Bioremediation of waste, process streams and soil to remove some heavy metal species has been demonstrated. The basic aim is to remove from the environment, or at least render immobile, the contaminating species. Such systems employ a microbial species which is contacte with the contamination to affect a chemical alteration. The chemical alteration of the contamination from a mobile form to an insoluble form, which can be retained at an intended location, and removed subsequently represents one such general technique.

The chemistry of the predominant forms in which certain metals occur in contamination and the chemistry of the forms following alteration of the type outlined above prevent the successful use of such systems in a number of cases. Extrapolation between contaminating species is also difficult due to variations in the valency and solution behaviour. It should also be noted that bioremediation processes need to be applicable to solutions carrying very low levels of contamination and yet remove a significant amount of the contamination. This is particularly so where radionuclides are being considered. Variations in the toxicity of contaminants to biological organisms also var-y quite considerably between species.

Particular difficulties are encountered with pentavalent form metal contamination. One such problematical example, an actinide, is 237Neptunium which represents a decay product of 241plutonium and 2'lAmericium. These are in turn common components of nuclear waste. The relative half lives, 237Np (2.1 million years), 24lpU (14.9 years) and 24lAm (433 years) mean that 237Np will become the increasingly predominant form in wastes. The chemistry of Np is such that it readily occurs as a highly soluble, and hence highly mobile form, which is biologically accessible. The result is a generally difficult to handle system.

Neptunium commonly occurs in the form Np (V) in the environment and as demonstrated below, Figure 1, attempts to convert this direct to an insoluble and recoverable form have not been successful. Bioremediation for Np wastes has not therefore been seen as a practical option.

The present invention aims to provide a method and system for the removal of pentavalent or higher valency forms of contamination, including Neptunium and/or other forms of contamination which are not susceptible to conventional bioremediation.

According to a first aspect of the invention we provide a method for extracting one or more selected species from a sample : providing the selected species in a dissolve form; contacting the selected species with a first agent, the first agent reducing the selected species valency; contacting the selected species with a second agent to convert the selected species to an insoluble form; and removing the insoluble species from the sample.

The selected species may be one or more metallic elements, or one or more ions incorporating one or more metallic elements.

The selected species may be a single metallic element or ions incorporating that metallic element. The metallic element may include all forms of that element or those forms in one or more specified valences. The selected species may refer to all isotopic forms of an element or may refer to an individual isotopic form.

The selected species may be in pentavalent or higher valency form. The selected species is preferably an actinide in penta- valent or higher valency form.

The selected species may be an actinide or ions incorporating an actinide. The selected species may be Neptunium and in particular may be 237Neptunium. Preferably the Neptunium is in penta-valent or higher valency form. The Neptunium may be present as NpO2+ ions.

The sample may comprise an aqueous sample. The sample may contain suspende solids. The sample may comprise a waste stream from a prior process, including a leach liquor extracted from a leaching stage. Preferably the sample is substantially free of solids on being fed to the first agent.

The selected species may be dissolve as elemental ions and/or as compound ions.

The sample containing the selected species may be contacte with the first agent in a reactor vessel. The first agent may ! De introduced to the reactor vessel alongside the sample, but is preferably present in the vessel on feeding the sample to the vessel.

Preferably the first agent is retained in the vessel and is not removed with the sample.

The sample may be fed to the vessel processed, retained therein and then extracted in a batch manner. Alternatively the sample may be fed to the vessel, remain in the vessel for the required time period and then be withdrawn, the residence time in the vessel depending on the through flow rate of the sample. A continuous process may thus be provided.

The sample may be contacte with the first agent by mixing the two components together.

The first agent may be contacte with the sample by causing the sample to flow over the first agent. The first agent may be provided on and/or in inert means in the vessel. The inert means may be fixed in position or free to move within a restricted space defined within the reactor vessel.

The inert means may be provided as a packed bed, fluidised bed, column or plug flow reactor.

The first agent may react with the selected species to effect the reduction in valency of the selected species.-The first agent may be a chemical reducing agent. The first agent may, for instance be an organic acid containing one or more carboxylate groups. The first agent may comprise ascorbic acid.

