Bioresource Technology 172 (2014) 449–452

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Bioresource Technology

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Short Communication

Photosynthetic biomineralization of radioactive Sr via microalgal CO2

absorption

http://dx.doi.org/10.1016/j.biortech.2014.09.0230960-8524/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +82 42 868 4735.E-mail addresses: seungylee@kaeri.re.kr (S.Y. Lee), sylee@sogang.ac.kr (S.-Y. Lee).

1 Equal contribution.

Seung Yeop Lee a,⇑, Kwang-Hwan Jung b,c, Ju Eun Lee d, Keon Ah Lee b, Sang-Hyo Lee c, Ji Young Lee a,Jae Kwang Lee a, Jong Tae Jeong a, Seung-Yop Lee c,d,1

a Korea Atomic Energy Research Institute, Yuseong-Gu, Daejeon 305-353, Republic of Koreab Department of Life Science, Sogang University, 35 Baekbeom-ro, Mapo-Gu, Seoul 121-742, Republic of Koreac Department of Interdisciplinary Program of Integrated Biotechnology, Sogang University, 35 Baekbeom-ro, Mapo-Gu, Seoul 121-742, Republic of Koread Department of Mechanical Engineering, Sogang University, 35 Baekbeom-ro, Mapo-Gu, Seoul 121-742, Republic of Korea

h i g h l i g h t s

� Photosynthetic biomineralization of 90Sr was performed by microalgae.� Sr was crystallized as strontianite (SrCO3) at the biomass/liquid interface.� Chlorella vulgaris carried out the highest 90Sr removal ever reported.� Microalgal carbonating process is one of innovative ways to effectively remove Sr.

a r t i c l e i n f o

Article history:Received 17 July 2014Received in revised form 1 September 2014Accepted 4 September 2014Available online 16 September 2014

Keywords:RadiostrontiumMicroalgaeBiomineralChlorella vulgarisPhotosynthesis

a b s t r a c t

Water-soluble radiostrontium (90Sr) was efficiently removed as a carbonate form through microalgalphotosynthetic process. The immobilization of soluble 90Sr radionuclide and production of highly-precip-itable radio-strontianite (90SrCO3) biomineral are achieved by using Chlorella vulgaris, and the biologicallyinduced mineralization drastically decreased the 90Sr radioactivity in water to make the highest 90Srremoval ever reported. The high-resolution microscopy revealed that the short-term removal of soluble90Sr by C. vulgaris was attributable to the rapid and selective carbonation of 90Sr together with the con-sumption of dissolved CO2 during photosynthesis. A small amount of carbonate in water could act as Sr2+

sinks through the particular ability of the microalga to make the carbonate mineral of Sr stabilized firmlyat the surface site.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Current growing interest in nuclear power as a potential solu-tion for global energy may also raise serious environmental andhealth concerns due to unanticipated nuclear accidents. An enor-mous amount of radioactive nuclides was released during theaccidents of Chernobyl and Fukushima, and some of these reachedthe stratosphere and caused widespread contamination of theenvironment (García-León and Manjón, 1997; Bolsunovsky andDementyev, 2011; Shimura et al., 2012). Of these radioactive nuc-lides, radiostrontium (90Sr) and radiocesium (137Cs) are importantbecause of their long half-life (around 30 years) and high water

solubility (Howard et al., 1991; Robison and Stone, 1992; Dupréde Boulois et al., 2008; Kuwahara et al., 2011; Thorpe et al.,2012). Water-soluble radioactive 90Sr can contaminate aquaticecosystems (Yablokov and Nesterenko, 2009). Radiocesium(137Cs) is also an important radionuclide because of its ability totransfer to living organisms through food chains (Avery, 1996).Thus, developing an effective and economical method for removingthe radionuclides from aquatic ecosystems and radioactivewastewater has become an increasingly important issue.

Conventional methods employed to remove the radionuclidesby mostly chemical precipitation and ion exchange, but they arecostly and/or ineffective for low concentrations with significantlarge volume. Therefore, there is an urgent and indispensable needfor development of innovative but low cost processes to efficientlyremove 90Sr and 137Cs.

There has been rare study for concentrating soluble radionuc-lides into hard materials (e.g., SrCO3) using living organisms for

450 S.Y. Lee et al. / Bioresource Technology 172 (2014) 449–452

the strategy of radioactive wastewater remediation (Krejci et al.,2011; Achal et al., 2012). Especially, the microalgae-driven radio-strontium (90Sr) sequestration by biomineral carbonation (90SrCO3)process has not been reported. The present study focuses on theelimination and mineralization of 90Sr utilizing microalgae, espe-cially at environmentally relevant strontium concentrations andin relation to their activity.

