WO2011121291A1

WO2011121291A1 - Decontamination method

Info

Publication number: WO2011121291A1
Application number: PCT/GB2011/000482
Authority: WO
Inventors: Marina Fomina; Geoffrey Gadd
Original assignee: University of Dundee
Current assignee: University of Dundee
Priority date: 2010-04-01
Filing date: 2011-03-30
Publication date: 2011-10-06
Anticipated expiration: 2012-10-01
Also published as: GB201216821D0; GB2493295A; GB2493295B

Abstract

A method of decontaminating a surface includes providing a discrete portion of a solid matrix and either; providing a microorganism capable of removing contaminants at or near the surface and inoculating the portion of solid matrix with the microorganism or; providing a decontaminating fluid capable of removing contaminants at or near the surface and soaking the portion of solid matrix with the decontaminating fluid. The discrete portion of solid matrix is placed in contact with the surface and removed after a selected period of time.

Description

Decontamination Method

Field of the Invention

The present invention relates to methods of decontaminating surfaces by use of fluids or microorganisms and provides fluids and matrices and matrices including microorganisms for use in the methods. The methods, fluids, microorganisms and matrices may be used for removing metals and other contaminants, for example radionuclides, from surfaces such as concrete and metal surfaces.

Background to the Invention

The use of fluids (e.g. water or water-based solutions) as cleaning agents is well known as a means for decontaminating surfaces. Generally the fluid may be applied by spraying, or wiping the surface with an absorbent material soaked in the fluid. The decontaminating effect depends on the dissolution or dispersion and uptake of the contaminants from the surface into the fluid. For example the decontamination of surfaces (e.g. of cementitious materials, rock and mineral-based building materials, metal surfaces) contaminated by nuclear wastes is based on (1 ) the ability of fluids such as water and organic and mineral acids to dissolve and leach contaminants from the surfaces of building and barrier materials and (2) the ability of water and organic and mineral acids to degrade the surfaces of building and barrier materials .

These methods make use of the penetration of the fluid into the contaminated region near the surface of the object being decontaminated and in the case of some examples the deterioration at and near contaminated surfaces, caused by e.g. water and/or acids that can dissolve and remove contaminants.

However, where the contaminating agents are especially hazardous, for example radionuclides or other toxic substances, decontaminating a surface with a fluid may present difficulties. When the decontaminating fluid is applied in excess, as for a typical cleaning operation, the contaminant becomes dispersed in a large volume of fluid and is difficult to contain/collect. The fluid will run from the surface and can thus result in the spread of the contaminant from the surface being treated to the surrounding area. Even if all the fluid is successfully collected after use the volume of fluid required to achieve decontamination may be large and so further processing by e.g. concentration of the fluid may be required to produce a manageable waste. If less decontaminating fluid is applied in an effort to reduce the waste volume/ the spread of contamination to the surrounding area, then the decontaminant may not be successfully removed from the surface, especially where the surface is porous and the contaminant has penetrated into a surface layer.

Thus where a decontaminating fluid is employed and especially where particularly hazardous contaminants are present there is a need for alternative methods that avoid or at least reduce some of the aforementioned problems. The use of microorganisms such as fungi or bacteria has also been suggested as a possible means of decontaminating surfaces. The decontaminating effect depends on the dissolution and uptake of the contaminants from the surface. For example the decontamination of surfaces (e.g. of cementitious materials, rock and mineral-based building materials, metal surfaces) contaminated by nuclear wastes is based on (1 ) the ability of microorganisms to transform toxic metals and radionuclides (e.g. Current Biology 18:R375 -R377, 2008 (Fomina et al)) and (2) the ability of microorganisms to degrade the surfaces of building and barrier materials (e.g. Geomicrobiology Journal 24:643-653, 2007 (Fomina et al)). These proposals make use of the deterioration, at and near contaminated surfaces, caused by microorganisms that can include dissolution and uptake of contaminants into the microorganism(s). Thus, for example radioactive and toxic metal contaminants, and non-metal radionuclides, can be accumulated and concentrated within the biomass of the microorganism(s), which on removal can be disposed of.

Potential difficulties associated with using a microorganism-based approach to decontamination include ensuring a good distribution of the microorganism across the contaminated surface, providing good growth conditions for the microorganism and the efficient and safe removal of the biomass and its load of accumulated contaminants.

In WO9603754 and GB2328783 the issue of distribution of microorganism(s) to a surface is addressed by providing the microorganism in a suitable carrier medium, such as a gel or other fluid composition which will adhere to a surface and can be applied by spraying, painting etc to it. Subsequent removal of the microorganism containing carrier medium can be for example by washing or scraping off.

While these proposals may improve the potential to use microorganisms in decontamination procedures there is still a need for alternative methods.

Description of the Invention

According to a first aspect the present invention provides a method of decontaminating a surface comprising:

providing a microorganism capable of removing contaminants at or near the surface;

providing a discrete portion of a solid matrix and inoculating the portion of solid matrix with the microorganism;

placing the discrete portion of solid matrix in contact with the surface; and removing the discrete portion of solid matrix from the surface after a selected period of time.

The microorganism removes the contaminant at or near the surface being treated by action of their growth and/or metabolism. It will be understood that more than one type or strain of microorganism may be employed to improve the effectiveness of the decontamination procedure and throughout this document the term "microorganism" includes the possibility of a method employing more than one type, species or strain of microorganism in a given solid matrix.

According to a second aspect the present invention provides a method of decontaminating a surface comprising:

providing a decontaminating fluid capable of removing contaminants at or near the surface;

providing a discrete portion of an absorbent solid matrix and soaking the portion of solid matrix with the decontaminating fluid;

placing the discrete portion of solid matrix in contact with the surface; and removing the discrete portion of solid matrix from the surface after a selected period of time. The decontaminating fluid removes the contaminant at or near the surface being treated by physical and/or chemical action. For example, penetration of the fluid carried within the applied matrix into porous (e.g. cementitious) materials (soaking), dissolution of solid contaminants, and leaching of the contaminant into the applied matrix, which may be by capillary action. It will be understood that more than one type of fluid may be employed to improve the effectiveness of the decontamination procedure and throughout this document the term "decontaminating fluid" includes the possibility of a method employing more than one type of decontaminating fluid in a mixture in a given solid matrix.

