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Aug 27, 2012 - The production of volatile polonium (Pov), a naturally occurring radioactive element, by pure cultures of aerobic marine tellurite-resi...
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Volatile Dimethyl Polonium Produced by Aerobic Marine Microorganisms Andrew 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 Université de Pau et des Pays de l’Adour, 64053 Pau, France S Supporting Information *

ABSTRACT: The production of volatile polonium (Pov), a naturally occurring radioactive element, by pure cultures of aerobic marine telluriteresistant microorganisms was investigated. Rhodotorula mucilaginosa, a carotogenic 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. Gas chromatography-inductively coupled plasma-mass spectrometry (GCICP-MS) analysis identified dimethyl polonide (DMPo) as the predominant volatile Po compound in culture headspace of the yeast. This species assignment is based on the exact relation between GC retention times and boiling points of this and other Group VI B analogues (S, Se, and Te). The extent of the biotic Pov production correlates exponentially with elevated particulate Po (Pop): dissolved Po (Poaq) ratios in the cultures, consistent with efficient Po bioaccumulation. Further experimentation demonstrated that some abiotic Pov generation is possible. However, high-level Pov generation in these cultures is predominantly biotic.

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and nonsterile Florida groundwater.3 Volatile Po has been trapped from the headspace of cultures inoculated with sediments16 and pure bacterial strains,17 though the molecular identities of Pov compounds has not yet been established. In nature, many heavy metal(loid)s undergo both biotic18,19 and abiotic20,21 trans-alkylation reactions leading to volatile, methylated species. This study investigated Po volatilization using pure cultures of tellurite (TeO32‑) resistant (TeR) microorganisms isolated from Delaware saltmarsh sediment. These organisms were utilized as previously demonstrated to produce volatile methyl compounds of Te, Se, and S (predominantly DMTe, DMSe, and DMS).22,23 Our hypothesis was that TeR microbes capable of volatile methyl Te production should also volatilize Po. In fact the organisms studied here that are robust Pov producers (the yeast R. mucilaginosa and the gram positive bacterium Bacillus sp.), are common members of saltmarsh microbial communities that could contribute to previously documented Pov presence in this atmospheric environment.5

INTRODUCTION Polonium-210 (210Po) occurs naturally as the last radioactive member of the Uranium-238 decay series and has proven to be useful as a tracer for environmental carbon cycles on time scales concordant with its half-life (T1/2 = 138 days). Polonium is subject to assimilation by biota and associated with the cycling of organic matter in aquatic systems serving as a tracer of downward particulate organic carbon flux and regeneration in the oceanic water column, as well as tracing the dynamics of S biogeochemistry in the aquatic carbon cycle.1−4 A correlation exists between excess atmospheric Po and maximum wind speed,5 similar to that of other biogenic volatile gases in the Group VIA series such as dimethyl sulfide (DMS) and dimethyl selenide (DMSe) where concentrations and fluxes in surface oceanic waters are comparable.6 As previously reported,7 radionuclide tracer applications for Po are complicated by surface water exchange mechanisms8−10 that could include biovolatilization.11 For example, Po biovolatilization, along with atmospheric input from volcanic activity12 or combustion,13 has been inferred from discrepancies between the atmospheric residence times of aerosols calculated with 210Po/210Pb ratios and those using other radon daughter pairs.14,15 Direct evidence for biological production of Pov is scarce, yet Po loss was observed from bread mold cultures11 © 2012 American Chemical Society

