WO2006087334A1

WO2006087334A1 - Use of bacterium strains for the preparation of metallic biocatalysts, in particular for the preparation of palladium biocatalysts

Info

Publication number: WO2006087334A1
Application number: PCT/EP2006/050942
Authority: WO
Inventors: Marc Rousset; Laurence Casalot; Pascale De Philip; Anne Belaich; Iryna MIKHEENKO; Lynne Macaskie
Original assignee: Centre National de la Recherche Scientifique CNRS
Current assignee: Centre National de la Recherche Scientifique CNRS
Priority date: 2005-02-21
Filing date: 2006-02-15
Publication date: 2006-08-24
Anticipated expiration: 2007-08-21

Abstract

The present invention relates to the use of a bacterium strain characterized in that its genome contains at least a gene coding for a membrane-bound [NiFe] hydrogenase, or of membrane extracts containing said membrane-bound [NiFe] hydrogenase of said bacterium strain, for the preparation of metallic biocatalysts, provided that in the case of the use of a bacterium strain the genome of which contains one or several genes coding for a soluble periplasmic hydrogenase, then said strain is transformed by inactivation, repression or deletion of at least one of said genes coding for a soluble periplasmic hydrogenase, the corresponding soluble periplasmic hydrogenase being then non produced or absent.

Description

USE OF BACTERIUM STRAINS FOR THE PREPARATION OF METALLIC BIOCATALYSTS, IN PARTICULAR FOR THE PREPARATION OF PALLADIUM BIOCATALYSTS

The present invention concerns the use of bacterium strains for the preparation of metallic biocatalysts, in particular for the preparation of palladium biocatalysts.

Palladium is one of the most expensive metals; the highest consumer worldwide is the automobile industry where this metal, together with other platinum group metals (PGM), is used in automobile catalytic converters to reduce the toxicity of vehicle exhaust gases. Palladium is also extensively used in the electronics industry and in chemical manufacturing and processing (Platinum, 2003). Palladium is a highly valuable metal with only limited world resources reported before the widespread implementation of automotive catalyst (Hoffman, 1988). The existing methods of Pd recovery from scrap, e.g. hydro- and pyrometallurgical routes, are either energy demanding or harmful to the environment, or both. Therefore alternative, clean methods of recovery are very important. It was shown by Lloyd et al., (1998), that palladium can be effectively recovered from solution by resting cells of Desulfovibrio desulfuricans at ambient temperature. As an alternative approach, Mertig, et al. (1998) and Wahl et al. (2001) described abiotic reduction of Pd(II) to Pd(O) onto a protein surface under H₂, but, unlike the use of bacteria (Yong et al., 2003) this has not been applied to the development of a flow-through metal recovery process.

Desulfovibrio sp. and other sulphate-reducing bacteria (SRB) are well-known as metal reducers (Lovely & Philips, 1992; Tucker et al.. 1998; Ganesh et al. 1999; Spear et al.. 1999; Lloyd et al., 1998, 1999, 1999a, 2001). Yong et al. (2002) showed that

Pd(II) can be reduced by D. desulfuricans over a wide pH range and this organism was effectively used to recover Pd from chemical processing waste (Yong, et al., 2002a) and leachates from spent automotive catalysts (Yong et al., 2003).

The biomineralised palladium (Bio-Pd) identified as Pd(O) by X-ray powder diffraction analysis (Lloyd et al., 1998) was found to have catalytic properties not seen in Pd(O) prepared by simple chemical reduction of Pd(II) salts under H₂. For example, Bio-Pd catalysed the reduction of Cr(VI) to Cr(III) (Mabbett et al., 2001), the reductive dehalogenation of chlorophenol and polychlorinated biphenyls (Baxter-Plant et al., 2003) and effected the hydrogenation of methylene succinic acid at rates comparable to a commercial supported palladium catalyst. The properties of a supported metal catalyst depend critically on both the size of the metal clusters (Henry, 1998) and the ability of supporting material to prevent their coalescence (Henry, 1998). Bacteria have both biomanufacturing and templating/supporting functions. It has been reported that Escherichia coli, like D. desulfuricans, possesses significant metal reducing capability (Lloyd et al., 1997, 1997a, 1999a). The physiology and genetics of this organism are well investigated (Menon et al., 1991; Sauter et al., 1992; Lloyd et al., 1997, 1999a) and, using these approaches, involvement of hydrogenase activity in Tc(VII) reduction by E. coli was attributed to the hydrogenase III component of the formate hydrogenlyase (FHL) complex (Lloyd et al., 1997), a rudimentary form of which has been proposed to exist in D. desulfuricans (Peck, 1993). The aim of the present invention is to provide a new process for the preparation of metallic biocatalysts, which implementation is easier and cheaper than industrial processes. The aim of the present invention is to provide a new process for the preparation of metallic biocatalysts, which comprises the use of transformed bacterium strains, in particular of transformed Desulfovibrio fructosovorans strains.

The aim of the present invention is to provide a metallic biocatalyst of smaller particle size than the catalysts of the prior art, said metallic biocatalyst thus having a higher catalytic activity.

The present invention relates to the use

- of a bacterium strain characterized in that its genome contains at least a gene coding for a membrane-bound [NiFe] hydrogenase, or

- of membrane extracts containing said membrane-bound [NiFe] hydrogenase of said bacterium strain, for the preparation of metallic biocatalysts, provided that in the case of the use of a bacterium strain the genome of which contains one or several genes coding for a soluble periplasmic hydrogenase, then said strain is transformed by inactivation, repression or deletion of at least one of said genes coding for a soluble periplasmic hydrogenase, the corresponding soluble periplasmic hydrogenase being then non produced or absent.

The expression "membrane-bound hydrogenase" designates any enzyme, attached directly or by an anchoring subunit to the membrane, which catalyses hydrogen production or oxidation. Membrane-bound hydrogenases are closely related to the proton-pumping NADH:quinone oxidoreductase (complex I): the small subunit is considerably smaller than that of other [NiFe]hydrogenases and contains only the cysteine ligands for the proximal [4Fe-4S] cluster (Classification and phylogeny of hydrogenases, Vignais et al., 2001). The corresponding hydrogenase activity is measured according to the method described in Fernandez et al. (1985).

The expression "soluble periplasmic hydrogenase" designates any enzyme containing iron and nickel or only iron, which catalyses hydrogen production or oxydation (Vignais et al., 2001). The corresponding hydrogenase activity is measured according to the method described in Fernandez et al. (1985). The expression "inactivation of said gene coding for a soluble periplasmic hydrogenase" corresponds to an insertion of a DNA fragment into said gene or to a mutation of the coding sequence of said gene resulting in the absence of expression or to the expression of a truncated or aberrant protein.

The expression "repression of said gene coding for a soluble periplasmic hydrogenase" corresponds to the absence of production of the corresponding protein due to growth conditions under which said gene is under the control of a regulatory element that prevents synthesis of the corresponding messenger RNA or the production of the translated protein.

The expression "deletion of said gene coding for a soluble periplasmic hydrogenase" corresponds to the total or partial removal of the coding sequence of said gene from its natural locus.

The present invention relates to the above-mentioned use of a bacterium strain, or of membrane extracts thereof, the genome of which contains a gene, represented by

SEQ ID NO: 1, coding for a membrane-bound [NiFe] hydrogenase represented by the sequence SEQ ID NO: 2, or contains a gene coding for a sequence having at least 30% of identity with sequence SEQ ID NO: 2.

The present invention relates to the above-mentioned use of a bacterium strain, or of membrane extracts thereof, chosen among Eubacteria or Archaeobacteria.

