Enzyme-mediated bioreduction of metals is known. For example, it is reported that Desulfovibrio desulfuricans has broad metal reducing specificity (Fe, Mn, U, Cr, Tc and Pd) via hydrogenase and/or cytochrome C3. Metal ions are reduced and precipitated in the periplasm (J. R. Lloyd et al, Applied and Environmental Microbiology ; (1998), 64 (11), p4607).

Such precipitation has been proposed as a means for recovering valuable palladium which is widely used in automobile catalysts.

It is an object of the present invention to provide a novel catalytic reduction method.

According to the present invention, there is provided a method of reducing a substrate in an aqueous medium in the presence of a pre-prepared catalyst, comprising the steps of:- (i) in a reaction vessel, contacting the substrate in the aqueous medium with the pre-prepared catalyst, and (ii) providing an electron donor to the aqueous medium, whereby to reduce the substrate, wherein the pre-prepared catalyst comprises a biological cell having palladium metal particles attached thereto.

Preferably, there is substantially no accumulation of the reduction product by the biological cells.

Preferably, the reduction product of the method is not precipitated in the aqueous medium. lut will be understood that the substrate may be dissolved in the aqueous medium or suspended or dispersed therein.

Preferably, said substrate is a metal ion having a relatively high oxidation state, and the reduction product is the metal ion having a relatively lower oxidation state. Examples of suitable metals for reduction include Cr, Tc, Np, U, Pu, Mn, Se, Au, Pd, Pt, Rh, Ir, Mo and V.

Alternatively, the substrate may be a halogenated aromatic compound (eg. a chlorophenol compound or a polychlorinated biphenyl compound (PCB)) or a salt of hypophosphorous acid (eg. NaH2PO2).

Preferably, the method includes the step of pre-preparing said catalyst, comprising attaching palladium metal particles to enzymatically active biological cells. It will be understood that said cells are not necessarily alive, i. e. viable.

Preferably, said catalyst pre-preparation step comprises suspending said cells in a Pd (ll) solution, followed, after a predetermined period, by introduction of an electron donor.

Preferably, said enzymatically active cell is a live resting cell. Preferably, said enzymatically active cell has metal reductase activity or cytochrome or hydrogenase activity functioning as a metal reductase. More preferably, said enzymatically active cell is the bacterium Desulfovibrio desulfuricans.

Most preferably, said enzymatically active cell is that deposited as ATCC 29577.

Preferably, the aqueous medium is buffered to a pH of 7 or less, and more preferably to a pH of about 3.

The electron donor of step (ii) may be, for example, hydrogen or a salt of formic acid or pyruvic acid such as sodium formate or sodium pyruvate respectively. Preferably, the electron donor is hydrogen, in which case step (ii) may be achieved by bubbling hydrogen gas into the medium in the reaction vessel (eg. from a hydrogen cylinder, from a metal hydride store such as LaNi5, or from an electrochemical cell).

Alternatively, the substrate itself may serve as the electron donor. For example the conversion of sodium hypophosphite (NaH2PO2) to sodium phosphite (NaH2PO3) involves donation of electrons from P (l) (which is oxidised to P (lil)) to protons, the reduced protons being evolved as hydrogen gas.

Step (i) may be achieved by suspending the catalyst in the aqueous medium in the reaction vessel. Alternatively, the method may include the prior step of immobilising the catalyst within the reaction vessel. Said immobilisation step may be achieved by retaining the catalyst within a matrix in the reaction vessel, the matrix being permeable to the aqueous medium so that the contacting step can occur. Suitable matrix materials include cotton wool and microfibre glass. The catalyst may be adhered to the matrix in order to retain the catalyst in the reaction vessel. However, it will be understood that the matrix may serve as a physical barrier to passage of catalyst out of reaction vessel, in which case such adherence is not required.

It will be understood that immobilising the catalyst within the reaction vessel enables the aqueous medium containing the substrate to be passed through the reaction vessel on a continuous basis, the catalyst remaining immobilised in the reaction vessel. This is particularly useful when the reduction product is dissolved in the aqueous medium.

