FR2512058A1 - Synthetic plasmids giving faster metal oxidn. - for extn. of copper, uranium and manganese from their ores - Google Patents

Synthetic plasmids giving faster metal oxidn. - for extn. of copper, uranium and manganese from their ores Download PDF

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FR2512058A1
FR2512058A1 FR8116232A FR8116232A FR2512058A1 FR 2512058 A1 FR2512058 A1 FR 2512058A1 FR 8116232 A FR8116232 A FR 8116232A FR 8116232 A FR8116232 A FR 8116232A FR 2512058 A1 FR2512058 A1 FR 2512058A1
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plasmid
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Claude Nicolau
Alexander Honigman
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Centre National de la Recherche Scientifique CNRS
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Abstract

Synthetic plasmids having at least one bacterial DNA fragment from a vector plasmid and a DNA fragment carrying the coding genes for the oxidation of metals, are new. The ligation technique used follows conventional practice in genetic engineering, and comprises binding DNA fragments, previously obtained by digestion of a plasmid having the metal oxidation coding genes with a restricting endonuclease, with DNA fragments obtained by digestion of a vector plasmid, in the presence of a ligase. The plasmids are sepd. from the ligation mixture by selection, using their oxidising capacity. The plasmids are used to transform bacteria, esp. Thiobacillus ferroxidans, in order to improve their ability to leach metals, such as Cu, U, and Mn from their cres.

Description

La présente invention concerne de
nouveaux plasmides synthétiques améliorant les capacités d'oxydation des bactéries, notamment du genre
Thiobacillus, les plasmides ainsi obtenus, les souches bactériennes transformées par ces plasmides et leur application à la lixiviation des métaux.The present invention relates to
new synthetic plasmids improving the oxidation capacities of bacteria, especially of the genus
Thiobacillus, the plasmids thus obtained, the bacterial strains transformed by these plasmids and their application to the leaching of metals.

Les bactéries Thiobacillus ferroxidans sont les principaux microorganismes lixiviant les sulfures métalliques : pyrites et CuS, par exemple, ainsi que les minerais diura- nium. Ce sont des bactéries autotrophes lorsqu'elles croissent en présence d'ions Fe et hétérotrophes lorsqu'elles croissent sur un-milieu organique (par exemple le glucose). I1 existe déjà quelques installations industrielles mettant en oeuvre cette technique de lixiviation bactérienne, essentiellement pour le cuivre (USA) et pour l'uranium (Afrique du Sud, Canada). The Thiobacillus ferroxidans bacteria are the main microorganisms leaching metal sulfides: pyrites and CuS, for example, as well as diuranium ores. They are autotrophic bacteria when they grow in the presence of Fe ions and heterotrophic when they grow on an organic medium (for example glucose). There are already some industrial installations using this technique of bacterial leaching, mainly for copper (USA) and for uranium (South Africa, Canada).

Les techniques industrielles de lixiviation de l'uranium ont pour but de transformer l'uranium qui est présent dans les minerais sous forme d'oxyde insoluble en sel d'uranium soluble dans l'eau. Le minerai d'uranium contient fréquemment de la pyrite (FeS2) et l'action de
Thiobacillus ferroxidans s'exerce de la façon suivante
FeS2 + 3/2 02 + H20

Figure img00010001

FeS04 + H2S04 (1) 2 FeS04 + 1/2 2 + H2S04
Figure img00010002
Industrial uranium leaching techniques aim to transform the uranium that is present in ores in the form of insoluble oxide into water-soluble uranium salt. Uranium ore frequently contains pyrite (FeS2) and the action of
Thiobacillus ferroxidans works as follows
FeS2 + 3/2 02 + H20
Figure img00010001

FeS04 + H2S04 (1) 2 FeS04 + 1/2 2 + H2S04
Figure img00010002

<tb> T-ferroxidans
<tb> Fe2(S04)3+H20 (2) et U02 + Fe2(S04)3

Figure img00010003
<tb> T-ferroxidans
<tb> Fe2 (S04) 3 + H20 (2) and U02 + Fe2 (S04) 3
Figure img00010003

U02S04 + 2FeS04 (3)
U02S04 est soluble dans l'eau.U02S04 + 2FeS04 (3)
U02S04 is soluble in water.