According to either the first or the second aspect, successive treatments of the surface with the same solid matrix/microorganism combination or with the same solid matrix/decontaminating fluid combination are possible. Similarly successive treatments of the surface with different solid matrix/ microorganism combinations or with different solid matrix/decontaminating fluid combinations are also possible and may be advantageous e.g. if more than one contaminant is to be removed. As a yet further option successive treatments with solid matrix/microorganism combinations and solid matrix/fluid combinations may be advantageous and each treatment applied may involve the use of a same or different matrix/microorganism or matrix/fluid combination.

According to a third aspect the present invention provides a discrete portion of a solid matrix comprising a microorganism for use according to the first aspect of the invention.

According to a fourth aspect the present invention provides a discrete portion of a solid matrix impregnated with a decontaminating fluid for use according to the second aspect of the invention. In contrast to previous methods, the method of the present invention, by making use of discrete portions of solid matrix, allows easy removal of the matrix, with contaminants attached or impregnated within, from the surface after the selected time period. As a discrete portion of a solid matrix has been employed it can removed in a unitary manner, making containment of the contaminants easier when compared with a matrix that has been sprayed, painted or otherwise coated onto a surface. The methods described herein are particularly suited to the decontamination of surfaces, especially porous surfaces that have been contaminated by metals, including metal radionuclides, or non-metallic radionuclides. For example, radionuclides of metals such as strontium, caesium, cobalt, zinc and actinides such as uranium.

The methods may also be applied to contaminants and substances other than metals. For example, non-metal radionuclides could include isotopes of iodine and carbon. Surfaces to be decontaminated can include cementitious materials (for example concretes and mortars), rock and mineral-based building materials and metal or plastic surfaces.

Where a matrix layer has been applied to a surface by coating with a flowable micro- organism containing matrix (as in the prior art), the continuous layer formed has to be removed by e.g. washing or scraping. With this method the area around the surface being decontaminated is highly likely to be contaminated by the dispersal of fragments of the contaminated microorganism containing matrix, especially if the matrix is allowed to dry before removal. Furthermore the resulting waste - fragments of microorganism matrix, water, etc (all contaminated) is relatively voluminous and liable to accidental dispersal if not very carefully contained.

Even if the continuous layer of contaminated matrix/microorganism is carefully scraped from the surface, small fragments/particles will be formed. Scraping from the surface will also result in considerable potential to expose personnel involved to the contaminants, and to the microorganism (which may itself be harmful in some instances).

In contrast, by providing discrete portions of a solid matrix with associated micro- organism in accordance with the first aspect of the present invention the removal process can be simplified and containment made easier. Each discrete portion utilised can be removed from the surface as a single piece or substantially as a single piece without fragmentation of the matrix and dispersal of the contaminant(s). This is particularly important with highly toxic or otherwise hazardous contaminants that are persistent. For example, radionuclides. Similar benefits can arise when using the method in accordance with the second aspect of the invention, especially if the decontaminating fluid is allowed to dry out before removal of the solid matrix from the contaminated surface. In use of the method according to the first aspect of the invention.the portions of solid matrix are placed in contact with the contaminated surface to allow the action of the microorganisms to cause decontamination. The portions of solid matrix are not required to be bonded continuously to the contaminated surface on application but are merely placed in contact with it. In use the microorganism may, on growing penetrate and bond to, and/or impregnate the contaminated surface as the decontaminating action occurs.

In use of the method according to the second aspect of the invention The portions of solid matrix are placed in contact with the contaminated surface to allow the action of the decontaminating fluid to cause decontamination. The portions of solid matrix are not required to be bonded continuously to the contaminated surface on application but are merely placed in contact with it. In use the decontaminating fluid contacts the surface and if the surface is porous, it penetrates it, allowing the physical and/or chemical decontaminating action discussed below.

The decontaminating fluid may be selected from the group consisting of water (that may include deionised or distilled water), buffered and non-buffered water or aqueous solutions, organic and mineral acids (such as phosphoric, hydrochloric, nitric and sulphuric acid, citric and oxalic acid) , sequestering agents (e.g. ethylenediamine tetraacetic acid (EDTA), hydroxyethylene diamine tetraacetic acid, nitrilotriacetic acid), ammonium bifluoride, and microbial metabolites containing mineral-dissolving and metal-sequestering agents. Ammonium bifluoride (ammonium hydrogen fluoride) is of particular utility for silicate types of building materials as it can act to dissolve the near surface region.

Thus for example water (deionised or distilled), oxalic and citric acid solutions (that may be from 1 to 100mM in concentration) and elin-Norkans salts solution [(NH₄)₂HP0₄ (0.50 g-r¹), KH2PO4 (0.30 g-r ), MgS0₄-7H₂0 (0.14 g l^"1), CaCI₂-6H₂0 (50 mg l^"1), NaCI (25 mg Γ¹)] have been shown to be effective in tests on metal ion contaminated surfaces as described in more detail hereafter. Similar mixtures to the Melin-Norkans salts solution described above, containing varying concentrations of the component salts employed or omitting one or more of the component salts may also be employed.

Examples of suitable microbial metabolites with decontaminating properties for metals, mineral phases or other substances include organic acids, metal-binding peptides, amino acids, polysaccharides, melanins and other complexing or chelating agents..

The decontaminating fluid may also contain electrolytes as well as clay minerals or other mineral colloids, e.g. sulfides or any other suitable materials and chemicals, which may have the effect of binding, precipitating or sorbing contaminants in solution.

