J . Chem. Tech. Biotechnol. 1990, 49, 381-394
Review of Biotechnology Applications to Nuclear Waste Treatment* Nicholas V. Ashley & Daniel J. W. Roach PA Consulting Group-Technology, Cambridge Laboratory, Melbourn, Royston, Hertfordshire SG8 6DP. UK (Received 29 September 1989; accepted 21 December 1989)
ABSTRACT This paper gives an overview of the feasibility of the application of biotechnology to nuclear waste treatment. The contents are based on a report which PA Technology carried out for the Department of the Environment (DOE Reference: DoEIR W/88.008 Sector N o 2.3). Many living and dead organisms accumulate heavy metals and radionuclides. The controlled use of this phenomenon forms the basis for the application of biotechnology to the removal of radionuclides ffom nuclear waste streams. Indeed, biotechnology offers a series of new opportunities for removal of radionuclides f f o m dilute aqueous process effluents. Such technology is already used for heavy metal removal on a commercial basis and could be optimised for radionuclide removal. An overview of biotechnology areas, namely the use of biopolymers and biosorption using biomass applicable to the removal of radionuclides ffom industrial nuclear effluents is given. The potential of biomagnetic separation technology, genetic engineering and monoclonal antibody technology is also to be examined. The most appropriate technologies to develop for radionuclide removal in the short term appear to be those based on biosorption of radionuclides by biomass and the use of modified and unmodified biopolymers in the medium term. Key words: biotechnology, nuclear waste, waste treatment, biosorption, biopolymers.
* Paper presented at the meeting ‘Recovery/Removalof Metals by Biosorption-A Commercial Reality or a Scientist’s Dream?’, organised by the Solvent Extraction and Ion Exchange Group of the Society of Chemical Industry and held in London on 18 May 1989. 381
J . Chem. Tech. Biotechnol. 0268-2575/90/$03.50 0 1990 SCI. Printed in Great Britain
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I INTRODUCI'ION The cocktail of metals in nuclear wastes is a function both of the fuel and of its containment materials; its processing presents special problems even though much of the chemistry is of a character familiar in other areas of industrial chemometallurgy . The nuclear industry is required to ensure that discharges of radioisotopes and other hazardous chemicals are minimised at the point of discharge. The present practice is to treat aqueous nuclear waste streams by means of large-scale inorganic ion-exchange or chelation sometimes coupled with flocculation and ultra-filtration. Low-volume, highly active aqueous wastes are stored in special tanks pending vitrification. Inorganic material bioprocessing is now receiving attention from industries where microorganisms and microbially based processes are increasingly perceived as potential lowenergy routes to recovering metals from low-grade ores, upgrading of ore waste and fossil fuels prior to conventional processing and combustion, and toxic and/or precious metal recovery from waste streams. Investigations into the range and potential for commercial microbial transformations of heavy elements are just beginning. Heavy metal uptake by microorganisms (and many higher organisms also) and bioproducts derived from them is a well-recognised phenomenon. There is a considerable body of research devoted to the use of biologically based systems for the accumulation of heavy, often precious, metals from a variety of aqueous waste sources. Part of this is concerned with the accumulation of radionuclides.
2 RADIONUCLIDE CONCENTRATIONS IN NUCLEAR INDUSTRY WASTE STREAMS Data were supplied by the DOEand from published reports on the major inactive cations, anions and organics in aqueous waste streams and significant active radionuclides and total element concentrations for a number of waste streams, for example, low active waste streams (Table 1). The data provided by the DOE and obtained from Harwell Report 12338 contained information as to the non-radioisotopic composition of some waste streams.' Such data are important because the concentration of some metal ions may be far higher than that shown if only the radioisotopes are considered. The total metal concentration is an important piece of data, for it should enable an estimate of the efficiency of a biotechnology based metal recovery system to be estimated.
2.1 Isotopic concentrations: A summary High- and medium-level activity waste streams: -mg to tens of mg dm-3
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TABLE 1 BNFL Reference Stream Three" ~~~
Concentration (g d m - j )
~
Activity (Ci d m - j )
Major inactive species
Fe Na K Ca U
015 04 003 001 001 001 003 003 17
Ms
TBP Kerosene NO;
so: -
05
CI
0007
Significant active species
Ru-106 Ce-144 Sr-90 Zr-95 Nb-95 CS-137 Tc-99 Pu alpha Am-241 Np-237 CO-60 Pu-241 (3-134
3.2 x 10-9 3.1 x 1O-I' 8.3 x 10-9 1.ox 10-10 2.3 x lo-'' 4.1 x lo-' 2.9 x 10-5 1.4~ 4.8 x lo-' 8.5 x
3.9 x 10-10 6 . 4 10-7 ~ 6.3 x lo-''
1.1 x 10-5 1.0 x 1.2x 10-6 2.2x 10-6 8.8 x 3.6 x s.ox 10-7 1.7 x loW6 1.7 x 6 . 0 lo-' ~ 4.3 x 10-7 6 4 10-5 ~ 8.4 x
Concentrations of total elements
Ru Ce Sr Zr Nb cs Tc PU Am NP co
1.2 x 10-7 7.ox 10-9 1.5 x 10-8 8.2 x lo-' 2.3 x lo-'' 1.2 x 10-7 3.ox 10-5 1.4 x 10-5 5.3 x 10-8 8.5 x 10-5 3.9 x 10-10
Source: DOE. *Volume, lo00 m3 dm-j; acidity, approximately 0.3 mol dm-3 as HNO,. Low-level activity waste streams: -vary depending on isotope; for example
95Zr 1 . 0 lo-'' ~ g dm-j 237Np8-5 x lo-' g dm-3 Uranium (mainly 238) 1 x
g dmP3
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Storage pond emuents: -vary depending on isotope; for example 13'Cs 1.8 x lo-" g dm-3 235U1 x g dmP3 Thus the concentrations of nuclides vary depending on the nature of the waste stream. The final concentration of the non-transuranic elements may be considerably higher than the radioisotope fraction of each element. The total metal ion concentration is a requirement which is necessary to judge which of the biotechnology approaches would be most effectiveand suitable for treating aqueous nuclear waste streams. This is not intended to be an exhaustive review of all possible nuclear waste streams but serves as an indicator as to the different varieties of radionuclides, other metal ions and their concentrations that may be found in typical reprocessing streams.