Iv L'oxydation de UIV à VI
L'oxydation de U à U étant plus rapide en présence des bactéries qu'en présence d'ions Fe 3+ seuls, des études récentes suggèrent une action directe de Thiobacillus ferrox-dans sur UIV 2 U02 + 2 + 2 H2 SO4

Figure img00010004
Iv Oxidation from UIV to VI
Since the oxidation of U to U is faster in the presence of bacteria than in the presence of Fe 3+ ions alone, recent studies suggest a direct action of Thiobacillus ferrox-in on UIV 2 U02 + 2 + 2 H2 SO4
Figure img00010004

<tb> T-ferroxidans
<tb> 2 uO2SO4 +2H2O (4)
En utilisant les capacités oxydantes de cette bactérie 85% de l'uranium d'un minerai pauvre (200 à 300 ppm d'uranium) peuvent être lixiviés en 20 semaines. Si le minerai est plus finement broyé, (dimension particulaire : 0,8 à 1,2mm) en 9 semaines 60 à 70 % de l'uranium peuvent être lixiviés par les bactéries.<tb> T-ferroxidans
<tb> 2 uO2SO4 + 2H2O (4)
By using the oxidizing capacities of this bacteria 85% of the uranium of a poor ore (200 to 300 ppm of uranium) can be leached in 20 weeks. If the ore is more finely ground, (particle size: 0.8 to 1.2mm) in 9 weeks 60 to 70% of the uranium can be leached by bacteria.

La réaction (2) est la base du procédé de lixiviation bactérien de l'uranium. De façon à rendre ce procédé économiquement plus intéressant, la phase de latence dans l'oxydation de Fe en Fe doit être aussi courte que possible, et le temps de demi-vie de la réaction doit être aussi bref que possible. En utilisant le procédé Bacfox, (E. Livesey-Goldbatt, T.H. Tunley, at I.F. Nagy : Bacterial
Leaching, Proc. of a Conference held in Brunschweig-Stockheim, ed. W. Schwartz, Verlag Chemie, Weinheim, New York, 1977, pp. 175-190) il est possible d'obtenir une oxydation rapide Fe 2+ de Fe2+ après un temps de latence de 48 h. Dans ces conditions en 6 jours, on peut obtenir une vitesse d'oxydation de 7,5 g de Fe2+/m2 de surface bactérienne par heure.Reaction (2) is the basis of the bacterial uranium leaching process. In order to make this process more economical, the lag phase in the oxidation of Fe to Fe should be as short as possible, and the half-life of the reaction should be as short as possible. Using the Bacfox process, (E. Livesey-Goldbatt, TH Tunley, at IF Nagy: Bacterial
Leaching, Proc. of a Conference held in Brunschweig-Stockheim, ed. W. Schwartz, Verlag Chemie, Weinheim, New York, 1977, pp. 175-190) it is possible to obtain a rapid Fe 2+ oxidation of Fe2 + after a lag time of 48 h. Under these conditions in 6 days, an oxidation rate of 7.5 g of Fe2 + / m2 of bacterial surface can be obtained per hour.

L'objet de l'invention est d'améliorer les capacités oxydantes des T-ferroxidans en diminuant le temps de latence et en augmentant la vitesse d'oxydation. The object of the invention is to improve the oxidative capacities of T-ferroxidans by reducing the latency time and by increasing the oxidation rate.

La présente invention repose sur la découverte que les capacités d'oxydation des bactéries T-ferroxidans sont codées par un ou plusieurs gènes qui se trouvent disposés sur un plasmide. Ces gènes seront dénommés ci-après "gènes codants pour l'activité d'oxydation des métaux. Le plasmide en cause a été nommé pBPl et il présente une dimension de l'ordre de 5 KB. The present invention is based on the discovery that the oxidation capacities of T-ferroxidans bacteria are encoded by one or more genes which are located on a plasmid. These genes will be called hereinafter "genes coding for the oxidation activity of metals. The plasmid in question has been named pBP1 and it has a dimension of the order of 5 KB.

