Volatile Dimethyl Polonium Produced by Aerobic MarineMicroorganismsAndrew S. Bahrou,† Patrick R. L. Ollivier,† Thomas E. Hanson,†,‡ Emmanuel Tessier,§ David Amouroux,§

and Thomas M. Church*,†

†School of Marine Science and Policy, College of Earth, Ocean, and Environment, University of Delaware, Newark, Delaware 19716,United States‡Delaware Biotechnology Institute, University of Delaware, Newark, Delaware 19711, United States§Laboratoire de Chimie Analytique BioInorganique et Environnement, IPREM UMR 5254 CNRS et Universite de Pau et des Pays del’Adour, 64053 Pau, France

*S Supporting Information

ABSTRACT: The production of volatile polonium (Pov), a naturallyoccurring radioactive element, by pure cultures of aerobic marine tellurite-resistant microorganisms was investigated. Rhodotorula mucilaginosa, acarotogenic yeast, and a Bacillus sp. strain, a Gram-positive bacterium,generated approximately one and 2 orders of magnitude, respectively,greater amounts of Pov compared to the other organisms tested. Gaschromatography-inductively coupled plasma-mass spectrometry (GC-ICP-MS) analysis identified dimethyl polonide (DMPo) as thepredominant volatile Po compound in culture headspace of the yeast.This species assignment is based on the exact relation between GCretention times and boiling points of this and other Group VI B analogues(S, Se, and Te). The extent of the biotic Pov production correlatesexponentially with elevated particulate Po (Pop): dissolved Po (Poaq) ratios in the cultures, consistent with efficient Pobioaccumulation. Further experimentation demonstrated that some abiotic Pov generation is possible. However, high-level Povgeneration in these cultures is predominantly biotic.

■ INTRODUCTION

Polonium-210 (210Po) occurs naturally as the last radioactivemember of the Uranium-238 decay series and has proven to beuseful as a tracer for environmental carbon cycles on time scalesconcordant with its half-life (T1/2 = 138 days). Polonium issubject to assimilation by biota and associated with the cyclingof organic matter in aquatic systems serving as a tracer ofdownward particulate organic carbon flux and regeneration inthe oceanic water column, as well as tracing the dynamics of Sbiogeochemistry in the aquatic carbon cycle.1−4 A correlationexists between excess atmospheric Po and maximum windspeed,5 similar to that of other biogenic volatile gases in theGroup VIA series such as dimethyl sulfide (DMS) and dimethylselenide (DMSe) where concentrations and fluxes in surfaceoceanic waters are comparable.6

As previously reported,7 radionuclide tracer applications forPo are complicated by surface water exchange mechanisms8−10

that could include biovolatilization.11 For example, Pobiovolatilization, along with atmospheric input from volcanicactivity12 or combustion,13 has been inferred from discrepanciesbetween the atmospheric residence times of aerosols calculatedwith 210Po/210Pb ratios and those using other radon daughterpairs.14,15 Direct evidence for biological production of Pov isscarce, yet Po loss was observed from bread mold cultures11

and nonsterile Florida groundwater.3 Volatile Po has beentrapped from the headspace of cultures inoculated withsediments16 and pure bacterial strains,17 though the molecularidentities of Pov compounds has not yet been established. Innature, many heavy metal(loid)s undergo both biotic18,19 andabiotic20,21 trans-alkylation reactions leading to volatile,methylated species. This study investigated Po volatilizationusing pure cultures of tellurite (TeO3

2‑) resistant (TeR)microorganisms isolated from Delaware saltmarsh sediment.These organisms were utilized as previously demonstrated toproduce volatile methyl compounds of Te, Se, and S(predominantly DMTe, DMSe, and DMS).22,23 Our hypothesiswas that TeR microbes capable of volatile methyl Te productionshould also volatilize Po. In fact the organisms studied here thatare robust Pov producers (the yeast R. mucilaginosa and thegram positive bacterium Bacillus sp.), are common members ofsaltmarsh microbial communities that could contribute topreviously documented Pov presence in this atmosphericenvironment.5