The first agent can be an electrochemical reduction.

The first agent may provide and/or produce a substance which acts and/or facilitates and/or promotes electron transfer in the selected species. The reduction may be effected enzymatically by the agent. Preferably the microorganism employs the contaminant species as an electron acceptor. The microorganism may use the electron acceptor in the oxidation of a nutrient/electron donor for the microorganism. H2 may be employed as an electron donor.

The first agent may be a microorganism. The microorganism may be selected from Shewanella sp, for instance Shewanella putrefaciens. The microorganism (s) may be sulphate reducing bac_erium, for instance selected from Desulfovibrio sp, such as Desulfovibrio desulfuricans. The microorganism may be selected from Clostridium sp. The microorganism (s) may be a fungus or fungi.

The valency of the selected species may be reduced by a single or plurality of oxidation states by the first agent. The reduction of the selected species to mixed valences and/or from mixed valences is envisage. The reduction may convert the selected species from the penta-valent to tetra-valent form. Preferably a still further reduction to a lower statu, for instance tri-valent, is provided. The reduction may be of Np (V) to Np (IV) and/or Np (III), particularly for z3'Np.

Additional species or components may be fed to the reactor and/or mixed with the first agent and/or selected species. The additional components may include pH buffers and micro-organism nutrients.

The sample may be contacte with the second agent in a reactor vessel. The vessel may be the same vessel or a different vessel to that in which the sample is contacte with the first agent. The contact with the first and second agent may occur, optionally within the same vessel, at substantially the same time.

The second agent may be provided in any of the manners outlined for the first agent. The first and second agents may be provided in the same manner. The first and second agents may be provided on and/or in the same inert support, potentially on the same discret support items or on different discret support items.

The second agent may react with the selected species to generate an insoluble form of that species.

Preferably the second agent is a micro-organism. The second agent may be selected from Citrobacter sp for instance Citrobacter N14. The second agent may be selected from Acinetobacter sp, for instance Acinetobacter calcoaceticus.

The second agent may be Escherichia coli bearing the gene phoN cloned from Salmonella typhimurium.

The second agent may produce by a biochemical component and/or through enzymatic and/or metabolic processes, species de- solubilising ligands. Preferably the ligands are produced at the surface of the micro-organism. The ligand production may be mediated by an enzyme, such as phosphatase or via biological H2Sproduction.

The second agent may produce the de-solubilising ligand through hydrolytic cleavage of a donor molecule, preferably an organic donor molecule. The donor molecule may be a glycerol phosphate molecule. <BR> <BR> <BR> <BR> <BR> <BR> <BR> <P>The de-solubilising ligand may be Ho2-, . The ligand may be produced from an organic phosphate molecule, such as a glycerol phosphate, for instance glycerol 2-phosphate or tributyl phosphate.

Additional species or components may be fed to the reactor and/or mixed with the second agent and/or selected species.

The additional components may include pH buffers and micro- organism nutrients and j or electron donors.

Precipitation and/or deposition assisting agents may be added. The addition of lanthanium ions, and in particular La~ ions, is envisage.

The in-soluble form may be amorphous, but is more preferably crystalline. The insoluble form may be deposited on the cells and/or support for the second agent. Seeding or other forms of crystallisation promotion may be provided.

The insoluble form may be one or more phosphate forms of the contaminant.

The insoluble form may be removed with the second agent and/or support therefore from the sampi. Centrifugation, filtration, flocculation or other forms of solid/liquid separation may be employed to separate the insoluble form from the sample.

The sample, depleted or substantially free of the selected species, may be subjected to further processing or discharged as waste. Further processing may, for instance, include the selective removal of other components, including the possibility of removal in a similar manner to the process outlined above.

The insoluble form may be further processed or stored-as waste.

In the case of the selected species being, or including, a radionuclide, conventional processing prior to disposa may be employed. The selected species may be dispose of together with the first and/or second agent or the agents maybe separated from the insoluble species.

