 μl in 96-well titration plates in Anaerocult® (Merck) containers, which provide anaerobic conditions for 168 hours.
The cultures carried out in the liquid medium were aimed to determine the abilities of the selected strains to dissimilatory reduce arsenates. After 5 days of incubation under anaerobic conditions, 100 μl of 0.1 M solution of silver nitrate were added to the cultures. The result of the reaction between AgNO3 and As (III) or As (V) is the formation of a coloured precipitate. A brown precipitate indicates the presence of Ag3AsO4 (silver orthoarsenate), while a yellow precipitate indicates the presence of Ag3AsO3 (silver arsenite). In case of testing for the ability to reduce arsenates, the presence of a yellow precipitate indicates that As(V) was reduced to As(III).
The cultures carried out in solid LB medium enriched with sodium arsenate were aimed to determine the resistance to As(V). In turn, the cultures carried out in solid LB medium (not enriched with additional substances) were aimed to secure the potential mutants (positive control). All the strains, which have grown in LB medium, and were not capable of growth in LB medium enriched with As(V) and in minimal R1-R2 medium containing arsenate (the final electron acceptor) and lactate (electron donor), have been designated as potential mutants, deprived of the plasmid pSheB.
In order to verify the selected consortia, the plasmid profile of the wild-type strain (Shewanella sp. O23S) and the potential plasmid-less mutants was checked. The plasmid DNA was isolated by alkaline lysis method, and electrophoretic analysis (0.8% agarose gel) was conducted. The comparison of the plasmid patterns of the selected strains allowed for the identification of the plasmid-less mutants. An additional confirmation of the absence of the plasmid pSheB in the cells of the constructed mutants was PCR analysis. PCR reaction was carried out in the genomes of the potential mutants, using the following primers:
Primer endA-L respectively corresponds to the position of the nucleotide 127, while endA-R—position 679 of the plasmid pSheB in reference to SEQ ID NO: 1. The strains (potential mutants) which gave a negative result in the PCR reaction, using the primers described above, were selected for further analysis.
The next step of the verification of the strains not-possessing the plasmid pSheB was their functional analysis. It was checked again whether the selected strains are capable of dissimilatory arsenate reduction. For this purpose, tests were carried out in minimal R1-R2 medium enriched with sodium arsenate and sodium lactate, and applying the test with 0.1 M solution of silver nitrate. The strains, which were not capable of reduction, were susceptible to As(V) and As(III), and did not possess the plasmid pSheB, turned out to be a proof that the plasmid pSheB determines the capability of arsenate respiration and resistance to arsenic.
In order to demonstrate, which genes located on the plasmid pSheB encode proteins responsible for dissimilatory arsenate reduction, the arr module, which may comprise i.a. genes for dissimilatory arsenate reductase arrAB, was cloned into the vector pBBR1-MCS2 (Km), in the Escherichia coli TOP 10 strain, and its functionality was tested.
In order to clone the arr module, amplification of a DNA fragment of the size 8634 by (which may comprise the region from position 63978 to 72599 in the genome of pSheB) was performed on a DNA template of the plasmid pSheB, isolated by alkaline lysis. For PCR reaction, the following oligonucleotides were used as primers:
She_Mph1103F: GAAATCTTGCAGTAGCGATGCATC (SEQ ID NO: 4) [position in the genome of the plasmid pSheB: 63978-64001; the underlined sequence is the restriction site recognized by the enzyme Mph 11031 (NsiI)], and
She_XmaJR: GTTGTTCCTAGGCTGGTGCCATATCAACCTCTAG (SEQ ID NO: 5) (position in the genome of the plasmid pSheB: 72578-72599; the sequence written in italic is an added sequence; the underlined site is recognized by the restriction enzyme XmaJI). For the amplification, Phusion® High-Fidelity DNA Polymerase (Thermo Scientific) was used.