Chemical solutions can be used in different combinations, sequences and concentrations. Chemicals can be introduced into two and more adjacent matrices (side-by-side or on top of each other) made from the same or different absorbing materials within complex matrix constructions.

Some examples of suitable chemicals are 1.5 mM oxalic and citric acid, de-ionised or distilled water, Melin-Norkrans salts solution [(NH₄)₂HP0₄ (0.50 g-Γ¹), KH₂P0₄ (0.30 g l^"1), MgS0₄-7H₂0 (0.14 g l^"1), CaCI₂-6H₂0 (50 mg l ¹), NaCI (25 mg l^"1)]

Different concentrations of oxalic and citric acids (for example up to 100 mM) may be used.

In some circumstances it may be advantageous to include a gelling agent in the decontaminating fluid, to reduce evaporation or other losses (leakage), such as a polysaccharide, gelatine, or agar for example.

The method can also provide advantages in comparison to the standard cleaning procedure of wiping a surface with a cloth carrying absorbed decontaminating fluid. A substantial area or even the whole of the surface to be contaminated may be contacted with portions of the matrix simultaneously, with the matrix being left in place in intimate contact with the contaminated surface for a significant period of time. This allows longer contact time between the decontaminating fluid and the surface to permit the cleaning action to occur. This beneficial prolonged contact can occur without the presence of an operative, especially useful where dangerous, toxic or radioactive contaminants are being removed.

For example the matrix and associated decontaminating fluid may be placed in contact with the contaminated surface for a period of several hours or even days. For example for a period of from 1 to 7 days or even from 1 to 6 weeks.

At the same time the decontaminating fluid is not dispersed away from the surface being decontaminated and the matrix portion. The decontaminating fluid can be contained within the absorbing porous matrix. The volume of fluid applied to the absorbing matrix material to provide adequate wetting of the contaminated surface, whilst avoiding substantial leakage to the surrounding area can be readily calculated and/or determined by testing. To assist in maintaining the fluid content of the matrix (for example where the fluid is liable to evaporate over time e.g. an aqueous fluid), a protective film may be provided over the outer surface (i.e. the surface not contacting the contaminated surface) of the matrix. This can prevent excessive evaporation and aid handling the matrix portion as it is being placed in position for use.

In some circumstances, where a high fluid content in the matrix is required to obtain the desired results and the surface being decontaminated is, e.g. a vertical wall then the method may include the use of drip catching trays. The trays are placed beneath the portions of matrix to catch any decontaminating fluid that is not successfully contained within the matrix as it is held in contact with the contaminated surface.

After use the portion of matrix is removed for disposal or processing to extract the decontaminant or to reduce the waste volume. For example drying may be used to reduce the volume of contaminant fluid associated with the matrix portion.

Advantageously when the matrix is applied to a contaminated surface in a method according to either the first or the second aspect of the invention, light mechanical pressure may be used to ensure good contact. At least the matrix should generally be held in good contact with the surface. Good contact and/or light mechanical pressure can be achieved in a number of ways. The portion of matrix may be laid on the contaminated surface and a weight or weights placed on top. For example a horizontally disposed contaminated surface may have a sheet of matrix applied with a number of weights on top. Alternatively a single sheet of a suitably heavy material is placed on top, thus providing a consistent pressure across the whole matrix sheet. For a vertically disposed surface the portion of matrix may be held vertical by a suitable frame and pushed against the surface. Rams may be used to supply light pressure, pressing the matrix to the contaminated surface. In some cases the matrix may be wrapped around an object with a contaminated surface, as a bandage. The tension applied when wrapping the bandage around the object supplies pressure to the contact between the contaminated surface and the matrix material.

Alternatively mechanical fixings may be employed to hold the portion of matrix in contact with the contaminated surface. For example clamps may be employed; or nails screws or bolts that pass through the matrix into the contaminated surface. As a yet further alternative suitable adhesives may be employed to form bonds between the matrix and the contaminated surface. For example adhesive can be applied between the portion of matrix and the contaminated surface at or near the edges of the portion and/or at selected places across the portion, on the face that contacts the contaminated surface in use. For example adhesive may be used across a surface of the portion of matrix in the form of a discontinuous layer i.e. the adhesive layer has breaks in it to allow contact between the matrix and the contaminated surface, thereby allowing the microorganism to interact with the contaminated surface. If an adhesive is employed it is preferably selected so as to allow removal of the matrix portion from the surface without tearing. For example it may be a peelable adhesive.

For the first aspect of the invention the discrete portion of matrix may be of any solid material that can support the microorganism's growth when in contact with a contaminated surface. Typically the matrix material is provided in the form of a sheet. The thickness of the sheet is selected for the ability to support the growth of the micro- organism and to allow easy removal (e.g. without tearing). Any shape or size of discrete portion of matrix may be employed. Advantageously the matrix material has some flexibility or resilience so that it can be held or pressed into good contact with a contaminated surface. For the second aspect of the invention the discrete portion of matrix may be of any solid material with suitable absorbent properties when in contact with a contaminated surface. As for the first aspect, typically the matrix material is provided in the form of a sheet. The thickness of the sheet is selected to allow easy removal (e.g. without tearing) and to retain sufficient decontaminating fluid for effective decontaminating action. Any shape or size of discrete portion of matrix may be employed. Advantageously the matrix material has some flexibility or resilience so that it can be held or pressed into good contact with a contaminated surface.

Advantageously the matrix comprises, or consists essentially of or, is a porous material, typically a porous water absorbing material. The porous matrix material provides a good environment for holding a microorganism and water and certain nutrients that are normally required to support the growth and activity of the microorganism(s) (unless, e.g. sufficient is present in the atmosphere or in the contaminated surface). Similarly the porous matrix material provides good absorbency for a decontaminating fluid if that is being employed. Other materials may be included in the matrix, for example fibres or wires to increase strength. The matrix may also be in the form of a laminate, for example a layer of porous material (for supporting the microorganism or holing the decontaminating fluid) backed by a sheet of a tougher plastics material to provide strength, especially for when the matrix is removed from a contaminated surface after use.