According to a second aspect of the invention we provide apparats for extracting one or more selected species from a sample, comprising a reactor vessel provided with an inlet through which the sample containing the selected species is fed in dissolve form and containing a first agent to reduce the selected species valency and a reactor vessel provided with an inlet through which the selected species is fed and containing a second agent to convert the selected species to an insoluble form, means being provided tc, separate the insoluble species from the sample.

The apparats may include the features options and possibilities set out elsewhere in this application, including the first aspect of the invention.

The reactor may be operated on a batch and/or semi continuous and/or continuous basins.

The reactor vessel containing the first agent and the reactor vessel containing the second agent may be a difserent vessel, but are preferably the same vessel.

The first agent and/or second agent may be provided in the reactor vessel on support means. The first and second agent may be provided on the same support particles, i. e. a bead carrying both agents on its surface, or the first and second agents may be provided on discrete support particles, i. e. one bead carrying one agent and another bead carrying the other agent.

Preferably the support means are inert in the environment encountered. The support means may be fixed in position within the vessel, for instance packed bed, or may be free to move within the confines of the vessel, for instance a fluidised bed. Preferably the supports define a series of flowpaths over the support surface between the reactor inlet and reactor outlet.

The support may be provided by zeolites, beads, foam or gel particles. The support may be formed of wood particles or stainless steel, but is more preferably formed of polyacrylamide or polyurethane. Polyacrylanide gel and/or polyurethane foam and/or ultrafiltration membranes may be used.

The support may provide the agent as a biofilm.

The outlet from the reactor vessel or vessels is preferably provided with a barrier which restrains the passage of the first and/or second agents but which allows the passage of the sample.

The reactor vessel may have the first and/or second agent and/or support removed on a continuous or periodic basis.

Various embodiments and results relating to the operation of the invention will now be described with reference to the accompanying drawings, in which :- Figure la illustrates the removal of La (III) and Th (IV) from contaminated samples by a biological agent; Figure lb illustrates Lhe ineffectiveness of removal of Np (V) by the same biological agent as for Figure la; Figure 2 schematically illustrates a process according to the present invention; Figure 3 illustrates the chromatographic spread of mixed valency ions; Figure 4 illustrates the chromatographic spread of 239Np (V) and 235Np (IV) i Figure 5 illustrates the counts per minute against Rf value for the starting sample of 233va and 237Np (V); Figure 6 illustrates the affect on the profile of Figure 5 of a chemicai reducing agent; Figure 7 illustrates the affect on the plot of Figure 5 of a biological reducing agent; Figure 8 illustrates the chromatographic spread of Np (IV) following treatment with a biological agent; and Figure 9 illustrates the counts per minute plot against Rf value for a process according to the invention.

The invention is now illustrated with reference to Neptunium contamination but applies equally to other similar systems such as Pu (V).

As an illustration of the problems involved in the recovery of a pentavalent species from contaminated environn ! ents a chemical precipitation route on Neptunium contamination is considered in comparison with the effectiveness on La (III) and Th (IV) amples. These two species are established models for the behaviour of 241Am (I I I) and 241Pu (IV) and by analogy for Np (III) and (IV).

A Citrobacter sp. strain N14 useful in such a comparison of the direct bioaccumulation of samples of La (III); Th (IV); 233Pa ; and 237Np (V) can be prepared in a minimal medium containing (g dm-3), Tris buffer, 12.0; (NH4)2SO4, 0.96; glycerol 2-phosphate (G2P, 5.5 H20; BDH Ltd), 0.67; KCl, 0.62; MgSO4 7H20,0.063; FeS04 7H2, 0.00032; glycerol, 2.0. The pH was adjusted to 7.0 with 2 mol dm-3 HCl. Cultures for free or immobilized cell preparations were grown in 3 dm3 batches (30°C)- with forced aeration, inoculated using 50cm-3 of overnight culture from cells previously maintained by two daily subcultures in the minimal medium, and harvested in stationary phase (24h, OD, oo-=-1.20).

The cultures were harvested by centrifugation, washed twice in isotonic saline (8.5 g dm-3 NaCl).

