Bioremediation of radioactive waste orbioremediation of radionuclides is an application ofbioremediation based on the use of biological agentsbacteria,plants andfungi (natural orgenetically modified) to catalyzechemical reactions that allow the decontamination of sites affected byradionuclides.[1] These radioactive particles are by-products generated as a result of activities related tonuclear energy and constitute a pollution and aradiotoxicity problem (with serioushealth andecological consequences) due to its unstable nature ofionizing radiation emissions.
The techniques of bioremediation of environmental areas assoil,water andsediments contaminated by radionuclides are diverse and currently being set up as an ecological and economic alternative to traditional procedures. Physico-chemical conventional strategies are based on the extraction of waste by excavating and drilling, with a subsequent long-range transport for their final confinement. These works and transport have often unacceptable estimatedcosts of operation that could exceed atrilliondollars in theUS and 50millionpounds in theUK.[2]
The species involved in these processes have the ability to influence the properties of radionuclides such assolubility,bioavailability andmobility to accelerate its stabilization. Its action is largely influenced byelectron donors andacceptors,nutrient medium, complexation of radioactive particles with the material andenvironmental factors. These are measures that can be performed on the source of contamination (in situ) or in controlled and limited facilities in order to follow thebiological process more accurately and combine it with other systems (ex situ).[3][4]
The presence of radioactive waste in the environment may cause long-term effects due to theactivity andhalf-life of the radionuclides, leading their impact to grow with time.[2] These particles exist in variousoxidation states and are found asoxides,coprecipitates, or asorganic orinorganic complexes, according to their origin and ways of liberation. Most commonly they are found in oxidized form, which makes them more soluble in water and thus more mobile.[4] Unlike organic contaminants, however, they cannot be destroyed and must be converted into a stable form or extracted from the environment.[5]
The sources of radioactivity are not exclusive of human activity. Natural radioactivity does not come from human sources: it covers up to three fourths of the total radioactivity in the world and has its origins in the interaction of terrestrial elements with high energycosmic rays (cosmogenic radionuclides) or in the existing materials onEarth since its formation (primordial radionuclides). In this regard, there are differences in the levels of radioactivity throughout theEarth's crust.India and mountains like theAlps are among the areas with the highest level of natural radioactivity due to their composition ofrocks andsand.[6]
The most frequent radionuclides in soils are naturallyradium-226 (226Ra),radon-222 (222Rn),thorium-232 (232Th),uranium-238 (238U) andpotassium-40 (40K). Potassium-40 (up to 88% of total activity),carbon-14 (14C),radium-226,uranium-238 andrubidium-87 (87Rb) are found inocean waters. Moreover, ingroundwater abound radius radioisotopes such as radium-226 andradium-228 (228Ra).[7][8] They are also habitual inbuilding materials radionuclides of uranium, thorium and potassium (the latter common towood).[8]
At the same time,anthropogenic radionuclides (caused by humans) are due tothermonuclear reactions resulting fromexplosions andnuclear weapons tests, discharges fromnuclear facilities, accidents deriving from the reprocessing of commercialfuel, waste storage from these processes and to a lesser extent,nuclear medicine.[9] Some polluted sites by these radionuclides are theUS DOE facilities (likeHanford Site), theChernobyl andFukushimaexclusion zones and the affected area ofChelyabinsk Oblast due to theKyshtym disaster.