La présente invention concerne des plasmides synthétiques incorporant tout ou partie des genes codants pour l'activité oxydante et qui sont portés notamment par le plasmide pBPl mentionné précédemment. The present invention relates to synthetic plasmids incorporating all or part of the genes coding for the oxidative activity and which are carried in particular by the plasmid pBPl mentioned above.

C'est pourquoi la présente invention concerne un plasmide synthétique susceptible d'augmenter l'activité oxydante pour les métaux d'une bactérie en particulier du genre
Thiobacillus caractérisé en ce qu'il comporte
- au moins un fragment d'ADN bactérien provenant d'un plasmide vecteur susceptible de se multiplier dans ladite bactérie et d'un fragment d'ADN comportant les genes codants pour l'activité d'oxydation des métaux.This is why the present invention relates to a synthetic plasmid capable of increasing the oxidative activity for the metals of a bacterium in particular of the genus.
Thiobacillus characterized in that it comprises
at least one fragment of bacterial DNA originating from a vector plasmid capable of multiplying in said bacteria and of a DNA fragment comprising the genes coding for the metal oxidation activity.

Parmi les plasmides vecteurs utilisables dans le procédé selon la présente invention, il faut mentionner plus particulièrement les plasmides susceptibles de se multiplier dans les bactéries du genre Thiobacillus tels que pTL 12 et pHV 23 décrits respectivement dans T. Tanaka and
N. Kawano : Gene 10, 131-136, 1980, et B. Michel, E. Palla,
B. Niaudet, a. S.D. Ehrlich : gene 12, 147-154, 1980.Among the vector plasmids which can be used in the process according to the present invention, mention should be made more particularly of the plasmids capable of multiplying in bacteria of the genus Thiobacillus such as pTL 12 and pHV 23 described respectively in T. Tanaka and
N. Kawano: Gene 10, 131-136, 1980, and B. Michel, E. Palla,
B. Niaudet, a. SD Ehrlich: gene 12, 147-154, 1980.

Par plasmide synthétique on entend désigner un plasmide non naturel obtenu par des techniques de génie génétique en particulier par ligation des fragments d'ADN adéquats. The term “synthetic plasmid” is intended to denote an unnatural plasmid obtained by genetic engineering techniques in particular by ligation of suitable DNA fragments.

La technique de ligation utilisée peut être l'une quelconque de celle utilisée en génie génétique. Elle consiste en général à lier - les fragments d'ADN préalablement obtenus par digestion
d'un plasmide portant les gènes codants pour l'activité
d'oxydation des métaux par une endonuclease de restric
tion, et - les fragments d'ADN obtenus par digestion d'un plasmide
vecteur, en présence d'une ligase. Les plasmides selon l'invention peuvent être séparés par une "sélection" à partir des mélanges de ligation en mettant en oeuvre la capacité oxydante de bacilles transformés par les plasmides obtenus.The ligation technique used can be any of those used in genetic engineering. It generally consists in binding - the DNA fragments previously obtained by digestion
of a plasmid carrying the genes coding for the activity
of metal oxidation by a restric endonuclease
tion, and - the DNA fragments obtained by digestion of a plasmid
vector, in the presence of a ligase. The plasmids according to the invention can be separated by a "selection" from the ligation mixtures by using the oxidizing capacity of bacilli transformed by the plasmids obtained.

La présente invention concerne également les bactéries transformées par les plasmides selon l'invention et en particulier les Thiobacillus, notamment T.ferroxidans transformés. The present invention also relates to the bacteria transformed by the plasmids according to the invention and in particular the Thiobacillus, in particular transformed T. ferroxidans.

L'invention concerne également l'application de ces bacilles à l'oxydation des métaux et en particulier au lessivage des minerais notamment de cuivre, d'uranium et de manganèse.  The invention also relates to the application of these bacilli to the oxidation of metals and in particular to the leaching of ores, in particular copper, uranium and manganese.

Les exemplés suivants sont destinés à illustrer certaines caractéristiques et avantages de la présente invention mais ne la limitent en aucune façon. The following examples are intended to illustrate certain characteristics and advantages of the present invention, but do not limit it in any way.