According to an advantageous embodiment, the use according to the present invention is characterized in that the bacterium strain, or the membrane extracts thereof, is chosen among: * the following Eubacteria:

- Desulfovibrio vulgaris, the genome of which contains a gene represented by SEQ ID NO: 1 coding for a membrane-bound [NiFe] hydrogenase represented by SEQ ID NO: 2, and a gene represented by SEQ ID NO: 3 coding for a membrane-bound [NiFe] hydrogenase represented by SEQ ID NO: 4,

- Desulfovibrio fructosovorans, the genome of which contains a gene represented by SEQ ID NO: 5 coding for a membrane-bound [NiFe] hydrogenase having 70% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 6,

- Desulfovibrio gigas, the genome of which contains a gene represented by SEQ ID NO: 7 coding for a membrane-bound [NiFe] hydrogenase having 74% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 8, - Thermoanaerobacter tengcongensis, the genome of which contains a gene represented by SEQ ID NO: 9 coding for a membrane-bound [NiFe] hydrogenase having 63% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 10, and contains a gene represented by SEQ ID NO: 11 coding for a membrane-bound [NiFe] hydrogenase having 38% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 12,

- Rhodospirillum rubrum, the genome of which contains a gene represented by SEQ ID NO: 13 coding for a membrane-bound [NiFe] hydrogenase having 41% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 14, - Escherichia coli, the genome of which contains a gene represented by

SEQ ID NO: 15 coding for a membrane-bound [NiFe] hydrogenase having 37% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 16, and contains a gene represented by SEQ ID NO: 17 coding for a membrane-bound [NiFe] hydrogenase having 35% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 18,

- Erwinia carotovora, the genome of which contains a gene represented by SEQ ID NO: 19 coding for a membrane-bound [NiFe] hydrogenase having 36% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 20,

- Salmonella typhi, the genome of which contains a gene represented by SEQ ID NO: 21 coding for a membrane-bound [NiFe] hydrogenase having 36% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 22,

- Salmonella typhimurium, the genome of which contains a gene represented by SEQ ID NO: 23 coding for a membrane-bound [NiFe] hydrogenase having 36% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 24,

- Shigella flexneri, the genome of which contains a gene represented by SEQ ID NO: 25 coding for a membrane-bound [NiFe] hydrogenase having 35% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 26,

- Wolinella succinogenes, the genome of which contains a gene represented by SEQ ID NO: 27 coding for a membrane-bound [NiFe] hydrogenase having 35% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 28, * the following Archaeobacteria:

- Methanosarcina mazei, the genome of which contains a gene represented by SEQ ID NO: 29 coding for a membrane-bound [NiFe] hydrogenase having 54% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 30, - Methanosarcina barkeri, the genome of which contains a gene represented by

SEQ ID NO: 31 coding for a membrane-bound [NiFe] hydrogenase having 53% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 32,

- Pyrococcus abyssi, the genome of which contains a gene represented by SEQ ID NO: 33 coding for a membrane-bound [NiFe] hydrogenase having 40% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 34, and contains a gene represented by SEQ ID NO: 35 coding for a membrane-bound [NiFe] hydrogenase having 38% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 36, - Pyrococcus horikoshii, the genome of which contains a gene represented by

SEQ ID NO: 37 coding for a membrane-bound [NiFe] hydrogenase having 39% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 38,

- Pyrococcus furiosus, the genome of which containing a gene represented by SEQ ID NO: 39 coding for a membrane-bound [NiFe] hydrogenase having 39% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 40,

- Methanococcus jannaschii, the genome of which contains a gene represented by SEQ ID NO: 41 coding for a membrane-bound [NiFe] hydrogenase having 36% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 42,

- Methanococcus maripuladis, the genome of which contains a gene represented by SEQ ID NO: 43 coding for a membrane-bound [NiFe] hydrogenase having 36% of identity with sequence SEQ ID NO: 1, and represented by SEQ ID NO: 44,

- Methanopyrus kandleri, the genome of which contains a gene represented by SEQ ID NO: 45 coding for a membrane-bound [NiFe] hydrogenase having 36% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 46, - Methanobacterium thermoautotrophicum, the genome of which contains a gene represented by SEQ ID NO: 47 coding for a membrane-bound [NiFe] hydrogenase having 37% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 48,

- Methanobacterium thermoformicium, the genome of which contains a gene represented by SEQ ID NO: 49 coding for a membrane-bound [NiFe] hydrogenase having 37% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 50, and contains a gene represented by SEQ ID NO: 51 coding for a membrane-bound [NiFe] hydrogenase having 33% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 52. The present invention relates to the above-mentioned use of a bacterium strain characterized in that its genome contains at least a gene coding for a membrane-bound [NiFe] hydrogenase, but does not contain genes coding for a soluble periplasmic hydrogenase.

According to an advantageous embodiment, the present invention relates to the use of a bacterium strain whose genome contains at least a gene coding for a membrane- bound [NiFe] hydrogenase, but does not contain genes coding for a soluble periplasmic hydrogenase, said bacterium strain being in particular E. coli.

The present invention also relates to the above-mentioned use, of a bacterium strain characterized in that its genome contains at least a gene coding for a membrane- bound [NiFe] hydrogenase, and one or several genes coding for a soluble periplasmic hydrogenase, said strain being transformed by inactivation or deletion of at least one of said genes coding for a soluble periplasmic hydrogenase, the corresponding soluble periplasmic hydrogenase being then inactivated or deleted. According to an advantageous embodiment, said gene is mutated by the use of a chemical agent such as acridine orange or nitomycine or by the insertion of a mobile element (transposons - insertion sequences) or by recombination, or by the use of ionising radiations (UV radiations). The present invention also relates to the above-mentioned use, of a bacterium strain characterized in that its genome contains at least a gene coding for a membrane- bound [NiFe] hydrogenase, and one or several genes coding for a soluble periplasmic hydrogenase, said strain being cultivated in such conditions that at least one of said genes coding for a soluble periplasmic hydrogenase is repressed, the corresponding soluble periplasmic hydrogenase being then not produced.

According to an advantageous embodiment, repression of some hydrogenase genes is observed under non-nitrogen fixing conditions, in aerobiosis or in the absence of formate or molecular hydrogen in the growth medium.

The present invention also relates to the use such as defined above, of a transformed bacterium strain chosen among Desulfovibrio vulgaris, Desulfovibrio fructosovorans, Desulfovibrio gigas, or Desulfovibrio desulfuricans, the genome of which being transformed by inactivation or deletion of at least one of said genes coding for a soluble periplasmic hydrogenase.

The present invention also relates to the use such as defined above, of a bacterium strain the genome of which being transformed by deletion of at least one of said genes coding for a soluble periplasmic hydrogenase.

The present invention also relates to the use as defined above, of a Desulfovibrio fructosovorans strain transformed in such way that the gene represented by SEQ ID

NO: 53 coding for the soluble periplasmic [NiFe] hydrogenase represented by SEQ ID NO: 54, and/or the gene represented by SEQ ID NO: 55 coding for the soluble periplasmic [Fe] hydrogenase represented by SEQ ID NO: 56, are deleted.

According to an advantageous embodiment, the present invention relates to the use of a transformed Desulfovibrio fructosovorans strain wherein the gene represented by SEQ ID NO: 53 is deleted. According to an advantageous embodiment, the present invention relates to the use of the transformed Desulfovibrio fructosovorans strain MR400, which was deposited on the January 11^th, 2005, at the Collection Nationale de Cultures de Microorganismes (CNCM) of the Institut Pasteur under the number 1-3349. According to an advantageous embodiment, the present invention relates to the use of a transformed Desulfovibrio fructosovorans strain wherein the genes represented by SEQ ID NO: 53 and SEQ ID NO: 55 are both deleted.

According to an advantageous embodiment, the present invention relates to the use of the transformed Desulfovibrio fructosovorans strain DF400, which was deposited on the January 11^th, 2005, at the Collection Nationale de Cultures de Microorganismes (CNCM) of the Institut Pasteur under the number 1-3350.

The present invention also relates to the above-mentioned use, of a bacterium strain, the genome of which being transformed by inactivation of at least one of said genes coding for a soluble periplasmic hydrogenase.

According to an advantageous embodiment, the present invention relates to the use of a bacterium strain, the genome of which being transformed by inactivation of at least one of said genes coding for a soluble periplasmic hydrogenase, said inactivation being obtained by mutation of at least one amino acid of said genes, and selection of the corresponding mutated proteins encoded by said mutated genes exhibiting from 0.1% to

30% of the native soluble periplasmic hydrogenase activity.