Suitable matrix materials include but are not limited to porous water absorbing fibrous framework materials or solid foams. They may be made, for example, from plastics (such as polyurethane, polyvinyl alcohol, rubbers (natural or synthetic), cellulose (e.g. viscose, paper).

Suitable foam materials include polyurethane (PU) polyvinyl alcohol (PVA) and cellulose foams. Polyurethane and polyvinyl alcohol have the advantages of being both non-biodegradable and suitable as a support for microbial growth in a surface decontamination matrix. Cellulose or viscose foam materials are biodegradable, sometimes over a relatively short period which may limit their use as a decontaminating solid matrix, especially if the conditions of use of the matrix are liable to cause rapid degradation (e.g. if biodegrading microorganisms are used). However for some indications, for example when the decontamination period - the time a given potion of the solid matrix is applied to a contaminated surface - is short then such materials may prove satisfactory. In the method according to the first aspect, in addition to the microorganism(s) the matrix may contain other items. For example the matrix may be impregnated with water and/or nutrients or other substances (such as an energy source) to support the growth of the microorganism during the decontamination process. Alternatively or additionally to placing these materials in the matrix before applying the matrix portion to a contaminated surface, they may be put into the matrix after it has been contacted to the decontaminated surface. The microorganism(s) may also be put into the matrix after it has been applied to the contaminated surface if desired.

The matrix may also be supplemented with microorganism(s), water, nutrients or other substances during the period it is placed in contact with the contaminated surface, to avoid the microorganism stopping its action and aid continued effective decontamination action. The time period of a decontamination treatment (i.e. one application of the matrix/microorganism combination) may be from one to two weeks or up to 6 weeks, even if the matrix is not supplemented with microorganism(s), water, nutrients or other substances. Where supplementation is employed the matrix may be kept almost indefinitely in contact with the surface, but its removal and replacement with further decontamination treatment (e.g. successive matrix/microorganism and /or matrix decontaminating fluid treatments) maybe more effective and faster.

The methods of the invention may be employed with any type of contaminant that can be taken into the matrix by the action of microorganism(s) or of the decontaminating fluid. For example, metals, organic substances, metal-organic compounds and radionuclides.

Any microorganism that has a decontaminating action may be employed. For example, a fungus or a bacterium, as well as other prokaryotes (e.g. archea) and eukaryotes (e.g. algae) with appropriate adjustment of operating conditions. Selection of a suitable microorganism can be by carrying out appropriate tests for the ability to solubilize, accumulate, or transform the contaminant and for penetrating the surface to be contaminated. Examples of a suitable selection procedure are given hereafter with reference to specific embodiments. When testing organisms for suitability especially, for example when dealing with mutagenic contaminants such as radionuclides, the originally applied organisms may mutate in response to the contaminant. Thus the testing procedure may result in the production of a new, particularly suitable microorganism for a given decontamination task.

Thus according to a third aspect the present invention provides a method for producing a microorganism for use in decontaminating a surface according to the first aspect of the invention. The method comprises: contacting a sample or samples of known microorganisms with a mutagenic contaminant, under conditions for growth of the microorganism; and selecting samples of mutated microorganisms displaying the ability to grow and to uptake or solubilise the contaminant. Where metal or radionuclide decontamination of a surface, for example a concrete surface, is contemplated the microorganism may be a fungus. Suitable fungi may operate by displaying the following activity:

(i) producing and excreting into their microenvironment metal sequestering or mineral dissolving agents such as organic acids (e.g. oxalic, citric acids) and other metabolites (e.g. H⁺, siderophores),

(ii) accumulating metals and radionuclides released from dissolved/deteriorated materials,

(iii) concentrating accumulated metals and radionuclides with subsequent organic and inorganic precipitation and formation of secondary minerals.

Examples of fungi that can display such activity include but are not limited to: Aspergillus niger, and species that belong to the genera, Penicillium, Beauveria, Serpula, Geomyces, Coniophora, Paecilomyces and Rhizopogon.. Examples of suitable fungi thus include Aspergillus niger, Beauveria caledonica, Coniophora puteana, Penicillium commune, Penicillium namyslowsky, Penicillium roqueforti, Paecilomyces lilacinus, Serpula himantioides, and Rhizopogon rubescens.

Suitable strains of fungi can be obtained from the environment (e.g. from soil) and cultured in the known manner or are commercially available. Growth of fungi within the matrix is promoted and maintained by favourable nutritional and environmental conditions, e.g. suitable liquid medium providing the organisms(s) with suitable energy source and nutrients and moisturizing the matrix/carrier. If necessary, water could be also used for moisturizing the matrix/carrier. The matrix can be designed to retain nutrients for periods of time adequate for the decontamination process. For example it may be made of a thicker layer of porous material and/or the pore size may be adjusted to suit.

