One of the issues facing the nuclear power industry is how to store spent nuclear fuel which is contaminated with radionuclides produced during nuclear fission, including caesium ((134)Cs(+), (135)Cs(+) and (137)Cs(+)) and cobalt ((60)Co(2+)). In this study, we have isolated Co(2+)- and Cs(+)-resistant bacteria from water collected from a nuclear fuel storage pond. The most resistant Cs(+) and Co(2+) isolates grew in the presence of 500 mM CsCl and 3 mM CoCl2. Strain Cs67-2 is resistant to fourfold more Cs(+) than Cupriavidus metallidurans str. CH34 making it the most Cs(+)-resistant strain identified to date. The Cs(+)-resistant isolates were closely related to bacteria in the Serratia and Yersinia genera, while the Co(2+)-resistant isolates were closely related to the Curvibacter and Tardiphaga genera. These new isolates could be used for bioremediation

Avery

Barceloux

Barras

Edgar

Joanne M. Santini

Kobayashi

Kuwahara

Lane

Liesegang

Linda Dekker

Melnikov

Mergeay

Monchy

Monsieurs

Nies

Paterson-Beedle

Ranquet

Reasoner

Rodrigue

Saitou

Tamura

Thomas H. Osborne

Wang

FEMS Microbiology Letters

English

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Isolation and identification of cobalt- and caesium-resistant bacteria from a nuclear fuel storage pond

One of the issues facing the nuclear power industry is how to store spent nuclear fuel which is contaminated with radionuclides produced during nuclear fission, including caesium ((134) Cs(+) , (135) Cs(+) and (137) Cs(+) ) and cobalt ((60) Co(2+) ). In this study we have isolated Co(2+) and Cs(+) resistant bacteria from water collected from a nuclear fuel storage pond. The most resistant Cs(+) and Co(2+) isolates grew in the presence of 500 mM CsCl and 3 mM CoCl2 . Strain Cs67-2 is resistant to 4-fold more Cs(+) than Cupriavidus metallidurans str. CH34 making it the most Cs(+) resistant strain identified to date. The Cs(+) resistant isolates were closely related to bacteria in the Serratia and Yersinia genera, while the Co(2+) resistant isolates were closely related to the Curvibacter and Tardiphaga genera. These new isolates could be used for bioremediation. This article is protected by copyright. All rights reserved