3 BIOTECHNOLOGY APPROACHES AND AQUEOUS RADIONUCLIDE EFFLUENT TREATMENT
The main biotechnologically based areas that will have an impact on aqueous nuclear emuent treatment are the use of biopolymers and biosorbants. Novel biotechnology-based opportunities are also described. These comprise biomolecular engineering (protein and genetic engineering), the use of monoclonal antibodies and biomagnetic separation. 3.1 Biopolymers
Many biopolymers display properties which make them very attractive as candidates for use in aqueous nuclear waste treatment (see Table 2). Many biopolymers carry some degree of charge and in some cases the charge density is TABLE 2 Properties and Costs of Materials which may be used ih Aqueous Nuclear Waste Treatment Material
Biopolymer
Ion-exchange
Functional property
Yes Yes Yes Yes
No Yes Yes Yes
Water soluble Insoluble Charge and chelation Charge and chelation
Properties Xanthan Chitin Amino phosphonic acid Iminodiacetate costs Chitin Chitosan Modified biopolymer Genetically engineered biopolymer
cost (E) 2000-6900 4000-12 500 64 OOO 5000
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high. In addition most biopolymers carry functional groups which have potential for co-ordination. The virtually infinite range of biopolymers found in nature display a complete range of solubilities. Whole unpurified biopolymers are insoluble and resemble conventional ionexchange resin systems. In addition many biopolymers are highly hydrophilic and permeable which would permit use of insoluble unpurified biopolymers in columns or fluidised beds or as an additive which can be removed by filtration or centrifugation in the treatment of aqueous nuclear waste. The solubility of individual biopolymers may not, however, be a disadvantage in a pure or semi-pure form. Solubility will vary with low to high molecular weights and the different natures of each polymer (and upon the conditions of pH and ion strength that prevail in the fluid undergoing treatment). The addition of a biopolymer solution to an aqueous nuclear waste stream would permit a more intimate mixing and increase the likelihood of interaction of solutes or colloids with target cations. Binding may then produce spontaneous precipitation (often observed with polysaccharide-transition element interactions). Alternatively a change in pH or ionic strength or some other parameter may be used to induce precipitation of the metal biopolymer/complex.
3.2 Opportunities for using biopolymers (1) From the work performed to date there is great potential for the use of biopolymers in aqueous nuclear waste treatment. The commercial/industrial application of biopolymers within ten years is possible but will demand major capital and personal commitment on a large but focused multidisciplinary research and development programme. Biopolymerscannot be considered to be an instant solution or even have immediate practical use in the nuclear industry for use in aqueous waste treatment. From both a technical and commercial point of view much research and development work still needs to be carried out. (2) There is potential in using unmodified biopolymers such as certain proteins, polysaccharides (extracellular bacterial mucilages, etc.), lignin and chitin as generalised radionuclide adsorbing materials. (3) Modified biopolymers in which the polymer is functionalised with a ligand such as an amine could be used to extract dilute radionuclides. (4) There is some potential for the utilisation of the diverse group of molecules called siderophores. The function of these low molecular weight organic compounds is to overcome the problem of obtaining iron from the environment where the element is an inaccessible form, e.g. ferric hydroxide. ( 5 ) The greatest long-term potential for biopolymers (more than 15 years from now, i.e. commissioning in the next century) probably lies with the ‘tailoring’ of biological compounds (genetic and protein engineering) based upon lessons learnt from molecules which have developed through evolution to specifically sequester metals.
3.3 Biosorption For some time it has been observed that aquatic microorganisms have the ability to
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concentrate metal ions from surrounding waters. This cation uptake is often significant and may also be selective. The term biosorption has been used to describe this process. Biosorption may be defined as the sequestering of metal ions by solid materials of biological origin.2 The mechanism of uptake of metal ions may be by means of one or more of the following p r o c e ~ s e s : ~ . ~ (a) Active transport of ions across the cell membrane; (b) Ionexchange processes; (c) Complexation and chelation; (d) Adsorption at the surface; (e) Precipitation (e.g. via hydrolysis of sorbed ions); (f) Particulate entrapment by extracellular organelles or exudates; (g) Particulate ingestion by a pinocytotic-like mechanism. Active transport and particulate ingestion would be carried out by certain types of living cells, while the other processes could be carried out by living or dead biomass or cellular components. Biosorption of heavy metals and radionuclides by live cells would be limited, owing to the accumulated ions engendering toxicity to the microorganisms. This would usually prevent further growth of the microorganisms once the toxic threshold of the metal was reached. Under selection pressure produced by the presence of one or more potentially toxic metal ions adsorptive microorganisms will become less sensitive to such toxic effects. 3.4 Practical biosorption using microorganisms For a practical process designed to remove nuclides from a waste stream it is not sufficient to use microbial suspensions or dispersed, easily lost biomass, with all the attendant pollution problems this would create. The biomass should be in a compact, accessible and recoverable form, probably as a pellet or granule for efficient use in a bioreactor or biocontractor system. 3.4.1 Immobilised microorganisms Immobilisation of biosorptive biomass is an important consideration both from the process and commercial viewpoint. The technology should enable a robust system to be created which does not suffer from problems inherent using very small microbial cells, parts of cells or tangled filaments but is based on uniform granules which can be optimised as to their size and constituency and hence diffusion characteristics.The loss of immobilised biomass will be minimal which will help to reduce cost-maintenance of such a process and remove the need for an organic treatment to clean up radioactive organic debris downstream from a commercial biosorption processer. Immobilised Streptomyces and Chlorella have been studied as to their ability to biosorb ~ r a n i u m . Here, ’ ~ ~ cells were immobilised in polyacrylamide gel beads. They had high adsorptive ability and good mechanical properties. The adsorption of the uranyl ions by the immobilised cells was reportedly not affected by pH values in the range 4 to 9, and thus seemingly independent of pH after immobilisation. This may be due to the buffering capacity of the microenvironment generated within the beads
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by the microorganisms. This may have implications for the treatment of very acid nuclear reprocessing waste streams. It was also observed that the uranyl ions could be very efficiently desorbed from the immobilised cells using a solution of sodium carbonate. Thus such immobilised systems have the potential to allow concentration of dilute nuclides from aqueous waste streams via biosorption and subsequent desorption and the immobilised cells may be reused. The extent of reusability is very important for a system such as this and greater efforts should be made in developing a cycling process optimised for adsorption and desorption of the biosorbent materials. There are many different techniques for the immobilisation of microbial cells.' Work on the stability and physiology of immobilised organisms has also been performed.8-' The diffusion characteristics of immobilised cells in gel beads has important implications for biosorptive processes. The properties of the bead, the biosorbent, the radionuclide ion and the desorbing ion must all be considered when planning a commercial-scale process. Molecular diffusion characteristics of such immobilised systems have been described quantitatively.' 3.5 Disposal
Although fungal biomass offers the potential to act as an efficient biosorbent of radionuclides contained in process waste streams, the nuclear industry would not want to be troubled with the disposal of large quantities of biodegradable and radioactive biomass. Any biosorptive system would have to be capable of the adsorbed ions being efficiently desorbed and the regenerated biosorbent reused many times. To this end a number of workers have demonstrated that adsorbed nuclides can be removed from biosorbents using a variety of treatments with chelating agents such as EDTA (ethylenediaminetetra-acetic acid) or sodium/and or ammonium carbonate solutions (notably for uranium cation removal). Thus there is the basis for the setting up a biosorbent recycling process where nuclides may be removed from waste streams, recovered and the biosorbent used to treat further efluent.'2-' The benefits from a cycling process where the adsorbent biomass can be used many times are: 0 0
0 0
Production of a concentrated eluate. Small volume of concentrated nuclide solution produced, facilitates disposal via vitrification or being put into concrete. No organic residues. Minimisation of biomass volume for ultimate disposal.