As discussed above the method of the invention may include multiple or sequential treatments of the contaminated surface. Each treatment may use the same or similar application of a discrete portion of solid matrix. Each treatment may use the same or different microorganisms and/or decontaminating fluid in an appropriate matrix. As discussed above with respect to the second aspect of the invention matrices containing microbial metabolites, rather than the microorganisms themselves, and/or their chemical analogues or simulants such as organic and mineral acids, and water can be also used for decontamination, e.g. as a part of sequential treatments. The term simulant as used herein means: substances such as organic and mineral acids which simulate, for example the ligand-promoted and proton-promoted microbial attack on contaminated surfaces. The decontamination method of the invention may include treatment of the surface with such a chemical treatment, which may be done making use of a solid matrix as with the use of the microorganism. Alternatively a chemical treatment may be applied to the surface by other means e.g. spraying or painting in addition to treatment with a microorganism/matrix or a decontaminating fluid/matrix combination or combinations

After surface decontamination and prior to disposal, matrices may be treated, for example by suitable chemicals or polymers, to make their disposal as safe as possible. For example, highly mobile caesium concentrated within matrices can be treated prior to disposal by using a flexible framework sulphide through selective incarceration of caesium ions by a "Venus flytrap" action as discussed in Nature Chemistry, Published online: 24 January 2010 doi:10.1038/nchem.519 (Ding & Kanatzidis)

When a fungus is employed the decontamination action can be as follows. When supplied with energy source and nutrients within the matrix, fungi perform biogeochemical attack on the surface layers by excretion of metabolites such as protons and ligands, e.g. oxalic and citric acids. The surfaces to be decontaminated can be, e.g. rock, mineral, metal, glass, plastic, as well as cementitious materials such as concrete. For example, cement and concrete are widely used in the nuclear and other industries as building materials and as barriers in all kinds of nuclear waste repositories. If brought into contact with cementitious materials, fungi dissolve the cement components leaching structural elements (mainly calcium and silicon) together with metal/radionuclide contaminants, and accumulate the leached elements within the fungal biofilms and associated microenvironment of the matrix. A property of the matrix can be biomineralization. Metal contaminants are not only absorbed, adsorbed and concentrated within the matrix but may also be transformed into biogenic minerals (e.g. metal oxalates, phosphates, oxides and/or secondary carbonates) which are a less bioavailable chemical form of metal in the waste.

The detachable matrix may be tested for all necessary parameters (e.g. metals/radionuclide content, biomass yield) after the anticipated time period required to carry out the amount of decontamination expected from a given matrix and microorganism or matrix and decontaminating fluid combination. Where a microorganism is employed, at the end of the decontamination process, the matrix may be nutrient- depleted which will slow or stop growth activity of the microorganism. Microbial activity can be stopped by drying the matrix. If desired, the microorganisms can be killed by applying sterilizing processes such as heat, pressure, biocides or fumigation.

After use in contact with a contaminated surface the discrete matrix portion is removed. It will contain at least some of the contaminants previously found on the contaminated surface. It can either be disposed of or regenerated, for example by washing out the contaminants and introducing fresh decontaminating fluid or microorganism, perhaps following a sterilizing step.

The contaminants may be recovered from the matrix for disposal, recycling or further treatment as appropriate.

Brief Description of the Drawings

Figures 1a to 1d illustrate schematically the application of the method of the invention in decontaminating a surface; and

Figure 2 shows decontamination of mortar test blocks. Detailed Description of Some Aspects of the Invention with Reference to Examples

Figure 1 a shows in schematic elevation two portions of a matrix material 1 ,2 attached, (adjacent to each other), to a contaminated surface, in this example a vertically disposed concrete wall 4 contaminated with radionuclides 6 (see figure 1 b). It will be understood that typically the whole of such a contaminated surface would be covered by matrix potions, each time a decontaminating treatment step is applied. For clarity only two matrix portions are shown here.

The matrix material portions 1 , 2 are sheets of open cell foam, for example a polyurethane foam, and have either : been inoculated with a microorganism and impregnated with water and nutrients to support the growth of the microorganism; or have been impregnated with a decontaminating fluid.

The portions of matrix material 1 , 2 are held in contact with the wall 4 by means of nails 8. Alternative fixings such as a border of a peelable adhesive (suggested by the dashed lines 10) around the edges of the portions 1 , 2 may be employed. Where a microorganism is employed, he growth and metabolism of the microorganism causes deterioration of the surface layer of the wall and leaching/sequestration of minerals including contaminant radionuclides 6 into the matrix portions 1 , 2. Where a decontaminating fluid is employed, The action of the decontaminating fluid causes deterioration of the surface layer of the wall and leaching/sequestration of minerals including contaminant radionuclides 6 into the matrix portions 1 , 2.

See the schematic sectional end elevation figure 1 b taken through line X-X of figure 1 a, where the entry into the surface layer of the wall 4 by the microorganism and/or its metabolites and the removal of minerals and contaminants 6 to the matrix portion 1 are indicated by the double headed arrows.

After a preselected time interval or when monitoring (e.g. of microorganism viability or matrix uptake of radioactivity) indicates that the decontamination process has reached a desired level; or has slowed or stopped, the matrix portions 1 , 2 are removed for disposal or reuse. Reuse would follow removal from the matrix material of the radionuclides that have been taken up from the contaminated wall 4. Fresh matrix portions 1 , 16 can then be applied as shown in schematic elevation figure 1c. The new matrix portions 14, 16 may include the same or a different microorganism or decontaminating fluid. The process can be repeated using either decontaminating fluids or microorganisms of different types as required.

In figure 1c the new portions of matrix 14, 16 are shown applied to the wall 4 in positions that are offset from the position of the originally applied matrix portions 1 , 2 (indicated by dashed lines 18). Offsetting successively applied matrix portions in this way assists in ensuring the treatment regime is evenly applied across the contaminated surface. It is an especially useful approach if an adhesive is used to hold the matrix to the contaminated surface as there may be little or no decontaminating effect at places where an adhesive contacts the contaminated surface. After a suitable time interval the matrix portions 14, 16 are themselves removed for disposal or reuse.

Figure 1d is a schematic sectional end elevation similar to that of figure 1 b but illustrating an alternative means of holding a portion of matrix 1 in good contact against the wall 4. In this example the matrix portion 1 is held in a frame 20 that is held in place by supports 22 and 24, mounted on a mass M located on a floor 26. Several alternative means of mounting the matrix portions in place can be employed. For example a frame holding a matrix portion may be pressed against a surface by means of a pneumatic or hydraulic ram.