In ocean waters, the presence oftritium (3H),cesium-137 (137Cs),strontium-90 (90Sr),plutonium-239 (239Pu) andplutonium-240 (240Pu) has significantly increased due to anthropogenic causes.[10][11] In soils,technetium-99 (99Tc), carbon-14, strontium-90,cobalt-60 (60Co),iodine-129 (129I),iodine-131 (131I),americium-241 (241Am),neptunium-237 (237Np) and various forms of radioactive plutonium and uranium are the most common radionuclides.[2][8][9]
| Frequency of occurrence of selected radionuclides atUS DOE facilities | |||||
| Ground water | Soils/Sediments | ||||
 |  | ||||
| Source:United States Department of Energy,US Government (1992)[12] | |||||
The classification of radioactive waste established by theInternational Atomic Energy Agency (IAEA) distinguishes six levels according toequivalent dose,specific activity,heat released andhalf-life of the radionuclides:[13]
Radioactive contamination is a potential danger for living organisms and results in external hazards, concerning radiation sources outside the body, and internal dangers, as a result of the incorporation of radionuclides inside the body (often byinhalation of particles oringestion ofcontaminated food).[14]
In humans, single doses from 0.25Sv produce first anomalies in the amount ofleukocytes. This effect is accentuated if theabsorbed dose is between 0.5 and 2 Sv, in whose first damage,nausea andhair loss are suffered. The strip ranging between 2 and 5 Sv is considered the most serious and includebleeding,ulcers and risk ofdeath; values exceeding 5 Sv involve immediate death.[14] If radiation, likewise, is received in small doses over long periods of time, the consequences can be equally severe. It is difficult to quantify the health effects for doses below 10mSv, but it has been shown that there is a direct relationship between prolonged exposure andcancer risk (although there is not a very clear dose-response relationship to establish clear limits of exposure).[15]
The information available on the effect of natural background radiation with respect anthropogenic pollution onwildlife is scarce and refers to very few species. It is very difficult to estimate from the available data the total doses that can accumulate during specific stages of thelife cycle (embryonic development or reproductive age), in changes inbehavior or depending onenvironmental factors such asseasonality.[16] The phenomena of radioactivebioaccumulation,bioconcentration andbiomagnification, however, are especially known to sea level. They are caused by the recruitment and retention of radioisotopes bybivalves,crustaceans,corals andphytoplankton, which then amounted to the rest of thefood chain at low concentration factors.[17]
Radiobiological literature andIAEA establish a safe limit of absorbed dose of 0.001Gy/d forterrestrial animals and 0.01 Gy/d forplants andmarine biota, although this limit should be reconsidered for long-lived species with low reproductive capacity.[18]
Radiation tests inmodel organisms that determine the effects of high radiation on animals and plants are:[18]
The effects of radioactivity onbacteria are given, as ineukaryotes, byionization of water and production ofreactive oxygen species. These compounds mutateDNA strands and producegenetic damage, inducing newlylysis and subsequentcell death.[20][21]
Its action on viruses, on the other hand, results in damagednucleic acids and viral inactivation.[22] They have asensory threshold ranging between 1000 and 10,000 Gy (range occupying most biological organisms) which decreases with increasinggenome size.[23]
The biochemical transformation of radionuclides into stable isotopes bybacterial species significantly differs from the metabolism oforganic compounds coming from carbon sources. They are highly energetic radioactive forms which can be converted indirectly by the process of microbialenergy transfer.[1]
Radioisotopes can be transformed directly through changes invalence state by acting asacceptors or by acting ascofactors toenzymes. They can also be transformed indirectly byreducing andoxidizing agents produced by microorganisms that cause changes inpH orredox potential. Other processes includeprecipitation and complexation ofsurfactants, orchelating agents that bind to radioactive elements. Human intervention, on the other hand, can improve these processes throughgenetic engineering andomics, or by injection of microorganisms ornutrients into the treatment area.[1][5]
According to the radioactive element and the specific site conditions, bacteria can enzymatically immobilize radionuclides directly or indirectly. Theirredox potential is exploited by some microbial species to carry out reductions that alter thesolubility and hence, mobility,bioavailability andradiotoxicity. This waste treatment technique called bioreduction or enzymatic biotransformation is very attractive because it can be done in mild conditions for the environment, does not produce hazardous secondary waste and has potential as a solution for waste of various kinds.[4]