The periplasmic hydrogenase activity is measured according to the method described in Activity measurements (Fernandez et al., 1985). H₂ uptake activity was measured with ImM methyl viologen as described in Fernandez et al. (1985). Briefly, oxygen was removed under vacuum and the cuvette was flushed with Ar. Enzyme (10 μg for wt (wild-type) and E25D mutated form and 1 mg for E25Q, and E25V mutated forms) was allowed to activate for Ih under H₂ in 2OmM Tris/Hcl buffer at pH 8. The activity was then measured in an H2-flushed UV-cuvette, containing 1 mL of the buffer, in which residual oxygen was eliminated by adding 1 μL of a lOOg.L^"1 dithionite solution. The reaction was started by the addition of 5 to 30 μL of activated enzyme and the kinetics of the reduction of methyl viologen was measured at 604 nm in a UV 1601 spectrophotometer (Shimadzu) at 30°C.

According to an advantageous embodiment, the genome of Desulfovibrio fructosovorans strain is transformed by inactivation of the gene represented by SEQ ID NO: 53 coding for the soluble periplasmic [NiFe] hydrogenase represented by SEQ ID

NO: 54, and/or of the gene represented by SEQ ID NO: 55 coding for the soluble periplasmic [Fe] hydrogenase represented by SEQ ID NO: 56. According to an advantageous embodiment, the invention relates to the use of a transformed Desulfovibrio fructosovorans strain wherein the gene represented by SEQ ID NO: 53 is inactivated by:

- mutation of the nucleotide(s) GAA in position 264 of SEQ ID NO: 53 by substitution of said nucleotide by GAC, leading to a mutated gene represented by SEQ

ID NO: 57 encoding a mutated soluble periplasmic [NiFe] hydrogenase represented by SEQ ID NO: 58 and corresponding to the native sequence SEQ ID NO: 54 wherein the glutamate in position 25 is substituted by an aspartate, said mutated soluble periplasmic [NiFe] hydrogenase SEQ ID NO: 58 exhibiting from 30% to 60%, more particularly 45%, of the native soluble periplasmic hydrogenase SEQ ID NO: 54 activity,

- and/or mutation of the nucleotide(s) GAA in position 264 of SEQ ID NO: 53 by substitution of said nucleotide by CAG, leading to a mutated gene represented by SEQ ID NO: 59 encoding a mutated soluble periplasmic [NiFe] hydrogenase represented by SEQ ID NO: 60 and corresponding to the native sequence SEQ ID NO: 54 wherein the glutamate in position 25 is substituted by a glutamine, said mutated soluble periplasmic [NiFe] hydrogenase SEQ ID NO: 60 exhibiting from 0.05% to 0.2%, more particularly 0.1%, of the native soluble periplasmic hydrogenase SEQ ID NO: 54 activity,

- and/or mutation of the nucleotide(s) GAA in position 264 of SEQ ID NO: 53 by substitution of said nucleotide by GTC, leading to a mutated gene represented by

SEQ ID NO: 61 encoding a mutated soluble periplasmic [NiFe] hydrogenase represented by SEQ ID NO: 62 and corresponding to the native sequence SEQ ID NO: 54 wherein the glutamate in position 25 is substituted by a valine, said mutated soluble periplasmic [NiFe] hydrogenase SEQ ID NO: 62 exhibiting from 8% to 15%, more particularly 10%, of the native soluble periplasmic hydrogenase SEQ ID NO: 54 activity.

The present invention also relates to the above-mentioned use , of membrane extracts containing at least one membrane-bound [NiFe] hydrogenase of bacterium strains as defined above. The present invention also relates to the use of membrane extracts such as obtained according to the following method, which comprises the following steps:

- centrifugation of fully grown cells and washing of these cells, in order to obtain a cell suspension, - breaking of said cell suspension to obtain a crude extract, which is centrifuged at low speed to collect the bacterial debris, and then centrifuged for two hours at 50,000 rpm, in order to obtain a supernatant and a membrane pellet,

- discarding of said supernatant containing soluble proteins, and resuspension of said membrane pellet.

Fully grown cells are harvested by centrifugation at 8000 rpm and washed twice in Tris/HCl buffer pH = 8. The cell suspension is then broken using a French pressure cell. The crude extract is first centrifuged at low speed (500 rpm for ten minutes) to collect the bacterial debris, and then centrifuged for two hours at 50,000 rpm. The supernatant containing soluble proteins is discarded, and the membrane pellet is resuspended and tested for hydrogenase activity and palladisation experiments.

According to an advantageous embodiment, the present invention relates to the use of membrane extracts of Desulfovibrio fructosovorans such as obtained according to the method such as defined above. The present invention also relates to the use as defined above, for the preparation of metallic biocatalysts chosen among the following precious metals: palladium, platinum, ruthenium, rhodium and iridium.

The present invention also relates to a process for the preparation of metallic biocatalysts comprising the following steps: - bringing together bacterium strains, or transformed bacterium strains, or membrane extracts such as defined above, with a solution of a metal in oxidized form, for a time period sufficient to allow the initial sorption of the metal in oxidized form in said, bacterium strains, or transformed bacterium strains, or membrane extracts,

- bubbling of hydrogen gas through the solution such as obtained in the previous step, in order to allow the formation of a metal precipitate corresponding to the metal in reduced form, and recovering of said metal in reduced form.

The expression "for a time period sufficient to allow the initial sorption of the metal in oxidized form" corresponds to a duration of a few minutes to about one hour.

According to an advantageous embodiment, the process of the invention is used for the preparation of metallic biocatalysts chosen among the following precious metals: palladium, platinum, ruthenium, rhodium and iridium. The present invention also relates to a process for the preparation of palladium biocatalyst comprising the following steps:

- bringing together the DF400 strain such as defined above with a solution of palladium in oxidized form Pd(II), especially a solution of Na₂PdCl₄, for a time period sufficient to allow the initial sorption of Pd(II) in said strain,

- bubbling of hydrogen gas through the solution such as obtained in the previous step, in order to allow the formation of a palladium precipitate corresponding to the metal in reduced form Pd(O), and recovering of said palladium.

DESCRIPTION OF FIGURES

Figure IA represents the deletion of the [NiFe] hydrogenase encoding genes and construction of the MR400 strain and derivatives. hynA,B,C corresponds to the [NiFe] hydrogenase operon and npt to the fragment containing the Kn^r gene.

Figure IB represents the deletion of the [Fe] hydrogenase encoding genes and construction of the DF400 strain and derivatives. hydA,B corresponds to the [Fe] hydrogenase operon and accl to a gentamicin resistance gene.

Figure 2 - Reduction of Pd(II) by D. fructosovorans, wild type (white circles o);

[Fe]-only hydrogenase negative mutant (white lozenges 0); [NiFe] hydrogenase negative mutant (black circles •); double [Fe]-[NiFe] hydrogenase negative mutant (black triangles A); control, no cells (black squares ■); control, heat -killed cells (white squares D). The mass ratio Pd(II) dry biomass was 1:3; the test solution was 2 mM Na₂PdCl₄, in 0.1 M HNO₃; the electron donor 0.25 mM sodium formate. The final pH of the mixture after adding electron donor and the sample was 2.3. The cells were suspended in the mixture for 1 h prior to formate addition: the difference between the dosed Pd (II) and that analysed at the time of formate addition was contributable to biosorption by the biomass and remained constant in the heat-killed cells.

Figure 3 - TEM micrograph of initial stages of Pd(O) clusters growth within the periplasm of D. fructosovorans wild type cell (a), [Fe] -only hydrogenase negative mutant cell (b), [NiFe] hydrogenase negative mutant cell (c) and double [Fe]-[NiFe] hydrogenase negative mutant cell (d). The reaction stopped after 5 min of hydrogen supply into the cell suspensions preincubated with 2 mM Na₂PdCl₄, pH 2.3. The magnification is 120 000^x.

Figure 4 - TEM micrograph of palladised 'periplasmically treated' wild type (a) and double mutant (b) D. fructosovorans cells. Pd(II) was reduced from 2 mM Na₂PdCU₁PH 2.3 with hydrogen. The magnification is 120 000^x.

EXPERIMENTAL PART

The ability of sulphate-reducing bacteria (SRB) to reduce metallic ion species is a well-investigated phenomenon, and the involvement of microbial enzymes in this process has been established for many metals. Microbial enzyme-mediated reduction of Cr(VI), Se(VI), As(V), Tc(VII) and other metals has been reported for several species of bacteria during the last decade.

