BIOREMEDIATION OF WASTE STREAMS CONTAINING DISSOLVED METALS SUCH AS NEPTUNIUM BY TWO-STAGE BIO-REDUCΗON/EXTRACTION
This invention concerns improvements in and relating to bioremediation, particularly, but not exclusively to the bioremediation of Neptunium and other pentavalent actinide species .
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 contacted 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 vary 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 241Americium. These are in turn common components of nuclear waste. The relative half lives, 237Np (2.1 million years), 41Pu (14.9 years) and 241Am (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 N (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 comprising : - providing the selected species in a dissolved 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 valencies. 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 pentavalent 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 23Neptunium. Preferably the Neptunium is in penta-valent or higher valency form. The Neptunium may be present as Np02 + ions .
The sample may comprise an aqueous sample. The sample may contain suspended 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 dissolved as elemental ions and/or as compound ions .
The sample containing the selected species may be contacted with the first agent in a reactor vessel. The first agent may be 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 contacted with the first agent by mixing the two components together.
The first agent may be contacted 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 Sh.ewa.ne 11 a sp, for instance Shewanella pu trefaciens . 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 Clos tridium 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 valencies and/or from mixed valencies is envisaged. The reduction may convert the selected species from the penta-valent to tetra-valent form. Preferably a still further reduction to a lower state, for instance tri-valent, is provided. The reduction may be of Np(V) to Np(IV) and/or Np(III), particularly for 237Np .
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 contacted 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 contacted 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 discrete support items or on different discrete 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 Ci trobacter sp for instance Ci trobacter N14 . The second agent may be selected from
Acinetojbacter 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 H2S production.
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 .
The de-solubilising ligand may be HPO2 4. 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 microorganism nutrients and / or electron donors.
Precipitation and / or deposition assisting agents may be added. The addition of lanthanium ions, and in particular La3" ions, is envisaged.
The insoluble form may be amorphous, but is more preferably crystalline. The insoluble form may be deposited on the cells and/or support for the second agen . 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 sample. 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 disposal may be employed. The selected species may be disposed 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 apparatus 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 dissolved 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 to separate the insoluble species from the sample.
The apparatus 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 basis.
The reactor vessel containing the first agent and the reactor vessel containing the second agent may be a different 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.
-Si- 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 the 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 239Np(IV) ;
Figure 5 illustrates the counts per minute against Rf value for the starting sample of 233Pa and 237Np (V) ;
Figure 6 illustrates the affect on the profile of Figure 5 of a chemical 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 environments a chemical precipitation route on Neptunium contamination is considered in comparison with the effectiveness on La (III) and Th(IV) samples. These two species are established models for the behaviour of 241Am(III) and 241Pu ( IV) and by analogy for Np(III) and N (IV) .
A Ci trobacter 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),S04, 0.96; glycerol 2-phosphate (G2P, 5.5 H20; BDH Ltd), 0.67; KC1 , 0.62; MgS04 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, OD600 = -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 40mM 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 lOOmM 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 Ci trobacter sp . and separating the cells from the supernatant following processing with Ci trobacter sp . Retention 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 233Pa 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 practicable 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 m 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 biiological 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 contacted with a second biological agent which again may be immobilised on beads 14. The second agent effects a further reaction of the contamination, again upon introduction of the necessary feed materials, stream 15. The reactions arising lead to the Neptunium's deposition as an insoluble form. Again the
reaction is discussed in more detail below. The immobilised contamination can be periodically removed in the resulting more concentrated form for disposal.
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 Al teromonas 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 phosphorlmager .
Other organisms include:- sulphate reducing bacterium, such as Desulfovibrio desulfuricans (available from various open culture collections) ; and Clostridiu sp (also available from various open culture collections) .
The species in solution can be assayed by radiography with a Phosphorlmager (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 collected by a fibreoptic bundle connected 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 (lOμl) 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 visualised 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 valencies of the species in question are separated by paper chromatography . Samples (lOμl) 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 valencies of the species were visualised and quantified using the Phosphorlmager 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/xml) 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 lOμl 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 valencies present by paper chromatography and with the valencies being quantified 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 reaction is thus attractive as presenting no hazard in itself.
An illustrative organism suitable for performing the second stage of the present invention is Ci trobacter 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 Escheri chia coli DH5α carrying the cloned gene phoN.
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 ImM 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.
In the case of Ci trobacter 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 reaction 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 :-
Np4+ + 2HP04 2" - Np(HP04)2
or in the further reduced form
Np3+ + HP04 2 " ► NpP04 + H+
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 reaction 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 reaction 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 reaction scheme; a continuous or semi-continuous reaction 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 HC1 and subsequently with several changes of distilled 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 - lOμg of each metal) was applied (volume 5μl) to the paper. The chromatographs were developed in tri ethylamine/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 0.15% 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 purified sample separated from all co- contaminants and daughter products.
237Np itself (alpha-emitter) is Phosphorlmager "silent" at the concentration used, when used as clingfilm-wrapped samples (irradiation 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 239Np(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 1cm strips. The strips were then cut into 1cm x 1cm 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 experiment 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, 1% final concentration, as a reducing agent lead to a reduction in the height of the 237Np(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 237Np(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 N2 and C02 (80:20) , passed through an oxygen trap. The growth medium contained lOOmM sodium lactate as the electron donor, with 50mM ferric citrate added as the electron acceptor. Medium was dispensed (10ml aliquot) into 12ml serum bottles and sealed with butyl rubber stoppers. Any dissolved oxygen was displaced by bubbling through the N2 : C02 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 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 NaHC03, 1.5g of NaH2P04, 0. lg of KC1 per litre of distilled water, deaerated) .
The material was then resuspended in 20mM MOPS buffer, pH 7 as required. A lOOμM 237Np feed level in 30μl of diluted stock
from the commercial source was then added to 300μl of the cells. The headspace of the reaction 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 lOμl spots, dried under N, between every addition , total load 30μl) was separated using chromatography as above and quantified by scintillation counting, expressed as a count rate versus Rf plot .
As the results, Figure 7, show a reduction in the 237Np (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 237Np, carbonate buffer 30 millimolar pH 7.5.
Similar tests were conducted on 2 9Np (V) samples. S. putrefaciens was prepared as above and resuspended in a 30mM carbonate buffer, pH 7.5. The feed consisted of cleaned ie. passed through an ion exchange column to remove contaminating daughter-species 237Np (V) , to 2μM, and 2 9Np (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 phosphorlmager as above. The results shown in Figure 8 clearly show the new spot D conforming to the expected location for 39Np(IV) .
This first stage alone, however, was not effective in removing Np(V) from the environment. Only 25% of the fed Np was retained with the cells following the biological reduction. Even though this was far higher than the retained level for the
chemical based reduction process alone, in which only 12% 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 experiment preparations of both Citrobacter S . 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 lOOmM 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 compared with Np(V) phosphates. In the experiments over 95% 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 with the CitroJacter 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) .