Examples using microorganisms

Preliminary screening of microorganisms

Fungal strains from the inventors own collection of microorganisms were maintained at 25°C on modified Melin-Norkrans (MMN) agar medium comprising: (NH₄)₂HP0₄ (0.50 g-r¹), KH₂P0₄ (0.30 g l^'1), MgS0₄-7H₂0 (0.14 g l^"1), CaCI₂-6H₂0 (50 mg-Γ¹), NaCI (25 mg l^'1), D-glucose (10 g Γ¹) and agar No. 1 (Lab M, Bury, UK) (14 g l^"1). Before adding the agar and prior to autoclaving, the liquid medium was adjusted to pH 5.5 using concentrated HCI. Fungi were grown in 90 mm diameter Petri dishes on 20 cm³ MN agar with a mixture of Cs, Co and Sr carbonate at concentration 8 mM each. Metal carbonates were oven- sterilized for 48 h at 100° C.

Prior to inoculation, 84 mm diameter discs of sterile cellophane membrane were placed aseptically on the surface of the agar in each Petri dish. Inoculations were carried out using 7mm diameter discs of mycelium cut from the leading edge of colonies which had been incubated on the modified Melin-Norkrans agar medium at 23° C for at least 14 days. The fungi were inoculated and incubated at 23° C for two months (at least three replicates).

Preliminary screening of microorganisms was based on such criteria as

• solubilization of metals and its efficiency,

growth and tolerance in the presence of metals,

• crystal formation (metal salts) in biomass and agar,

• metal accumulation within biomass (by MS), and

• reproducibility of results for key abilities (standard error of mean and/or

confidence interval calculations).

Growth and Tolerance: Growth of the fungi was determined by dry weight. Colonies were removed from replicate agar plates by peeling the biomass from the dialysis membrane. The mycelia were oven-dried at 105° C until reaching constant weight. Tolerance to Cs, Co and Sr carbonate mixture was expressed in terms of a tolerance index (Tl) based on the dry weights (DW) of fungal biomass where:

DW of metal - treated mycelium

Dw - _r : ; 7. x iuu( /o)

DW oj control mycelium

Solubilization: The diameter of the solubilization area (a clear halo) in agar was the main criterion for estimating the solubilizing ability of tested fungi against metal carbonates. The greater the diameter of the halo around the sample of fungus the greater the solubilization of the metal carbonate achieved.

Additionally or as an alternative an indirect index for fungal solubilizing ability can be obtained by measuring the amount of Co, Cs and Sr accumulated by the biomass. From these results fungal strains were selected for further study, in tests against contaminated mortar samples as discussed below. The selected strains included Aspergillus niger, Beauveria caledonica, Coniophora puteana, Penicillium commune, Penicillium namyslowsk and Penicillium roqueforti.

Immobilization of fungi within a matrix

Fungi were immobilized within a selected matrix material by physical entrapment within the open pore network. Polyurethane (PU [a white polyurethane foam stopper, from Fisher Scientific, Loughborough, United Kingdom, catalogue number FB68838]), polyvinyl alcohol (PVA) [Ramer PVA sponge samples: types CYLO and # EL2b from Ramer Sponges, Wintney, Hampshire, United Kingdom]) or cellulose (Spontex® brand super absorbent sponges, from apa Spontex UK Ltd. Worcester, United Kingdom). Foam blocks with dimensions ranging from 10 to 25 mm (length of sides) were tested. Of the two types of PVA foam tested samples of type #EL2b were found to be best for immobilization of organisms and so this form of PVA foam was used for subsequent experiments.

PU blocks were sterilized by autoclaving, and PVA and cellulose blocks were sterilized by treatment with ethanol.

(i) Starter culture: Prior to immobilization microbial biomass (starter culture) was grown for about 4-6 days in 70-100 ml liquid MMN medium in 250-ml flasks under submerged conditions at 160 rpm and 25° C.

(ii) Maceration: Biomass from the starter culture was rinsed with sterile water and resuspended in fresh MMN medium. 10 ml of suspension was added to 70 ml of MMN in 500 ml flasks with 4 mm glass beads (15 ml). Flasks were shaken at 300 rpm and 25° C for 30-45 min.

(iii) Entrapment: After maceration the suspension (10-15ml) was added to 250-ml flasks with 70 ml MMN and blocks of matrix (e.g. 4 cubes of side 10mm). Flasks were shaken for 24 hours at 160 rpm and 25° C.

(iv) Pre-growth: The blocks of matrices were removed from the flasks and rinsed with sterile water to remove non-immobilized biomass. The blocks of matrices were then put into 250-ml flasks with 70-100 ml of fresh MMN medium. Flasks were shaken for 5-7 days at 160 rpm and 25° C. After that the blocks of matrix containing immobilized and pre-grown microbial biomass were ready to be used in decontamination process. Preparation of contaminated test surfaces - Mortar

Mortar cuboids (2.5x2.5x1.5 cm) to be used as a model of cementitious building mortar cuboids were manufactured under sterile conditions.

Portland cement powder was mixed with sand and water in ratio 1 :2.5:0.7 (w/w) (e.g. 90g cement paste mixed with 225g sand and 70ml water). Cement powder and sand were oven-sterilized at 105° C for at least 3 days. Water was autoclaved for 15-20 min at pressure 15psi and temperature 121° C.

The mixture of cement powder, sand and water was put into silicone moulds, covered with glass plates and left for hardening. Total curing time was 28 days.

Radionuclides were simulated by the use of corresponding non-radioactive metals. Radionuclides of interest include medium-lived fission products strontium-90 and caesium-137 and the activation product cobalt-60 which are of concern in the nuclear industry.

To simulate the surface contamination of cementitious building materials, metals were introduced into a thin surface layer of the mortar samples. For multiple metal incorporation mortar cubes were soaked in water-based solutions of cobalt, caesium and strontium nitrates having metal concentrations of 100mM.

Decontamination tests

Decontamination tests were performed by bringing in contact the contaminated surface of a mortar cuboid (see above) with a block of the porous matrix containing immobilized and pre-grown microbial biomass (the "bio-matrix").