The growth of anaerobic sulphate-reducing Desulfovibrio species is based on the reduction of sulphate as the terminal electron acceptor and these organisms can utilise hydrogen as the sole source of electrons and energy (Brandis et al., 1981 ). Hydrogen metabolism plays a central role in the energy-generating mechanisms of the SRB. The key enzymes in hydrogen metabolism are hydrogenases which catalyse the reversible redox reaction: H₂ <→ 2H⁺ + 2e^~(Fauque et al., 1988).

Three types of hydrogenase have been isolated from various Desulfovibrio species and have been characterised in detail (Casalot et al., 2002; Fauque et al., 1988). The presence of one periplasmic [NiFe] hydrogenase, which represents about 1 % of the total periplasmic proteins, one periplasmic [Fe] hydrogenase and one cytoplasmic heterotetrameric NADP-reducing hydrogenase (Malki et al., 1995) was previously established in D. fructosovorans.

In order to elucidate the importance of these hydrogenases in the energy- generating metabolism of D. fructosovorans, mutant strains were produced by marker exchange mutagenesis of the genes encoding [NiFe] hydrogenase (Rousset et al., 1991) and the NADP-reducing hydrogenase (Malki et al., 1997). It was demonstrated that the single- and double- mutants were able to grow on organic substrates and on medium containing hydrogen as the sole energy source (Malki et al., 1997). It was suggested that growth under these conditions was possible due to the presence of the periplasmic [Fe]- only hydrogenase. Later a strain of D. fructosovorans lacking all three hydrogenases was constructed using marker exchange mutagenesis (Casalot et al., 2002). The triple hydrogenase negative mutant grew more slowly than the wild type although there were no major differences in growth compared to the double mutant strain (Malki et al.,

1997). The growth of the triple mutant in medium with H₂ as the sole electron donor can only be explained by the presence of an enzyme able to split hydrogen. This helped to establish the presence of a fourth hydrogenase in D. fructosovorans.

A focus of the present invention is to investigate the hydrogenase-dependent reduction of Pd(II) under acidic conditions using wild type D. fructosovorans and to study the involvement of hydrogenase in Pd(II) reduction using specific periplasmic hydrogenase-negative mutant strains ([Fe] -hydrogenase, [NiFe] -hydrogenase and double [Fe]-[NiFe] negative mutant).

MATERIAL AND METHODS

Preparation of the mutant strains

1) Deletion of the [NiFe] hydrogenase encoding genes and construction of the MR400 strain and derivatives (Figure IA) The 7-kb fragment that contains the [NiFe] hydrogenase operon (hynA,B,C), was digested with MIuI, blunt-end repaired and ligated with the Stoal-digested 1.4-kb fragment containing the Kn^r gene (npt). The resulting construction was cloned into a suicide vector and introduced into Desulfovibrio frustosovorans wild-type strain (DSM3604). Recombinant strains were selected on culture medium containing Kanamycin. One clone, named MR400, has been isolated and characterised.

2) Deletion of the [Fe] hydrogenase encoding genes and construction of the DF400 strain and derivatives (Figure IB)

The gentamicin resistance gene (accl) was obtained by PCR amplification performed on using two oligonucleotides designed to introduce the BspEI and BstEII restriction sites. The 5-kb fragment that contains the [Fe] hydrogenase operon (hydA,B), was digested with BspEI and BstEII and ligated with the ifapEI/itøEII-digested 1.4-kb fragment containing the Gm^r gene (accl). The resulting construction was cloned into a suicide vector and introduced into D. frustosovorans wild -type strain (DSM3604) and D. fructosovorans MR400 strain described above. Recombinant strains were selected on culture medium containing Genatamicin. One clone, named SF, deriving from the wild- type strain has been isolated and characterised. Another clone, named DF400, deriving from the MR400 strain has been isolated and characterised.

Bacterial Strains and Growth Conditions

Mutants of D. fructosovorans were constructed using marker exchange mutagenesis (Rousset et al., 1991). D. fructosovorans wild type and strains carrying a deletion in the [NiFe] hydrogenase operon (Rousset et al., 1991) and [Fe] hydrogenase operon and the double [Fe]-[NiFe] hydrogenase-negative mutant were grown anaerobically at 37°C in SOS medium (Rousset et al., 1998). Cells were grown in 2 litre septum- stoppered serum bottles.

Preparation of resting cells The cells were harvested at 48 hours after inoculation (OD₆₀₀ = 2.100) by centrifugation in air (8000 rpm, Beckman G2-21M/E centrifuge, 20 min). The obtained pellet was washed in 20 mM N-morpholinopropanesulfonic acid (MOPS)-NaOH buffer, pH 7.0, three times. After the final wash the cells were centrifuged as before and the pellets were resuspended in 50 ml of 20 mM MOPS-NaOH buffer, pH 7.0. Aliquots of the suspension were used for assay of hydrogenase activity, Pd(II) reduction tests and for preparation of "periplasmically treated cells" (see below).

Pd(II) assay

Pd(II) concentration in solutions was estimated spectrophotometrically by measuring the concentration of the yellow coloured [PdCl₄]^"2 complex ion at A₄₂₀. The maximum height of the specific absorption peak of [PdCl₄]^2" at 420 nm is proportional to the palladium concentration. This method was confirmed using an assay based on SnCl₂ZHCl (Dasages, 1978) with the latter method in turn cross-checked using polarographic analysis (Yong et al., 2002). The three methods gave identical results.

Pd(II) reduction

A solution OfNa₂PdCl₄ (10 ml of 2 mM in 0.01 M HNO₃, pH 2.2) was degassed under oxygen free nitrogen (OFN) in butyl rubber septum-sealed vials. Resting cell suspensions of each strain were added anaerobically to the Pd(II) solution in the ratio of 3 parts dry weight of biomass (estimated by a pre-determined conversion from the OD₆₀₀ of the suspension) to 1 one part of Pd(II) by mass and left to biosorb Pd(II) at 30°C. After one hour, aliquots of the cell suspensions in the Pd(II) solution were withdrawn and centrifuged (13000 rpm for 3 min, Heraeus Biofuge pico). The supernatants were analysed to quantify Pd(II) remaining in solution after biosorption but prior to reduction. Pd(II) bioreduction was initiated via addition of the electron donor (20 mM sodium formate solution, pH 7.0). The final pH of the reaction mixture was 2.3. The reaction was carried out anaerobically at 30°C with samples withdrawn at 20 min intervals. The residual concentration of Pd in the supernatant was measured and plotted as a function of time. All experiments were done in triplicate; the experimental error was within 5% of the mean throughout.

Preparation of 'periplasmically-treated' (PT) cells

In order to prepare the wild type D. fructosovorans cells containing only membrane-bound hydrogenase, the periplasmic proteins were extracted from the cells using a modification of the procedure developed by Badzoing and Thauer as described in Hatchikian et al. (1990). Cells of wild type D. fructosovorans and the double mutant, used as a reference strain, were treated simultaneously. Approximately 1 g wet weight of freshly harvested cells (by centrifugation: 8 000 rpm at 4°C, 25 min Beckman G2- 21M/E centrifuge) was resuspended in 100 ml of 50 mM Tris-HCl buffer, pH 9, containing 50 mM EDTA and 170 mM Na₂CO₃. The ratio of wet cell mass to the volume of the buffer was 10 times higher compared to the original method to ensure maximum extraction of periplasmic proteins. The suspensions were stirred gently for 30 min at 35°C and then centrifuged at 25 000 g for 30 min. The cells were resuspended in the same buffer and the treatment was repeated. The periplasmic proteins were retained in the combined supernatants (Hatchikian et al., 1990). The periplasmically-treated cells were resuspended in 20 mM MOPS buffer, pH 7.0, and used to reduce palladium (II) in parallel with untreated controls. The supernatants were concentrated (from 200 ml to 5 ml) using Millipore ultrafiltration polyethersulfone 76 mm diameter membranes (nominal molecular weight cut-off 30 000) under nitrogen pressure for estimation of extracted periplasmic hydrogenase activity. The hydrogenase activity of suspensions of 'periplasmically' treated cells was also measured. Preparation of membrane extracts

Freshly thawed cells were passed twice in a French pressure cell at 1,000 lb/in² pressure in the presence of a few crystals of DNase. Cell debris were removed by centrifugation at 4,000 x g for 30min, and the supernatant (crude extract) was then centrifuged at 120,000 x g for 2 h. The membrane fraction that was collected in the pellet was used to reduce palladium (II).