EXEMPLE 1 : Préparation des plasmides selon l'invention.EXAMPLE 1 Preparation of the plasmids according to the invention.

Des échantillons de plasmide pBP1 isolés d'une souche de Thiobacillus ferroxidans sont mis à digérer séparément avec sept enzymes de restriction
Bam HI, Eco RI, Hpa I, Kpn I, Pst I, Sal I et Xba I.Plasmid pBP1 samples isolated from a Thiobacillus ferroxidans strain are digested separately with seven restriction enzymes
Bam HI, Eco RI, Hpa I, Kpn I, Pst I, Sal I and Xba I.

D'un autre côté, des échantillons des plasmides avec les mêmes enzymes, les enzymes en question ont été choisies car les plasmides vecteurs ne possèdent qu'un seul rite de restriction correspondant pour chacune de ces enzymes.On the other hand, samples of the plasmids with the same enzymes, the enzymes in question were chosen because the vector plasmids have only one corresponding restriction rite for each of these enzymes.

Avant ligation les morceaux de restriction sont traités avec du phosphate de sodium
On effectue ensuite la ligati9n des différents fragments d'ADN de pB1 et des plasmides vecteurs par les procédés connus du génie génétique. On obtient ainsi un ensemble de plasmides qu'il faut sélectionner.Before ligation the restriction pieces are treated with sodium phosphate
The different fragments of pB1 DNA and vector plasmids are then ligated by known methods of genetic engineering. This gives a set of plasmids which must be selected.

EXEMPLE 2 : Isolation de cellules transformées.EXAMPLE 2: Isolation of transformed cells.

On effectue la transformation, par des procédés connus, d'une souche E. Coli rec A-600. Les souches E. Coli transformées sont sélectionnées sur plaque d'agar LB contenant de l'ampicilline (les plasmides pTL 12 et pHV 23 confèrent le phenotype Amer). L'identification des clones bactériens est effectuée par hybridation en utilisant une sonde marquée au p32. Chaque clone E. Coli est purifié et les plasmides hybrides des différents clones sont comparés l'un à l'autre en utilisant "l'essai rapide pour l'isolation des plasmides" décrits dans H.C. Birnboisn et J. Doly
Nucleic Acids Res. 7, 1513-1523, 1979.Transformation is carried out, by known methods, of an E. Coli rec A-600 strain. The transformed E. Coli strains are selected on an LB agar plate containing ampicillin (the plasmids pTL 12 and pHV 23 confer the Amer phenotype). The identification of the bacterial clones is carried out by hybridization using a probe labeled with p32. Each E. Coli clone is purified and the hybrid plasmids of the different clones are compared with one another using the "rapid assay for the isolation of plasmids" described in HC Birnboisn and J. Doly
Nucleic Acids Res. 7, 1513-1523, 1979.

Chacun des plasmides est utilisé, après purification dans CsC12, pour transformer une souche de T.ferroxidans. Each of the plasmids is used, after purification in CsC12, to transform a strain of T. ferroxidans.

La sélection des transformants de T.ferroxidans est effectuée sur milieu minimum (comme indiqué dans l'article de
E. Livesey-Goldblatt et al) contenant de l'ampicilline et un indicateur de pH (rougecresol ; pH : 0-1,7). The selection of transformants of T. ferroxidans is carried out on a minimum medium (as indicated in the article of
E. Livesey-Goldblatt et al) containing ampicillin and a pH indicator (rougecresol; pH: 0-1.7).

EXEMPLE 3 : Détermination de l'activité oxydante.EXAMPLE 3 Determination of the oxidizing activity.

Le procédé mis en oeuvre pour déterminer l'activité oxydante est celui décrit par Livesey-Goldblatt et col. The method used to determine the oxidizing activity is that described by Livesey-Goldblatt et al.