The spent biomass generated after the optimum cycling events have been achieved could be easily sterilised (such as by microwave irradiation) and suitably enrobed in inert material for efficient final disposal. Sterilising the biomass should prevent decay and therefore leakage from the containment vessel. Some examples of radionuclide removal by microorganisms are given in Table 3. 3.6 Biosorption process system
The biosorbent could be prepared as a pelleted or particulate bead of an alga or
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TABLE 3 Comparable Uranium Recovery by Microbial Cells or Microbial Cell Products Organism Rhizopus arrhizus (f) Acinetobacter RAG (b) Penicillium digitatum (f) Pseudomonas aeruginosa (b) Saccharomyces cereviseae (y) Zoogloea ramigera (b) Streptomyces viride chromogenes (b) Chlorella regularis (a) Citrobacter sp. (b)
Method of U uptake
Adsorption to cell wall Binding to extracellular polymer Adsorption to cell wall Intracellular Adsorption to wall Binding to extracellular polysaccharide Adsorption to cell wall Adsorption to cell wall Binding to cell surface via enzyme action
Accumulation Reference (mg-' g dry wt)
180
800
17 18
150 150 500-2500
19 20 20 21,22
312
5
I59 9000
23, 25, 26
5.7
5
a = Alga, b = bacterium, f =fungus (filamentous), y = yeast.
filamentous fungus or bacterium depending on the eMuent type involved. A continuous upflow reactor acting as a fluidised bed is one approach that has been described (Fig. 1). Here, the influent waste stream enters at the base of the biosorption reactor and the outflow from the biosorption reactor leaves at the top and would be recycled with more inflowing effluent or passed to a second-stage bioreactor. Nuclide-laden biosorbent would be removed from the system automatically and desorbed via a separate cycle. The concentrated nuclide solution produced at this stage would be transferred to the usual long-term storage tank. Fresh biosorbent would be added to the system on a balanced basis and the whole process would act to produce a cleaned up final aqueous effluent and a concentrated radionuclidecontaining product which could be stored or treated further. Similar systems have been considered6**'and there is a commercial system available from Advanced Mineral Technologies (Golden, Colorado), the AMT Bioclaim biosorbent system, for heavy metal removal from effluent streams.
4 NOVEL BIOTECHNOLOGY APPROACHES TO AQUEOUS NUCLEAR EFFLUENT TREATMENT
4.1 Biomagnetic separation There is potential for the use of microorganisms to actively (and passively) separate radioactive metal ions from dilute streams. It has also been shown that microorganisms can be made to take up metal ions from solution and can acquire significant magnetic moments which allow them to be captured using the technique of high gradient magnetic separation (HGMS) (Watson, J . H. P. &
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FOR RECYCLE ADSORPTION
-
CONTAMINATED LIQUID IN
DESORPTION -EXPENDED
BIOSORBENT
Fig. 1. Continuous countercurrent contactor for removal of heavy metals.
Ellwood, D.C., 1987, pers. comm.). Thus there appears to be potential in synergistically combining the use of microorganisms (bacteria) in conjunction with HGMS to separate radioactive metal ions from dilute waste streams. 4.2 Monoclonal antibody technology Monoclonal antibodies specific to cell types used in a biomagnetic separation process could be used to aid the selective recovery of specific radionuclides. For example, two nuclides, 1 and 2, respectively, may be adsorbed by organisms A and B, giving 1A and 2B complexes. If one has immobilised monoclonal antibodies to organism 2 and exposes the mixture to such a treatment then the eluate would be made up entirely of 1A with 2B retained. Complex 1A could then be concentrated by a magnetic field and 2B eluted separately, thus giving two separate and concentrated nuclide ‘pools’ from the original mixture.
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4.3 Selection of strongly biosorbent microorganisms Thus the use of relatively simple microbiological selection techniques such as exposure to sequentially increasing levels of radionuclides or continuous fermentation culture can be employed to select for highly biosorptive strains of bacteria, yeasts, fungi and algae. 4.4 Biomolecular engineering Various microbial and other polymers plus whole microorganisms and enzyme systems having the capacity for radionuclide biosorption or aiding such biosorption via augmentation of other metal accumulation processes could be modified to give inproved performance by genetic engineering and protein engineering techniques. 4.4.1 Genetic engineering of proteins Metallothioneins, siderophores and some enzymes have been mentioned earlier as having cation affinity or the ability to remove cations, including radionuclides from solution. Such proteins could be produced in quantity using DNA cloning and expression techniques. The DNA coding for the protein is isolated from the genome of a microorganism which produces it. This DNA is placed directly into a selfreplicating plasmid vector and transferred into another microorganism to yield many tens or hundreds of copies of the recombinant plasmid. A chemical trigger or a temperature shift could then be used to stimulate the production of the desired protein. The desired protein could be extracted from microbial cells, or the organism containing the high levels of protein could be used directly.’ 4.4.2 Protein engineering Nuclear waste streams, as described earlier, may be hostile to life processes, regarding pH. However, there are numerous organisms that live in hot, acid springs. Their proteins are resistant to strong acids even at temperatures up to 80°C plus. A knowledge of their protein structure should enable design changes in pre-existing useful biosorptive proteins which do not possess these useful acid and/or thermal resistance characteristics. Based on a knowledge or amino acid sequence and protein folding of acid-resistant proteins, subtle changes can be induced in proteins to try to instil some characteristics of greater acid resistance or thermal stability. This process is achieved by isolating the cloned DNA of the protein in question. The structural sequence of nucleotide bases would be ascertained and hence the amino acid sequence can be worked out. This, together with information on the way the protein folds up (coils on itself to produce a complex three-dimensional structure which enables it to perform its biological function) enables protein engineering to be performed. Pieces of DNA similar to that coding for the protein in question, but which are slightly different, are placed into the DNA fragment as a substitute for the natural corresponding piece. Thus when the protein is produced from this DNA there will be a change in one (or more depending on the changes to the DNA) amino acid. The protein will then be examined as to changes in properties engendered by the changes in structure.
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Protein engineering is rapidly becoming a sophisticated branch of molecular biology and enables proteins with novel characteristics to be constructed. Indeed, such proteins could be ‘designer proteins’ with tailor-made, specified, properties. Thus the potential is there for a significant impact to be made in radionuclide adsorption by suitably modified radionuclide adsorptive proteins, although the time-scales for realisation of this may be quite long (1&15 years). 4.4.3 Genetic engineering of whole organisms Essentially this is very similar to the process described in Section 4.4.1, except the objective is not to isolate the protein but to transfer its activity and hence a new characteristic to the host organism. Thus, since metal accumulation has been shown to be under genetic control, the genetic information controlling this function could be transferred to another organism to render the second organism of greater use in biosorptive process. For example, if a characteristic is to be transferred from a higher organism, such as a flowering plant or animal, the RNA molecule (produced from DNA) that codes for the protein molecule required is isolated (messenger RNA) and converted into a DNA copy, called cDNA. This is placed into a suitable plasmid vector. As described earlier, DNA from a microorganism (bacterium) can be used directly for cloning purposes. The plasmid could be introduced into a suitable filamentous fungus which already has a noted ability to accumulate radionuclides. When cultured with the cloned gene and activated, an augmentation of the biosorptive function might ensue. This does not confine itself to just producing a metal accumulating protein. The cloned protein may improve a naturally occurring system, e.g. such that more cell wall biopolymer (such as chitin, a noted radionuclide adsorbent) could be synthesised. Thus augmentation of characteristics already present as well as the transfer of novel ones are potential approaches to the area of radionuclide biosorption. Indeed, even enabling metal accumulating biomass to be grown on a cheaper carbon source may be a cost-effective application of genetic engineering. Since the biomass would be killed by heat and/or chemical treatment prior to use, genetic modification would pose no threat with respect to ‘release’ of modified genetic material to the environment. The effort versus the risk of development for these various biotechnology applications are summarised in Fig. 2.