As a result of the decontamination process, extensive leaching of calcium from cement with consequent degradation of the mortar surface and accumulation of metal contaminants (caesium, cobalt and strontium) within the bio-matrix occurred.

For example, degradation of mortar with an approximate 90% reduction of cobalt contamination in the surface layer ( mm depth) was observed and confirmed by scanning electron microscopy coupled with energy dispersive X-ray microanalysis, after 3 weeks of decontamination. Atomic absorption spectrophotometry of samples of the bio-matrices showed that approximately 8-10 mg Cs, 0.5-0.8 mg Sr and 0.13- 0.17mg Co were removed from each mortar cuboid and were accumulated within the adjacent bio-matrix after a 3- week decontamination period. These results were obtained using samples of Beauveria caledonica, Penicillium namyslowsky and

Penicillium roqueforti. All these strains are from the inventors own collection of microorganisms isolated from soil or rock (UK).

Further tests using more strains from a collection of microorganisms of inventors e.g. Aspergillus niger, Coniophora puteana, Penicillium commune demonstrated visual discoloration of the dark-brown coloured surface of cobalt-contaminated mortar indicating successful cobalt decontamination. These further tests demonstrated that the decontamination cycle could be reduced to only one or two weeks i.e. a similar level of decontamination can be achieved in an even shorter period, using the same testing protocols.

Examples using decontaminating fluids

Decontaminating fluids tested

The solutions used in the these experiments were:

• water (distilled deionized H?Q)

• oxalic acid 1.5 mM

• citric acid 1.5 mM

• Melin Norkrans salts: (NH₄)₂HP0₄ (0.50 g-Γ¹), KH₂P0₄ (0.30 g l^'1), MgSCv7H₂0 (0.14 g l^'1), CaCI₂-6H₂0 (50 mg l^'1), NaCI (25 mg l^'1).

Soaking matrix with a solution

The selected fluids were absorbed by a selected matrix material within an open pore network. These were polyurethane (PU [a white polyurethane foam stopper, from Fisher Scientific, Loughborough, United Kingdom, catalogue number FB68838]), polyvinyl alcohol (PVA) [Ramer PVA sponge samples: types CYLO and # EL2b from Ramer Sponges, Wintney, Hampshire, United Kingdom]) or cellulose (Spontex® brand super absorbent sponges, from Mapa Spontex UK Ltd. Worcester, United Kingdom). Foam blocks with dimensions ranging from 10 to 25 mm (length of sides) were tested. Matrices were sterilized prior to use to prevent from growth of microorganisms to ensure that the decontamination process is not affected by microbial growth and activity. PU blocks were sterilized by autoclaving, and PVA and cellulose blocks were sterilized by treatment with ethanol.

Preparation of contaminated test surfaces - Mortar

Portland cement powder was mixed with sand and water in a ratio of 1 :2.5:0.7 (w/w) (e.g. 90g cement paste mixed with 225g sand and 70ml water). Cement powder and sand were oven-sterilized at 105° C for at least 3 days. Water was autoclaved for 15-20 min at pressure 15psi and temperature 121° C.

The mixture of cement powder, sand and water was put into silicone moulds, covered with glass plates and left for hardening. Total curing time was 28 days. Radionuclides were simulated by the use of corresponding non-radioactive metals. Radionuclides of interest include medium-lived fission products strontium-90 and caesium-137 and the activation product cobalt-60 which are of concern in the nuclear industry. To simulate the surface contamination of cementitious building materials, metals were introduced into a thin surface layer of the mortar samples. For multiple metal incorporation mortar cubes were soaked in water-based solutions of cobalt, caesium and strontium nitrates having metal concentrations of 100mM. Decontamination tests

Decontamination tests were performed by bringing in contact the contaminated surface of a mortar cuboid (see above) with a block of the porous matrix containing the decontaminating fluid. As a result of the decontamination process, extensive leaching of calcium from cement with consequent degradation of the mortar surface and accumulation of metal contaminants (caesium, cobalt and strontium) within the matrix occurred. Atomic absorption spectrophotometry of samples of the matrices showed that the ranges of amount of contaminants that were removed from each mortar cuboid and accumulated within the adjacent matrix after a 6 week decontamination period were 0.57-11.73 mg Cs, 0.15-2.6 mg Sr and 0.001-0.02 mg Co for water matrices, and 1.12- 19.71 mg Cs, 0.03-1 mg Sr and 0.005-0.03 mg Co for Melin Norkrans salts matrices. The range of Ca amount leached into the matrices was 0.26 - 1.9 mg and 0.067 - 4.5 mg for water and Melin Norkrans salts matrices, respectively. These results were obtained using samples of PU, PVA and cellulose sponges, with no significant difference in decontamination was found for different materials. Further examples

Further tests with PU and PVA sponges soaked with water, Melin Norkrans salts, oxalic and citric acid demonstrated that the decontamination cycle could be reduced to only one week i.e. a similar level of decontamination can be achieved in an even shorter period, using the same protocols. It was demonstrated that the amount of cobalt leached from mortar cuboids and concentrated within the PU or PVA matrix was in the following ranges (after 1 week of treatment): 0.009 - 0.024 mg for 1.5 mM citric acid, 0.013 - 0.056 mg for 1.5 mM oxalic acid, 0.0086 - 0.067 mg for water and 0.002 - 0.093 mg for Melin Norkrans salts. Yet further experiments tested different concentrations of citric and oxalic acids (from 5 to 100 mM) absorbed in a PVA matrix. Preliminary visual observations indicate that that the decontamination process might be reduced to 3 days or even less (see Fig. 2).

