Biomineralization of Gold in Biofilms of Cupriavidus metallidurans

Feb 13, 2013 - While Au coatings on quartz grains were detected throughout the section, higher concentrations of Au were observed in the top 10 mm (Fi...
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Biomineralization of Gold in Biofilms of Cupriavidus metallidurans L. Fairbrother,†,‡ B. Etschmann,§ J. Brugger,§,∥ J. Shapter,‡ G. Southam,⊥ and F. Reith‡,§,* †

School of Chemical and Physical Sciences, Flinders University, Adelaide, SA 5001, Australia CSIRO Land and Water, Environmental Biogeochemistry, PMB2, Glen Osmond, SA 5064, Australia § Centre for Tectonics, Resources and Exploration (TRaX), School of Earth and Environmental Sciences, The University of Adelaide, North Terrace, SA 5005, Adelaide, Australia ∥ Division of Mineralogy, South Australian Museum, Adelaide, SA 5000, Australia ⊥ School of Earth Sciences, The University of Queensland, Brisbane, QLD 4072, Australia ‡

S Supporting Information *

ABSTRACT: Cupriavidus metallidurans, a bacterium capable of reductively precipitating toxic, aqueous gold(I/III)-complexes, dominates biofilm communities on gold (Au) grains from Australia. To examine the importance of C. metallidurans biofilms in secondary Au formation, we assessed the biomineralization potential of biofilms growing in quartz-sandpacked columns to periodic amendment with Au(I)-thiosulfate. In these experiments, >99 wt % of Au, was retained compared to 99 wt % Au, compared to primary Au that forms from hydrothermal fluids in the deep subsurface and contains 5−30 wt % Ag.13 Secondary Au is finely crystalline (0.01 to 109 cfu mL−1, and remained constant thereafter. The increased concentrations of cells in outlet solutions may represent a transition from a biofilm to planktonic growth strategy, to avoid the toxic effect of the Au, or could be an effect

3. RESULTS AND DISCUSSION 3.1. Retention of Au(I)-Thiosulfate in Columns. Each column was amended with 10.775 μmol of Au(I)-thiosulfate. Overall, >99 wt % of Au were retained in columns containing viable biofilms compared to 2 μm (Figures 4C,D, 5A−D). They occurred as isolated nanoparticles, as conglomerates of nanoparticles directly associated with cells (Figure 4C) and as larger (μm-scale) extracellular spheroidal and framboidal particles. These larger particles appeared to consist of conglomerates of nanoparticles (Figure 4D), and were more abundant than conglomerates directly associated with cells. Numerous, proteinaceous pili, i.e., putative nanowires, were connected to the larger extracellular particles and cells (Figure 4B). Often clusters of up to six cells were connected to an aggregate of Au via these putative nanowires (Figure 4C). Recent research on mechanisms of electron transport in bacteria living in soil and biofilm communities has shown that electrically conductive nanowires similar to those observed in this study, can be used to transfer excess electrons to remote electron acceptors. 36,37 While we have not demonstrated that the “nanowires” connected to extracellular Au particles are conductive, their presence suggests that they contribute to the growth of C. metallidurans biofilms under microaerophilic or anaerobic conditions in the lower segments

Figure 4. (A) Secondary electron micrographs of area of the biofilm showing its overall morphology, overlain is red-green map of Au and C, respectively, showing the distribution of Au in the biofilm; (B) individual cells and associated Au biominerals; and (C) SE micrograph showing the connection of cells to extracellular Au aggregates via nanowires. (D) BSE micrographs showing the large abundance of metallic spheroidal Au in the biofilms.

of the columns.36,37 Sectioning and analyzes of the biofilm using FIB-SEM showed that Au particles were dispersed throughout the biofilms (Figure 5A−D). Extracellular particles displayed internal growth structures with numerous voids and channels (Figure 5D,E) consistent with their formation via conglomeration of nanoparticles. In some instances, larger particles were rod-shaped and hollow (Figure 5B) confirming earlier assumptions that cellular processes led to Au nanoparticle formation, eventually encapsulating cells and ultimately replacing them, yet preserving their morphology. 3.4. The Role of Biofilms in Au Biomineralization. The current study extends our understanding concerning the role of E

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of the interaction of chemical, physical, physiological, and biochemical factors that govern biofilm activities.16 For example, extracellular signaling by quorum sensing systems improves the response of biofilms to oxidative stress caused by Au-complexes and other metals.40,41 The presence of extensive extracellular polymeric layers, observed in these biofilms and on natural Au grains (Figures 4, 5, and 6B,C respectively), leads to the temporary or permanent immobilization of metal ions and complexes, decreasing the amount of Au-complexes reaching