5 CONCLUSIONS There are a number of opportunities for the various aspects of biotechnology considered during this project for the removal of radionuclides from aqueous nuclear waste streams. 5.1 Biopolymers
Although there is apparent potential for the use of biopolymers per se for radionuclide accumulating systems it would appear that in general the probable time-scale for this industrial application is around 5-1 0 years hence. The
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High Intensity of Effort
Monoclonal antibody technology Genetic Engineering
Biomaanetic Separation LOW
I
LOW
Risk of Project Failure
High
Fig. 2. Effort versus risk of development.
biopolymers appear to offer few short-term applications benefits except perhaps for lignin, chitin and modified cellulosics. The long-term potential for biopolymers (into the next century) probably lies with the ‘tailoring’ of properties by the production of ‘designer’ molecules generated by application of genetic and protein engineering, e.g. for metallothioneins, and, siderophores. Chemically modified biopolymers, where a polymer such as lignin or chitin has a speciality radionuclide binding group chemically added could also find applications in the short term. 5.2 Biosorptive biomass
Biosorption offers a potentially eficient route to the removal of radionuclides from aqueous nuclear waste streams. Selected fungi, bacteria, yeasts and algae can all function as effective biosorbents and can be used as immobilised systems for adsorption of radionuclides followed by desorption and thus form part of a cyclic process. The major opportunities in the short term lie with dead biomass but there could be potential in the longer term for more active/living systems based on extra cellular polysaccharide production or enzyme p r o d ~ c t i o n . ~ ~ . ’ ~ There are some commercial applications of biosorption available at present and it is envisaged that cost-effective, efficient systems could be designed specifically for dilute nuclide streams within the five-year time horizon. 5.3 New applications and potential applications of biotechnology
Biomagnetic separation of radionuclides adsorbed to microorganisms appears to offer potential for the removal, concentration and separation of radionuclides in waste streams. With sufficient support it is envisaged that a pilot-scale process based on this technique could be available within a five-year time-scale. The use of immobilised monoclonal antibodies coupled with a selective microbial radionuclide adsorption could also be applied and achieve essentially similar separations without reliance on magnetic field-generating hardware.
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Genetic engineering is a rapidly expanding area of biotechnology and offers the potential to modify microorganisms such that highly efficient radionuclide bioaccumulation systems could be developed from preexisting organisms and biopol ymers.
REFERENCES 1. Hooper, E. W., Seminar on long term research into liquid emuent treatment. Harwell report 12338, AERE R12338 unclassified, Department of the Environment, 1987. 2. Volesky, B., Biosorbent materials. Biotechnol. Bioeng. Syrnp., 16 John Wiley and Sons Inc., New York, 1986. 3. Andleman, J. B., Trace Metals and Metalkorganic Interactions in Natural Waters, ed. P. C. Singer. Ann Arbor Science, Ann Arbor, MI, USA, 1973, pp. 57-8. 4. Shumate, S. E., Strandberg, G. W. & Parrott, J. R., Biotech. Bioeng. Symp., 8, John Wiley and Sons Inc., New York, 1978, pp. 13-20. 5. Najakima, A., Horikoshi, T. & Sakaguchi, T., Recovery of uranium by immobilized microorganisms. Eur. J . Appl. Microbiol. Biotechnol., 16 (1982) 88-91. 6. Darnall, D. W., Greene, B., Hosea, M., McPerson, R. A., Henzl, M. & Alexander, M. D., Recovery of heavy metals by immobilized algae. In Trace Metal Removal from Aqueous Solution, Proceedings of the Royal Society of Chemistry, ed. R. Thompson, University of Warwick, UK, 9-10 April 1986, Special Publication No. 61, pp. 1-24. 7. Robinson, P. K., Mak, A. L. & Trevan, M. D., Immobilized algae: A review. Process Biochem., 21 (4) (1986) 122-7. 8. Dainty, A. L., Goulding, K. H., Robinson, P. K., Simpkins, I. & Trevan, M. D., Stability of alginate immobilized algal cells. Biotechnol. Bioeng., 28 (1986) 21&16. 9. Robinson, P. K., Dainty, A. L., Goulding, K. H., Simpkins, I. & Trevan, M. D., Physiology of alginate immobilized Chlorella. Enzyme Microbial Technol., 7 (1986) 21 2-1 6. 10. Robinson, P. K., Goulding, K. H., Mak, A. L. & Trevan, M. D., Factors affecting the growth characteristics of alginate-entrapped Chlorella. Enzyme. Microbial. Technol., 8 (1986) 729-33. 1 1 . Tanaka, H., Matsumura, M. & Veliky, I. A., Diffusion characteristics of substrates in Ca-alginate gel beads. Biotechnol. Bioeng., 26 (1984) 53-8. 12. Galun, M., Keller, P., Malki, D., Feldstein, H., Galun, E., Siegel, S. & Siegel, B., Removal of uranium(V1) from solution by fungal biomass and fungal wall related biopolymers. Science, 219 (1982) 285-6. 13. Galun, M., Keller, P., Feldstein, H., Galun, E., Siegel, S. & Siegel, B., Recovery of uranium(1V) from solution using fungi 11. Release from uranium loaded Penicillium biomass. Water, Air and Soil Pollution, 20 (1983) 277-85. 14. Galun, M., Keller, P., Malki, D., Feldstein, H., Galun, E., Siegel, S. & Siegel, B., Recovery of uranium(V1) from solution using precultured Penicillium biomass. Water, Air and Soil Pollution, 20 (1983) 221-32. 15. Tsezos, M., Recovery of uranium from biological adsorgents. Desorption equilibrium. Biotechnol. Bioeng., 26 (1984) 873-81. 16. Tsezos, M., Baird, M. H. I. & Shemilt, L. W., The elution of radium adsorbed by microbial biomass. Chem. Eng. J., 34 (1987) B57-B64. 17. Tsezos, M. & Volesky, B., Biosorption of uranium and thorium. Biotechnol. Bioeng,, 23 (1981) 583-604. 18. Zosim, Z., Gutnick, D. & Rosenberg, E., Uranium binding by emulsan and emulsanols. Biotechnol. Bioeng., 25 (1983) 1725-35. 19. Galun, M., Keller, P., Feldstein, H., Galun, E., Siegel, S. & Siegel, B., Removal of uranium(V1) from solution by fungal biomass: Inhibition by iron. Water, Air and Soil Pollution, 21 (1984) 411-14.