.png)
Direct enzymatic reduction is the change of radionuclides of a higher oxidation state to a lower one made byfacultative andobligate anaerobes. The radioisotope interact with binding sites of metabolically active cells and is used asterminal electron acceptor in theelectron transport chain where compounds such asethyl lactate act aselectron donors underanaerobic respiration.[4]
Theperiplasm plays a very important role in these bioreductions. In the reduction ofuranium (VI) to insoluble uranium (IV), made byShewanella putrefaciens,Desulfovibrio vulgaris,Desulfovibrio desulfuricans andGeobacter sulfurreducens, the activity of periplasmiccytochromes is required. The reduction oftechnetium (VII) to technetium (IV) made byS. putrefaciens,G. sulfurreducens,D. desulfuricans,Geobacter metallireducens andEscherichia coli, on the other hand, requires the presence of the complexformate hydrogenlyase, also placed in this cell compartment.[2]
Other radioactiveactinides such asthorium,plutonium,neptunium andamericium are enzymatically reduced byRhodoferax ferrireducens,S. putrefaciens and several species ofGeobacter, and directly form an insolublemineral phase.[2]
The phenomenon of indirect enzymatic reduction is carried out bysulfate-reducing anddissimilatory metal-reducing bacteria onexcretion reactions ofmetabolites and breakdown products. There is a coupling of theoxidation oforganic acids —produced by the excretion of theseheterotrophic bacteria— with the reduction ofiron or othermetals and radionuclides, which forms insoluble compounds that can precipitate asoxide andhydroxide minerals. In the case of sulfate-reducing bacteria hydrogen sulfide is produced, promoting increased solubility of polluting radionuclides and theirbioleaching (as liquid waste that can then be recovered).[2][4]
There are several species of reducing microorganisms that produce indirectsequestering agents and specificchelators, such assiderophores. These sequestering agents are crucial in the complexation of radionuclides and increasing their solubility and bioavailability.Microbacterium flavescens, for example, grows in the presence of radioisotopes such as plutonium, thorium, uranium or americium and produces organic acids and siderophores that allow the dissolution and mobilization of radionuclides through the soil. It seems that siderophores on bacterial surface could also facilitate the entry of these elements within the cell as well.Pseudomonas aeruginosa also secretes chelating agents out that meet uranium and thorium when grown in a medium with these elements. In general, it has also been found thatenterobactin siderophores are extremely effective in solubilizing actinide oxides of plutonium.[2][4]
Citrate is a chelator which binds to certaintransition metals and radioactive actinides. Stable complexes such asbidentate,tridentate (ligands with more than one atom bound) and polynuclear complexes (with several radioactive atoms) can be formed with citrate and radionuclides, which receive a microbial action. Anaerobically,Desulfovibrio desulfuricans and species of the generaShewanella andClostridium are able to reduce bidentate complexes ofuranyl-citrate (VI) to uranyl-citrate (IV) and make them precipitate, despite not being able to degrade metabolically complexed citrate at the end of the process.[2] In denitrifying and aerobic conditions, however, it has been determined that it is not possible to reduce or degrade these uranium complexes. Bioreduction do not get a head when they are citrate complex mixed metal complexes or when they are tridentate, monomeric or polynuclear complexes, since they becomerecalcitrant and persistent in the environment.[4][24] From this knowledge exists a system that combines the degradation of radionuclide-citrate complex with subsequentphotodegradation of remaining reduced uranyl-citrate (previously not biodegradated but sensitive tolight), which allows for stable precipitates of uranium and also of thorium, strontium or cobalt fromcontaminated lands.[4]
.png)
The set of strategies that comprise biosorption, bioaccumulation and biomineralization are closely related to each other, because one way or another have a direct contact between the cell and radionuclide. These mechanisms are evaluated accurately using advanced analysis technologies such aselectron microscopy,X-ray diffraction andXANES,EXAFS andX-ray spectroscopies.[1][25]
Biosorption and bioaccumulation are two metabolic actions that are based on the ability to concentrate radionuclides over a thousand times the concentration of the environment. They consist of complexation of radioactive waste withphosphates, organic compounds andsulfites so that they become insoluble and less exposed to radiotoxicity. They are particularly useful inbiosolids foragricultural purposes andsoil amendments, although most properties of these biosolids are unknown.[26]