Assay of Hydrogenase Activity

The total hydrogenase activity (as H₂ uptake) of D. fructosovorans resting cell suspension was quantified by spectrophotometric measurement of the reduction of methyl viologen at 30°C (Fernandez et al., 1985). One ml of 1 mM methyl viologen in 50 mM Tris-HCl buffer, pH 8.0, containing 1 mM EDTA (to render the cell outer membrane permeable) was degassed in an air-tight quartz cuvette. The solution was then saturated with H₂ and 1 μl of sodium dithionite saturated aqueous solution was added to eliminate the traces of O₂. The reaction was initiated by adding 2 μl of sample and the reaction was followed at A₆₀₄. One unit of hydrogenase activity is the amount of enzyme which catalyses the reduction of 2 μM methyl viologen per minute. Alternatively, in the case where hydrogenase activity of the cell suspension was estimated, enzyme activity was expressed in μM H₂ consumed per minute per 10⁹ cells. In order to establish whether the periplasmic [NiFe] hydrogenase from

D. fructosovorans retains its activity at low pH, a purified (following the procedure reported previously (Rousset et al., 1998) periplasmic [NiFe] hydrogenase was used in the following experiments. A solution of purified enzyme (initial protein concentration 26 mg/ml) was mixed with 20 mM glycine-HCl buffer, pH 2.2, in a 1:10 (v/v) ratio, degassed, and left to stand. After one hour, the hydrogenase activity was measured with

1 mM methyl viologen as above in 50 mM Tris-NCl buffer, pH 8.0. Hydrogenase activity was also measured in the 20 mM glycine-HCl buffer, pH 2.2. In this case, reduction of the high-potential compound phenazine methosulfate was used instead of low-potential methyl viologen, and reduction was monitored spectrophotometrically at 387 nm.

Electron microscopy and Energy dispersive X-ray (EDS) analysis

Samples of Pd-loaded bacteria were fixed in 2.5% (wt/vol) aqueous glutaraldehyde and were resin-embedded following a standard dehydration procedure (Yong et al., 2002a). Microtome-cut sections 100-150 nm thick were placed on copper grids and viewed with a JEOL 120CX2 transmission electron microscope fitted with a Link ISI EDX system for Pd detection (Lloyd et al., 1999).

RESULTS

Stability of hydrogcnasc activity at acidic pH

Biorecovery of Pd(II) from solution was shown previously to be optimal if an initial biosorption step at pH 2-3 (30-60 min) preceded hydrogenase mediated metal reduction (Yong et al., 2002). The conditions for biosorption were defined by de Vargas et al. (2004), but the stability of the bacterial hydrogenase to exposure to acidic pH has not been established previously.

Measurement of hydrogen consumption by purified periplasmic [NiFe] hydrogenase from D. fructosovorans (pre -incubated in 20 mM glycine-HCl buffer, pH

2.2, for 1 hour) showed that the enzyme retained approximately 32 % of its initial activity following exposure to acidic conditions, but no enzyme activity was detected following 2 hours of pre-incubation in the acidic buffer. The enzyme activity in this case was estimated via reduction of methyl viologen in Tris-HCl buffer, pH 8.0. The purified hydrogenase was also active in the test with 25 μM phenazine methosulfate in 20 mM glycine-HCl buffer pH 2.2, where the activity was 8.23 ± 0.21 mA₃₈₇/min/mg protein.

Hydrogenase activity of periplasmic extracts and 'periplasmically treated' cells

The hydrogenase activity of the periplasmic protein extracted from the wild type cells was 0.416 μM H₂ consumed/min/mg protein whereas that of the extract from the double mutant was zero, confirming the lack of contamination of the periplasmic extract by the membrane-bound enzyme and confirming maintenance of cellular integrity. The hydrogenase activity of wild type and double mutant cell suspensions after extraction of periplasmic proteins ('periplasmically treated cells') was 0.00701 ± 0.0015 and 0.00661 ± 0.0018 respectively, which is comparable with the activity of untreated double mutant cell suspension (00684 ± 0.0004). Hydrogenase activity and Pd(II) reduction by parent and mutant strains

The total hydrogenase activity of resting cell suspensions of the wild type and mutant strains was estimated (Table 1).

Table 1

The hydrogenase activity of D. fructosovorans resting cell suspensions used for Bio-Pd reduction (H₂ uptake methyl viologen test).

Hydrogenase activity A_HD = μmol H₂ consumed/min/10⁹ cells. OD₆₀₀ = 1.00 corresponds to 10⁸ cells/ml.

The enzyme activity was consistent with the amount of enzyme expressed according to the genetic structure of the strains. The total hydrogenase activity of [Fe]- and [NiFe] -hydrogenase negative mutants was, accordingly, 83.7 % and 13.5 % of the hydrogenase activity of wild type cell suspension. The hydrogenase activity of the double mutant suspension, attributable to cytoplasmic-bound hydrogenase, comprised 3.4 % of that of wild type strain and agreed with residual amount in 'periplasmically treated' cells. The bioreduction of Pd(II) by the parental strain of D. fructosovorans and its [Fe]- only, [NiFe]- and double [Fe] -[NiFe] -hydrogenase negative was compared using Na₂PdCl₄ at pH 2.3 (Figure 2). Reduction of Pd by sodium formate alone was used as a control. Figure 2 shows that the wild type and all mutant strains initially reduced Pd(II) at approximately the same rate but after approximately 40 minutes Pd(II) reduction by the [NiFe]- and double mutant strains was slightly higher. Identical results were obtained using cell suspension sparged with H₂. Sodium formate was used in order to quantify the amount of reducing agent in the reaction mixture, which was more difficult to achieve using hydrogen. Examination of palladised cells

Palladised biomass was obtained by reducing palladium (II) using the wild type and [Fe]-, [NiFe]- and [Fe]-[NiFe] double hydrogenase negative mutants under acidic conditions (mimicking the pH of industrial waste solutions) from 2 mM Na₂PdCl₄ at pH

2, with hydrogen as a reducing agent. In order to observe the initial stage of Pd(O) cluster growth in the wild type and double mutant cells Pd(II) reduction was stopped after 5 minutes via termination of hydrogen supply through the solution and substituting with N₂. Pd(O) was visible within the periplasmic space of the parent and [Fe] hydrogenase mutant strains (Fig. 3a,b). EDX analyses of all preparations confirmed that the black precipitate associated with the cells was Pd(0)(Lloyd et al., 1998). Pd(O) clusters were distributed randomly through the whole width of the periplasm in the wild type and [Fe] -hydrogenase negative mutant cells (Fig. 3a, 3b), whereas in the [NiFe]- hydrogenase negative mutant (Fig. 3c) and double mutant cells (Fig. 3d) they were localised on the cytoplasmic membrane.

In both the double mutant and the wild type "periplasmically-treated" cells, from which soluble periplasmic hydrogenases had been extracted, Pd(O) clusters were associated with the inner cytoplasmic membrane (Fig. 4). These are similar to the Pd(O) deposits observed on the inner cytoplasmic membrane of intact double mutant cells (Fig. 3d).

DISCUSSION

Microbial Pd(II) reduction is not only of theoretical interest but also has a potential application in palladium recovery from waste solutions. Industrial Pd(II)- containing waste solutions are usually fairly acidic since in many cases they are prepared using aqua regia (mixture of hydrochloric acid and nitric acid) for precious metal leaching from solid wastes (Yong et al., 2002). This study showed that 30% of the activity of purified [NiFe] hydrogenase from D. fructosovorans is retained after one hour preincubation at pH 2.2; although whether the similar decreasing of activity under the same conditions was attributable equally to other two hydrogenases was not tested

(Table 1 shows the relative activities of the three hydrogenases in untreated cells).