Received: Revised: Accepted: Published: 11402

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EXPERIMENTAL SECTION Microorganisms, Growth Medium, and Reagents. The TeR microbes used in this studyBacillus spp. strains 6A and 28A, Virgibacillus halodenitrif icans (strain 14B), and Rhodotorula mucilaginosa (strains 1A, 13B, and 30B)and the culture medium, Luria−Bertani (LB)-Marine, are described in detail elsewhere.22 Bacillus subtilis (strain FB17) and Escherichia coli (strain DH5α) were obtained from the laboratory collections of Harsh Bais (University of Delaware) and Thomas Hanson, respectively. Polonium-209 (t1/2 = 103 years) is an artificial isotope that was obtained in soluble nitrate form in 5 M nitric acid from Eckert & Ziegler Isotope Products (Valencia, California), and the pure quantity certified to 3% as 209Po . A secondary dilution used for culture amendment was also made also in 5 M nitric acid. The 209Po solution (about 10 Bq per culture) was evaporated at 80 °C to a volume less than 1 mL, diluted with water, and pH adjusted to approximately 8.1 using 3 M NaOH. This dilution is assumed to have retained the original nitrate speciation that was added to LB-Marine medium and sterilized by filtration prior to inoculation. The 210 Po, prepared from IAEA RGU-1 Standard, was used as the yield tracer for the radiometric assays. Incubating and Trapping Volatile Polonium. The volatile-trapping incubation apparatus was a series of four tubes capped with pierced silicone stoppers and connected by flexible FEP tubing as diagramed in Figure S1 of the Supporting Information (SI). Pure cultures and reaction mixtures (10 mL) were amended with approximately 10 Bq of 209Po activity (600 dpm or 78 femto moles) and processed as described under Section 1 of the SI. The exact amendment with the respective phase distributions for each experiment is given in Table S1 of the SI. The cultures were incubated in the apparatus for 10 days (20-day incubation period for Bacillus sp. strain 28A). All culture samples were inoculated to a density of approximately 5 × 105 cells/mL. To quantify the concentration of viable cells at the end of each experiment, the number of colony forming units (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 cell pellet (particulate fraction), cotton vessel (droplet trap), and nitric acid traps (volatile fraction, Pov) (see SI for additional detail). Polonium was plated from acidified solution (see SI sec. 2) onto silver discs using standard methodology24 and quantified by α particle spectrometry. Counts were background-corrected and 210Po was corrected for decay between the plating and midcount dates. Propagated error was calculated based on 1σ counting statistics (i.e., N1/2/N). Volatile Species Identification. The chemical identification was made of the volatile Po (Pov) produced in the headspace during the incubation of the microbial yeast culture (Rhodotorula mucilaginosa, strain 13B). A headspace (HS)-solid microfiber extraction (SPME) technique was used to collect semiquantitatively the Pov and introduced to a highly sensitive gas chromatography (GC) hyphenated to an inductively coupled plasma mass spectrometer (ICPMS) as detailed in Section 2 of the SI. Inhibitor and Cell-Free Experiments. The chemical inhibition experiment took place in two stages, where stage one was identical to the standard incubation (above). For stage two, the culture was transferred into an identical sterile FEP

culture tube that was amended with either bronopol (2-bromo2-nitro-1,3-propanediol, Sigma-Aldrich, 1% w/v) or CTAB (cetyl trimethylammonium bromide, Acros Organics, 0.1% w/v final); control samples received no biocide (i.e., untreated cultures) 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 were autoclaved for 15 min at 121 °C. After cooling, they were amended with sterile 209Po and attached to the apparatus for 10 days. Cell extract (CE) was prepared from R. mucilaginosa 13B cells grown in 2 L of LB-Marine disrupted by sonication (see SI). Cell-free reaction mixtures contained 40 μg protein/mL as determined by Bradford assay (Bio-Rad) and were prepared as follows: CE + MeI (methyl iodide, Sigma-Aldrich, 100 μM); Po + CE; Po + CE + MeI; Po + Boiled CE (to denature proteins in the CE); and, Po + MeI and incubated as above.