In these experiments 4 individual pieces of matrix with 50 mM citric acid absorbed as decontaminating fluid were applied to the top surface of a mortar test block containing cobalt as a contaminant. The four matrix pieces were removed after 1 ,3,5 and 7 days respectively with decontamination assessed by visual appearance (extent of decolouration achieved). A mortar test block is shown, after this treatment, in figure 2a. The edges of the block are dark as a consequence of the original cobalt contamination. The top surface of the block is lighter as a result of the decontamination action. Figure 2b shows the arrangement of four matrix pieces that were applied to the block of figure 2a with the circled numbers 1 ,3,5 and 7 indicating the number of days each matrix portion was left in contact with the mortar surface.

After 3 days significant decolouration is observed. Even after one day (bottom left quarter of the block surface in figure 2a) the treatment shows decolouration albeit weaker than for a three day treatment. No further visual difference was observed in the mortar over 3-7 days of treatment.

Claims

1. A method of decontaminating a surface comprising:

providing a discrete portion of a solid matrix and either;

providing a microorganism capable of removing contaminants at or near the surface and inoculating the portion of solid matrix with the microorganism; or

providing a decontaminating fluid capable of removing contaminants at or near the surface and soaking the portion of solid matrix with the decontaminating fluid;

placing the discrete portion of solid matrix including the microorganism or the decontaminating fluid in contact with the surface; and

removing the discrete portion of solid matrix from the surface after a selected period of time.

2. The method of claim 1 wherein the solid matrix comprises, consists essentially of, or is a porous material.

3. The method of claim 2 wherein the porous material is a porous water absorbing fibrous framework material or a solid foam.

4. The method of claim 3 wherein the porous material is a solid foam selected from the group consisting of polyurethane, polyvinyl alcohol, viscose and cellulose foams.

5. The method according to any preceding claim wherein the portion of solid matrix is held in contact with the surface being decontaminated by means selected from: the application of a weight, supporting in a frame pushed against the surface, a ram pressing the matrix portion to the contaminated surface, wrapping the matrix portion around the surface, mechanical fixing to the surface, and adhesive bonding to the surface.

6. The method according to any preceding claim, further comprising the application of a chemical treatment.

7. The method according to any preceding claim further comprising; the application of at least one more portion of a solid matrix inoculated with a microorganism or soaked with decontaminating fluid to the surface for a selected period of time.

8. The method of any preceding claim, when a microorganism is provided, further comprising: maintaining the inoculated portion of solid matrix under conditions that support growth of the microorganism, for a selected period, prior to its placement in contact with the contaminated surface

9. The method of any preceding claim, when a microorganism is provided, wherein more than one type, species or strain of microorganism is provided in a said discrete portion of a solid matrix.

10. The method of any preceding claim, when a microorganism is provided, wherein the surface is decontaminated by successive treatments using the same solid matrix/microorganism combination or with different solid matrix/ microorganism combinations.

11. The method according to any preceding claim, when a microorganism is provided, wherein the solid matrix is impregnated with water and/or other nutrients and/or an energy source to support the growth of the microorganism during the decontamination process.

12. The method according to any preceding claim, when a microorganism is provided, wherein the microorganism is selected from the group consisting of fungi, bacteria, archea and algae.

13. The method according to claim 12 wherein the microorganism is a fungus selected from the group consisting of species that belong to the genera, Aspergillus, Penicillium, Beauveria, Serpula, Geomyces, Coniophora, Paecilomyces and Rhizopogon..

14. The method according to claim 13 wherein the fungus is selected from the group consisting of Aspergillus niger, Beauveria caledonica, Coniophora puteana, Penicillium commune, Penicillium namyslowsky, Penicillium roqueforti, Paecilomyces lilacinus, Serpula himantioides, and Rhizopogon rubescens.

15. The method according to any preceding claim, when a microorganism is provided, further comprising the application of a chemical treatment incorporated in a portion of a solid matrix.

16. The method according to any preceding claim wherein a fungus is provided as microorganism, the surface being decontaminated is of a cementitious material and the contaminants are metals and/or radionuclides.

17. The method according to any one of claims 1 to 7 wherein a decontaminating fluid is provided and is selected from the group consisting of:

water, aqueous solutions, organic and mineral acids, sequestering agents, ammonium bifluoride, and microbial metabolites containing mineral-dissolving and metal-sequestering agents.

18. The method according to claim 17 wherein the decontaminating fluid is selected from the group consisting of:

water, oxalic acid solutions, citric acid solutions and elin-Norkans salts solution.

19. The method according to any one of claims 1 to 7, when a decontaminating fluid is provided, and 17 and 18,

wherein the decontaminating fluid further includes a gelling agent.

20. The method according to any one of claims 1 to 7, when a decontaminating fluid is provided, and 17 to 19,

wherein the selected period of time is from 1 to 6 weeks.

21. The method according to any one of claims 1 to 7, when a decontaminating fluid is provided, and 17 to 20,

wherein a protective film is provided over the outer surface of the matrix.

22. The method according to any one of claims 1 to 7, when a decontaminating fluid is provided, and 17 to 21 ,

wherein the surface being decontaminated is of a cementitious material and the contaminants are metals and/or radionuclides.

23. A discrete portion of a solid matrix comprising a microorganism for use according to the method of any one of claims 1 to 7, when a microorganism is provided, and 8 to 16.

24. A method for producing a microorganism for use in decontaminating a surface in a method according to any one of claims 1 to 16, the method comprising:

contacting a sample or samples of known microorganisms with a mutagenic contaminant, under conditions for growth of the microorganism; and

selecting samples of mutated microorganisms displaying the ability to grow and to uptake or solubilise the contaminant.

25. The method of claim 24 wherein the contacting of a sample or samples of known microorganisms with the mutagenic contaminant is carried out when the microorganisms are in a discrete portion of a solid matrix being employed in a method of decontaminating a surface according to any one of claims 1 to 15.

26. A discrete portion of a solid matrix comprising a decontaminating fluid for use according to the method of any one of claims 1 to 7, when a decontaminating fluid is provided, and 1 to 21.