In one embodiment, said immobilisation step may be achieved by adhering the catalyst to a first surface of a support, preferably an active support, in the reaction vessel. As used herein, an active support is defined as a support which effects the electrochemical injection of hydrogen or homolytic fission of hydrogen molecules into hydrogen atoms. Preferably, said active support is a palladium-based alloy (eg. Pd- Ag, Pd-Y and Pd-Ce). More preferably, said alloy is a Pd-Ag, Pd-Y or Pd- Ce alloy containing from 20 to 25 atomic% Ag, from 8 to 10 atomic% Y or 6 atomic% Ce respectively. A particularly preferred alloy is 0.77% Pd- 0.23% Ag.

Preferably, hydrogen is supplied to the first surface of the support through the support from an opposite second surface. It will be appreciated that, in the case of an active support, hydrogen will be supplied to the first surface, and hence the cells attached thereto, as nascent hydrogen atoms.

In a particularly preferred version of this embodiment, the active support serves as a cathode of an electrolytic cell and is in the form of a tube having a closed lower end, the outer surface of the tube defining the first surface to which the cells are attached in use. In use, electrolyte solution within the tube and in contact with the second surface is separated from the substrate-containing aqueous medium within the reaction vessel by the active support which serves as a hydrogen permeable membrane between the aqueous medium and the electrolyte solution. Thus, it will be appreciated that the electrolyte solution can be optimised for hydrogen generation without an adverse effect on the reduction.

Embodiments of the present invention will now be described by way of example only with reference to the accompanying drawings in which:- Figures 1A, 1 B and 2 are plots of Cr (VI) concentration against time under various reduction conditions, Figure 3 is a schematic view of a flow-through bioreactor used to prepare a catalyst for use in the present invention, Figures 4 and 5 are diagrammatic views of part of an apparatus for carrying out the method of the present invention, Figure 6 is a diagrammatic view of part of another apparatus for carrying out the method of the present invention, Figure 7 is a plot of Cl-concentration against time for the reductive dehalogenation of 2-chlorophenol according to the present invention, and Figure 8 is a plot indicating time for onset of hydrogen evolution at various temperatures in the conversion of NaH2PO2 to NaH2PO3.

A. Preparation of catalysts in accordance with the invention "Bio-Pd" (prep A) Desulfovibrio desulfuricans (ATCC 29577) was grown for 24 hrs in anaerobic 100 ml serum bottles in Postgate's medium C and harvested by centrifugation for 30 mins under oxygen free nitrogen (OFN) at 3, 000 rpm and ambient temperature, the bottles being kept on ice both before and after centrifugation. The resultant pellet was washed anaerobically under OFN in the serum bottle with OFN pre-bubbled 20 mM morpholinopropane-sulphonic acid (MOPS)-NaOH buffer at pH 7 (50 ml) and the cells were suspended at a density of 0.25 mg dry weight/ml in 2 mM (Pd (II) solution (0.589 g Na2PdCì4 per litre in 0.01 M HN03 ; pH 2).

This was achieved by placing 5 mi of the Pd (ll) solution (or other volume as appropriate) in small anaerobic bottles under OFN, followed by addition of sufficient cells to give a biomass: Pd ratio of 1: 1 on a weight basis. The resultant cell suspension was left in contact with the Pd (If) solution for 2 hrs, this being the time required to biosorb Pd (li) onto the biomass, achieving saturation of biosorption sites. The cells were not left longer than this to contact the Pd (II) since extended times can result in less active biomass. After the 2hrs contacting time, the nitrogen was gassed- out by the introduction of H2, and the suspension was stored under H2 until all of the residual Pd (fol) had precipitated onto the biomass as Pd°.

The resultant'active biomass'or'bio-Pd'had a Pd: biomass dry weight ratio of 1 : 1. The identity of the precipitated material was confirmed as Pd° by X-ray powder diffraction analysis.

"Bio-Pd" (prep B) The cells were pre-grown as described above for prep A. The cells were then immobilised in a flow-through bioreactor described with reference to Figure 3. The flow-through bioreactor comprises a reaction vessel 2 (20moi flow volume) having an inlet 4 and an outlet 6, a reservoir 8 for feedstock solution and a reservoir 10 for treated solution connected by tubing 12 to the inlet 4 and outlet 6 of the reaction vessel 2 respectively, and a pump 14 directly upstream of the reaction vessel 2 for effecting and controlling flow of solution through the reaction vessel 2. In use, solution within the reaction vessel 2 is stirred by means of a magnetic stirrer unit 16 and follower 18. The bioreactor also incorporates an electrolytic cell comprising a dc power supply 20, a 0.76% Pd-0. 24% Ag alloy cathode 22 in the form of a hollow tube (length 3.2cm, diameter 1.0cm, approximate surface area 12cm2) closed at its bottom end and defining an electrolyte chamber 24, and a platinum anode 26 extending into the electrolyte chamber 24. The electrolyte is 1M HNO3. In other embodiments (not shown), other supports for cell attachment are used, such as a carbon matrix.