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20. Strandberg, G. W., Shumate, S. E. & Parrott, J. R., Microbial cells as biosorbents for heavy metals. Accumulation of uranium by Saccharomyces cereoiseae and Pseudomonas aeruginosa. Appl. Environ. Microbiol., 41 (1981) 237-45. 21. Strandberg, G. W. & Shumate, S. E., Accumulation of uranium, cesium and radium by microbial cells- bench-scale studies. USA Dept of Energy Contract W-7405-eng-26, Washington, 1982. 22. Norberg, A. B. & Person, H., Accumulation of heavy metal ions by Zoogloea ramigera. Biotechnol. Bioeng., 26 (1984) 23946. 23. Macaskie, L. E. & Dean, A. C. R., Uranium accumulation by immobilized cells of a Citrobacter sp. Biotechnol. Letters, 7(7) (1985) 457-62. 24. Chakrabarty, A. M., Genetic mechanisms in metal-microbe interaction. In Metallurgical Applications of Bacterial Leaching and Related Microbiological Phenomena, ed. L. E. Murr, A. E. Torma & J. A. Brierley. Academic Press, NY, 1978, pp. 137-49. 25. Macaski, L. E. & Dean, A. C. R., Uranium accumulation by a Citrobacter sp. immobilized as a biofilm on various support materials. In Proc. 4th European Congress in Biotechnology, Vol. 2, ed. 0. M. Neijssel, R. K. van der Moer & K. C. A. M. Luyben. Elsevier Science, Amsterdam, 1987, pp. 37-40. 26. Macaskie, L. E., Watts, J. M. & Dean, A. C. R., Cadmium accumulation by a Citrobacter sp. immobilized on gel and solid supports: Applicability to the treatment of liquid wastes containing heavy metal cations. Biotechnol. Bioeng., 29 (1987) (in press).
Review of Biotechnology Applications to Nuclear Waste Treatment* Nicholas V. Ashley & Daniel J. W. Roach PA Consulting Group-Technology, Cambridge Laboratory, Melbourn, Royston, Hertfordshire SG8 6DP. UK (Received 29 September 1989; accepted 21 December 1989)
ABSTRACT This paper gives an overview of the feasibility of the application of biotechnology to nuclear waste treatment. The contents are based on a report which PA Technology carried out for the Department of the Environment (DOE Reference: DoEIR W/88.008 Sector N o 2.3). Many living and dead organisms accumulate heavy metals and radionuclides. The controlled use of this phenomenon forms the basis for the application of biotechnology to the removal of radionuclides ffom nuclear waste streams. Indeed, biotechnology offers a series of new opportunities for removal of radionuclides f f o m dilute aqueous process effluents. Such technology is already used for heavy metal removal on a commercial basis and could be optimised for radionuclide removal. An overview of biotechnology areas, namely the use of biopolymers and biosorption using biomass applicable to the removal of radionuclides ffom industrial nuclear effluents is given. The potential of biomagnetic separation technology, genetic engineering and monoclonal antibody technology is also to be examined. The most appropriate technologies to develop for radionuclide removal in the short term appear to be those based on biosorption of radionuclides by biomass and the use of modified and unmodified biopolymers in the medium term. Key words: biotechnology, nuclear waste, waste treatment, biosorption, biopolymers.
* Paper presented at the meeting ‘Recovery/Removalof Metals by Biosorption-A Commercial Reality or a Scientist’s Dream?’, organised by the Solvent Extraction and Ion Exchange Group of the Society of Chemical Industry and held in London on 18 May 1989. 381
J . Chem. Tech. Biotechnol. 0268-2575/90/$03.50 0 1990 SCI. Printed in Great Britain
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I INTRODUCI'ION The cocktail of metals in nuclear wastes is a function both of the fuel and of its containment materials; its processing presents special problems even though much of the chemistry is of a character familiar in other areas of industrial chemometallurgy . The nuclear industry is required to ensure that discharges of radioisotopes and other hazardous chemicals are minimised at the point of discharge. The present practice is to treat aqueous nuclear waste streams by means of large-scale inorganic ion-exchange or chelation sometimes coupled with flocculation and ultra-filtration. Low-volume, highly active aqueous wastes are stored in special tanks pending vitrification. Inorganic material bioprocessing is now receiving attention from industries where microorganisms and microbially based processes are increasingly perceived as potential lowenergy routes to recovering metals from low-grade ores, upgrading of ore waste and fossil fuels prior to conventional processing and combustion, and toxic and/or precious metal recovery from waste streams. Investigations into the range and potential for commercial microbial transformations of heavy elements are just beginning. Heavy metal uptake by microorganisms (and many higher organisms also) and bioproducts derived from them is a well-recognised phenomenon. There is a considerable body of research devoted to the use of biologically based systems for the accumulation of heavy, often precious, metals from a variety of aqueous waste sources. Part of this is concerned with the accumulation of radionuclides.
2 RADIONUCLIDE CONCENTRATIONS IN NUCLEAR INDUSTRY WASTE STREAMS Data were supplied by the DOEand from published reports on the major inactive cations, anions and organics in aqueous waste streams and significant active radionuclides and total element concentrations for a number of waste streams, for example, low active waste streams (Table 1). The data provided by the DOE and obtained from Harwell Report 12338 contained information as to the non-radioisotopic composition of some waste streams.' Such data are important because the concentration of some metal ions may be far higher than that shown if only the radioisotopes are considered. The total metal concentration is an important piece of data, for it should enable an estimate of the efficiency of a biotechnology based metal recovery system to be estimated.
2.1 Isotopic concentrations: A summary High- and medium-level activity waste streams: -mg to tens of mg dm-3
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TABLE 1 BNFL Reference Stream Three" ~~~
Concentration (g d m - j )
~
Activity (Ci d m - j )
Major inactive species
Fe Na K Ca U
015 04 003 001 001 001 003 003 17
Ms
TBP Kerosene NO;
so: -
05
CI
0007
Significant active species
Ru-106 Ce-144 Sr-90 Zr-95 Nb-95 CS-137 Tc-99 Pu alpha Am-241 Np-237 CO-60 Pu-241 (3-134
3.2 x 10-9 3.1 x 1O-I' 8.3 x 10-9 1.ox 10-10 2.3 x lo-'' 4.1 x lo-' 2.9 x 10-5 1.4~ 4.8 x lo-' 8.5 x
3.9 x 10-10 6 . 4 10-7 ~ 6.3 x lo-''
1.1 x 10-5 1.0 x 1.2x 10-6 2.2x 10-6 8.8 x 3.6 x s.ox 10-7 1.7 x loW6 1.7 x 6 . 0 lo-' ~ 4.3 x 10-7 6 4 10-5 ~ 8.4 x
Concentrations of total elements
Ru Ce Sr Zr Nb cs Tc PU Am NP co
1.2 x 10-7 7.ox 10-9 1.5 x 10-8 8.2 x lo-' 2.3 x lo-'' 1.2 x 10-7 3.ox 10-5 1.4 x 10-5 5.3 x 10-8 8.5 x 10-5 3.9 x 10-10
Source: DOE. *Volume, lo00 m3 dm-j; acidity, approximately 0.3 mol dm-3 as HNO,. Low-level activity waste streams: -vary depending on isotope; for example
95Zr 1 . 0 lo-'' ~ g dm-j 237Np8-5 x lo-' g dm-3 Uranium (mainly 238) 1 x
g dmP3
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Storage pond emuents: -vary depending on isotope; for example 13'Cs 1.8 x lo-" g dm-3 235U1 x g dmP3 Thus the concentrations of nuclides vary depending on the nature of the waste stream. The final concentration of the non-transuranic elements may be considerably higher than the radioisotope fraction of each element. The total metal ion concentration is a requirement which is necessary to judge which of the biotechnology approaches would be most effectiveand suitable for treating aqueous nuclear waste streams. This is not intended to be an exhaustive review of all possible nuclear waste streams but serves as an indicator as to the different varieties of radionuclides, other metal ions and their concentrations that may be found in typical reprocessing streams.