The obtained PCR product (8634bp) was cloned into a plasmid vector: pBBR1MCS-2 (Km1) [Kovach et al., 1995] digested (linearized) with SmaI. The ligation mixture of the PCR product and the vector pBBR1MCS2 digested with the enzyme SmaI was introduced, by means of chemical transformation, using the calcium-rubidium method according to Kushner (1978), into the cells of Escherichia coli Top10 strain [mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara-leu)7697 galU galK rpsL endA1 nupG]. Complete LB medium with kanamycin (30 μg/ml), IPTG (0.5 μg), and X-gal (40 μg/ml) was used as the selection medium.
From the pool of the obtained transformants (white colonies resistant to kanamycin) strains that were harbouring a plasmid of the appropriate size: 13778 bp (pBBR1MCS2-5144bp +arr module−8634 bp). The presence of the constructed plasmid was confirmed by restriction analysis (digestion with the enzymes XmaJI and Mph11031), electrophoretic analysis and sequencing. The Escherichia coli MR1 strain (derivative of the E. coli TOP10 strain) harbouring the plasmid pARR1A (derivative of pBBR1MCS2 with cloned arr module), was selected for further analysis.
In order to verify the functionality of the constructed plasmid pARR1A, phenotypic analysis of the E. coli MR1 strain was carried out in minimal R1-R2 medium enriched with 2 mM sodium arsenate and 5 mM sodium lactate and supplemented with 0.004% yeast extract. The culture was carried out for 120 h under anaerobic conditions (in CO2:N2 atmosphere) at 37° C. After five days of culturing, the test with 0.1 M solution of silver nitrate was carried out, which showed that the investigated strain has reduced arsenates to arsenites. A confirmation of the reduction of arsenates to arsenites by the E. coli MR1 (pARR1 A) strain, was the qualitative analysis of arsenic speciation by HPLC. The conducted analysis showed that after 120 h of incubation of the E. coli MR1 strain, arsenites were identified in the medium. In the control reaction with the E. coli TOP10 strain (without the plasmid), reduction of As(V) to As(III) was not observed.
The obtained results showed that the introduction of the genes of the arr module, originating from the genome of the plasmid pSheB, on the vector pBBR1MCS into the E coli TOP10 strain, leads to the acquisition of the capability of dissimilatory arsenate reduction. The module introduced to E. coli TOP10 corresponded to the fragment of the nucleotide sequence from 63978 to 72599 of the plasmid pSheB, having the sequence shown in SEQ ID NR: 1. In this way it was demonstrated that this is the fragment of the plasmid responsible for the capability of dissimilatory arsenate reduction. It was also demonstrated, that a bacterium other than that, from which the plasmid pSheB originates, that is another species of bacteria or a bacterial strain, into which the fragment of the sequence which may comprise the fragment of the nucleotide sequence from 63978 to 72599 of the plasmid pSheB, having the sequence shown in SEQ ID NO: 1 has been introduced, acquires the ability to dissimilatory reduce arsenates.
Removal of Arsenic from Copper Minerals using the Shewanella sp. O23S Strain
In order to demonstrate that a strain which may comprise the plasmid pSheB (SEQ ID NO: 1), for example the Shewanella sp. O23S strain, harbouring the plasmid pSheB, can be used in biometallurgy, in the processes of selective removal of arsenic from mineral resources, an experiment was carried out, using two types of minerals: copper-bearing bituminous shale of the “Kupferschiefer” type and flotation tailings from the first series, obtained from the Mining Plant “Lubin” (KGHM, Poland). The average arsenic content in the copper-bearing bituminous shale designated as “Shales” was 1000-3000 mg/kg of the shale, whereas the arsenic content in the flotation tailings designated as “Middlings” was 250-350 mg/kg of the tailings. Apart from arsenic, both types of minerals contain Cu in the range of 35000-110000 mg/kg of dry mass, and other precious metals, e.g. Co—500-2500 mg/kg, Zn—15-2800 mg/kg, Ni—250-500 mg/kg,
The experiment of arsenic removal from the mineral resources described above was conducted in R1-R2 medium enriched with 5 mM sodium lactate as the source of carbon and energy, Tuovinen salts (2 ml/l) and yeast extract at a final concentration of 0.004%. The mineral substrate (shales or flotation tailings) having the fraction size of 125-250 mm, was added to a final concentration of 10%. The cultures were carried out in 100-ml bottles under anaerobic conditions, in the atmosphere of the mixture of gases N2:CO2 (4:1) for 21 days at 22° C. with shaking (160 rpm). In order to obtain an inoculum of the Shewanella sp. O23S strain, overnight cultures were started in liquid complete LB medium. The overnight culture of the O23S strain was centrifuged several times and flushed with saline solution, and then passaged to an appropriate medium, to obtain a density of approximately 106 cells/ml. Sterile medium supplemented with an appropriate substrate, not inoculated with bacteria, was used as the control.