Received: February 21, 2012Revised: August 21, 2012Accepted: August 27, 2012

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■ EXPERIMENTAL SECTION

Microorganisms, Growth Medium, and Reagents. TheTeR microbes used in this studyBacillus spp. strains 6A and28A, Virgibacillus halodenitrif icans (strain 14B), and Rhodotorulamucilaginosa (strains 1A, 13B, and 30B)and the culturemedium, Luria−Bertani (LB)-Marine, are described in detailelsewhere.22 Bacillus subtilis (strain FB17) and Escherichia coli(strain DH5α) were obtained from the laboratory collections ofHarsh Bais (University of Delaware) and Thomas Hanson,respectively. Polonium-209 (t1/2 = 103 years) is an artificialisotope that was obtained in soluble nitrate form in 5 M nitricacid from Eckert & Ziegler Isotope Products (Valencia,California), and the pure quantity certified to 3% as 209Po . Asecondary dilution used for culture amendment was also madealso in 5 M nitric acid. The 209Po solution (about 10 Bq perculture) was evaporated at 80 °C to a volume less than 1 mL,diluted with water, and pH adjusted to approximately 8.1 using3 M NaOH. This dilution is assumed to have retained theoriginal nitrate speciation that was added to LB-Marinemedium and sterilized by filtration prior to inoculation. The210Po, prepared from IAEA RGU-1 Standard, was used as theyield tracer for the radiometric assays.Incubating and Trapping Volatile Polonium. The

volatile-trapping incubation apparatus was a series of fourtubes capped with pierced silicone stoppers and connected byflexible FEP tubing as diagramed in Figure S1 of the SupportingInformation (SI). Pure cultures and reaction mixtures (10 mL)were amended with approximately 10 Bq of 209Po activity (600dpm or 78 femto moles) and processed as described underSection 1 of the SI. The exact amendment with the respectivephase distributions for each experiment is given in Table S1 ofthe SI.The cultures were incubated in the apparatus for 10 days

(20-day incubation period for Bacillus sp. strain 28A). Allculture samples were inoculated to a density of approximately 5× 105 cells/mL. To quantify the concentration of viable cells atthe end of each experiment, the number of colony formingunits (CFUs) was assayed from a culture subsample (10 μL),which was used for dilution series spot plating.Polonium Analyses. Four separate fractions were assayed

for 209Po: culture supernatant (dissolved fraction), culture cellpellet (particulate fraction), cotton vessel (droplet trap), andnitric acid traps (volatile fraction, Pov) (see SI for additionaldetail). Polonium was plated from acidified solution (see SI sec.2) onto silver discs using standard methodology24 andquantified by α particle spectrometry. Counts were back-ground-corrected and 210Po was corrected for decay betweenthe plating and midcount dates. Propagated error wascalculated based on 1σ counting statistics (i.e., N1/2/N).Volatile Species Identification. The chemical identifica-

tion was made of the volatile Po (Pov) produced in theheadspace during the incubation of the microbial yeast culture(Rhodotorula mucilaginosa, strain 13B). A headspace (HS)-solidmicrofiber extraction (SPME) technique was used to collectsemiquantitatively the Pov and introduced to a highly sensitivegas chromatography (GC) hyphenated to an inductivelycoupled plasma mass spectrometer (ICPMS) as detailed inSection 2 of the SI.Inhibitor and Cell-Free Experiments. The chemical

inhibition experiment took place in two stages, where stageone was identical to the standard incubation (above). For stagetwo, the culture was transferred into an identical sterile FEP

culture tube that was amended with either bronopol (2-bromo-2-nitro-1,3-propanediol, Sigma-Aldrich, 1% w/v) or CTAB(cetyl trimethylammonium bromide, Acros Organics, 0.1% w/vfinal); control samples received no biocide (i.e., untreatedcultures) for stage two of the experiment (see SI).For sterilization, 10 mL of R. mucilaginosa sp. strain 13B and

Bacillus sp. strain 6A cultures in stationary phase wereautoclaved for 15 min at 121 °C. After cooling, they wereamended with sterile 209Po and attached to the apparatus for 10days.Cell extract (CE) was prepared from R. mucilaginosa 13B

cells grown in 2 L of LB-Marine disrupted by sonication (seeSI). Cell-free reaction mixtures contained 40 μg protein/mL asdetermined by Bradford assay (Bio-Rad) and were prepared asfollows: CE + MeI (methyl iodide, Sigma-Aldrich, 100 μM); Po+ CE; Po + CE + MeI; Po + Boiled CE (to denature proteins inthe CE); and, Po + MeI and incubated as above.