3 BIOTECHNOLOGY APPROACHES AND AQUEOUS RADIONUCLIDE EFFLUENT TREATMENT
The main biotechnologically based areas that will have an impact on aqueous nuclear emuent treatment are the use of biopolymers and biosorbants. Novel biotechnology-based opportunities are also described. These comprise biomolecular engineering (protein and genetic engineering), the use of monoclonal antibodies and biomagnetic separation. 3.1 Biopolymers
Many biopolymers display properties which make them very attractive as candidates for use in aqueous nuclear waste treatment (see Table 2). Many biopolymers carry some degree of charge and in some cases the charge density is TABLE 2 Properties and Costs of Materials which may be used ih Aqueous Nuclear Waste Treatment Material
Biopolymer
Ion-exchange
Functional property
Yes Yes Yes Yes
No Yes Yes Yes
Water soluble Insoluble Charge and chelation Charge and chelation
Properties Xanthan Chitin Amino phosphonic acid Iminodiacetate costs Chitin Chitosan Modified biopolymer Genetically engineered biopolymer
cost (E) 2000-6900 4000-12 500 64 OOO 5000
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high. In addition most biopolymers carry functional groups which have potential for co-ordination. The virtually infinite range of biopolymers found in nature display a complete range of solubilities. Whole unpurified biopolymers are insoluble and resemble conventional ionexchange resin systems. In addition many biopolymers are highly hydrophilic and permeable which would permit use of insoluble unpurified biopolymers in columns or fluidised beds or as an additive which can be removed by filtration or centrifugation in the treatment of aqueous nuclear waste. The solubility of individual biopolymers may not, however, be a disadvantage in a pure or semi-pure form. Solubility will vary with low to high molecular weights and the different natures of each polymer (and upon the conditions of pH and ion strength that prevail in the fluid undergoing treatment). The addition of a biopolymer solution to an aqueous nuclear waste stream would permit a more intimate mixing and increase the likelihood of interaction of solutes or colloids with target cations. Binding may then produce spontaneous precipitation (often observed with polysaccharide-transition element interactions). Alternatively a change in pH or ionic strength or some other parameter may be used to induce precipitation of the metal biopolymer/complex.
3.2 Opportunities for using biopolymers (1) From the work performed to date there is great potential for the use of biopolymers in aqueous nuclear waste treatment. The commercial/industrial application of biopolymers within ten years is possible but will demand major capital and personal commitment on a large but focused multidisciplinary research and development programme. Biopolymerscannot be considered to be an instant solution or even have immediate practical use in the nuclear industry for use in aqueous waste treatment. From both a technical and commercial point of view much research and development work still needs to be carried out. (2) There is potential in using unmodified biopolymers such as certain proteins, polysaccharides (extracellular bacterial mucilages, etc.), lignin and chitin as generalised radionuclide adsorbing materials. (3) Modified biopolymers in which the polymer is functionalised with a ligand such as an amine could be used to extract dilute radionuclides. (4) There is some potential for the utilisation of the diverse group of molecules called siderophores. The function of these low molecular weight organic compounds is to overcome the problem of obtaining iron from the environment where the element is an inaccessible form, e.g. ferric hydroxide. ( 5 ) The greatest long-term potential for biopolymers (more than 15 years from now, i.e. commissioning in the next century) probably lies with the ‘tailoring’ of biological compounds (genetic and protein engineering) based upon lessons learnt from molecules which have developed through evolution to specifically sequester metals.
3.3 Biosorption For some time it has been observed that aquatic microorganisms have the ability to
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concentrate metal ions from surrounding waters. This cation uptake is often significant and may also be selective. The term biosorption has been used to describe this process. Biosorption may be defined as the sequestering of metal ions by solid materials of biological origin.2 The mechanism of uptake of metal ions may be by means of one or more of the following p r o c e ~ s e s : ~ . ~ (a) Active transport of ions across the cell membrane; (b) Ionexchange processes; (c) Complexation and chelation; (d) Adsorption at the surface; (e) Precipitation (e.g. via hydrolysis of sorbed ions); (f) Particulate entrapment by extracellular organelles or exudates; (g) Particulate ingestion by a pinocytotic-like mechanism. Active transport and particulate ingestion would be carried out by certain types of living cells, while the other processes could be carried out by living or dead biomass or cellular components. Biosorption of heavy metals and radionuclides by live cells would be limited, owing to the accumulated ions engendering toxicity to the microorganisms. This would usually prevent further growth of the microorganisms once the toxic threshold of the metal was reached. Under selection pressure produced by the presence of one or more potentially toxic metal ions adsorptive microorganisms will become less sensitive to such toxic effects. 3.4 Practical biosorption using microorganisms For a practical process designed to remove nuclides from a waste stream it is not sufficient to use microbial suspensions or dispersed, easily lost biomass, with all the attendant pollution problems this would create. The biomass should be in a compact, accessible and recoverable form, probably as a pellet or granule for efficient use in a bioreactor or biocontractor system. 3.4.1 Immobilised microorganisms Immobilisation of biosorptive biomass is an important consideration both from the process and commercial viewpoint. The technology should enable a robust system to be created which does not suffer from problems inherent using very small microbial cells, parts of cells or tangled filaments but is based on uniform granules which can be optimised as to their size and constituency and hence diffusion characteristics.The loss of immobilised biomass will be minimal which will help to reduce cost-maintenance of such a process and remove the need for an organic treatment to clean up radioactive organic debris downstream from a commercial biosorption processer. Immobilised Streptomyces and Chlorella have been studied as to their ability to biosorb ~ r a n i u m . Here, ’ ~ ~ cells were immobilised in polyacrylamide gel beads. They had high adsorptive ability and good mechanical properties. The adsorption of the uranyl ions by the immobilised cells was reportedly not affected by pH values in the range 4 to 9, and thus seemingly independent of pH after immobilisation. This may be due to the buffering capacity of the microenvironment generated within the beads
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by the microorganisms. This may have implications for the treatment of very acid nuclear reprocessing waste streams. It was also observed that the uranyl ions could be very efficiently desorbed from the immobilised cells using a solution of sodium carbonate. Thus such immobilised systems have the potential to allow concentration of dilute nuclides from aqueous waste streams via biosorption and subsequent desorption and the immobilised cells may be reused. The extent of reusability is very important for a system such as this and greater efforts should be made in developing a cycling process optimised for adsorption and desorption of the biosorbent materials. There are many different techniques for the immobilisation of microbial cells.' Work on the stability and physiology of immobilised organisms has also been performed.8-' The diffusion characteristics of immobilised cells in gel beads has important implications for biosorptive processes. The properties of the bead, the biosorbent, the radionuclide ion and the desorbing ion must all be considered when planning a commercial-scale process. Molecular diffusion characteristics of such immobilised systems have been described quantitatively.' 3.5 Disposal
Although fungal biomass offers the potential to act as an efficient biosorbent of radionuclides contained in process waste streams, the nuclear industry would not want to be troubled with the disposal of large quantities of biodegradable and radioactive biomass. Any biosorptive system would have to be capable of the adsorbed ions being efficiently desorbed and the regenerated biosorbent reused many times. To this end a number of workers have demonstrated that adsorbed nuclides can be removed from biosorbents using a variety of treatments with chelating agents such as EDTA (ethylenediaminetetra-acetic acid) or sodium/and or ammonium carbonate solutions (notably for uranium cation removal). Thus there is the basis for the setting up a biosorbent recycling process where nuclides may be removed from waste streams, recovered and the biosorbent used to treat further efluent.'2-' The benefits from a cycling process where the adsorbent biomass can be used many times are: 0 0
0 0
Production of a concentrated eluate. Small volume of concentrated nuclide solution produced, facilitates disposal via vitrification or being put into concrete. No organic residues. Minimisation of biomass volume for ultimate disposal.