At the beginning of the experiment, and every 7 days, samples for the determination of copper and arsenic content were collected, which was performed using atomic absorption spectroscopy—flame technique; (AA Solaar M6 Spectrometer, TJA Solutions, UK).
The conducted analyses showed, that the highest concentration of arsenic in culture fluids in medium enriched with flotation tailings was noted after 14 days of incubation (324.75 μg/l) (
The conducted experiment allowed to demonstrate that the strain which may comprise the plasmid pSheB (SEQ ID NO: 1), i.e. the Shewanella sp. O23S strain, deposited as KKP2045p, removes arsenic from copper-bearing bituminous shales, as well as from flotation tailings in a specific way, without simultaneous mobilization of copper from copper deposits,
Mobilization of Arsenic from the Soils Contaminated with Arsenic Using the Shewanella sp. O23S Strain
In order to demonstrate that a strain harbouring the plasmid pSheB, having the SEQ ID NO: 1 or its functional derivative, such as Shewanella sp. O23S, harbouring the plasmid pSheB, can be used in bioremediation, in the processes of selective arsenic removal from the soils contaminated with arsenic, an experiment using the soil originating from the vicinity of a gold mine in Zloty Stok was conducted. The average arsenic content in the soil was 17955.7 mg/kg of soil.
The experiment of arsenic removal from the soil was conducted in R1-R2 medium enriched with 5 mM sodium lactate (carbon and energy source), Tuovinen* salts (2 ml) and yeast extract at a final concentration of 0.004%. The fragmented sample (having the fraction size of <3 mm) was added to a final concentration of 10%. The cultures were carried out in 100-ml bottles under anaerobic conditions in the atmosphere of the mixture of gases N2:CO2 (4:1) for 21 days at 22° C. with shaking (160 rpm). In order to obtain an inoculum of the Shewanella sp. O23S strain, overnight cultures were started in liquid complete LB medium. The overnight culture of the O23S strain was centrifuged several times and flushed with saline solution, and then passaged to an appropriate medium to obtain a density of approximately 106 cells/ml. Sterile medium with the addition of the soil, not inoculated with bacteria, was used as the control.
At the beginning of the experiment, and every 7 days, samples were collected for: (i) determination of the arsenic content (determination by atomic absorption spectroscopy—flame technique) in culture fluids (ii) monitoring the growth of bacteria, by the analysis of the number of colony forming units (cfu) (plating on solid LB medium enriched with 5 mM sodium arsenate). The conducted analyses showed, that the highest concentration of arsenic in culture fluids was noted after 14 days of incubation (166.53 mg/l), whereas after a further 7 days of incubation, concentration of arsenic has decreased slightly (123.03 mg/l), which may be associated with the precipitation of arsenic in the form of secondary minerals. In the control samples, concentration of arsenic was at much lower level (it did not exceed 25 mg/l), and reflected the chemical processes of leaching. It was demonstrated that a strain which may comprise the plasmid pSheB, having the SEQ IN NO:1, i.e. the Shewanella sp. O23S strain, removes arsenic from the soils contaminated with this element and can be used in bioremediation, e.g. in the recultivation of arsenic contaminated soils or the removal of arsenic from other contaminated environments.