Cells were prepared by diluting them (~0. 25mg dry weight of cells/ml of solution) with a 2mM solution of Na2PdCI4 in HN03 at pH 2 (pre-gassed with N2). The mixture was allowed to stand for 2 hours and poured into the reaction vessel 2 so as to partially submerge the Pd-Ag cathode 22.

After overnight stirring with hydrogen generated from the electrolytic cell (3V, 20mA), Pd° was precipitated on the cells, causing them to become attached to the surface of the Pd-Ag cathode 22. Thus, the Pd-Ag cathode 22 also serves as a support for cell attachment. The level of attachment was calculated by assay of residual protein in the solution following adhesion (protein accounting for 50% of the dry weight of the cells) and found to be 1.5mg dry weight of cells/cm2 surface area of cathode 22.

The flow-through bioreactor was then challenged with a flow of NaaPdCl2 in HN03 (2 mM Pd (II), pH 2, pregassed with OFN) supplemented with D. desulfuricans ATCC 29577 (to give a biomass dry weight to Pd ratio of 1: 1 in the input solution) at an appropriate flow rate, with the biomass Pd (II) adhering to the electrode. The metal-containing biomass (a black solid) was recovered from the floor of the flow-through bioreactor, having fallen from the electrode surface under gravity and/or scraped from the electrode surface.

"Industrial waste bio-Pd" Biomass prepared as for prep A was added to a vessel containing a processing waste solution diluted 1: 500 with water (supplied by Degussa Ltd, Germany) supplemented with laboratory waste solution containing Pd (II) (total volume 2 I, pH 2.5). The amount of biomass used was the same as the mass of Pd ( !)) in the laboratory waste solution (0.2 mg/ml biomass (dry weight), i. e. a total of 0.4 g dry weight of biomass in the vessel. The waste solution contained Pd (il), Rh (lil) and Pt (lV). The electrolytic cell described with reference to Figure 3 was immersed in the vessel and hydrogen generated (3V, 20 mA) while the Pd (II) and cell suspension mixture was stirred. After 48 hours, biomass was harvested from the electrode by scraping and from the solution by centrifugation.

The harvested material was washed with water and then acetone and dried at 80 °C. The resultant material is hereinafter referred to as"industrial waste bio-Pd"and comprised Pd, Rh and Pt precipitated on the biomass (approximate 1: 1 ratio of biomass to metal).

B. Preparation of comparative catalysts "Chemical-Pd" (Prep A) A 2 mM solution of Na2PdCI4 in 0.01 M HN03 at pH 2 was allowed to stand under H2 in the absence of cells. Although slower than in the presence of cells, precipitation of Pd (0) did eventually occur as evidenced by the appearance of a black precipitate. This constituted a chemical preparation of Pd° (hereinafter referred to as chemical-Pd).

"Chemical Pd" (Prep B) A second preparation of chemical-Pd was achieved by following the methodology for the preparation of Bio-Pd (prep B) using the flow through bioreactor of Figure 3 in the absence of cells.

Reduction of Cr (VI) to Cr (ill) Examples 1A and 1B A solution (5 ml) was prepared under OFN in 20 mM MOPS-NaOH buffer, pH 7. To this was added 2 mg of bio-Pd (prep A). Two treatments were carried out. In the first case (1A) the solution was outgassed with OFN for 5 mins and then hydrogen for 5 mins and allowed to stand under H2 (1 atmosphere). In the second case (1 B) sodium formate (pH 7) to a final concentration of 25 mM was added to the solution which was allowed to stand under OFN. The reaction was started by the addition of 700u. m Cr (VI) as sodium chromate. In each case, samples were removed periodically and centrifuged in air. The residual Cr (VI) in the sample supernatants was determined by reaction with diphenyl carbazide, a reagent specific for Cr (VI), followed by a comparison of absorbance at 540 nm with standardised solutions of Na2CrO4 reacted in the same way. The results are shown in Figures 1A and 1 B. comparative examples 1A and 1B The methods of Examples 1 A and 1 B were repeated with the bio-Pd replaced by chemical-Pd. The results are shown in Figures 1A and 1 B. comparative examples 2A and 2B The methods of Examples 1A and 1 B were repeated with the bio-Pd replaced by cells of Desulfovibrio desulfuricans (ATCC 29577), Desulfovibrio vulgaris (ATCC 29579) or Desulfovibrio sp. strain"Oz 7" cells which had not been subjected to the Pd biosorption/precipitation methodology. The results for the ATCC 29579 cells are shown in Figures 1A and 1 B.