The spent biomass generated after the optimum cycling events have been achieved could be easily sterilised (such as by microwave irradiation) and suitably enrobed in inert material for efficient final disposal. Sterilising the biomass should prevent decay and therefore leakage from the containment vessel. Some examples of radionuclide removal by microorganisms are given in Table 3. 3.6 Biosorption process system
The biosorbent could be prepared as a pelleted or particulate bead of an alga or
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TABLE 3 Comparable Uranium Recovery by Microbial Cells or Microbial Cell Products Organism Rhizopus arrhizus (f) Acinetobacter RAG (b) Penicillium digitatum (f) Pseudomonas aeruginosa (b) Saccharomyces cereviseae (y) Zoogloea ramigera (b) Streptomyces viride chromogenes (b) Chlorella regularis (a) Citrobacter sp. (b)
Method of U uptake
Adsorption to cell wall Binding to extracellular polymer Adsorption to cell wall Intracellular Adsorption to wall Binding to extracellular polysaccharide Adsorption to cell wall Adsorption to cell wall Binding to cell surface via enzyme action
Accumulation Reference (mg-' g dry wt)
180
800
17 18
150 150 500-2500
19 20 20 21,22
312
5
I59 9000
23, 25, 26
5.7
5
a = Alga, b = bacterium, f =fungus (filamentous), y = yeast.
filamentous fungus or bacterium depending on the eMuent type involved. A continuous upflow reactor acting as a fluidised bed is one approach that has been described (Fig. 1). Here, the influent waste stream enters at the base of the biosorption reactor and the outflow from the biosorption reactor leaves at the top and would be recycled with more inflowing effluent or passed to a second-stage bioreactor. Nuclide-laden biosorbent would be removed from the system automatically and desorbed via a separate cycle. The concentrated nuclide solution produced at this stage would be transferred to the usual long-term storage tank. Fresh biosorbent would be added to the system on a balanced basis and the whole process would act to produce a cleaned up final aqueous effluent and a concentrated radionuclidecontaining product which could be stored or treated further. Similar systems have been considered6**'and there is a commercial system available from Advanced Mineral Technologies (Golden, Colorado), the AMT Bioclaim biosorbent system, for heavy metal removal from effluent streams.
4 NOVEL BIOTECHNOLOGY APPROACHES TO AQUEOUS NUCLEAR EFFLUENT TREATMENT
4.1 Biomagnetic separation There is potential for the use of microorganisms to actively (and passively) separate radioactive metal ions from dilute streams. It has also been shown that microorganisms can be made to take up metal ions from solution and can acquire significant magnetic moments which allow them to be captured using the technique of high gradient magnetic separation (HGMS) (Watson, J . H. P. &
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FOR RECYCLE ADSORPTION
-
CONTAMINATED LIQUID IN
DESORPTION -EXPENDED
BIOSORBENT
Fig. 1. Continuous countercurrent contactor for removal of heavy metals.
Ellwood, D.C., 1987, pers. comm.). Thus there appears to be potential in synergistically combining the use of microorganisms (bacteria) in conjunction with HGMS to separate radioactive metal ions from dilute waste streams. 4.2 Monoclonal antibody technology Monoclonal antibodies specific to cell types used in a biomagnetic separation process could be used to aid the selective recovery of specific radionuclides. For example, two nuclides, 1 and 2, respectively, may be adsorbed by organisms A and B, giving 1A and 2B complexes. If one has immobilised monoclonal antibodies to organism 2 and exposes the mixture to such a treatment then the eluate would be made up entirely of 1A with 2B retained. Complex 1A could then be concentrated by a magnetic field and 2B eluted separately, thus giving two separate and concentrated nuclide ‘pools’ from the original mixture.
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4.3 Selection of strongly biosorbent microorganisms Thus the use of relatively simple microbiological selection techniques such as exposure to sequentially increasing levels of radionuclides or continuous fermentation culture can be employed to select for highly biosorptive strains of bacteria, yeasts, fungi and algae. 4.4 Biomolecular engineering Various microbial and other polymers plus whole microorganisms and enzyme systems having the capacity for radionuclide biosorption or aiding such biosorption via augmentation of other metal accumulation processes could be modified to give inproved performance by genetic engineering and protein engineering techniques. 4.4.1 Genetic engineering of proteins Metallothioneins, siderophores and some enzymes have been mentioned earlier as having cation affinity or the ability to remove cations, including radionuclides from solution. Such proteins could be produced in quantity using DNA cloning and expression techniques. The DNA coding for the protein is isolated from the genome of a microorganism which produces it. This DNA is placed directly into a selfreplicating plasmid vector and transferred into another microorganism to yield many tens or hundreds of copies of the recombinant plasmid. A chemical trigger or a temperature shift could then be used to stimulate the production of the desired protein. The desired protein could be extracted from microbial cells, or the organism containing the high levels of protein could be used directly.’ 4.4.2 Protein engineering Nuclear waste streams, as described earlier, may be hostile to life processes, regarding pH. However, there are numerous organisms that live in hot, acid springs. Their proteins are resistant to strong acids even at temperatures up to 80°C plus. A knowledge of their protein structure should enable design changes in pre-existing useful biosorptive proteins which do not possess these useful acid and/or thermal resistance characteristics. Based on a knowledge or amino acid sequence and protein folding of acid-resistant proteins, subtle changes can be induced in proteins to try to instil some characteristics of greater acid resistance or thermal stability. This process is achieved by isolating the cloned DNA of the protein in question. The structural sequence of nucleotide bases would be ascertained and hence the amino acid sequence can be worked out. This, together with information on the way the protein folds up (coils on itself to produce a complex three-dimensional structure which enables it to perform its biological function) enables protein engineering to be performed. Pieces of DNA similar to that coding for the protein in question, but which are slightly different, are placed into the DNA fragment as a substitute for the natural corresponding piece. Thus when the protein is produced from this DNA there will be a change in one (or more depending on the changes to the DNA) amino acid. The protein will then be examined as to changes in properties engendered by the changes in structure.
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Protein engineering is rapidly becoming a sophisticated branch of molecular biology and enables proteins with novel characteristics to be constructed. Indeed, such proteins could be ‘designer proteins’ with tailor-made, specified, properties. Thus the potential is there for a significant impact to be made in radionuclide adsorption by suitably modified radionuclide adsorptive proteins, although the time-scales for realisation of this may be quite long (1&15 years). 4.4.3 Genetic engineering of whole organisms Essentially this is very similar to the process described in Section 4.4.1, except the objective is not to isolate the protein but to transfer its activity and hence a new characteristic to the host organism. Thus, since metal accumulation has been shown to be under genetic control, the genetic information controlling this function could be transferred to another organism to render the second organism of greater use in biosorptive process. For example, if a characteristic is to be transferred from a higher organism, such as a flowering plant or animal, the RNA molecule (produced from DNA) that codes for the protein molecule required is isolated (messenger RNA) and converted into a DNA copy, called cDNA. This is placed into a suitable plasmid vector. As described earlier, DNA from a microorganism (bacterium) can be used directly for cloning purposes. The plasmid could be introduced into a suitable filamentous fungus which already has a noted ability to accumulate radionuclides. When cultured with the cloned gene and activated, an augmentation of the biosorptive function might ensue. This does not confine itself to just producing a metal accumulating protein. The cloned protein may improve a naturally occurring system, e.g. such that more cell wall biopolymer (such as chitin, a noted radionuclide adsorbent) could be synthesised. Thus augmentation of characteristics already present as well as the transfer of novel ones are potential approaches to the area of radionuclide biosorption. Indeed, even enabling metal accumulating biomass to be grown on a cheaper carbon source may be a cost-effective application of genetic engineering. Since the biomass would be killed by heat and/or chemical treatment prior to use, genetic modification would pose no threat with respect to ‘release’ of modified genetic material to the environment. The effort versus the risk of development for these various biotechnology applications are summarised in Fig. 2.