Dekker, L

Osborne, TH

Santini, JM

UCL Discovery

R E S EA RCH L E T T E RIsolation and identification of cobalt- and caesium-resistantbacteria from a nuclear fuel storage pondLinda Dekker, Thomas H. Osborne & Joanne M. SantiniInstitute of Structural and Molecular Biology, University College London, London, UKCorrespondence: Joanne M. Santini,Institute of Structural and Molecular Biology,University College London Gower Street,London, WC1E 6BT, UK.Tel.: +44 2076316675;fax: +44 2076316803;e-mail: j.santini@ucl.ac.ukReceived 21 May 2014; revised 25 July 2014;accepted 27 July 2014. Final versionpublished online 2 September 2014.DOI: 10.1111/1574-6968.12562Editor: Aharon OrenKeywordscobalt; caesium; resistance; radionuclide.AbstractOne of the issues facing the nuclear power industry is how to store spentnuclear fuel which is contaminated with radionuclides produced during nuclearfission, including caesium (134Cs+, 135Cs+ and 137Cs+) and cobalt (60Co2+). Inthis study, we have isolated Co2+- and Cs+-resistant bacteria from water col-lected from a nuclear fuel storage pond. The most resistant Cs+ and Co2+ iso-lates grew in the presence of 500 mM CsCl and 3 mM CoCl2. Strain Cs67-2 isresistant to fourfold more Cs+ than Cupriavidus metallidurans str. CH34 mak-ing it the most Cs+-resistant strain identified to date. The Cs+-resistant isolateswere closely related to bacteria in the Serratia and Yersinia genera, while theCo2+-resistant isolates were closely related to the Curvibacter and Tardiphagagenera. These new isolates could be used for bioremediation.IntroductionNuclear power is a major contributor to electrical energyproduction in many countries; however, it produces a sig-nificant amount of toxic environmental waste. The radio-nuclide 60Co2+ has a half-life of 5.3 years and is producedduring the nuclear fission process by thermal neutronbombardment of the natural isotope, which is present ina number of steel containing components of nuclear reac-tors. Radioactive Cs+ is a fission product and its isotopes134Cs+, 135Cs+ and 137Cs+ have half-lives of 2.1 years,2.3 million years and 30 years, respectively (Kobayashi &Shimizu, 1999). One of the major problems at nuclearpower plants is the disposal of spent nuclear fuel that isno longer effective for producing a nuclear reaction andhence needs to be safely disposed. Spent nuclear fuelmust be kept in underwater racks to cool prior to finalstorage. Storage ponds use deionized water to cool thespent fuel and protect against radiation.The main health concern associated with these radio-nuclides is the increased risk of cancer due to the effectsof beta and gamma radiation. In addition to radiation,the toxicity of Co2+ and Cs+ is also detrimental to humanhealth. Cs+ enters the body through ingestion, and due toits physiochemical resemblance to K+, it is transportedaround the body via K+ transport systems (Kuwaharaet al., 2011) interfering with K+ homeostasis. It has beenproposed that the mode of toxicity of Cs+ is by the deple-tion of K+ (Avery, 1995). Co2+ also enters the body viaingestion, where it competes with Fe during synthesis ofFe-S clusters in essential metabolic proteins, resulting intheir inactivation (Ranquet et al., 2007; Barras & Fonte-cave, 2011). Co2+ toxicity can cause various health prob-lems such as contact dermatitis, pneumonia, allergicasthma and lung cancer (Barceloux, 1999), while Cs+ tox-icity is associated with fatigue, muscle weakness, palpita-tions and arrhythmia (Melnikov & Zanoni, 2013). Thepotential negative health effects associated with Co2+ andCs+ from spent nuclear fuel necessitate the requirementfor removal strategies; bacteria that can survive in envi-ronments with high concentrations of Co2+ or Cs+ radio-nuclides could be useful for nuclear fuel remediation.Materials and methodsSample siteA water sample from an external storage pond at SellafieldLtd (Cumbria, UK) was obtained from 5 m below the sur-face to enrich and isolate bacteria resistant to Co2+ and Cs+.FEMS Microbiol Lett 359 (2014) 81–84 ª 2014 The Authors. FEMS Microbiology Letterspublished by John Wiley & Sons Ltd on behalf of Federation of European Microbiological Societies.This is an open access article under the terms of the Creative Commons Attribution License, which permits use,distribution and reproduction in any medium, provided the original work is properly cited.MICROBIOLOGY LETTERSIsolation of Co- and Cs-tolerantmicroorganisms from enrichment culturesDuplicate enrichment cultures were set up in 10 mL ofR2A medium (Reasoner & Geldreich, 1985) where eitherCoCl2 was added