Referring to Figures 1A and 1 B, it can be seen that ATCC 29579 ce. lis alone did not reduce Cr (VI) to any appreciable extent at the expense of either H2 or formate as electron donors under comparable conditions to Example 1 (plots C2A and C2B respectively, ATCC 29577 and Oz 7 cells give comparable results-data not shown). Similarly, chemical-Pd reduced little Cr (VI) with H2 (plot C1A) or formate (plot C1 B). However, bio-Pd (prep A) was effective in the reduction of Cr (VI), with approximately 68% reduction being achieved within 3 hours. The rate was comparable using H2 (plot 1A) or formate (plot 1B) as the electron donor. Bio-Pd prepared by prep B gives comparable results (data not shown).

Referring to Figure 2, the results for reduction of Cr (VI) using bio-Pd under conditions similar to Example 1 in the absence of electron donor are shown. Substantially no reduction of Cr (VI) was observed. Similarly, substantially no reduction was achieved by the addition of H2 as electron donor in the absence of catalyst (not shown).

Example 2 A solution (5ml) was prepared under OFN comprising a mixture of sodium formate and sodium acetate (1 M each, pH 3). To this was added 2 mg of bio-Pd (prep A). The solution was allowed to stand under OFN and the reaction was started by the addition of 700pm Cr (Vl) as sodium chromate.

Samples were taken periodically for Cr (VI) analysis as described above with reference to Example 1. In this case, substantially all of the Cr (VI) was reduced within 3 hours (not shown). This Example demonstrates that much faster rates of reduction can be obtained under acidic conditions in the presence of a high concentration of the organic acid electron donor.

Slower reaction rates were observed under similar reaction conditions at pH1, 5and7.

Example 3 and comparative example 3 The protocol of Example 2 was followed, using 2 mg of industrial waste bio-Pd (Example 3) or 2mg of chemical-Pd (prep A, comparative example 3) and 500 u. M of Cr (VI). In Example 3, the industrial waste bio-Pd removed 95% of the Cr (VI) within 1 hr and all of the Cr (VI) by 2 hrs, whereas the chemical-Pd (comparative example 3) removed only 34% of the Cr (VI) after 2 hrs, with approximately 2% residual Cr (VI) remaining in the solution after 24 hrs.

Example 3 shows that the presence of other metals on the cells does not poison the catalyst. In addition, two objectives can be achieved, namely the recovery of waste precious metals, and their use in the catalytic reaction of the present invention. In view of the fact that most waste precious metals are not recovered, such waste provides a cheap source of Pd for use in the method of the present invention.

Example 4 Referring to Figures 4 and 5, industrial waste bio-Pd (50 mg) was sprinkled in a line 30 between opposite top and bottom edges 32 and midway between opposite side edges 34 of a square plate of absorbent cotton wool 36 (4cm x 4cm; total weight of cotton wool 0.29 g). This was rolled up into a cylinder such that the line 30 of waste bio-Pd extended substantially up the centre of the cylinder between the top and bottom end faces of the cylinder. The cotton wool cylinder was inserted into a tubular fibre filter 38 and the cylinder-filter assembly was inserted into a tubular plastic column 40.

A Cr (VI) solution containing 1 mM Cr (VI) in a carrier of 1 M sodium formate and 1 M acetate (pH 3) was pumped into the base of the assembly (indicated by arrow A) and out of the top of the assembly (indicated by arrow B). It will be understood that the solution passed through the cotton wool (serving as a matrix for the waste bio-Pd) and contacted the waste bio-Pd, but the waste bio-Pd was retained within the cotton wool cylinder.

The removal of Cr (VI) by the column was 100% at a flow rate of 7 cumin and 96% at a flow rate of 20 ul/min (1.2 ml/hr). The fluid volume of the column was 14 ml therefore the flow residence time at the latter flow rate was about 33 hr.