The prepared agent was re-suspended in a 4OmM MOPS buffer, pH7 at a level of 0.47mg dry weight/ml. The feed was prepared and added at the levels discussed below for Figure 7. The headspace was not flushed with H2, but instead 5mM glycerol 2- phosphate and 100mM ammonium acetate was added. Removal of the reduced Np (or Pu) is promoted if La3+ ions (100mm) are also incorporated into the solution, to facilitate deposition of Np and Pu phosphates.

The samples were processed in batches in a reactor containing the immobilised biological agent and produced the results illustrated in Figures la and b for the assay of take up by the biomass (measured as the count level) over time of exposure of the contamination to the biomass.

Figure la illustrates that only trace activity levels will remain in the supernatant following processing with Citrobacter sp. and separating the cells from the supernatant following processing with Ci trobacter sp. ReXention of the insoluble phosphates of La (III) and of Th(IV) is evident from the high count levels, solid and open dots respectively, as measured using the colorimetric reagent arsenazo III.

Similarly in Figure lb the z33Pa was readily converted to insoluble protactinium phosphate which was retained with the cells as a result, so giving a high count, solid dots. The 237Np on the other hand remained highly soluble and was still present in the form Np (V) in the supernatant, giving a low count, open dots. Such precipitation techniques do not, therefore, offer a prdcticable technique for removing Np (V).

As demonstrate by these results attempts to directly immobilise Np (V) are not successful.

The embodiment of the invention, illustrated schematically in Figure 2, uses a two part process to effect a separation of the Np (V) contamination from its environment.

The system employs leaching and/or washing of the contaminated material 2 to remove the contamination as its naturally occurring highly mobile form in process stream 4.

The stream 4 is fed to a first reactor 6 to effect the first stage of the process. The reactor contains an immobilised biological reducing agent which, when presented with the desired feed materials, stream 7, reduces the Np (V) to Np (IV) (or Np (III)) by virtue of a process discussed in more detail below. The biological agent is fixed on beads 8 around which the process stream flows.

The process stream 10 leaving this reactor contains the Np contamination in predominantly the reduced tetravalent form.

The stream 10 is then fed to a second reactor 12 for the second stage of the process. Here the process stream is contacte with a second biological agent which again may be immobilised on beads 14. The second agent effects a furtner rection of the contamination, again upon introduction of the necessary feed materials, stream 15. The rections arising lead to the Neptunium's deposition as an insoluble form. Again the rection is discussed in more detail below. The immobilised contamination can be periodically removed in the resulting more concentrated form for disposa.

The process stream 16 exiting the second reactor 12 is, therefore, depleted or free from Np species.

The process stream 16 may be returned to the contaminated material 2 for removing still further amounts of contamination.

The process stream 4 may be subjected to processing steps before its introduction to reactor 6. For instance the material may be treated to another bioremediation stage to remove another species present which can be removed by a technique which leaves the Np in solution. Similarly further processing of the stream 16 leaving the second reactor can be provided.

An illustrative organism suitable for performing the first reduction stage of the present invention is Shewanella putrefaciens (formerly Alteromonas putrefaciens) available in open cultures, including American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, USA, as ATCC 8071.

Other suitable organisms exist and the suitability of any microorganism for use as a reducing agent for the species in question can, however, readily be determined using the following procedure, based on the use of a phosphorImager.

Other organisms include:- sulphate reducing bacterium, such as Desulfovibrio desulfuricans (available from various open culture collections); and Clostridium sp (also available from various open culture collections).

The species in solution can be assayed by radiography with a PhosphorImager (Molecular Dynamic's, Sevenoaks, Kent, UK). A storage phosphor screen, highly sensitive to beta particles, gamma rays and X-rays is used to trap energy from the radioactive decay. The trapped energy is stable until scanned with an 88mm laser beam which releases the stored energies' blue light. The resulting emissions are collecte by a fibreoptic bundle connecte to a filter multiplier and digitised. Scanning and data analysis are managed through the Molecular Dynamic's software package Image Quant.