■ RESULTS AND DISCUSSIONVolatile Speciation Indicates Microbial Formation of

DMPo (Dimethyl Polonide). Volatile speciation wasperformed on headspace of R. mucilaginosa 13B culturesusing SPME-HRGC-ICPMS as described in Section 3 of the SI.The species identification is based on (1) mass 209 tracesshowing the proposed DMPo species from R. mucilaginosaliquid culture headspace, yet lacking in culture controlssupplemented with Bi, a potential monoisotopic mass 209interfering element (SI Figure S2), and (2) highly linear GCretention times versus measured boiling points of knownmethylated Group 6A species for S, Se and Te closely matchthat predicted for DMPo (SI Figure S3). The HS-SPME-GC-ICPMS analysis of headspace confirms the identity of dimethylpolonide (DMPo) as the primary volatile species (see SI).Organo-compound identification based on retention time

boiling point relationship has also been performed in previousworks for which natural concentration levels were extremelylow and standards not available.25 Some of these compoundswere then later confirmed by GCMS techniques whenconcentrations were high enough.26 Analogous experimentsdone using the similar18 or same organisms amended with Teor Se addition have produced relatively large amounts of DMTeor DMSe.22,23 This confirms that the Po analogue is the mostprobable compound that could be produced under theseculture conditions. While the chemical speciation of Po in theculture medium is unknown, it is likely to include bothinorganic species as nitrate used to amend the cultures, andorganic/alkylated species including the volatile methyl species(DMPo) identified in the headspace.

Microbes Convert Dissolved Po to Both Particulateand Volatile forms. The amended activity of approximately10 Bq (600 dpm or 78 femto moles) was chosen to be sufficientfor quantifying within reasonable counting periods the Podistribution between all phases. While the amended activityexceeds the 210Po activity of natural samples (atto moles for amuch shorter half-life of 138 days), both are sufficiently small inconcentration to be representative. The activity for eachamendment in the culture and that of the distributed phases isgiven in Table S1 of the SI. The percentage activity yieldsreported as follows are equivalent to mole percent for the given209 isotope.Polonium phase partitioning was quantified in pure cultures

of six strains of TeR microbes (Figure 1): three isolates of theyeast Rhodotorula mucilaginosa (strains 1A, 13B, and 30B; mean

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values shown) and three distinct Bacillus/Virgibacillus spp.(strains 6A, 14B, 28A). Wild type strains of B. subtilis and E. coliwere included in the study as controls for comparison to a priorstudy.27 There was considerable variation in particulate andvolatile phase partitioning observed between strains. Theparticulate fraction contained 47−90% of the amended Po,which is consistent with the tendency of Po to be stronglyassimilated in aquatic ecosystems and enriched in biogenicparticles.3,7,8 The particulate fraction includes Po within cells orbound to extracellular material, but these two fractions were notdistinguishable in these experiments. Based on the fact thatthese strains precipitate Te(0) as intracellular particles,22 wehypothesize that the Po has been internalized and precipitated.The most prolific Po volatilizing strains were Bacillus sp.

strain 6A and R. mucilaginosa strains 1A, 13B and 30 B. TheBacilus sp. strain 6A generated about 8% of amended Po(Figure 1). The three R. mucilaginosa strains were similaramong each other in Pov productivity, which averaged 1.6% ofamended Po. E. coli DH5α was the only other strain thatproduced significant quantities of Pov (0.2%). Volatilizationrates were normalized to culture volume to compare withprevious rates determined with bread mold (femto moles Pov/day/g bread)11 and microbial cultures (% Pov/day).