to a final concentration of 0.5, 0.75, 1or 2 mM, or CsCl was added to a final concentration of25, 50, 75 or 100 mM. Escherichia coli str. K38 is consid-ered to be neither metal resistant nor sensitive and has aminimum inhibitory concentration (MIC) of 1 mM forCoCl2 and > 50 mM for CsCl (Nies, 1999); therefore,representative concentrations were used for the enrich-ments. One milliliter of the pond water sample was addedto each tube which was incubated at either 10 or 28 °C.As the storage pond is outside, the temperature is notregulated and the water temperature is affected by theweather. The water temperature at the time of samplingwas 21.2 °C; enrichments were conducted at 10 and28 °C to account for seasonal changes in temperatureassociated with the UK climate and the heating of thepool caused by the spent fuel. Following incubation, a1% inoculum from the enrichment culture containing1 mM CoCl2 or 100 mM CsCl in which growth wasobserved (by turbidity) was transferred to fresh brothcontaining the same and a twofold higher concentrationof CoCl2 or CsCl and incubated at the same temperature.The subsequent enrichments were plated onto R2A agarcontaining the same concentration of CoCl2 or CsCl asthe original culture. Colonies of unique morphology werepicked and streaked onto fresh R2A agar containingCoCl2 or CsCl. This process was repeated twice more toensure pure cultures were obtained.Restriction fragment length polymorphism(RFLP) analysesPCR was undertaken using the universal primers 63f and1387r (Lane, 1991) to amplify the 16S rRNA gene of theisolates. PCR products were microdialysed against MQwater for 45 min using a MFTM-Millipore membrane filterwith a pore size of 0.025 lm to remove salts from thesolution. Each isolate was digested with the 4-bp cutterrestriction enzymes HhaI, MspI and RsaI (Promega) fol-lowing the manufacturer’s guidelines. Following digestion,PCR products (10 lL) were visualized by electrophoresison 2% agarose gels and the restriction profiles analysed.16S rRNA gene sequencingDNA sequencing was performed by Source Bioscienceusing an ABI 3730xl 96 capillary Genome Analyser analy-sis system. The template of each isolate was provided as apurified PCR product at a concentration of 15 ng lL1and in a volume of 5 lL. For all isolates, the primers 27fand 1492r (Lane, 1991) were used for PCR amplificationand sequencing.Sequence alignment and phylogenetic analysisNucleotide sequences were trimmed and aligned usingMUSCLE (Edgar, 2004) using default settings. BLAST searchesof the 16S rRNA gene sequence against the 16S ribosomalRNA sequences (Bacteria and Archaea) database wereused to determine which bacteria the isolates were closelyrelated to. Using the CLASSIFIER tool of the RibosomalDatabase Project (Wang et al., 2007) and the EZTAXON-EDatabase (Kim et al., 2012), isolates were identified to thefamily or genus level. Phylogenetic analysis and trees wereconstructed with MEGA 5.05 (Tamura et al., 2011). Phylo-genetic trees were constructed using the kimura-2-param-eter algorithm and neighbour-joining method (Saitou &Nei 1987). Bootstrap values were from 100 resamples.MIC of CsCl and CoCl2 for water sampleisolatesCultures of the Co- and Cs-resistant bacterial isolateswere grown in 10 mL of R2A medium and incubated at28 °C until turbid. A dilution of the culture (25 lL) wasspread plated onto half an R2A agar plate or R2A agarplates supplemented with either 100, 200, 300, 400, 500,1000 mM CsCl, 0.5, 1, 2, 3, 4, 5 mM CoCl2 or NiCl2 orZnCl2; or 0.25, 1, 2, 3, 4 mM CdCl2 or CuCl2. The plateswere incubated at 28 °C until colonies were visible. Theeffect of osmotic stress on the Cs-resistant isolates wastested in 10 mL R2A medium containing 300, 400, 500and 700 mM NaCl.Results and discussionEight Co2+-resistant and four Cs+-resistant isolates werepurified from R2A agar containing 2 mM Co2+ or100 mM Cs+, respectively. One isolate (Cs60-2) was iso-lated from 10 °C, while the remainder were isolated from28 °C enrichments. Three different RFLP profiles wereobserved with the Co2+ water sample isolates, and two dif-ferent RFLP profiles were seen with the Cs+ water sampleisolates. Representatives of each phylotype were identifiedby sequencing a 1465-bp region of the 16S rRNA gene.The 16S rRNA gene sequences were used to construct aphylogenetic tree (Fig. 1) and submitted to the EZTAXON-EDatabase (Kim et al., 2012) for taxonomic identification.All isolates were members of the Proteobacteria, with theCs+-resistant isolates belonging to the Gammaproteobacte-ria, whereas the Co2+-resistant isolates were members ofthe