The fact that purified [Ni-Fe] hydrogenase was exposed to glycine-HCl buffer, pH 2.2, for one hour and remained active demonstrated in principle its resistance to acidic conditions; the enzymes could be more pH-stable in their cellular locations. The one hour period was chosen because it was estimated to be the optimal time for preincubation of the resting cells with Pd(II)-containing industrial waste solution (pH about 2) to achieve biosorption of the [PdCl₄]^2" complex onto the protonated biomass prior to bio-reduction (Yong et al., 2002). The working pH 2.2 is consistent the usual pH of precious metal wastes. According to de Vargas et al. (2004), biosorption of Pd (II) onto D. desulfuricans at pH 0.1-3.0 in HNO₃ background was achieved after 30 min, the time taken to reach equilibrium. During the pre-incubation period, the hydrogenase retained its structure, which was monitored visually as the brown colour of the enzyme solution (the denaturated enzyme loses Fe atoms and becomes colourless) . After two hours exposure, the enzyme demonstrated no hydrogenase activity, possibly due to degradation. The residual activity (if to assume that hydrogenase was affected by low pH within the resting cell) after one hour was sufficient to achieve good Pd(II) removal.

The purified [Ni-Fe] hydrogenase was also active at pH 2.2 when its activity was measured using the high potential phenazine-methosulfate. In all cases prior to activity measurements the enzyme was activated with hydrogen as in Hatchikian et al. (1990). These results suggest that periplasmic hydrogenases in vivo within resting cells remain active during pre-incubation of the biomass with low pH palladium-containing solutions and can work effectively at pH 2.3. Comparison of Pd(II) bioreduction by wild type D. fructosovorans and the [Fe]- only, [NiFe]- and double [Fe] -[NiFe] -hydrogenase negative mutants after preincubation at low pH showed that the initial rate of Pd(II) reduction was similar. After approximately 1 hour in the presence of formate, Pd(II) reduction by [NiFe]- and double mutants was slightly higher (Figure 2) than that by the wild type and [Fe] -only negative mutant. No correlation was observed between the initial hydrogenase activity of the strains and their Pd(II) reducing activity. This to be expected since only nucleation and initiation of the palladium crystals is dependent on hydrogenase activity whereas subsequent crystal growth is due to auto-catalytic reduction of palladium (Yong et al., 2002). Electron microscopy of the initial stage of cellular palladisation revealed peculiarities of palladium deposition onto the cell surface depending on the mutant used. The most obvious difference was between wild type and [NiFe] -hydrogenase negative mutant and double mutant. In the wild type cells, Pd(O) clusters were distributed randomly throughout the periplasm (Fig. 3 a) whereas in the double mutant cells, which lack soluble periplasmic hydrogenases and contain only a single cytoplasmic membrane bound [NiFe] -hydrogenase (Casalot et al., 2002), Pd clusters were clearly associated with the inner cytoplasmic membrane (Fig. 3d). A similar pattern of Pd crystal distribution was observed in the [NiFe] hydrogenase negative mutant (Fig. 3c), which retained only 13.5 % of its periplasmic hydrogenase activity compared to the parental strain. Removal of the minor periplasmic [Fe] -only hydrogenase appeared to have little effect on the localisation of Pd(O), probably attributable to the predominance of the periplasmic [NiFe] hydrogenase, but quantitative assays were not attempted since the retention of activity of each of the enzymes at pH 2.2 was not attempted.

The localisation of palladium crystals on the inner cytoplasmic membrane is coincident with the localisation of this hydrogenase suggesting that the enzyme serves as a nucleation site for palladium particle growth, probably by supplying the electrons for the initial Pd(II) reduction. In order to verify this suggestion, 'periplasmically -treated' cells of the wild type and double mutant were used for palladisation. These cells were depleted of periplasmic soluble proteins and left with only membrane-bound periplasmic hydrogenase. Analysis of hydrogenase activity of the soluble proteins extract from the parental strain demonstrated hydrogenase activity whereas the double mutant extract did not. This indicated that the inner cytoplasmic membrane remained intact and prevented extraction of cytoplasmic hydrogenase and membrane-bound hydrogenases.

Periplasmically-treated cells were palladised at pH 2.3 and the distribution of Pd clusters was observed using TEM. In periplasmically-treated cells of both the wild type and the double mutant Pd(O) clusters were associated with the inner cytoplasmic membrane (Fig. 4 ), similar to the pattern observed in intact double mutant cells

(Fig. 3d). This again suggests that the localisation of Pd(II) reduction is associated with the localisation of membrane -bound hydrogenase in the double mutant cells and the periplasmically treated wild type cells.

In conclusion, the data obtained show that in the parent strain Pd(O) nucleation could occur at both membrane and periplasmic loci, whereas in the double mutant the nucleation sites would be confined to the cytoplasmic membrane (Fig. 3a,d). REFERENCES

- Baxter-Plant VS, Mikheenko IP, Macaskie LE (2003) Sulphate reducing bacteria, palladium and reductive dehalogenation of chlorinated aromatic compounds. Biodegradation 14: 83-90,

- Brandis A , and Thauer RK (1981) Growth of Desulfovibrio species on hydrogen and sulfate as sole energy source. J. Gen. Microbiol. 126: 249-252, - Casalot L, de Luca G, Dermoun Z, Rousset M and de Philip P (2002) Evidence for a fourth hydrogenase in Desulfovibrio fructosovorans. J. Bacteriol. 184: 853-856,

- Dasages GC (1978) Absorptiometriques des elements mineraux. Masson Ed. Paris,

- de Vargas I, Macaskie LE and Guibal E (2004) Biosorption of palladium and platinum by sulfate-reducing bacteria. J. Chem. Technol. Biotechnol. 79: 49-56,

- Fauque G, Peck DH, Huynh BH, Berlier Y, Dervatanian DV, Texieira M, Przybyla AE, Lespinat PA, Moura I and LeGaIl J (1988) The three classes of hydrogenases from sulfate-reducing bacteria of the genus Desulfovibrio. FEMS Microbiol. Rev. 54: 299-344, - Fernandez VM, Hatchikian EC and Cammack R (1985) Properties and reactivation of two different deactivated forms of Desulfovibrio gigas hydrogenase. Biochim. Biophys. Acta. 832: 69-79,

- Ganesh R, Robinson KG, Chu L, Kucsmas D, Reed GD (1999) Reductive precipitation of uranium by Desulfovibrio desulfuricans: Evaluation of contaminant effects and elective removal. Water Research 33: 3447-3458,

- Hatchikian CE, Traore A, Fernandez VM and Cammack R (1990) Characterization of the nickel-iron periplasmic hydrogenase from Desulfovibrio fructosovorans. Europ. J. ofBiochem. 187: 635-643,

- Hatchikian CE, Traore A, Fernandez VM and Cammack R (1990). Characterization of the nickel-iron periplasmic hydrogenase from Desulfovibrio fructosovorans. Europ. J. ofBiochem. 187: 635-643, - Henry CR ( 1998) Surface studies of supported model catalysts. Surf Science Reports 31:235-325,

- Hoffman JE ( 1988) Recovering platinum-group metals from auto catalysts. J Metals 88: 40-44, - Lloyd JR, Cole JA, Macaskie LE (1997) Reduction and removal of heptavalent technetium from solution by Escherichia coli. J. Bacteriol 179: 2014 -2021,

- Lloyd JR, Harding CL, Macaskie LE (1997a) Reduction and accumulation of Tc(VII) by immobilized cells of Escherichia coli in a hollow fibre reactor. Biotechnol Bioeng 55: 505-510, - Lloyd JR, Nolting HF, Sole VA, Bosecker K, Macaskie LE (1998) Technetium reduction and precipitation by sulphate-reducing bacteria. GeomicrobiolJ 15: 43-58,

- Lloyd JR, Yong P, Macaskie LE ( 1998) Enzymatic recovery of elemental palladium by using sulfate-reducing bacteria. Appl Environ Microbiol 64: 4607-4609,

- Lloyd JR, Ridley J, Khizniak T, Lyalikova NN, Macaskie LE (1999) Reduction of technetium by Desulfovibrio desulfuricans: biocatalyst characterization and use in a flowthrough bioreactor. Appl Environ Microbiol 65: 2691-2696,

- Lloyd JR, Thomas GH, Finlay JA, Cole JA, Macaskie LE ( 1999a) Microbial reduction of technetium by Escherichia coli and Desulfovibrio desulfuricans: enhancement via the use of high-activity strains and effect of process parameters. Biotechnol Bioeng 66: 122- 130,

- Lloyd JR, Mabbett AN, Williams DR, Macaskie LE (2001) Metal reduction by sulphate-reducing bacteria: physiological diversity and metal specificity. Hydrometallurgy 59: 327-337.