Les souches de T.ferroxidans sont cultivées sur solution de Silverman 9K (Silverman M.B. et Lungden D.G,
Journal of Bacteriology 77, 642 - 1959), contenant 30 g/l de (NH 2
8,5 g/l de Fe
à pH 1,7.The T. ferroxidans strains are cultured on Silverman 9K solution (Silverman MB and Lungden DG,
Journal of Bacteriology 77, 642 - 1959), containing 30 g / l of (NH 2
8.5 g / l Fe
at pH 1.7.

Les souches sont incubées à 340C.The strains are incubated at 340C.

La figure annexee représente les résultats du pourcentage d'oxydation de Fe2+ en Fie3+ en fonction du temps en heure. The appended figure represents the results of the percentage of oxidation of Fe2 + to Fie3 + as a function of time in hours.

La courbe a) représente l'activité de la souche sauvage de T.ferroxidans et également de souches transformées avec pTL 12 et pHV 23, qui donnent des résultats équivalents. Curve a) represents the activity of the wild strain of T. ferroxidans and also of strains transformed with pTL 12 and pHV 23, which give equivalent results.

La courbe b) représente l'activité d'une souche de T.ferroxidans transformée par un plasmide selon la présente invention comportant l'ADN du plasmide vecteur pTL 12 nommé pNH1. Curve b) represents the activity of a strain of T. ferroxidans transformed by a plasmid according to the present invention comprising the DNA of the vector plasmid pTL 12 called pNH1.

La courbe c) représente l'activité d'une souche de T.ferroxidans transformée par un plasmide selon l'invention comportant 1'ADN du plasmide vecteur pHV 23 nommé pNH2. Curve c) represents the activity of a strain of T. ferroxidans transformed with a plasmid according to the invention comprising the DNA of the vector plasmid pHV 23 called pNH2.

La courbe b) montre un temps de latence diminuée (20 heures) par rapport à la souche sauvage. Mais le courbe c) qui ne montre pas de reduction significative du temps de latence (35 heures) montre par contre une oxydation beaucoup plus rapide que celle de la souche sauvage.  Curve b) shows a reduced latency time (20 hours) compared to the wild strain. However, curve c), which does not show a significant reduction in the latency time (35 hours), shows on the other hand a much faster oxidation than that of the wild strain.

Ainsi, on constate une amélioration très nette de l'oxydation de Fe2+ grâce aux souches selon la présente invention.  Thus, there is a very clear improvement in the oxidation of Fe2 + thanks to the strains according to the present invention.

Claims (12)