A calibration curve is prepared by placing samples of various concentrations (ranging from 1 to O. OlmM) in sequential spots (10y1) on a strip of Whatman 3mM cellulose chromatographic paper which was then wrapped in cling film. Cling film wrapped samples exposed to a storage phosphor screen for 16 hours and the spots of radioactivity were visualise and quantified. A densitometer scan of the resulting image was made with an Image Quant software package run on a Del 466/ME personal computer.

Image density and subsequent peak area values from the scan were used to construct a standard curve.

Different valences of the species in question are separated by paper chromatography. Samples (10µl) are then spotted on to a Whatman 3MM chromatographic paper and separated with trimethylamine/actetone/formic acid 2: 6: 2 v/v as a mobile phase. Papers are acid washed in 2M HC1 prior to use. Air dried chromatographs are wrapped in cling film and different valences of the species were visualise and quantifie using the PhosphorImager described above. The preparation of standards of a known valency can be used as a comparison.

In identifying the valency of a species present in the supernatant, samples (150. m1) are removed from the cultures periodically and assayed for the total species content.

Samples are centrifuged prior to analysis with a Heraeus Sepatech Biofuge 13 microfuge set at 13,000 rpm for 20 minutes.

A total of 10y1 of culture supernatant is then placed on a 3mm cellulose chromatographic paper wrapped in cling film. The wrapped filter paper impregnated with sample is exposed to the phosphorimager screen for 16 hours prior to visualisation as above. Separation of the different valences present by paper chromatography and with the valences being quantifie with the phosphorimager, for beta and/or gamma emitters, was performed.

The effect of the micro-organism on the sample is thus determined so determining its suitability for performing the first stage function of the present invention.

Advantageously the reduction may be effected as part of the growth activity of the organism in a number of cases. Thus the metal species is used as an electron acceptor, with H2 acting as the electron donor, in an enzymatic mechanism. In generating the reduction the biological agent consumes H2 which it oxidises to a non-toxic product. The by product of the rection is thus attractive as presenting no hazard in itself.

An illustrative organism suitable for performing the second stage of the present invention is Citrobacter sp N14 of ISIS Innovations Ltd deposited by Dr A. C. R. Dean on 16 February 1990 with National Collection of Industrial and Marine Bacteria, at NCIMB Ltd, 23 St Machar Drive, Aberdeen, AB2 1RY, UK, under NCIMB no. 40259 under the Budapest Treaty, the applicant has been authorised by the depositor to the deposit and a copy of that permission accompanies the application.

Other organisms include Acinetobacter SP, including the strains deposited by The University of Birmingham on 5 November 1993 on behalf of the applicant with NCIMB at NCIMB Ltd, 23 St Machar Drive, Aberdeen, AB2 1RY, UK, under deposit nos. NCIMB 40594 and 40595 under the Budapest Treaty. The applicant has been authorised by the depositor to the deposit and a copy of that permission accompanies the application, and a reference strain, Acinetobacter calcoaceticus, open samples of which are available, for instance as ATCC 23055 and NCIMB 10694; as well as Escherichia coli DHSU carrying the cloned gene phot.

The suitability of any microorganism for achieving the desired precipitation/crystallisation can readily be determined, however, by experimentation. For instance, by providing the cation to be considered at a level of lmM in a 2mM citrate buffer together with 5mM glycerol 2-phosphate and by measuring the radioactivity for the biomass and effluent following passage of the sample the take up through precipitation can be determined. Using such a procedure organisms capable of precipitating the contaminant can be determined. <BR> <BR> <BR> <BR> <BR> <BR> <P>In the case of Citrobacter Sp. the biological agent possesses a surface enzyme which is active in achieving hydrolytic cleaving of organic phosphates fed to the system. The phosphate ligand is able to react with the Np cation as a result. The rection is mediated by a metal resistant phosphatase. In the reduced form the Neptunium phosphate produced is largely insoluble and as a consequence is precipitated/crystallises on the surface of the cells. It is believed that the process:- or in the further reduced form occurs.