Alphaproteobacteria and Betaproteobacteria (Fig. 1).FEMS Microbiol Lett 359 (2014) 81–84ª 2014 The Authors. FEMS Microbiology Letterspublished by John Wiley & Sons Ltd on behalf of Federation of European Microbiological Societies.82 L. Dekker et al.The Co2+ isolates Co1 and Co64-2 are both closely relatedto Tardiphaga robiniae, while Co12 is closely related tomembers of the Curvibacter genera (Fig. 1). Cs+ isolatesCs60-2 and Cs67-2 are closely related to members of theSerratia and Yersinia genera, respectively (Fig. 1). Strainshave been sent to the DSMZ for deposition.The MIC for CoCl2 and CsCl of the Co2+ and the Cs+isolates was determined. The Co2+-resistant isolates wereall resistant to 2 mM CoCl2, and one isolate (Co64-2)was able to grow in the presence of 3 mM CoCl2. One ofthe Cs+-resistant isolates (Cs60-2) grew in the presence of0.5 mM CoCl2; however, Cs67-2 was unable to grow inthe presence of 0.5 mM CoCl2. The Co concentration inthe external storage pond was not measured. The Cs+-resistant isolates grew in the presence of 300 mM (Cs60-2) and 500 mM (Cs67-2) CsCl; there are no knownorganisms able to tolerate these concentrations. BothCs67-2 and Cs60-2 grew in the presence of 700 mM and500 mM NaCl, respectively, indicating that Cs+ toxicitywas not due to osmotic stress. Co2+ resistance is generallyassociated with Ni2+ and/or Zn2+ resistance via an effluxpump mechanism and can be either chromosomally orplasmid-encoded (Nies, 2003; Rodrigue et al., 2005),while the mechanism of resistance to Cs+ is currentlyunknown. Apart from Serratia, which is known to beresistant to Cs+ (Paterson-Beedle et al., 2006), none ofthe closest relatives of the identified isolates have beenshown to be resistant to either Cs+ or Co2+.All of the other identified isolates in this work arerelated to genera that have been commonly isolated fromwater samples. The highly metal-resistant bacterium Cu-priavidus metallidurans str. CH34 has a MIC of 25 mMfor CoCl2 and 125 mM for CsCl (Monsieurs et al., 2011).The genome of C. metallidurans str. CH34 contains twochromosomes and two megaplasmids that contain a largenumber of genes implicated in the resistance to heavymetals (Mergeay et al., 2003). It has been shown thatgenes on the megaplasmids can be activated by more thanone metal; metal response genes are found on both mega-plasmids pMOL28 and pMOL30 for Cd2+, Ni2+, Cu2+,Pb2+, Zn2+ and Co2+ (Monsieurs et al., 2011). Cupriavi-dus metallidurans str. CH34 contains two clusters ofheavy metal-resistance genes, czc located on pMOL30(Liesegang et al., 1993) and cnr located on pMOL28, thathave been shown to be involved in Co2+ resistance whichmay explain its elevated MIC (Mergeay et al., 1985; Nieset al., 1987). In E. coli, Co2+ is transported into the cellby constitutively expressed divalent cation uptake systemsof broad specificity, for example Mg2+ and Zn2+ transportsystems (Nies, 1992); the rcnA gene encodes a membrane-bound protein that confers Ni2+ and Co2+ resistance andacts as an efflux pump to export the metals (Rodrigueet al., 2005). Given the MIC to Co2+ of the isolates inthis study (Table 1), it is possible that the mechanism forresistance for isolate Co12 is similar to that of E. coli as itcannot grow in the presence of Zn2+. Although Cs+ isconsidered to be relatively nontoxic to microorganisms(Avery, 1995), isolate Cs67-2 grew in a medium withfourfold more CsCl than C. metallidurans str. CH34,identifying it as the most Cs+-resistant bacterial strainknown to date.With the renewed interest in the nuclear fuel industry,there is also the need to develop technologies for theremediation of nuclear waste and contaminated materials.The nuclear industry needs to resolve the problem oflong-term containment of radionuclide wastes and theSerratia fonticola AJ233429 Serratia glossinae FJ790328Cs60-2 KF811206Serratia proteamaculans AJ233434Cs67-2 KF811209Yersinia intermedia AF36680Yersinia aldovae AF366376 Yersinia pekkanenii GQ451990Shewanella putrefaciens X81623Alcaligenes faecalis D88008Nitrosomonas europaea AB070982 Cupriavidus metallidurans D87999Curvibacter gracilis AB109889Curvibacter delicatus AF078756Co12 KF811208Curvibacter fontanus AB120963Caulobacter vibrioides AJ009957Xanthobacter autotrophicus X94201Co64-2 KF811210Tardiphaga robiniae FR753034Co1 KF811211Bradyrhizobium elkanii AY6241351001001001009997100100100100921001000.05αβγFig. 