16 Theresults indicate that the average Po volatilization rates from theprevious two studies fall within the range of those observedhere. Momoshima et al.16 also observed order of magnitudedifferences between strains, in the order: B. subtilis < E. coli <Chromobacterium violaceum and a similar range of values acrossdistinct strains were observed here, with B. subtilis/V.halodenitrif icans strain 14B < Bacillus sp. strain 28A < E. coli< R. mucilaginosa < Bacillus sp. strain 6A.The incubation apparatus included a cotton droplet trap to

prevent the liquid phase aerosols from transferring to the nitricacid traps. Analysis of Po retained by the cotton droplet trapsindicate that the amount of Pov recovered in the nitric acidtraps is a conservative estimate of the total Pov for R.mucilaginosa. First, Po trapped by the R. mucilaginosa cottondroplet traps was between 8 and 19% of amended Po, whereasfor other strains this trap always contained less than 5% ofamended Po, with less than 1% in most cases. When trapped

liquid and the cotton plug were assayed separately, nearly all Powas recovered from the cotton in R. mucilaginosa traps whilethe opposite was true for the bacterial cultures. Furthermore,the Po mass balance (Po recovered versus Po amended) wasgenerally poorer from R. mucilaginosa cultures (84 ± 11%) thanfrom the other strains (92 ± 4%), suggesting that theunaccounted activity probably remained bound to the cottonafter the HCl rinsing. This difference corroborates the previousobservation that R. mucilaginosa strains produced higherquantities and a wider variety of methyl Te and S compoundswhen grown in the presence of tellurite.22 We hypothesize thatthe same is true for volatile Po species produced by R.mucilaginosa, and that some of these species may be morereactive with organic materials (cotton) than those produced bybacterial strains.The dissolved and particulate fractions are traditionally the

two phases differentiated in environmental water samples fortrace element analysis. Therefore, the relationship betweenthese media, specifically the particulate-Po:dissolved-Po ratio(P:D ratio), and the Po volatilization rate was examined forthese experiments. Exponential regression analysis shows apositive curvilinear correlation (r2 = 0.85; p < 0.01) betweenPov amount and P:D ratio (Figure 2). While the cause of this

correlation is not clearly understood, it can be hypothesizedthat microbial reductive processes leading to particulate Poproduction, simultaneously and/or resultantly, promote theproduction of reduced volatile compounds as well. Thisrelationship suggests that when only the particulate anddissolved phases are measured, and the P:D ratios are elevated,there may be significant Pov production. On a Pacific Oceantransect that measured Po in the surface water (0−1 m), theP:D ratios clustered from 0.5 to 1,28 while in salt marshes muchhigher P:D ratios have been reported.29 Based on theobservation here that P:D ratios >10 show high Pov production,salt marshes that host the microbes studied here could berobust effusive sources of biogenic Pov species. This isconsistent with excess 210Po relative to 222Rn support observed

Figure 1. Polonium phase partitioning in pure liquid cultures ofmarine microorganisms. N = 2 for all cultures except R. mucilaginosa, N= 6, and the cell-free control, N = 1. The Po mass balance (Porecovered versus Po amended) was generally poorer from R.mucilaginosa cultures (84 ± 11%) than from the other strains (92 ±4%).

Figure 2. Particulate: Dissolved Po ratios correlate with volatile Po. Aregression analysis was performed based on exponential statistics(LOGEST), and a positive curvilinear correlation (r2 = 0.85) wasfound to be significant (p < 0.01). Error bars represent the standarddeviation for R. mucilaginosa cultures (N = 6) and the ranges for allothers (N = 2). Please note that both axes are on a logarithmic scale.