Microalgae (�lm in size) represent an important group in theaquatic environment given their abundance and broad occurrencein fresh and sea water as well as in soils and extreme habitats (Heet al., 2013; Ji et al., 2014). Chlorella is unicellular green microalgaubiquitously present all over the aquatic world. They are autotrophand proliferate well in waters containing low concentrations ofinorganic nutrients. Thus, these species would exhibit great poten-tial in wastewater treatment.

2. Experimental

2.1. Microalgae culture

Chlorella vulgaris (UTEX 26, UTEX Culture Collection of Algae)was cultured in 1.5% Modified Basal Medium (MBM) (Shi et al.,1997) agar plate for 1 month with continuous light at 30 �C.Chlorella sorokiniana ANA9 was cultured in 1.5% YPG media (pep-tone, 10 g; yeast extract, 5 g; glucose, 10 g) agar plate (Yoshidaet al., 2006) for 3 days with continuous light at 30 �C. Each strainwas washed 3 times with 3 mM of NaHCO3 to remove and min-imize any residual media and trace elements for subsequentexperiments. Finally, cells were resuspended in 3 mM ofNaHCO3. Cell viability and pH stability of cell suspension for acertain period of time, 14 days for example, were investigated,confirming that cells could survive in simple sodium bicarbonatesolution, and alkaline pH was maintained around 7.8 to 8.6 (datanot shown).

2.2. Radioisotopes (90Sr and 137Cs) uptake in cell suspension

For the kinetics study, aqueous media (3 mM of NaHCO3) con-taining radionuclides (90Sr and 137Cs) were prepared. Prior to theaddition of radionuclides into the media, their stock solutions for90Sr and 137Cs were prepared in 74,000 and 37,000 Bq/ml radioac-tivity, respectively. From the stock solutions, 90Sr and 137Cs wereaseptically and separately injected as 90SrCl2 (200 and 2000 Bq/ml)and 137CsCl (210 and 2100 Bq/ml). Microalgal cells weredispensed into the radioactive media (30 ml of NaHCO3 3 mM) toprovide ca. 0.1–0.2 million cells/ml in a near-neutral pH condition.The cell suspensions were incubated at 30 �C for 6 days underillumination (1800 lux) of LED. Periodically, liquid radioisotopicsamples were aseptically removed by syringe and needle through0.2 lm cellulose acetate filters and analyzed to determine thechange of radioactivity in the solution using beta or gamma-rayspectroscopy.

2.3. Radioactivity measurement and microscopic analysis

The radioactivity (Bq/ml) of 90Sr in the liquid sample was mea-sured by a liquid scintillation counter (LSC, Perkin Elmer, Tri-Carb2910 TM) in the range of 0–2000 keV for 600 s. A diluted solutionthat has liquid sample (3.9 ml) and distilled water (1.1 ml) in a vialwas mixed with 15 ml of cocktail solution (Ultima Gold™). Theprepared solution was kept in a cold and dark place for 24 h beforea beta-ray measurement by the LSC. The 137Cs radioactivity in adiluted solution that was prepared by mixing liquid sample(1 ml) and distilled water (9 ml) was measured using a gamma-ray spectroscopy (Canberra, Ge detector GC2018) at 661 keV for

120 s. The change of cell numbers in the media with time wasfrequently measured using UV–Vis absorption spectrophotometer(Cary 300, Agilent Technologies) at 686 nm.

To microscopically identify the existence of radionuclides inand around the cellular body, Sr2+ (as stable Sr(NO3)2) or Cs+ (asstable CsCl) was added in the aqueous media to achieve 2.0 mMconcentration. Solid samples (cell + nuclides) were frequentlyremoved by syringe and needle, and then the samples were inves-tigated by using X-ray diffractometer and other microscopic ana-lyzers. For X-ray diffraction (XRD) analysis, the solid phaseseparated from solution by centrifugation (4000 rpm for 10 min)was frozen and freeze-dried for 48 h by a freeze dryer (Bondiro,Ilshin Co). The XRD analysis was performed with a Bruker D8Advance automatic horizontal goniometer diffractometerequipped with a scintillation counter and a Cu X-ray tube operat-ing at 40 kV/30 mA in a continuous scan mode. For the electronmicroscopic observation, the microalgal suspension was centri-fuged, frozen, and dried in the freeze-dryer. The microalgal samplewas uniformly sprayed on a carbon tape pasted on the specimenholder. In order to avoid charging during observation, the samplewas coated with a thin OsO4 (�10 nm) layer. The sample wasobserved using field emission scanning electron microscopy(FESEM; S-4700, Hitach) to acquire its morphological and struc-tural information in detail. Chemical analysis was also carriedout using an energy dispersive X-ray spectrometer (EDS; EMAX,Horiba). HRTEM (high-resolution transmission electron micros-copy) was used to examine the cell and its metabolic byproductin detail, especially for the biogenic structure and crystallinity(Lee et al., 2014). The collected sample was fixed in 2% paraformal-dehyde and 2% glutaraldehyde. After washing it with Na-cacodyl-ate, it was gradually dehydrated by ethanol series and theninfiltrated into Spurr’s resin (see Table S1 for the detailed proce-dure). Cured blocks of the resin sample were sectioned by theultramicrotome diamond knife (MT-X RMC). Ultrathin sections(50–70 nm in thickness) were mounted on copper grids withFormvar support film coated with carbon. Samples were examinedby the JEOL JEM 2100F high-resolution field emission TEM at200 kV. An energy dispersive X-ray spectrometer (EDS) was alsoused to analyze a chemical distribution of isotopes.