Figure 5. Secondary electron and BSE micrographs of FIB-milled sections of the biofilms. (A) Abundance of microparticulate conglomerates of Au in biofilms; (B) hollow conglomerates of Au replacing bacteria cells in the biofilm; and (C, D) ultrafine structure of Au conglomerates formed via aggregation of approximately 50 nm sized particles, displaying concentric growth structures.

biofilms in Au biomineralization. Recent research has shown that biofilms are less susceptible to metal toxicity compared to planktonic cells.38,39 For example, single-species biofilms of Burkholderia cepacia, a relative of C. metallidurans, and Escherichia coli have shown resistance to five times higher concentrations of Ag nanoparticles and Ga3+, respectively, compared to planktonic cells.16,40 Biofilms of C. metallidurans displayed a similar behavior, while the minimum bactericidal concentrations for planktonic C. metallidurans with aqueous Au(I)-thiosulfate approximates 100 μM, biofilm communities remained viable after amendment with solutions containing more than 6 times this concentration of Au.5 The increased resistance of biofilm communities to metal toxicity is the result

Figure 6. Biofilms, polymorphic layers, and associated Au particles from the surfaces of natural secondary Au grains from Australian sites. (A) Au-particle containing polymorphic layer on the surface of grains from New Zealand;12 (B) biofilms displaying numerous nanowires on Au grains from the Flinders Ranges in South Australia; (C) spheroidal secondary Au growing on Au grains from Kilkivan, Queensland; and (D) nanocrystalline structure of neoformed Au replacing a bacterial cell from Kilkivan grains. F

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the cells.16,42 The number of toxic metal-ions reaching viable cells is reduced through the reactivity of dead cells contained in the biofilms, which have been shown to rapidly accumulate transition metals.43,44 These processes do not apply to planktonic cells, which predominately rely on their inherent or accumulated genetic resistance mechanisms, as expressed through the number of specific and unspecific metal-resistance gene clusters, to counteract the toxic effects of metals.16,20 Biomineralization in C. metallidurans biofilms resulted in Au biominerals that are morphologically analogous to those observed on natural Au grains (see Figure 6). For example, spherical Au nanoparticles were observed in biofilms on Au grains from New Zealand (Figure 6A);19 microbial biofilms containing abundant nanowires as well as Au particles were observed on Au grains from the Flinders Ranges, Australia (Figure 6B,C); and nanoparticles, spheroidal and bacteriomorphic Au was observed in polymorphic layers on Au grains from Kilkivan, Australia (Figure 6C,D).9 In particular, bacteriomorphic Au aggregates, composed of nanoparticles that encapsulated void areas the size and shape bacterial cells, were observed in the lower section of the polymorphic layer covering the grains from Kilkivan (Figure 6D). These aggregates, which were common in our biofilms, likely form around cells that have actively precipitated Au nanoparticles. Due to the intrinsic electrochemical affinity of complexed as well as particulate Au to Au-surfaces, which is commonly known as the “nugget effect”, Au particles precipitated by cells will act as nuclei for further aggregation. Gold aggregates around cells will keep growing by biomineralization and electrochemical aggregation, ultimately leading to encapsulation and replacement of cells. This effect may be even more pronounced in biofilms, in which conductive nanowires enable electron transfer from cells to Au aggregates, promoting the reductive precipitation Au from aqueous Au(I)-complexes and growth of existing aggregates. The data present herein provides experimental verification for the importance of biofilms of C. metallidurans for the detoxification of Au-complexes, and confirms the central role of bacterial biomineralization in the formation of highly pure Au in surface environments.

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No. 239); CSIRO Land and Water; The South Australian Museum; Newmont Mining Corp.; Barrick Gold of Australia; Adelaide Microscopy, esp. L. Green; The Australian Synchrotron, esp. Dr. D. Patterson; J. Parsons (Kilkivan), Marg and Doug Sprigg at Arkaroola Sanctuary. Part of this research was undertaken on the X-ray Fluorescence Microscopy beamline at the Australian Synchrotron. This work was supported by the Australian Microscopy and Microanalysis Research Facility (AMMRF).

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ASSOCIATED CONTENT

S Supporting Information *

Figure S1 (A and B) Backscatter electron micrographs of sand grains from columns incubated under sterilized conditions and with viable biofilms, respectively, showing that metallic Au was only present in columns with viable biofilms. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] Present Address ▽

CSIRO Land and Water PMB 2 Glen Osmond, 5064 South Australia, Australia. Phone: +61 8 8303 8469; fax: +61 8 8303 8550; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the following individuals and institutions: the Australian Research Council (LP100200102 to FR); Flinders University; The University of Adelaide (TRaX. G

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