In general the lower the valency of the ions reacting with the phosphate the more crystalline the product was found to be. A highly crystalline deposit is more resilient and hence tends to be retained more readily on the cells both during the rection and during the subsequent separation. Formation of a crystal can be"helped"by the incorporation of La3+ ions into the solution to promote the formation of a mixed lanthanide/ actinide phosphate.

The cells employed in either or both stages of the process can be provided in free form and separated from the liquid in a subsequent process stage; a batch rection situation.

Alternatively the cells can be immobilised, in a polyacrylamide gel for instance, or otherwise retained, on inert supports as a biofilm for instance, so as to allow a flow past rection scheme; a continuous or semi-continuous rection system.

To demonstrate the effectiveness of the biological agent in reducing the Np (V) to lower valency, and Np (IV) in particular the following procedure was employed.

Figure 3 provides an illustration spread for surrogate metal species, in this case La (III), Th (IV) and U (VI), illustrated by an application of arsenazo III, but also indicative of the anticipated Rf values for the general valency ranges under consideration in this work. This illustration is used in interpreting the count plots obtained in subsequent experiments involving reduction of the valency of the contamination present.

The results were obtained using a chromatographic separation to visualise the species from a mixed valency solution as follows.

A Whatman 3MM filter paper was first washed with 2M HCl and subsequently with several changes of distille water to ensure that it was free from Cl- (tested against Ag+). The paper was then dried at room temperature. A mixture of the La (III), Th (IV) and U (VI) (0.5-long of each metal) was applied (volume 51il) to the paper. The chromatographs were developed in trimethylamine/acetone/formic acid: 2: 6: 2 v/v for approximately 1 hour and then air dried. The spots were detected by spraying the paper with a solution of 0.15t arsenazo III (w/v, aq).

The spread of the species with valency is clearly illustrated.

Two samples containing 237Np were employed in the experimental demonstration of the reduction and precipitation effects of the present inventions method.

The first was obtained from a commercially available source and provided a radioactive spot in the lower Rf range attributed to 233Protactinium, a beta emitting daughter product of Np.

The second was a chromatographically purifie sample separated from all co-contaminants and daughter products.

237Np itself (alpha-emitter) is PhosphorImager"silent"at the concentration used, when used as clingfilm-wrapped samples (a- radiation does not penetrate the clingfilm wrap) Confirmation for the Np (V) as locating in the appropriate part of the range displayed in Figure 3 was obtained by employing a 237Np sample spiked with gamma emitting 23yNp (V). The results are shown in Figure 4, with a spot corresponding to the (IV) and (V) valency and arising from the alpha and gamma emissions from the 239Np and suggest 239Np (IV) was also present.

Figure 5 illustrates an initial plot for an alpha and beta discriminated count result for the commercially obtained sample following chromatographic separation on 12 x lcm strips. The strips were then cut into lcm x lcm pieces with each piece added to Ultima Gold scintillation cocktail (Packard Instrument Co, Meriden CT 06450 USA) and counted using a Tri-Carb 2700TR Liquid Scintillation Analyzer (Packard Instrument Company).

The results for this expriment and subsequent experiments are present as count rates (counts per minute) against Rf values (cm).

The Figure 5 plot clearly shows the beta curve (solid dots) attributable to the Protactinium, with peak A, and the alpha curve (open dots) attributable to the 237Np (V) with peak B. The peak B generally corresponds to the (V) valency area as illustrated above.

As an initial technique for obtaining the desired reduction in valency chemical treatment was employed. The treatment of the sample by adding ascorbic acid, 1t final concentration, as a reducing agent lead to a reduction in the height of the 23'Np (V), peak B, and the generation of a new peak, peak C, attributed to 237Np (IV) formation, Figure 6. The positioning of the peak is due to the formation of colloidal, non-migrating species of 23'Np (IV). Th (IV) does not tend to form such colloids giving rise to a different position in Figure 3.

As an alternative to chemical reduction a micro-organism based process was also demonstrated. Figure 7 illustrates the plot arising from treatment of the sample with a biological reducing agent, Shewanella putrefaciens.

The biological agent was prepared under an anaerobic atmosphere consisting of NZ and COZ (80: 20), passed through an oxygen trap.