Analysis of Arsenic Accumulation by the Shewanella sp. O23S Strain
The growth experiment and the dissimilatory arsenate reduction performance analysis, carried out in R1-R2 medium (
The conducted analyses did not confirm the growth experiments in R1-R2 medium and revealed that arsenic compounds may be partially accumulated inside the cells of the Shewanella sp. O23S strain. The efficiency of this process, however is very low, less than 3% for As(III) compounds, and less than 8% for As(V) compounds. The obtain results are shown in Table 2.
Analysis of the Production of Volatile Arsenic Compounds by the Shewanella sp. O23S Strain
In order to verify whether the Shewanella sp. O23S strain which may comprise the plasmid pSheB is capable of producing volatile arsenic compounds, an anaerobic (in N2:CO2 atmosphere; 4:1) culture in minimal R1-R2 medium, enriched with 5 mM sodium lactate and 2.5 mM sodium arsenate, was carried out. After 5 days of incubation at 22° C. gas samples were collected for the chemical composition analysis in a gas chromatograph GC-MS and GC-AED. The conducted analyses revealed that the only volatile compound produced by the Shewanella sp. O23S strain with the plasmid pSheB is dimethyl monosulfide (DMS). The production of volatile arsenic compounds was not observed.
Precipitation of As2S3 (Arsenic(III) Sulfide) by the Shewanella sp. O23S Strain in Medium Containing As (V) and Thiosulfate
In order to demonstrate that a strain harbouring the plasmid pSheB, having the SEQ ID NO: 1 or its functional derivative, such as Shewanella sp. O23S, harbouring the plasmid pSheB can be used in bioremediation, in the processes of selective arsenic removal from the soils contaminated with arsenic, an experiment, in which arsenic was precipitated from the medium in the form of arsenic (III) sulfide (As2S3) was carried out. For this purpose, an experiment of arsenic removal from the soil was conducted in R1-R2 medium enriched with 5 mM sodium lactate (as the source of carbon and energy), Tuovinen salts (2 ml/l), and 2.5 mM sodium arsenate and 5 mM sodium thiosulfate as the final electron acceptors. The medium was inoculated to obtain the initial culture density of 106 cells/ml, The cultures were carried out under anaerobic conditions, at room temperature. The arsenic content in the solution was measured at time T0, and on days 1, 2 ,3, 4, and 7, 14 and 21 of the culture. The appearance of a yellow-orange arsenic (III) sulfide (As2S3) precipitate was also observed. After 7 days, a loss of 47.8% of arsenic in the solution, and the appearance of a characteristic precipitate, insoluble in water, were recorded. After 21 days of cultivation, the loss of arsenic reached 82.3% (
Capability of Dissimilatory Arsenate Reduction by the Shewanella sp. O23S Strain under Slightly Acidic Conditions
In order to verify whether the Shewanella sp. O23S strain is capable of dissimilatory arsenate reduction under slightly acidic conditions, culture was carried out in minimal R1-R2 medium having a pH 4, enriched with 2.5 mM sodium arsenate, 5 mM sodium lactate and 0.004% yeast extract. The culture was carried out for 168 h at room temperature. An analogous experiment in medium having a pH 8 was carried out as the control. The conducted research revealed, that in the most optimal conditions, in medium having a pH 8, Shewanella sp. O23S has completely reduced As(V) present in the medium within 4 h (
Resistance to Heavy Metals of the Shewanella sp. O23S Strain
In order to use a strain in mobilization of arsenic from polymetallic deposits, copper concentrates or flotation tailings, the used strain must be characterized by a high resistance to heavy metals. In accordance with the above, Shewanella sp. O23S capability of tolerating the presence of heavy metals, and capability of reducing As(V) to As(III) in the presence of heavy metals was verified.