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in the atmosphere downwind of Delaware salt marshes.7 Thedifference between the P:D ratios observed in ocean surfacewaters,30 and that observed here in culture or in salt marshes,may be due to Po scavenged by bacteria and plankton that aresubsequently be removed by grazers and undergo furthertrophic transfer.8

Po Volatilization Largely Requires Metabolic Turn-over. The data above clearly indicate Po volatilization ismediated by tellurite resistant microbes, but do not addresswhether the formation of Pov requires active metabolism or canbe mediated simply by reacting with organic matter.Momoshima, et al.16 concluded that Pov was produced bymetabolic activity after hydrogen peroxide, low temperature (0°C), and antibiotics (penicillin G) inhibited its production bymicrobial cultures. However, penicillin G gave a mixed result,and there was no demonstration that Pov compounds are stablein the presence of hydrogen peroxide or significantly volatile at0 °C.The quaternary ammonium compound, CTAB, was found to

be an effective biocide against both Bacillus sp. strain 6A and R.mucilaginosa 13B. CTAB induces membrane permeability andleakage;31 therefore, it might also facilitate abiotic reactions byincreasing the availability of cellular material to interact directlywith Po while inhibiting biotic reactions that rely onmetabolism. The CTAB addition dramatically decreased therates of Pov formation in replicated cultures of R. mucilaginosa(Figure 3a) and Bacillus sp. strain 6A (Figure 3b), while

untreated cultures maintained or increased the rate of Povolatilization. As untreated cultures generated similar Povquantities over two consecutive two-week intervals, thevolatilization is expected to be a sustained process (e.g., notlimited to exponential growth phase). One of the Bacillus sp.strain 6A replicates showed poor rates of Po volatilization priorto CTAB addition. Spread plating of cultures after CTABaddition confirmed that no viable cells remained in the treatedcultures.A separate experiment tested the possibility that CTAB

might directly react with Pov compounds by passing Povcontaining headspace from cultures through an intermediate

tube containing CTAB-amended medium prior to the acidtraps. For the control sample (medium only), the intermediatetube contained approximately 10% of the total Pov. The CTAB-amended medium contained 38% of the total Pov. Thus, whileCTAB did prevent some transfer of Pov, the result indicatesthat this cannot account for the total inhibition of Povproduction in treated cultures (Figure 3). This was not thecase with Bronopol, another inhibitor evaluated, which wassimilarly found to inhibit Po volatilization in live cultures.However, in the associated control experiment, Bronopolamended medium in the intermediate tube trapped 100% ofPov.The ability of autoclaved cultures to form Pov was also tested

(Figure 4, solid bars). Viability testing confirmed the absence of

live cells at the beginning and end of the incubations.Autoclaved samples of R. mucilaginosa 13B and Bacillus sp.strain 6A generated 15 and 8.3 mBq Pov, respectively. Thesevalues are roughly one and 2 orders of magnitude less than theamounts observed in the respective live cultures of these strains(Figure 4, striped bars). This result indicates that while abioticreactions can produce Pov, sustained production requiresmetabolic turnover.The particulate fractions from the autoclaved cultures

contained 57 and 66% of the total amended spike forautoclaved R. mucilaginosa 13B and Bacillus sp. strain 6A,respectively, which falls at the lower range for their respectivelive cultures. Therefore, viability is not required for the transferof dissolved Po to the particulate fraction. These observationsraise the question whether in live cultures the Po is actuallyassimilated or whether it merely adsorbs externally and/orcomplexes with cellular material. The variability in theparticulate fraction of live cultures suggests different scavengingmechanisms exist among the organisms (Figure 1).An attempt was made to reconstitute Pov formation in cell

free extracts, based on prior studies of mercury methylation incell extracts.21 Here, it was hypothesized that cell extract (CE)would react with inorganic Po to form Pov, and that thisreaction would be stimulated by the addition of methyl iodide(MeI) as a methyl donor. Viability plating before and after the10-day incubation period confirmed that no cells were present

Figure 3. CTAB inhibits polonium volatilization. Volatile Po activity(mBq) is represented as bars and numbers indicate experimentalreplicates; + and − signs refer to the presence and absence of CTAB.During stage 1, all cultures were incubated without CTAB (−) for twoweeks. In stage 2, CTAB was added to the cultures (+) and theincubation continued for an additional two weeks. Error bars representthe 1σ counting statistics propagated error. Please note that the y-axisis a logarithmic scale.