3. Results and discussion

3.1. Uptake of radionuclides by microalgae

C. vulgaris and C. sorokiniana cultivated in MBM and YPG media,respectively, were collected by centrifugation and washed threetimes with NaHCO3, 3 mM. The viability of microalgae interactedwith intense radioactive 90Sr and 137Cs was evaluated in differentlevels of radioactive solutions for 6 days. As a result, their survivalpatterns were different from each other according to the radiationeffect on the organisms (Fig. S1). With high radioresistance,C. vulgaris has doubled its biomass within 2 days in a malnutritivecondition. When cultured under illumination, uptake of radio-strontium and radiocesium by the microalgae was observed withtime after the addition of 90SrCl2 and 137CsCl, and maximum rem-ovals of 90Sr and 137Cs by C. vulgaris were P90% and 70% of thetotal 90Sr and 137Cs, respectively (Figs. 1 and S2). Both the microal-gal cultures containing C. vulgaris and C. sorokiniana demonstrateddifferent efficiency for strontium uptake. These results indicatethat the removal rates of strontium highly depend on microalgaestrains. The removal of radiostrontium by C. vulgaris was very fastin 24 h, in which almost 80% of the aqueous 90Sr was removed fromthe solution. Even in 2000 Bq/ml radiation, the dissolved 90Srdecreased quickly and the 90Sr radioactivity was little detectedin solution after several days. However, 90Sr sequestration by

Fig. 2. Conceptual model for a microalgal photosynthetic CO2 absorption (attrac-tion) and surface-carbonating process that largely cause a particular 90Sr carbon-ation, which specifically stabilizes soluble 90Sr by nucleating it with carbonateavailable on the surface.

Fig. 1. Comparative radioactive 90Sr removal by two microalgal strains, C. vulgaris and C. sorokiniana, in different radioactivity levels of (a) 200 Bq/ml and (b) 2000 Bq/ml.

S.Y. Lee et al. / Bioresource Technology 172 (2014) 449–452 451

C. sorokiniana was a little inefficient in higher 90Sr radioactivitywith lower removal rate of 90Sr.

3.2. Photosynthetic biocrystallization of soluble Sr2+

To probe the sequestered Sr kept by the cell, the centrifuged cellsuspension interacted with soluble Sr was pretreated by fixationand dehydration and subsequent polymerization with resin forHRTEM investigation. A cross-section of the polymerized samplewas made using a microtome to a thickness of 50–70 nm. Adetailed procedure for the HRTEM sample preparation is describedin Table S1.

The strontium trapped by C. vulgaris was overall examinedusing SEM-EDS, and a trace amount of Sr (3.47 wt%) was detectedon the cell body (Fig. S3). A byproduct derived from the interactionbetween cell and Sr was grown to micrometer sizes, and it wasfound as inorganic particles including considerable contents ofstrontium (average 40 wt%) within them. The biologically inducedbyproduct had a characteristic feature of elliptical cone morphol-ogy with averages of 5 lm in length and 1 lm in thickness.

C. vulgaris successfully survived in the highly radioactive condi-tions of Sr, displaying excellent biomineralization of soluble Sr2+. Itseems that the organism effectively alleviated high toxic 90Sr bypreventing its inward diffusion and synthesizing a solid phase atthe outer surface. To elucidate the biological removal of Sr, HRTEMwas used to inspect the Sr concentration and distribution over thecell body at the subcellular scale. A nanoscale probe for the sec-tioned specimen has made possible to find numerous fine Sr parti-cles outside rather than inside of the cell body (Fig. S4). The fineparticles seem to be an indirect evidence that microalgae coulddexterously nucleate soluble 90Sr to solid 90Sr aiming at outlivingin the severe radioactive environment.