Biosorption method is based on passive sequestration of positively charged radioisotopes bylipopolysaccharides (LPS) on thecell membrane (negatively charged), either live or dead bacteria. Its efficiency is directly related to the increase in temperature and can last for hours, being a much faster method than direct bioreduction. It occurs through the formation ofslimes and capsules, and with a preference for binding to thephosphate andphosphoryl groups (although it also occurs withcarboxyl,amine orsulfhydryl groups).Bacillota and other bacteria likeCitrobacter freudii have significant biosorption capabilities;Citrobacter does it throughelectrostatic interaction of uranium with phosphates of their LPS.[2][3]
Quantitative analyzes determine that, in the case of uranium, biosorption may vary within a range between 45 and 615milligrams per gram of celldry weight. However, it is a technique that requires a high amount of biomass to affect bioremediation; it presents problems ofsaturation and other cations that compete for binding to the bacterial surface.[3]
Bioaccumulation refers to uptake of radionuclides into the cell, where they are retained by complexations with negatively charged intracellular components, precipitation orgranules formations. Unlike biosorption, this is anactive process: it depends on an energy-dependent transport system.[citation needed] Some metals or radionuclides can be absorbed by bacteria accidentally because of its resemblance todietary elements formetabolic pathways. Several radioisotopes ofstrontium, for example, are recognized as analogs ofcalcium and incorporated withinMicrococcus luteus.[4]Uranium, however, has no known function and is believed that its entry into the cell interior may be due to its toxicity (it is able to increasemembrane permeability).[3]
Furthermore, biomineralization —also known as bioprecipitation— is theprecipitation of radionuclides through the generation of microbial ligands, resulting in the formation of stablebiogenic minerals. These minerals have a very important role in the retention of radioactive contaminants. A very localized and produced enzymatically ligand concentration is involved and provides anucleation site for the onset of biomineral precipitation.[27] This is particularly relevant in precipitations ofphosphatase activity-derivate biominerals, which cleavage molecules such asglycerol phosphate onperiplasm. InCitrobacter andSerratia genera, this cleavage liberates inorganic phosphates (HPO42−) that precipitates with uranyl ion (UO22+) and cause deposition ofpolycrystalline minerals around the cell wall.[2][28]Serratia also formbiofilms that promote precipitation of chernikovite (rich in uranium) and additionally, remove up to 85% ofcobalt-60 and 97% ofcesium-137 byproton substitution of this mineral.[25] In general, biomineralization is a process in which the cells do not have limitations of saturation and can accumulate up to several times its own weight as precipitated radionuclides.[4]
Investigations of terrestrial and marine bacterial isolates belonging to the generaAeromonas,Bacillus,Myxococcus,Pantoea,Pseudomonas,Rahnella andVibrio have also demonstrated the removal of uranium radioisotopes as phosphate biominerals in bothoxic andanoxic growth conditions.[25]
Aside from bioreduction, biosorption, bioaccumulation and biomineralization, which are bacterial strategies for natural attenuation of radioactive contamination, there are also human methods that increase the efficiency or speed of microbial processes. This accelerated natural attenuation involves an intervention in the contaminated area to improve conversion rates of radioactive waste, which tend to be slow. There are two variants: biostimulation and bioaugmentation.[30]
Biostimulation is the addition of nutrients withtrace elements,electron donors orelectron acceptors to stimulate activity and growth of naturalindigenous microbial communities.[4][30] It can range from simplefertilization or infiltration (called passive biostimulation) to more aggressive injections to the ground, and is widely used atUS DOE sites.[26]Nitrate is used as nutrient to biostimulate the reduction ofuranium, because it serves as very energetically favorableelectron acceptor formetal-reducing bacteria. However, many of these microorganisms (Geobacter,Shewanella orDesulfovibrio) exhibitresistance genes toheavy metals that limit their ability to bioremediate radionuclides. In these particular cases, a carbon source such asethanol is added to the medium to promote the reduction of nitrate at first, and then ofuranium. Ethanol is also used in soil injection systems withhydraulicrecirculations: it raises thepH and promotes the growth ofdenitrifying and radionuclide-reducing bacteria, that producebiofilms and achieve almost 90% decrease in the concentration of radioactive uranium.[2]
A number ofgeophysical techniques have been used to monitor the effects of in situ biostimulation trials including measurement of:spectral ionization potential,self potentials,current density,complex resistivity and alsoreactive transport modelling (RTM), which measureshydrogeological andgeochemical parameters to estimate chemical reactions of the microbial community.[3]