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RESULTS AND DISCUSSION Volatile Speciation Indicates Microbial Formation of DMPo (Dimethyl Polonide). Volatile speciation was performed on headspace of R. mucilaginosa 13B cultures using SPME-HRGC-ICPMS as described in Section 3 of the SI. The species identification is based on (1) mass 209 traces showing the proposed DMPo species from R. mucilaginosa liquid culture headspace, yet lacking in culture controls supplemented with Bi, a potential monoisotopic mass 209 interfering element (SI Figure S2), and (2) highly linear GC retention times versus measured boiling points of known methylated Group 6A species for S, Se and Te closely match that predicted for DMPo (SI Figure S3). The HS-SPME-GCICPMS analysis of headspace confirms the identity of dimethyl polonide (DMPo) as the primary volatile species (see SI). Organo-compound identification based on retention time boiling point relationship has also been performed in previous works for which natural concentration levels were extremely low and standards not available.25 Some of these compounds were then later confirmed by GCMS techniques when concentrations were high enough.26 Analogous experiments done using the similar18 or same organisms amended with Te or Se addition have produced relatively large amounts of DMTe or DMSe.22,23 This confirms that the Po analogue is the most probable compound that could be produced under these culture conditions. While the chemical speciation of Po in the culture medium is unknown, it is likely to include both inorganic species as nitrate used to amend the cultures, and organic/alkylated species including the volatile methyl species (DMPo) identified in the headspace. Microbes Convert Dissolved Po to Both Particulate and Volatile forms. The amended activity of approximately 10 Bq (600 dpm or 78 femto moles) was chosen to be sufficient for quantifying within reasonable counting periods the Po distribution between all phases. While the amended activity exceeds the 210Po activity of natural samples (atto moles for a much shorter half-life of 138 days), both are sufficiently small in concentration to be representative. The activity for each amendment in the culture and that of the distributed phases is given in Table S1 of the SI. The percentage activity yields reported as follows are equivalent to mole percent for the given 209 isotope. Polonium phase partitioning was quantified in pure cultures of six strains of TeR microbes (Figure 1): three isolates of the yeast Rhodotorula mucilaginosa (strains 1A, 13B, and 30B; mean 11403

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liquid and the cotton plug were assayed separately, nearly all Po was recovered from the cotton in R. mucilaginosa traps while the opposite was true for the bacterial cultures. Furthermore, the Po mass balance (Po recovered versus Po amended) was generally poorer from R. mucilaginosa cultures (84 ± 11%) than from the other strains (92 ± 4%), suggesting that the unaccounted activity probably remained bound to the cotton after the HCl rinsing. This difference corroborates the previous observation that R. mucilaginosa strains produced higher quantities and a wider variety of methyl Te and S compounds when grown in the presence of tellurite.22 We hypothesize that the same is true for volatile Po species produced by R. mucilaginosa, and that some of these species may be more reactive with organic materials (cotton) than those produced by bacterial strains. The dissolved and particulate fractions are traditionally the two phases differentiated in environmental water samples for trace element analysis. Therefore, the relationship between these media, specifically the particulate-Po:dissolved-Po ratio (P:D ratio), and the Po volatilization rate was examined for these experiments. Exponential regression analysis shows a positive curvilinear correlation (r2 = 0.85; p < 0.01) between Pov amount and P:D ratio (Figure 2). While the cause of this

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

values shown) and three distinct Bacillus/Virgibacillus spp. (strains 6A, 14B, 28A). Wild type strains of B. subtilis and E. coli were included in the study as controls for comparison to a prior study.27 There was considerable variation in particulate and volatile phase partitioning observed between strains. The particulate fraction contained 47−90% of the amended Po, which is consistent with the tendency of Po to be strongly assimilated in aquatic ecosystems and enriched in biogenic particles.3,7,8 The particulate fraction includes Po within cells or bound to extracellular material, but these two fractions were not distinguishable in these experiments. Based on the fact that these strains precipitate Te(0) as intracellular particles,22 we hypothesize 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. The Bacilus sp. strain 6A generated about 8% of amended Po (Figure 1). The three R. mucilaginosa strains were similar among each other in Pov productivity, which averaged 1.6% of amended Po. E. coli DH5α was the only other strain that produced significant quantities of Pov (0.2%). Volatilization rates were normalized to culture volume to compare with previous rates determined with bread mold (femto moles Pov/ day/g bread)11 and microbial cultures (% Pov/day).16 The results indicate that the average Po volatilization rates from the previous two studies fall within the range of those observed here. Momoshima et al.16 also observed order of magnitude differences between strains, in the order: B. subtilis < E. coli < Chromobacterium violaceum and a similar range of values across distinct 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 nitric acid traps. Analysis of Po retained by the cotton droplet traps indicate that the amount of Pov recovered in the nitric acid traps is a conservative estimate of the total Pov for R. mucilaginosa. First, Po trapped by the R. mucilaginosa cotton droplet traps was between 8 and 19% of amended Po, whereas for other strains this trap always contained less than 5% of amended Po, with less than 1% in most cases. When trapped