In order to verify the range of tolerance to the presence of heavy metals, the Shewanella sp. O23S strain was cultured in liquid complete LB medium supplemented with the appropriate heavy metals solutions (Table 3):
The overnight culture of Shewanella sp. O23S was passaged to LB medium enriched with the appropriate metal or metalloid compound (to obtain a density of approximately 106 cfu/ml) and was incubated for 24 hours at 22° C. Subsequently, OD600 measurements were carried out and the values of minimal inhibitory concentration (MIC), which is defined as the lowest Men+ concentration that completely inhibits the growth of bacteria, were determined. In order to verify whether the presence of heavy metals inhibits the capability of reducing arsenates to arsenites, a test was carried out in minimal R1-R2 medium enriched with 2.5 mM sodium arsenate, 5 mM sodium lactate, 0.004% yeast extract and the addition of the appropriate heavy metals solutions.
The conducted experiments revealed that the Shewanella sp. O23S strain, apart from the resistance to As(III) and As(V) is also resistant to Cu, Cd, Cr, Co, Mn, Zn, Se, V (Table 4). Furthermore, it is also capable of dissimilatory arsenate reduction in the presence of heavy metals. Only in the presence of iron (III) the capability of dissimilatory reduction was not demonstrated, because this strain is capable of Fe(III) reduction and using it as the final electron acceptor (Table 4).
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1. An isolated plasmid comprising the fragment of the nucleotide sequence from 63978 to 72599 of the plasmid pSheB, having the sequence shown in SEQ ID NO: 1 or its functional derivative.
2. An isolated plasmid pSheB, having the sequence shown in SEQ ID NO: 1 or its functional derivative.
3. A bacterial strain comprising the plasmid defined in paragraph 1 or 2, or comprising the nucleotide sequence comprising the nucleotides from 63978 to 72599 of SEQ ID NO: 1 or its functional derivative.
4. The bacterial strain according to paragraph 3, characterised in that it is the Shewanella sp. O23strain deposited in the IAFB Collection of Industrial Microorganisms in Warsaw, under the deposit number KKP 2045p.
5. A composition comprising the isolated plasmid defined in paragraphs 1-2 or the bacterial strain defined in paragraphs 3-4 or combination thereof.
6. Use of the bacterial strain defined in paragraphs 3-4 and/or the composition defined in paragraph 5 for the selective removal of arsenic from mineral resources, raw materials industry waste or soil.
7. The use according to paragraph 6, characterised in that the mineral resources are polymetallic copper deposits.
8. A method for selective removal of arsenic from mineral resources, raw materials industry waste or soil, wherein the
9. The method, according to paragraph 8, wherein the step of dissimilatory arsenate reduction is carried out under neutral or slightly alkaline conditions.
10. The method according to paragraphs 8-9, characterised in that the mineral resources are copper deposits.
11. A method for selective arsenic removal from a variety of mineral resources, raw materials industry or soils, wherein the removal of arsenic is carried out by dissimilatory arsenate reduction using a bacterial strain comprising the plasmid pSheB, having the sequence shown in SEQ ID NO: 1 or its functional derivative, preferably the Shewanella sp. O23strain deposited as KKP2045p, which method comprises the following steps:
12. The method according to paragraph 11, characterized in that, step b) is followed by step
13. The method according to paragraph 12, characterized in that, the removal of the released arsenic is carried out by flotation, preferably by selective precipitation of arsenites with sulfides.
14. The method according to paragraphs 11-13, characterized in that:
15. The method according to paragraphs 11-14, characterized in that the culture medium is R1-R2 medium, supplemented with sodium lactate, yeast extract and Tuovinen salts.
16. The method according to paragraphs 11-15, characterized in that, the culture medium does not contain NO3−and Fe3+.
17. The method according to paragraphs 11-16, characterized in that in step b), the culture is carried out under anaerobic atmosphere conditions, and is carried out with flushing of the medium with a mixture of gases N2:CO2, preferably in a ratio 4:1.
Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| P404376 | Jun 2013 | PL | national |
This application is a continuation-in-part application of international patent application Ser. No. PCT/IB2013/059773 filed 30 Oct. 2013, which published as PCT Publication No. WO 2014/203046 on 24 Dec. 2014, which claims benefit of Polish patent application Serial No. P.404376 filed 19 Jun. 2013.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/IB2013/059773 | Oct 2013 | US |
| Child | 14678143 | US |