Bioaugmentaton, on the other hand, is the deliberated addition to the environment of microorganisms with desired traits to accelerate bacterial metabolic conversion of radioactive waste. They are often added when necessary species for bioremediation do not exist in the treatment place.[4][30] This technique has shown in field trials over the years that it does not offer better results than biostimulation; neither it is clear that introduced species can be distributed effectively through the complex geological structures of mostsubsurface environments or that can compete long term with the indigenous microbiota.[1][26]
Omics, especially genomics and proteomics, allow identifying and evaluatinggenes,proteins andenzymes involved in radionuclide bioremediation, apart from the structural and functional interactions that exist between them and other metabolites.Genome sequencing of various microorganisms has uncovered, for example, thatGeobacter sulfurreducens possess more than 100coding regions forc-type cytochromes involved in bioremediation radionuclide, or thatNiCoT gene is significantly overexpressed inRhodopseudomonas palustris andNovosphingobium aromaticivorans when grown in medium with radioactivecobalt.[1][2]
From this information, different genetic engineering andrecombinant DNA techniques are being developed to generate specific bacteria for bioremediation. Someconstructs expressed in microbial species arephytochelatins,polyhistidines and otherpolypeptides byfusion-binding domains to outer-membrane-anchored proteins.[2] Some of these genetically modified strains are derived fromDeinococcus radiodurans, one of the most radiation-resistant organisms.D. radiodurans is capable to resistoxidative stress andDNA damage from radiation, and reducestechnetium,uranium andchromium naturally as well. Besides, through insertion of genes from other species it has been achieved that it can also precipitatesuranyl phosphates and degradesmercury by usingtoluene as an energy source to grow and stabilize other priority radionuclides.[1][3]
Directed evolution of bacterial proteins related to bioremediation of radionuclides is also a field research.YieF enzyme, for example, naturally catalyzes the reduction ofchromium with a very wide range ofsubstrates. Followingprotein engineering, however, it has also been able to participate inuranyl ion reduction.[31]
The use of plants to remove contaminants from the environment or to render them less harmful is called phytoremediation. In the case of radionuclides, it is a viable technology when decontamination times are long and waste are scattered at low concentrations.[32][33]
Some plant species are able to transform the state of radioisotopes (without suffering toxicity) concentrating them in different parts of their structure, making them rush through the roots, making them volatile or stabilizing them on the ground. As in bacteria, plantgenetic engineering procedures and biostimulation —calledphytostimulation— have improved and accelerate these processes, particularly with regard tofast-growing plants.[33] The use ofAgrobacterium rhizogenes, for example, is quite widespread and significantly increases radionuclide uptake by theroots.[citation needed]
In phytoextraction (also phytoaccumulation, phytosequesteration or phytoabsorption)[34] plants carry radioactive waste from theroot system to thevascular tissue and become concentrated in thebiomass of shoots. It is a technique that removes radionuclides without destroying the soil structure, with minimal impact onsoil fertility and valid for large areas with a low level of radioactivity. Its efficiency is evaluated throughbioaccumulation coefficient (BC) or total removal of radionuclides perm2, and is proven to attractcesium-137,strontium-90,technetium-99,cerium-144,plutonium-240,americium-241,neptunium-237 and various radioisotopes ofthorium andradium.[33] By contrast, it requires large biomass production in short periods of time.[citation needed]
Species likecommon heather oramaranths are able to concentrate cesium-137, the most abundant radionuclide in theChernobyl Exclusion Zone. In this region ofUkraine,mustard greens could remove up to 22% of average levels of cesium activity in a single growing season. In the same way,bok choy and mustard greens can concentrate 100 times moreuranium than other species.[33]
Rhizofiltration is the adsorption and precipitation of radionuclides in plant roots or absorption thereof if soluble in effluents. It has great efficiency in the treatment ofcesium-137 andstrontium-90, particularly byalgae andaquatic plants, such asCladophora andElodea genera, respectively. It is the most efficient strategy for bioremediation technologies inwetlands,[34] but must have a continuous and rigorous control ofpH to make it an optimal process.[35]