The growth medium contained 100mM sodium lactate as the electron donor, with 50mM ferric citrate added as the electron acceptor. Medium was dispense (10ml aliquot) into 12mol serum bottles and sealed with butyl rubber stoppers. Any dissolve <BR> <BR> <BR> <BR> oxygen was displaced by bubbling through the N2: Co, mixture for 10 minutes. A 10% v/v inoculum of a culture (pre-grown for 2 days in the same media) was added to the bottle after <BR> <BR> <BR> <BR> autoclaving (15min at 121°C). After 2 days of static culture at 30°C the stationary phase cells were centrifuged in the bottles at 5000rpm for 20 min with an MSE Super Minor bench top centrifuge and washed three times with carbonate buffer (2.5g of NaHCO3, 1.5g of NaH2PO4, O. lg of KCl per litre of distille water, deaerated).

The material was then resuspended in?. OmM DZOPS buffer, pH 7 as required. A 100µM 237Np feed level in 30µl of diluted stock from the commercial source was then added to 300µl of the cells. The headspace of the rection vessel was flushed with H2 for 5 minutes and the biological reduction allowed to proceed for 24 hours.

At the end of that period the cells and supernatant were separated by centrifugation. The supernatant (3 x 1OAl spots, dried under N2 between every addition, total load 30au) was separated using chromatography as above and quantifie by scintillation counting, expressed as a count rate versus Rf plot.

As the results, Figure 7, show a reduction in the 23'Np (V) peak B occurs and a new peak, peak C, is once more generated. The activity of the biological agent in achieving reduction of the valency of the 237Np is thus shown.

Similar results, not illustrated, were achieved with Np (V) in a carbonate buffer at a concentration mirroring that of contaminated natural water, 2 picomolar 239Np in 2 micromolar 237NPCarbonate buffer 30 millimolar pH 7.5.

Similar tests were conducted on 239NnV) samples. S. putrefaciens was prepared as above and resuspended in a 30mM carbonate buffer, pH 7.5. The feed consiste of cleaned ie. passed through an ion exchange column to remove contaminating daughter-species 237Np (V), to 2yM, and 239Np (V), to 2pM. The bioreduction was allowed to proceed for 48 hours. Subsequent separation of the 239Np species was followed by visualisation of the results using a phosphorImager as above. The results shown in Figure 8 clearly show the new spot D conforming to the expected location for 239Np (IV).

This first stage alone, however, was not effective in removing Np (V) from the environment. Only 25t of the fed Np was retained with the cells following the biological reduction.

Even though this was far hi. gher than the retained level for the chemical based reduction process alone, in which only 12t was recovered, the single stage is not sufficiently effective to form a practicable technique.

As previously illustrated above, Figure lb, the treatment of the Np feed with a biological agent intended to precipitate it direct from the Np (V) form also fails to form the basis of a practicable technique.

The effectiveness of the two stage process of the present invention is, however, clearly indicated in the-results displayed in Figure 9. In this expriment preparations of both Citrobacter Sp. and S. putrefaciens were prepared as above and both presented in 40mM MOPS buffer. The headspace was flushed with H2 and 5mM glycerol 2-phosphate and 100mM ammonium acetate were added. The mixture was allowed to process for 24 hours prior to separation.

Figure 9 illustrates the pre-treatment plot (open circles) showing the Np (V) present. Following treatment both the 233pa as above, and significantly the 237Np were retained in the cell mass and removed from the liquid. The increased retention of the Np is believed to arise from the far lower solubility of Np (IV) phosphates as compare with Np (V) phosphates. In the experiments over 95k of the feed Np was removed with the cells.

The remaining Np (IV) is still present in the supernatant and gives rise to a trace plot only (closed circles).

A similar separation of the Np (V) from the environment was obtained by reducing the Np chemically to the Np (IV) form and subsequently processing it together witn the Citrobacter Sp.

Once again the insoluble Np accumulated in the cells.

The above results therefore demonstrate the successful application of the invention to the environmental problem presented by a number of species, including Np (V). Other species for which the technique is applicable include Pu (V).