- Lovely DR, Phillips EJP ( 1992) Reduction of uranium by Desulfovibrio desulfuricans. Appl Environ Microbiol 54 : 1472- 1480,

- Mabbett NA (2001) The reduction and recovery of Cr(VI) from industrially polluted waste waters using sulphate-reducing bacteria, PhD Thesis, University of Birmingham, UK,

- Malki S, Saimmaime I, De Luca G, Rousset M, Dermoun Z and Belaich J-P (1995) Characterization of an operon encoding a NADP-reducing hydrogenase in

Desulfovibrio fructosovorans. J. Bacteriol. Ill: 2628-2636,

- Malki S , de Luca G , Fardeau ML , Rousset M , Belaich J -P and Dermoun Z (1997) Physiological characteristics and growth behavior of single and double hydrogenase mutants of Desulfovibrio fructosovorans. Arch, of Microbiol. 167: 38-45, - Menon NK, Robbins J, Wendt JC, Shanmugam, KT, Przybyla AE ( 1991) Mutational analysis and characterization of Escherichia coli hya operon, which encodes [NiFe] hydrogenase 1. JBacteriol 173: 4851-4861,

- Mertig M, Kirsch R, Pompe W ( 1998) Biomolecular approach to nanotube fadrication. Appl Phys A 66: S723 -727,

- Peck HD ( 1993) Bioenergetic strategies of the sulphate-reducing bacteria. In Odom JM, Singleton R. (eds.). Sulfate-Reducing Bacteria: Contemporary Perspectives. New York: Springer- Verlag,

- Platinum (2003) Interim Review. Johnson Matthey. November, 2003, - Postgate JR (1979) The Sulphate-Reducing Bacteria. Cambridge, UK:

University Press,

- Rousset M, Dermoun Z, Chippaux M and Belaich J-P (1991) Marker exchange mutagenesis of the hydN genes in Desulfovibrio fructosovorans. Molec. Microbiol. 5: 1735-1740, - Rousset M, Casalot L, Rapp-Giles BJ, Dermoun Z, de Philip P, Be'laich J-P and Wall JD ( 1998) New shuttle vectors for the introduction of cloned DNA in Desulfovibrio. Plasmid. 39: 114-122,

- Sauter M, Bohm R, Bock A ( 1992) Mutation analysis of the operon (hyc) determining hydrogenase 3 formation in Escherichia coli. MoI Microbiol 6: 1523-1532, - Spear JR, Figueroa LA, Honeyman BD ( 1999) Modelling the removal of uranium U(VI) from aqueous solutions in the presence of sulphate-reducing bacteria. Environ Science Technol 33: 2667-2675,

- Tucker MD, Barton LL, Thomson BM (1998) Reduction of Cr, Mo, Se and U by Desulfovibrio desulfuricans immobilized in polyacrylamide gels. J of Industrial Microbiol Biotechnol 20: 13 - 19,

- Vignais PM, Billoud B, Meyer M (2001) Classification and phylogeny of hydrogenases. FEMS Microbiol. Rev. 25: 455-501,

- Wahl R, Mertig M, Raff J, Selenska-Pobell S, Pompe W (2001) Electron-beam induced formation of highly ordered palladium and platinum nanoparticle arrays on the S layer of Bacillus sphaericus NCTC 9602. Adv Mater 13: 736-74025,

- Yong P, Rowson NA, Farr J.P, Harris IR, Macaskie LE (2002) Bioaccumulation of palladium by Desulfovibrio desulfuricans. J Chem Technol Biotechnol 77:1-9, - Yong P, Farr JPG, Harris IR, Macaskie LE ( 2002a) Palladium recovery by immobilized cells of Desulfovibrio desulfuricans using hydrogen as the electron donor in a novel electrobioreactor. Biotechnol Lett 4: 205-212,

- Yong P, Rowson NA, Farr J.P, Harris IR, Macaskie LE (2002b) Bioreduction and bio -crystallisation of palladium by Desulfovibrio desulfuricans NCIMB 8307. Biotechnol Bioeng 80: 369-379,

- Yong P, Rowson NA, Farr J.P, Harris IR, Macaskie LE ( 2003) A novel biotechnology for the recovery of precious metals from spent automotive catalyst. Environ Technol 24:289-297.

Claims

1. The use of a bacterium strain characterized in that its genome contains at least a gene coding for a membrane-bound [NiFe] hydrogenase, or of membrane extracts containing said membrane-bound [NiFe] hydrogenase of said bacterium strain, for the preparation of metallic biocatalysts, provided that in the case of the use of a bacterium strain the genome of which contains one or several genes coding for a soluble periplasmic hydrogenase, then said strain is transformed by inactivation, repression or deletion of at least one of said genes coding for a soluble periplasmic hydrogenase, the corresponding soluble periplasmic hydrogenase being then non produced or absent.

2. The use according to claim 1, of a bacterium strain, or of membrane extracts thereof, the genome of which containing a gene, represented by SEQ ID NO: 1, coding for a membrane-bound [NiFe] hydrogenase represented by the sequence SEQ ID NO: 2, or containing a gene coding for a sequence having at least 30% of identity with sequence SEQ ID NO: 2.

3. The use according to claim 1 or 2, of a bacterium strain, or of membrane extracts thereof, chosen among Eubacteria or Archaeobacteria.

4. The use according to any of claims 1 to 3, of a bacterium strain, or of membrane extracts thereof, chosen among:

* the following Eubacteria: - Desulfovibrio vulgaris, the genome of which contains a gene represented by

SEQ ID NO: 1 coding for a membrane-bound [NiFe] hydrogenase represented by SEQ

ID NO: 2, and a gene represented by SEQ ID NO: 3 coding for a membrane-bound

[NiFe] hydrogenase represented by SEQ ID NO: 4,

- Desulfovibrio fructosovorans, the genome of which contains a gene represented by SEQ ID NO: 5 coding for a membrane-bound [NiFe] hydrogenase having 70% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 6, - Desulfovibrio gigas, the genome of which contains a gene represented by SEQ ID NO: 7 coding for a membrane-bound [NiFe] hydrogenase having 74% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 8,

- Thermoanaerobacter tengcongensis, the genome of which contains a gene represented by SEQ ID NO: 9 coding for a membrane-bound [NiFe] hydrogenase having 63% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 10, and contains a gene represented by SEQ ID NO: 11 coding for a membrane-bound [NiFe] hydrogenase having 38% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 12, - Rhodospirillum rubrum, the genome of which contains a gene represented by

SEQ ID NO: 13 coding for a membrane-bound [NiFe] hydrogenase having 41% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 14,

- Escherichia coli, the genome of which contains a gene represented by SEQ ID NO: 15 coding for a membrane-bound [NiFe] hydrogenase having 37% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 16, and contains a gene represented by SEQ ID NO: 17 coding for a membrane-bound [NiFe] hydrogenase having 35% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 18, - Erwinia carotovora, the genome of which contains a gene represented by SEQ

ID NO: 19 coding for a membrane-bound [NiFe] hydrogenase having 36% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 20,

- Salmonella typhimurium, the genome of which contains a gene represented by SEQ ID NO: 23 coding for a membrane-bound [NiFe] hydrogenase having 36% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 24, - Shigella flexneri, the genome of which contains a gene represented by SEQ ID

NO: 25 coding for a membrane-bound [NiFe] hydrogenase having 35% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 26,

- Wolinella succinogenes, the genome of which contains a gene represented by SEQ ID NO: 27 coding for a membrane-bound [NiFe] hydrogenase having 35% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID

NO: 28,

* the following Archaeobacteria:

- Methanosarcina mazei, the genome of which contains a gene represented by SEQ ID NO: 29 coding for a membrane-bound [NiFe] hydrogenase having 54% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 30,

- Methanosarcina barkeri, the genome of which contains a gene represented by SEQ ID NO: 31 coding for a membrane-bound [NiFe] hydrogenase having 53% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID

NO: 32,

- Pyrococcus abyssi, the genome of which contains a gene represented by SEQ ID NO: 33 coding for a membrane-bound [NiFe] hydrogenase having 40% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 34, and contains a gene represented by SEQ ID NO: 35 coding for a membrane-bound

[NiFe] hydrogenase having 38% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 36,

- Pyrococcus horikoshii, the genome of which contains a gene represented by SEQ ID NO: 37 coding for a membrane-bound [NiFe] hydrogenase having 39% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID

NO: 38,

- Methanococcus jannaschii, the genome of which contains a gene represented by SEQ ID NO: 41 coding for a membrane-bound [NiFe] hydrogenase having 36% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 42, - Methanococcus maripuladis, the genome of which contains a gene represented by SEQ ID NO: 43 coding for a membrane-bound [NiFe] hydrogenase having 36% of identity with sequence SEQ ID NO: 1, and represented by SEQ ID NO: 44, - Methanopyrus kandleri, the genome of which contains a gene represented by SEQ ID NO: 45 coding for a membrane-bound [NiFe] hydrogenase having 36% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 46, - Methanobacterium thermoautotrophicum, the genome of which contains a gene represented by SEQ ID NO: 47 coding for a membrane-bound [NiFe] hydrogenase having 37% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 48,

- Methanobacterium thermoformicium, the genome of which contains a gene represented by SEQ ID NO: 49 coding for a membrane-bound [NiFe] hydrogenase having 37% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 50, and contains a gene represented by SEQ ID NO: 51 coding for a membrane-bound [NiFe] hydrogenase having 33% of identity with sequence SEQ ID NO: 2, said hydrogenase being represented by SEQ ID NO: 52.

5. The use according to any of claims 1 to 4, of a bacterium strain characterized in that its genome contains at least a gene coding for a membrane-bound [NiFe] hydrogenase, but does not contain genes coding for a soluble periplasmic hydrogenase.

6. The use according to claim 5, of E. coli.

7. The use according to any of claims 1 to 4, of a bacterium strain characterized in that its genome contains at least a gene coding for a membrane-bound [NiFe] hydrogenase, and one or several genes coding for a soluble periplasmic hydrogenase, said strain being transformed by inactivation or deletion of at least one of said genes coding for a soluble periplasmic hydrogenase, the corresponding soluble periplasmic hydrogenase being then inactivated or deleted.

8. The use according to any of claims 1 to 4, of a bacterium strain characterized in that its genome contains at least a gene coding for a membrane-bound [NiFe] hydrogenase, and one or several genes coding for a soluble periplasmic hydrogenase, said strain being cultivated in such conditions that at least one of said genes coding for a soluble periplasmic hydrogenase is repressed, the corresponding soluble periplasmic hydrogenase being then not produced.

9. The use according to claim 7, of a transformed bacterium strain chosen among Desulfovibrio vulgaris, Desulfovibrio fructosovorans, Desulfovibrio gigas, or

Desulfovibrio desulfuricans, the genome of which being transformed by inactivation or deletion of at least one of said genes coding for a soluble periplasmic hydrogenase.

10. The use according to any of claims 7 to 9, of a bacterium strain the genome of which being transformed by deletion of at least one of said genes coding for a soluble periplasmic hydrogenase.

11. The use according to any of claims 7 to 10, of a Desulfovibrio fructosovorans strain transformed in such way that the gene represented by SEQ ID NO: 53 coding for the soluble periplasmic [NiFe] hydrogenase represented by SEQ ID

NO: 54, and/or the gene represented by SEQ ID NO: 55 coding for the soluble periplasmic [Fe] hydrogenase represented by SEQ ID NO: 56, are deleted.

12. The use according to any of claims 7 to 11, of a transformed Desulfovibrio fructosovorans strain wherein the gene represented by SEQ ID NO: 53 is deleted.

13. The use according to claim 12, of the transformed Desulfovibrio fructosovorans strain MR400, which was deposited on the January 11^th, 2005 at the Collection Nationale de Cultures de Microorganismes (CNCM) of the Institut Pasteur under the number 1-3349.

14. The use according to any of claims 7 to 11, of a transformed Desulfovibrio fructosovorans strain wherein the genes represented by SEQ ID NO: 53 and SEQ ID NO: 55 are both deleted.

15. The use according to claim 14, of the transformed Desulfovibrio fructosovorans strain DF400, which was deposited on the January 11^th, 2005 at the Collection Nationale de Cultures de Microorganismes (CNCM) of the Institut Pasteur under the number 1-3350.

16. The use according to claims 7 to 9, of a bacterium strain the genome of which being transformed by inactivation of at least one of said genes coding for a soluble periplasmic hydrogenase.

17. The use according to claim 16, of a bacterium strain the genome of which being transformed by inactivation of at least one of said genes coding for a soluble periplasmic hydrogenase, said inactivation being obtained by mutation of at least one amino acid of said genes, and selection of the corresponding mutated proteins encoded by said mutated genes exhibiting from 0.1% to 30% of the native soluble periplasmic hydrogenase activity.

18. The use according to claim 16 or 17, of a Desulfovibrio fructosovorans strain the genome of which being transformed by inactivation of the gene represented by SEQ ID NO: 53 coding for the soluble periplasmic [NiFe] hydrogenase represented by SEQ ID NO: 54, and/or of the gene represented by SEQ ID NO: 55 coding for the soluble periplasmic [Fe] hydrogenase represented by SEQ ID NO: 56.

19. The use according to any of claims 16 to 18, of a transformed Desulfovibrio fructosovorans strain wherein the gene represented by SEQ ID NO: 53 is inactivated by:

- mutation of the nucleotide(s) GAA in position 264 of SEQ ID NO: 53 by substitution of said nucleotide by GAC, leading to a mutated gene represented by SEQ ID NO: 57 encoding a mutated soluble periplasmic [NiFe] hydrogenase represented by SEQ ID NO: 58 and corresponding to the native sequence SEQ ID NO: 54 wherein the glutamate in position 25 is substituted by an aspartate, said mutated soluble periplasmic

[NiFe] hydrogenase SEQ ID NO: 58 exhibiting from 30% to 60%, more particularly 45%, of the native soluble periplasmic hydrogenase SEQ ID NO: 54 activity,

- and/or mutation of the nucleotide(s) GAA in position 264 of SEQ ID NO: 53 by substitution of said nucleotide by GTC, leading to a mutated gene represented by SEQ ID NO: 61 encoding a mutated soluble periplasmic [NiFe] hydrogenase represented by SEQ ID NO: 62 and corresponding to the native sequence SEQ ID NO: 54 wherein the glutamate in position 25 is substituted by a valine, said mutated soluble periplasmic [NiFe] hydrogenase SEQ ID NO: 62 exhibiting from 8% to 15%, more particularly 10%, of the native soluble periplasmic hydrogenase SEQ ID NO: 54 activity.

20. The use according to any of claims 1 to 4, of membrane extracts containing at least one membrane-bound [NiFe] hydrogenase of bacterium strains as defined in claims 1 to 4.

21. The use according to claim 20, of membrane extracts such as obtained according to the following method, which comprises the following steps:

22. The use according to claim 20 or 21, of membrane extracts of Desulfovibrio fructosovorans such as obtained according to the method defined in claim 21.

23. The use according to any of claims 1 to 22, for the preparation of metallic biocatalysts chosen among the following precious metals: palladium, platinum, ruthenium, rhodium and iridium.

24. A process for the preparation of metallic biocatalysts comprising the following steps:

- bringing together bacterium strains, or transformed bacterium strains, or membrane extracts such as defined in any of claims 1 to 22, with a solution of a metal in oxidized form, for a time period sufficient to allow the initial sorption of the metal in oxidized form in said, bacterium strains, or transformed bacterium strains, or membrane extracts,

25. A process according to claim 24, for the preparation of metallic biocatalysts chosen among the following precious metals: palladium, platinum, ruthenium, rhodium and iridium.

26. A process according to claim 24 or 25, for the preparation of palladium biocatalyst comprising the following steps:

- bringing together the DF400 strain such as defined in claim 15 with a solution of palladium in oxidized form Pd(II), especially a solution of Na₂PdCl₄, for a time period sufficient to allow the initial sorption of Pd(II) in said strain,