From this process, some strategies have been designed based on sequences ofponds with a slowflow of water to clean polluted water with radionuclides. The results of these facilities, for flows of 1000 liters of effluent are about 95% retention of radiation in the first pond (by plants andsludge), and over 99% in three-base systems.[33]
The most promising plants for rhizofiltration aresunflowers. They are able to remove up to 95% ofuranium of contaminated water in 24 hours, and experiments inChernobyl have demonstrated that they can concentrate on 55 kg of plantdry weight all the cesium and strontium radioactivity from an area of 75 m2 (stabilized material suitable for transfer to a nuclear waste repository).[33]
Phytovolatilization involves the capture and subsequenttranspiration of radionuclides into theatmosphere. It does not remove contaminants but releases them in volatile form (less harmful). Despite not having too many applications for radioactive waste, it is very useful for the treatment oftritium, because it exploits plants' ability to transpire enormous amounts of water.[33][34]
The treatment applied to tritium (shielded by air produces almost no external radiation exposure, but its incorporation in water presents a health hazard when absorbed into the body) uses polluted effluents toirrigatephreatophytes. It becomes a system with a low operation cost and low maintenance, with savings of about 30% in comparison to conventional methods ofpumping and covering withasphalt.[33]
Phytostabilization is an specially valid strategy for radioactive contamination based on the immobilization of radionuclides in the soil by the action of the roots. This can occur by adsorption, absorption and precipitation within root zone, and ensures that radioactive waste can not be dispersed becausesoil erosion orleaching. It is useful in controlling tailings from strip and open pit uranium mines, and guarantees to retrieve theecosystem.[33][34] However, it has significant drawbacks such as large doses offertilizer needed to reforest the area, apart from radioactive source (which implies long-term maintenance) remaining at the same place.[citation needed]
Several fungi species have radioactive resistance values equal to or greater than more radioresistant bacteria; they perform mycoremediation processes. It was reported that some fungi had the ability of growing into, feeding, generatingspores and decomposing pieces ofgraphite from destroyedreactor No. 4 at theChernobyl Nuclear Power Station, which is contaminated with high concentrations ofcesium,plutonium andcobalt radionuclides. They were calledradiotrophic fungi.[36]
Since then, it has been shown that some species ofPenicillium,Cladosporium,Paecilomyces andXerocomus are able to useionizing radiation as energy through the electronic properties ofmelanins.[36][37] In their feeding they bioaccumulate radioisotopes, creating problems onconcrete walls ofdeep geological repositories.[38] Other fungi likeoyster mushrooms can bioremediateplutonium-239 andamericium-241.[39]
Current research on bioremediation techniques is fairly advanced and molecular mechanisms that govern them are well known. However, there are many doubts about the effectiveness and possible adversities of these processes in combination with the addition ofagrochemicals. In soils, the role ofmycorrhizae on radioactive waste is poorly described and sequestration patterns of radionuclides are not known with certainty.[40]
Longevity effects of some bacterial processes, such as maintenance of uranium in insoluble form because of bioreductions or biomineralizations, are unknown. There are not clear details about theelectronic transfer from some radionuclides with these bacterial species either.[3]
Another important aspect is the change ofex situ or laboratory scale processes to their real applicationin situ, in which soil heterogeneity and environmental conditions generate reproduction deficiencies of optimal biochemical status of the used species, a fact that decreases the efficiency. This implies finding what are the best conditions in which to carry out an efficient bioremediation with anions, metals, organic compounds or other chelating radionuclides that can compete with the uptake of interest radioactive waste.[2] Nevertheless, in many cases research is focused on the extraction of soil and water and itsex situ biological treatment to avoid these problems.[4]
Finally, the potential ofGMOs is limited byregulatory agencies in terms ofresponsibility andbioethical issues. Their release require support on the action zone and comparability with indigenous species. Multidisciplinary research is focused on defining more precisely necessarygenes andproteins to establish newcell-free systems which may avoid possible side effects on the environment by the intrusion oftransgenic orinvasive species.[2]
{{cite book}}:|journal= ignored (help){{cite book}}:|journal= ignored (help){{cite journal}}:Cite journal requires|journal= (help){{cite journal}}:Cite journal requires|journal= (help)