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

correlation is not clearly understood, it can be hypothesized that microbial reductive processes leading to particulate Po production, simultaneously and/or resultantly, promote the production of reduced volatile compounds as well. This relationship suggests that when only the particulate and dissolved phases are measured, and the P:D ratios are elevated, there may be significant Pov production. On a Pacific Ocean transect that measured Po in the surface water (0−1 m), the P:D ratios clustered from 0.5 to 1,28 while in salt marshes much higher P:D ratios have been reported.29 Based on the observation here that P:D ratios >10 show high Pov production, salt marshes that host the microbes studied here could be robust effusive sources of biogenic Pov species. This is consistent with excess 210Po relative to 222Rn support observed 11404

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in the atmosphere downwind of Delaware salt marshes.7 The difference between the P:D ratios observed in ocean surface waters,30 and that observed here in culture or in salt marshes, may be due to Po scavenged by bacteria and plankton that are subsequently be removed by grazers and undergo further trophic transfer.8 Po Volatilization Largely Requires Metabolic Turnover. The data above clearly indicate Po volatilization is mediated by tellurite resistant microbes, but do not address whether the formation of Pov requires active metabolism or can be mediated simply by reacting with organic matter. Momoshima, et al.16 concluded that Pov was produced by metabolic activity after hydrogen peroxide, low temperature (0 °C), and antibiotics (penicillin G) inhibited its production by microbial cultures. However, penicillin G gave a mixed result, and there was no demonstration that Pov compounds are stable in the presence of hydrogen peroxide or significantly volatile at 0 °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 and leakage;31 therefore, it might also facilitate abiotic reactions by increasing the availability of cellular material to interact directly with Po while inhibiting biotic reactions that rely on metabolism. The CTAB addition dramatically decreased the rates of Pov formation in replicated cultures of R. mucilaginosa (Figure 3a) and Bacillus sp. strain 6A (Figure 3b), while

tube containing CTAB-amended medium prior to the acid traps. For the control sample (medium only), the intermediate tube contained approximately 10% of the total Pov. The CTABamended medium contained 38% of the total Pov. Thus, while CTAB did prevent some transfer of Pov, the result indicates that this cannot account for the total inhibition of Pov production in treated cultures (Figure 3). This was not the case with Bronopol, another inhibitor evaluated, which was similarly found to inhibit Po volatilization in live cultures. However, in the associated control experiment, Bronopol amended medium in the intermediate tube trapped 100% of Pov. The ability of autoclaved cultures to form Pov was also tested (Figure 4, solid bars). Viability testing confirmed the absence of

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

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. These values are roughly one and 2 orders of magnitude less than the amounts observed in the respective live cultures of these strains (Figure 4, striped bars). This result indicates that while abiotic reactions can produce Pov, sustained production requires metabolic turnover. The particulate fractions from the autoclaved cultures contained 57 and 66% of the total amended spike for autoclaved R. mucilaginosa 13B and Bacillus sp. strain 6A, respectively, which falls at the lower range for their respective live cultures. Therefore, viability is not required for the transfer of dissolved Po to the particulate fraction. These observations raise the question whether in live cultures the Po is actually assimilated or whether it merely adsorbs externally and/or complexes with cellular material. The variability in the particulate fraction of live cultures suggests different scavenging mechanisms 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 in cell extracts.21 Here, it was hypothesized that cell extract (CE) would react with inorganic Po to form Pov, and that this reaction would be stimulated by the addition of methyl iodide (MeI) as a methyl donor. Viability plating before and after the 10-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 experimental replicates; + and − signs refer to the presence and absence of CTAB. During stage 1, all cultures were incubated without CTAB (−) for two weeks. In stage 2, CTAB was added to the cultures (+) and the incubation continued for an additional two weeks. Error bars represent the 1σ counting statistics propagated error. Please note that the y-axis is a logarithmic scale.