1. Phylogenetic tree of the 16S rRNA genes from Co2+- and Cs+-resistant isolates and their phylogenetic relatives. Bootstrap values(per 100 trials) are shown. a – Alphaproteobacteria; b –Betaproteobacteria; c – Gammaproteobacteria. Sequences werealigned with MUSCLE (Edgar, 2004) and the tree constructed using thekimura-2-parameter algorithm and neighbour-joining method withMEGA 5.05 (Tamura et al., 2011). The tree was rooted with the 16SrRNA gene sequence of Aeropyrum pernix (not shown). Barrepresents 0.05 substitutions per nucleotide position. GenBankaccession numbers are shown.Table 1. MIC of metals for growth of Cupriavidus metallidurans, theCo2+- and Cs+-resistant isolatesIsolateMIC (mM)CoCl2 CsCl2 NiCl2 ZnCl2 CuCl2 CdCl2C. metallidurans 25* 125* 13† 12† 3† 4†Cs60-2 1 400 1 5 1 1Cs67-2 0.5 1000 2 0.5 1 0.5Co1 3 100 3 3 2 0.5Co12 3 100 2 0 1 0.5Co-64-2 4 100 5 5 2 0.5*Values taken from Monsieurs et al. (2011).†Values taken from Monchy et al. (2007).FEMS Microbiol Lett 359 (2014) 81–84 ª 2014 The Authors. FEMS Microbiology Letterspublished by John Wiley & Sons Ltd on behalf of Federation of European Microbiological Societies.Cobalt- and caesium-resistant bacteria 83environmental impact of radionuclide migration.Microbial metabolism has the potential to significantlyalter the chemistry of radionuclide-contaminated environ-ments and control radionuclide speciation and mobility,and therefore has applications in waste storage and man-agement. It is now widely considered that the large metaluptake capacity and cheap availability of many microor-ganisms make them ideal candidates for industrial metalremoval, and several commercial operations have adoptedmicroorganisms-mediated systems as an important partof their detoxification process (Avery, 1995). The isola-tion of novel bacteria that are resistant to either Co2+ orCs+ could prove useful in the bioremediation of nuclearfuel storage ponds.AcknowledgementsWe would like to thank Lizzie Anderson and LorraineHarvey from Sellafield Ltd for obtaining the water sam-ple. This work was funded by the EPSRC DIAMONDUniversity Consortium (EP/G055412/1).ReferencesAvery SV (1995) Microbial interactions with caesium –implications for biotechnology. J Chem Technol Biotechnol62: 3–16.Barceloux DG (1999) Cobalt. J Toxicol Clin Toxicol 37: 201–206.Barras F & Fontecave M (2011) Cobalt stress in Escherichia coliand Salmonella enterica: Molecular bases for toxicity andresistance. Metallomics 3: 1130–1134.Edgar RC (2004) MUSCLE: multiple sequence alignment withhigh accuracy and high throughput. Nucleic Acids Res 32:1792–1797.Kim OS, Cho YJ, Lee K et al. (2012) Introducing EZTAXON-E: aprokaryotic 16S rRNA gene sequence database withphylotypes that represent uncultured species. 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FEMS Microbiol Rev 27: 313–339.Nies D, Mergeay M, Friedrich B & Schlegel HG (1987)Cloning of plasmid genes encoding resistance to cadmium,zinc, and cobalt in Alcaligenes eutrophus CH34. J Bacteriol169: 4865–4868.Paterson-Beedle M, Macaskie LE, Lee CH, Hriljac JA, Jee KY& Kim WH (2006) Utilisation of a hydrogen uranylphosphate-based ion exchanger supported on a biofilm forthe removal of cobalt, strontium and caesium from aqueoussolutions. Hydrometallurgy 83: 141–145.Ranquet C, Ollagnier-de-Choudens S, Loiseau L, Barras F &Fontecave M (2007) Cobalt stress in Escherichia coli. Theeffect on the iron-sulphur proteins. J Biol Chem 282: 30442–30451.Reasoner DJ & Geldreich EE (1985) A new medium for theenumeration and subculture of bacteria from potable water.Appl Environ Microbiol 49: 1–7.Rodrigue A, Effantin G & Mandrand-Berthelot MA (2005)Identification of rcnA (yohM), a nickel and cobalt resistancegene in Escherichia coli. J Bacteriol 187: 2912–2916.Saitou N & Nei M (1987) The neighbour-joining method: anew method for reconstructing phylogenetic trees. Mol BiolEvol 4: 406–425.Tamura K, Peterson D, Peterson N, Stecher G, Nei M & KumarS (2011) MEGA5: molecular evolutionary genetics analysisusing maximum likelihood, evolutionary distance, andmaximum parsimony methods. Mol Biol Evol 28: 2731–2739.Wang Q, Garrity GM, Tiedje JM & Cole JR (2007) NaiveBayesian classifier for rapid assignment of rRNA sequencesinto new bacterial taxonomy. Appl Environ Microbiol 73:5261–5267.FEMS Microbiol Lett 359 (2014) 81–84ª 2014 The Authors. FEMS Microbiology Letterspublished by John Wiley & Sons Ltd on behalf of Federation of European Microbiological Societies.84 L. Dekker et al.

Isolation and identification of cobalt and caesium resistant bacteria from a nuclear fuel storage pond.

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Isolation and identification of cobalt- and caesium-resistant bacteria from a nuclear fuel storage pond

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