REVENDICATIONS 1. Plasmide synthétique caractérisé en ce qu'il comporte au moins un fragment d'ADN bactérien provenant d'un plasmide vecteur et un fragment d'ADN comportant les gènes codants pour l'activité d'oxydation des métaux. 1. Synthetic plasmid characterized in that it comprises at least one bacterial DNA fragment originating from a vector plasmid and a DNA fragment comprising the genes coding for the metal oxidation activity. 2. Plasmide selon la revendication 1, caractérisé en ce que le plasmide vecteur est un plasmide se multipliant dans les souches bacteriennes du genre Thiobacillus. 2. Plasmid according to claim 1, characterized in that the vector plasmid is a multiplying plasmid in bacterial strains of the genus Thiobacillus. 3. Plasmide selon l'une des revendications 1 ou 2, caractérisé en ce que le fragment d'ADN du plasmide vecteur porte un gène de résistance à un antibiotique. 3. Plasmid according to one of claims 1 or 2, characterized in that the DNA fragment of the vector plasmid carries a gene for resistance to an antibiotic. 4. Plasmide selon l'une des revendications 1 à 3, caractérisé en ce que le fragment d'ADN comportant les gènes codants pour l'activité d'oxydation des métaux est obtenu par clivage enzymatique du plasmide naturel pBP1.  4. Plasmid according to one of claims 1 to 3, characterized in that the DNA fragment comprising the genes coding for the metal oxidation activity is obtained by enzymatic cleavage of the natural plasmid pBP1. 5. Plasmide selon l'une des revendications 1 à 4, caractérisé en ce que le plasmide vecteur est choisi parmi pTL 12 et pHv 23. 5. Plasmid according to one of claims 1 to 4, characterized in that the vector plasmid is chosen from pTL 12 and pHv 23. 6. Plasmide synthétique selon la revendication 5, choisi parmi pNHl et pNH2. 6. Synthetic plasmid according to claim 5, chosen from pNHl and pNH2. 7. Bactérie transformée par un plasmide selon l'une des revendications 1 à 6. 7. A bacterium transformed with a plasmid according to one of claims 1 to 6. 8. Bactérie selon la revendication 7, caractérisée en ce qu'il s'agit d'un Thiobacillus. 8. Bacterium according to claim 7, characterized in that it is a Thiobacillus. 9. Bactérie selon la revendication 8, caractérisée en ce qu'il s'agit d'un T.ferroxidans. 9. Bacterium according to claim 8, characterized in that it is a T. ferroxidans. 10. Application des bactéries selon l'une des revendications 7 à 9 à l'oxydation des métaux. 10. Application of bacteria according to one of claims 7 to 9 to the oxidation of metals. 11. Application selon la revendication 12 à la lixiviation des minerais métalliques. 11. Application according to claim 12 for the leaching of metallic ores. 12. Application selon la revendication 11, caractérisée en ce que les minerais sont des minerais de cuivre ou d'uranium et de manganèse.  12. Application according to claim 11, characterized in that the ores are copper or uranium and manganese ores.
FR8116232A 1981-08-25 1981-08-25 Synthetic plasmids giving faster metal oxidn. - for extn. of copper, uranium and manganese from their ores Granted FR2512058A1 (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2551086A1 (en) * 1983-08-29 1985-03-01 Gen Mining Union Corp
FR2551767A1 (en) * 1983-09-09 1985-03-15 Gen Mining Union Corp ARSENIC RESISTANCE PLASMIDS AND METHOD FOR THEIR CONSTRUCTION
FR2555605A1 (en) * 1983-11-24 1985-05-31 Centre Nat Rech Scient PROCESS FOR INCREASING THE RESISTANCE TO ASIII AND ASV OF BACTERIAL STRAINS IN PARTICULAR THIOBACILLUS FERROOXIDANS
EP0347965A2 (en) * 1988-06-20 1989-12-27 "Dredging International" A method and a device for purifying dredgings or ground contaminated with polluants

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FR2281426A1 (en) * 1974-08-08 1976-03-05 Ici Ltd PROCESS FOR MODIFICATION OF DESOXY-RIBONUCLEIC ACID AND PRODUCTS OBTAINED

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FR2281426A1 (en) * 1974-08-08 1976-03-05 Ici Ltd PROCESS FOR MODIFICATION OF DESOXY-RIBONUCLEIC ACID AND PRODUCTS OBTAINED

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2551086A1 (en) * 1983-08-29 1985-03-01 Gen Mining Union Corp
US4711849A (en) * 1983-08-29 1987-12-08 General Mining Union Corporation, Limited Construction of selectable shuttle cloning vectors for Thiobacillus ferrooxidans
FR2551767A1 (en) * 1983-09-09 1985-03-15 Gen Mining Union Corp ARSENIC RESISTANCE PLASMIDS AND METHOD FOR THEIR CONSTRUCTION
US4748118A (en) * 1983-09-09 1988-05-31 General Mining Union Corporation Limited Construction of arsenic resistance vectors for Thiobacillus ferrooxidans
FR2555605A1 (en) * 1983-11-24 1985-05-31 Centre Nat Rech Scient PROCESS FOR INCREASING THE RESISTANCE TO ASIII AND ASV OF BACTERIAL STRAINS IN PARTICULAR THIOBACILLUS FERROOXIDANS
WO1985002412A1 (en) * 1983-11-24 1985-06-06 Centre National De La Recherche Scientifique (Cnrs PROCESS INTENDED TO IMPROVE THE RESISTANCE TO AsIII AND AsV OF BACTERIAL STRAINS PARTICULARLY THIOBACILLUS FERROOXIDANS
EP0347965A2 (en) * 1988-06-20 1989-12-27 "Dredging International" A method and a device for purifying dredgings or ground contaminated with polluants
EP0347965A3 (en) * 1988-06-20 1990-10-17 "Dredging International" A method and a device for purifying dredgings or ground contaminated with polluants

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