The Cr (VI) removed from the column inflow was quantitatively recovered as Cr (ill) in the column outflow and therefore the catalyst did not accumulate soluble Cr ions or Cr (0), Cr203 or Cr (OH) 3 or any Cr (VI) or Cr (lit) precipitate. Presence of Cr (ill) was determined by comparing the maximum peaks in the uv/vis spectrum (280-800 nm) of the column outflow with the peaks of a stock solution of Cri3. 6H2O (1 mM in 1 M Na formate/1 M Na acetate at pH 3).

In a subsequent experiment it was found that by increasing the fluid volume to 24ml, 100% removal could be obtained at a flow rate of 23.5 u,)/min (1.4ml/hr), with a residence time of 17 hr. comparative example 4 Example 4 was repeated using chemical-Pd (prep A). Negligible Cr (VI) was removed from solution under the same conditions.

Example 5 Referring to Figure 6, 50 mg bio-Pd 50 (prep A) was sandwiched at the mid-point of a Perspex tube 52 between two micro-fibre glass filter pads 54. The sandwich arrangement was held in place within the tube 52 by cotton wool pads 56. The tube 52 was closed at each end with a pair of rubber bungs 58, each having a hole therethrough to serve as an inlet 60 or outlet port 62 respectively. A Cr (VI) solution containing 1 mM Cr (VI) in a carrier solution of 1M sodium formate and 1M acetate (pH 3) was pumped through the inlet port 60, up through the tube 52 and out of the outlet port 62 at a flow rate of 9 ml/hr (flow residence time 20 min). The presence of Cr (III) was measured in the outflow as for Example 4, indicating 100% Cr (VI) reduction to Cr (III). 100% reduction was maintained at a flow rate of 13ml/hr (flow resistance time 12.5 min).

In slight variations of the above Examples, it was found that comparable results for the reduction of Cr (Vf) were obtained when no precautions were taken to exclude air (Examples 2 and 3) or when sodium acetate was omitted (Examples 2 to 5).

Reduction of 2-chlorophenol to phenol Example 6 A solution (10 ml) was prepared under OFN comprising 5 mM 2 : chlorophenol in 20 mM MOPS-NaOH buffer, pH 7. To this was added 4 mg of bio-Pd (prep A). Sodium formate (pH 7) to a final concentration of 50 mM was added to the solution which was allowed to stand under OFN at 30 °C. Samples were removed periodically and centrifuged in air.

Reductive dehalogenation of the 2-chlorophenol occurred, with the amount of chloride released being determined by the thiocyanate method (spectrophotometric assay based on the displacement of thiocyanate by chloride ions from mercury (III) thiocyanate in the presence of Fe (III) ion.

Absorbance at 460nm of treated sample aliquot is fitted on a calibration curve constructed using standardised solutions of NaCI). As shown in Figure 7, the rate of chloride release was about 40 pg/hr/ml, corresponding to a specific rate of chloride release of 100 Fglhrlmg bio-Pd. 3-and 4- chlorophenol were also dehalogenated, but at slightly slower rates (data not shown).

Conversion of NaH2PO2 to NaH2PO3 Example 7 and comparative example 5 4 mg of bio-Pd (prep A) (Example 6) or 4 mg of chemical-Pd (prep A) (comparative example 5) was placed in a test tube to which 10 ml aqueous sodium hypophosphite solution (2% wt/vol) was added. The time taken for the onset of hydrogen evolution (conversion of NaH2PO2 to NaH2PO3) at various temperatures was determined, using a water trap to visualise hydrogen release. The results are shown in Figure 8 (plot A-bio-Pd: plot B chemical-Pd). For chemical-Pd (comparative example 5) 13 minutes were required at room temperature, falling to 1.5 minutes at 65 °C. For bio-Pd (Example 7), hydrogen was evolved within 1 minute at room temperature and instantaneously at temperatures of 50 °C or more.

It will be understood that similar results to Examples 1 to 7 can be obtained by preparing the appropriate catalyst in the bioreactor described with reference to Figure 3, flushing the depleted palladium-containing solution from the reaction vessel and replacing it with solution containing the substrate. Hydrogen as electron donor can be supplied by the electrochemical cell.

It will also be understood that although the catalysts used in the Examples were 1: 1 biomass to Pd, other ratios are possible such as 3: 1 or 6: 1 biomass to Pd.