Figure 4. Volatile polonium production under biotic conditionsgreatly exceeds abiotic conditions, compared using live and cell-free ordead cultures. Methyl iodide (MeI); Cell extract (CE); error barsrepresent 1σ counting statistics propagated error (see text for details).Please note that the y-axis is a logarithmic scale.

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in these experiments. Reaction mixtures containing MeI indeedproduced Pov (Figure 4, MeI, 6.5 ± 0.7 mBq 209Po), butproduced less than 1% of the level of live cultures and CE didnot influence this reaction (Figure 4, MeI + CE, 6.3 ± 0.5mBq). In a previous study, an increase in Pov production wasobserved by adding methylcobalamin (MeB12) to a microbialculture, but not from the addition of MeI.32 Thus, theseexperiments may need to be extended to include additionalmethyl donors or alternative conditions to replicate the abioticproduction of Pov suggested by CTAB and autoclave inhibitionexperiments.Interestingly, the amounts of Pov produced by autoclaved

cultures and CE mixtures (i.e., abiotic conditions) did not differstatistically (p = 0.37, 1-tailed t-test) from those produced bythe group of live culture low-level Po volatilizing strains (i.e.,Bacillus sp. strain 28A, B. subtilis, V. halodenitrif icans, and E.coli). In contrast, the Pov amounts from the pair of high-levelvolatilizing strains (R. mucilaginosa and Bacillus sp. Strain 6A)together are significantly different from amounts generated bythe aforementioned abiotic conditions and low-level livecultures (p = 0.014 and 0.013, respectively, 1-tailed t test).This result suggests that some microbes may support a basallevel of Pov production based on abiotic reactions, whereasothers, such as R. mucilaginosa and Bacillus sp. strain 6A, haveadditional metabolic mechanisms that dramatically enhance Povolatilization (Figure 4).Several analogous mechanisms have been proposed for

DMSe and DMTe formation,18 but none have been clearlyidentified or demonstrated for microorganism production.Generally abiotic formation is largely overwhelmed by bioticprocesses, and thus not further considered. While there is noclear hypothesis how DMPo could be produced by either bioticor abiotic means, similarly to Hg or Sn compounds, someabiotic production has always been detected.21

Nonetheless, one of the important findings of this work is theobservation of abiotic production of volatile Pov. Another is thesimilarity in Pov output observed among abiotic conditions andsome live cultures. There is also a clear distinction between livecultures of certain microbes examined (R. mucilaginosa andBacillus sp. strain 6A), demonstrating order(s) of magnitudehigher output. The authors conclude that the results yield atleast the following two categories of Po volatilization: (i) low,background levelproduced under abiotic and/or bioticconditions; and, (ii) high levelmediated exclusively bycertain organisms under biotic conditions.The results of this study as presented offer merit for further

investigation, for example:(i) Conclusive evidence presented for volatile Po production

by two cosmopolitan microbes from saltmarsh sediment needsto be expanded to additional organisms that are more broadlyrepresentative of marine ecosystems. These could includenitrogen fixing cyanobacteria typical of the oligotrophic openocean that show a preponderance of particulate Po production.7

As demonstrated in this study readily measured physicochem-ical parameters such as particulate-Po: dissolved-Po (P:D)ratios may also serve as a proxy for Pov production. (ii)Determination of the specific biotic involvement (e.g.,enzymatic action, metabolic turnover, genetic markers.). (iii)Designing field experiments, such as in salt marshes where thetwo model organisms were isolated, in order to extrapolate thefindings of this study to actual measured fluxes of volatile Po tothe atmosphere as already in evidence.5

■ ASSOCIATED CONTENT*S Supporting InformationAdditional details are provided: (1) the incubation apparatus,operation and analyses, (2) culture experimental procedures,(3) activity data for the cultures, (4) species identification ofDMPo, (5) mass spectral operating parameters, (6) massspectral chromatograms, and (7) GC retention and boilingpoints. This material is available free of charge via the Internetat http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*Phone: 302-831-2558; fax: 302-831-4575; e-mail: tchurch@udel.edu.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis research was supported by a National Science Foundationgrant (OCE-0425199) to T.M.C. and T.E.H.

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