untreated cultures maintained or increased the rate of Po volatilization. As untreated cultures generated similar Pov quantities over two consecutive two-week intervals, the volatilization is expected to be a sustained process (e.g., not limited to exponential growth phase). One of the Bacillus sp. strain 6A replicates showed poor rates of Po volatilization prior to CTAB addition. Spread plating of cultures after CTAB addition confirmed that no viable cells remained in the treated cultures. A separate experiment tested the possibility that CTAB might directly react with Pov compounds by passing Pov containing headspace from cultures through an intermediate 11405

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in these experiments. Reaction mixtures containing MeI indeed produced Pov (Figure 4, MeI, 6.5 ± 0.7 mBq 209Po), but produced less than 1% of the level of live cultures and CE did not influence this reaction (Figure 4, MeI + CE, 6.3 ± 0.5 mBq). In a previous study, an increase in Pov production was observed by adding methylcobalamin (MeB12) to a microbial culture, but not from the addition of MeI.32 Thus, these experiments may need to be extended to include additional methyl donors or alternative conditions to replicate the abiotic production of Pov suggested by CTAB and autoclave inhibition experiments. Interestingly, the amounts of Pov produced by autoclaved cultures and CE mixtures (i.e., abiotic conditions) did not differ statistically (p = 0.37, 1-tailed t-test) from those produced by the 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-level volatilizing strains (R. mucilaginosa and Bacillus sp. Strain 6A) together are significantly different from amounts generated by the aforementioned abiotic conditions and low-level live cultures (p = 0.014 and 0.013, respectively, 1-tailed t test). This result suggests that some microbes may support a basal level of Pov production based on abiotic reactions, whereas others, such as R. mucilaginosa and Bacillus sp. strain 6A, have additional metabolic mechanisms that dramatically enhance Po volatilization (Figure 4). Several analogous mechanisms have been proposed for DMSe and DMTe formation,18 but none have been clearly identified or demonstrated for microorganism production. Generally abiotic formation is largely overwhelmed by biotic processes, and thus not further considered. While there is no clear hypothesis how DMPo could be produced by either biotic or abiotic means, similarly to Hg or Sn compounds, some abiotic production has always been detected.21 Nonetheless, one of the important findings of this work is the observation of abiotic production of volatile Pov. Another is the similarity in Pov output observed among abiotic conditions and some live cultures. There is also a clear distinction between live cultures of certain microbes examined (R. mucilaginosa and Bacillus sp. strain 6A), demonstrating order(s) of magnitude higher output. The authors conclude that the results yield at least the following two categories of Po volatilization: (i) low, background levelproduced under abiotic and/or biotic conditions; and, (ii) high levelmediated exclusively by certain 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 needs to be expanded to additional organisms that are more broadly representative of marine ecosystems. These could include nitrogen fixing cyanobacteria typical of the oligotrophic open ocean that show a preponderance of particulate Po production.7 As demonstrated in this study readily measured physicochemical 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 the two model organisms were isolated, in order to extrapolate the findings of this study to actual measured fluxes of volatile Po to the atmosphere as already in evidence.5

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ASSOCIATED CONTENT

S Supporting Information *

Additional details are provided: (1) the incubation apparatus, operation and analyses, (2) culture experimental procedures, (3) activity data for the cultures, (4) species identification of DMPo, (5) mass spectral operating parameters, (6) mass spectral chromatograms, and (7) GC retention and boiling points. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 302-831-2558; fax: 302-831-4575; e-mail: tchurch@ udel.edu. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by a National Science Foundation grant (OCE-0425199) to T.M.C. and T.E.H. REFERENCES

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dx.doi.org/10.1021/es3006546 | Environ. Sci. Technol. 2012, 46, 11402−11407