5 CONCLUSIONS There are a number of opportunities for the various aspects of biotechnology considered during this project for the removal of radionuclides from aqueous nuclear waste streams. 5.1 Biopolymers
Although there is apparent potential for the use of biopolymers per se for radionuclide accumulating systems it would appear that in general the probable time-scale for this industrial application is around 5-1 0 years hence. The
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High Intensity of Effort
Monoclonal antibody technology Genetic Engineering
Biomaanetic Separation LOW
I
LOW
Risk of Project Failure
High
Fig. 2. Effort versus risk of development.
biopolymers appear to offer few short-term applications benefits except perhaps for lignin, chitin and modified cellulosics. The long-term potential for biopolymers (into the next century) probably lies with the ‘tailoring’ of properties by the production of ‘designer’ molecules generated by application of genetic and protein engineering, e.g. for metallothioneins, and, siderophores. Chemically modified biopolymers, where a polymer such as lignin or chitin has a speciality radionuclide binding group chemically added could also find applications in the short term. 5.2 Biosorptive biomass
Biosorption offers a potentially eficient route to the removal of radionuclides from aqueous nuclear waste streams. Selected fungi, bacteria, yeasts and algae can all function as effective biosorbents and can be used as immobilised systems for adsorption of radionuclides followed by desorption and thus form part of a cyclic process. The major opportunities in the short term lie with dead biomass but there could be potential in the longer term for more active/living systems based on extra cellular polysaccharide production or enzyme p r o d ~ c t i o n . ~ ~ . ’ ~ There are some commercial applications of biosorption available at present and it is envisaged that cost-effective, efficient systems could be designed specifically for dilute nuclide streams within the five-year time horizon. 5.3 New applications and potential applications of biotechnology
Biomagnetic separation of radionuclides adsorbed to microorganisms appears to offer potential for the removal, concentration and separation of radionuclides in waste streams. With sufficient support it is envisaged that a pilot-scale process based on this technique could be available within a five-year time-scale. The use of immobilised monoclonal antibodies coupled with a selective microbial radionuclide adsorption could also be applied and achieve essentially similar separations without reliance on magnetic field-generating hardware.
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Genetic engineering is a rapidly expanding area of biotechnology and offers the potential to modify microorganisms such that highly efficient radionuclide bioaccumulation systems could be developed from preexisting organisms and biopol ymers.
REFERENCES 1. Hooper, E. W., Seminar on long term research into liquid emuent treatment. Harwell report 12338, AERE R12338 unclassified, Department of the Environment, 1987. 2. Volesky, B., Biosorbent materials. Biotechnol. Bioeng. Syrnp., 16 John Wiley and Sons Inc., New York, 1986. 3. Andleman, J. B., Trace Metals and Metalkorganic Interactions in Natural Waters, ed. P. C. Singer. Ann Arbor Science, Ann Arbor, MI, USA, 1973, pp. 57-8. 4. Shumate, S. E., Strandberg, G. W. & Parrott, J. R., Biotech. Bioeng. Symp., 8, John Wiley and Sons Inc., New York, 1978, pp. 13-20. 5. Najakima, A., Horikoshi, T. & Sakaguchi, T., Recovery of uranium by immobilized microorganisms. Eur. J . Appl. Microbiol. Biotechnol., 16 (1982) 88-91. 6. Darnall, D. W., Greene, B., Hosea, M., McPerson, R. A., Henzl, M. & Alexander, M. D., Recovery of heavy metals by immobilized algae. In Trace Metal Removal from Aqueous Solution, Proceedings of the Royal Society of Chemistry, ed. R. Thompson, University of Warwick, UK, 9-10 April 1986, Special Publication No. 61, pp. 1-24. 7. Robinson, P. K., Mak, A. L. & Trevan, M. D., Immobilized algae: A review. Process Biochem., 21 (4) (1986) 122-7. 8. Dainty, A. L., Goulding, K. H., Robinson, P. K., Simpkins, I. & Trevan, M. D., Stability of alginate immobilized algal cells. Biotechnol. Bioeng., 28 (1986) 21&16. 9. Robinson, P. K., Dainty, A. L., Goulding, K. H., Simpkins, I. & Trevan, M. D., Physiology of alginate immobilized Chlorella. Enzyme Microbial Technol., 7 (1986) 21 2-1 6. 10. Robinson, P. K., Goulding, K. H., Mak, A. L. & Trevan, M. D., Factors affecting the growth characteristics of alginate-entrapped Chlorella. Enzyme. Microbial. Technol., 8 (1986) 729-33. 1 1 . Tanaka, H., Matsumura, M. & Veliky, I. A., Diffusion characteristics of substrates in Ca-alginate gel beads. Biotechnol. Bioeng., 26 (1984) 53-8. 12. Galun, M., Keller, P., Malki, D., Feldstein, H., Galun, E., Siegel, S. & Siegel, B., Removal of uranium(V1) from solution by fungal biomass and fungal wall related biopolymers. Science, 219 (1982) 285-6. 13. Galun, M., Keller, P., Feldstein, H., Galun, E., Siegel, S. & Siegel, B., Recovery of uranium(1V) from solution using fungi 11. Release from uranium loaded Penicillium biomass. Water, Air and Soil Pollution, 20 (1983) 277-85. 14. Galun, M., Keller, P., Malki, D., Feldstein, H., Galun, E., Siegel, S. & Siegel, B., Recovery of uranium(V1) from solution using precultured Penicillium biomass. Water, Air and Soil Pollution, 20 (1983) 221-32. 15. Tsezos, M., Recovery of uranium from biological adsorgents. Desorption equilibrium. Biotechnol. Bioeng., 26 (1984) 873-81. 16. Tsezos, M., Baird, M. H. I. & Shemilt, L. W., The elution of radium adsorbed by microbial biomass. Chem. Eng. J., 34 (1987) B57-B64. 17. Tsezos, M. & Volesky, B., Biosorption of uranium and thorium. Biotechnol. Bioeng,, 23 (1981) 583-604. 18. Zosim, Z., Gutnick, D. & Rosenberg, E., Uranium binding by emulsan and emulsanols. Biotechnol. Bioeng., 25 (1983) 1725-35. 19. Galun, M., Keller, P., Feldstein, H., Galun, E., Siegel, S. & Siegel, B., Removal of uranium(V1) from solution by fungal biomass: Inhibition by iron. Water, Air and Soil Pollution, 21 (1984) 411-14.
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