The exposed cell surface can function to provide proper sites totrap toxic Sr that diffuses into the cell body by combining stron-tium and carbonate. Cell walls are usually composed of heteropoly-saccharides and offer metal-binding functional groups such ascarboxyl, hydroxyl, sulfhydryl, phosphoryl and amino groupswhich cause a negatively charged or polar cell surface (Kalinet al., 2005; Bansal et al., 2012). The functional groups could afforda place for the mobile and toxic Sr ions to be captured and storedup efficiently, limiting the free-diffusion of Sr into the body.

A direct HRTEM probe on the Sr solid associated with the cellsurface wall reveals a way of Sr crystallization facilitated on thesurface as a biological pathway of Sr sequestration (Fig. S5). Sr par-ticles that were observed on the cell wall showed a characteristicfeature of multi-nucleated nanoparticles with typical lattice

fringes of strontianite. HRTEM mapping for the strontium clearlyshowed that most of Sr was locally enriched around the cell wall,while a small fraction of Sr was only detected inside of the cyto-plasm. This result implies that Sr was removed by the microalgaethrough two processes: firstly, Sr is rapidly and efficiently biosyn-thesized as a solid phase on the cell wall, and secondly, a part of Sris taken up into the cytoplasm (Krejci et al., 2011).

3.3. Microalgae-driven Sr solid formation as radio-strontianite(90SrCO3)

Conceptual model for the Sr enrichment at the outer surface ofcell reveals that soluble and toxic Sr2+ ions are trapped by the cellsurface, where 90SrCO3 nucleation and growth are facilitated ratherthan in normal aqueous solution (Fig. 2). The carbonate (e.g., CO3

2�)ions can be directly incorporated into the framework of biologi-cally-forming 90Sr-carbonate phase. This procedure will be termed‘‘biological 90Sr synthesis’’ and the carbonate species that reactwith Sr2+ ions can be available from water-dissolved CO2 chemicals(HCO3

� and CO32�) during active photosynthesis (Nonova and

Tosheva, 2014). There may be an unknown mechanism for promot-ing nucleation and/or facilitating crystal growth of Sr at the

452 S.Y. Lee et al. / Bioresource Technology 172 (2014) 449–452

surface, which convert dissolved species to solid phases. For exam-ple, local supersaturation of carbonate and subsequent pH increaseat the surface of cell could facilitate the growth of crystal nucleiand carbonate-binding process incorporating Sr as a solid phase.It is believed that a specialized photosynthetic Sr carbonating abil-ity and membrane-bound protein of C. vulgaris are responsible forstoring up strontium and carbonate and directing their surfaceassembly into high-ordered structure. Such a microalgae-drivencrystallization is potentially important as a course of photosyn-thetic Sr carbonation to steadily remove 90Sr from radioactivewastewater.

Using X-ray diffraction technique, it was found that a residualsolid obtained from the microalga-free solution was not only abit small in quantity but also a mixture of amorphous strontium-carbonate and sodium-bicarbonate (Fig. S6). However, the biologi-cally produced SrCO3 in the presence of C. vulgaris occurred as awell crystalline phase with highly diffracted intensity of XRD. Itis certain that the biologically-formed strontium carbonate is morestable and crystalliferous than those created by chemically precip-itable Sr from aqueous solution. The structure of biosynthesized Srbyproduct by C. vulgaris was identified as a pure and high denseSrCO3 crystal. However, the solid phase produced by C. sorokinianawas a little amorphous with lower XRD intensity and larger back-ground as compared with that by C. vulgaris. This result shows thatthe uptake and subsequent biocrystallization of Sr are muchaffected by an innate character of specific microalgae.

4. Conclusions

Living C. vulgaris had a potential to remove 90Sr to very low con-centrations in highly radioactive conditions. The strontium trappedby C. vulgaris was confirmed as a carbonate solid phase, strontian-ite (SrCO3). The 90Sr biocrystallization process that becomes struc-turally denser by microalgae is very important to effectively graspsoluble Sr ions and to prevent Sr resolubilization later on. Thisresult manifests that the environment-friendly innovative methodto sequester soluble radiostrontium as an immobile biomineralthrough the microalgae-driven carbonating process could play animportant role to prevent radiostrontium spread to ecologicalenvironments.

Acknowledgements

This research work was supported by the National NuclearR&D Program (2012M2A8A4055325) and (2012M2A8A5025589)through the National Research Foundation (NRF) funded by TheMinistry of Science ICT & Future Planning of Korea. The authorsacknowledge the late Jungmin Kim for motivating us to initiatethis work.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.biortech.2014.09.023.

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