Applying the Midas touch: Differing toxicity of mobile gold and platinum complexes drives biomineralization in the bacterium Cupriavidus metallidurans

Highlights

• Aqueous Au/Pt-complex stability, biotoxicity, biomineralization and environmental mobility are linked.

• Toxicity of Au- vs. Pt-complexes a key factor for nano-particle biomineralization.

• Metabolic state of cells a key factor for Au nano-particle biomineralization.

• Different biogenic Au/Pt turnover results in different environmental mobility.

Abstract

The β-Proteobacterium Cupriavidus metallidurans CH34, which dominates biofilm communities on natural gold (Au) grains, is a key species involved in their (trans)formation. Gold(III)-chloride complexes, with toxicity levels similar to those of Hg- and Ag-ions, are rapidly sorbed by C. metallidurans cells and detoxified by active reductive precipitation to metallic Au nanoparticles. In this study, we exposed C. metallidurans CH34 to a range of environmentally-relevant Au(I)- and Pt(II/IV)-complexes with differing toxicity levels, i.e., Au(I)-thiosulfate > Au(I)-cyanide, and cisplatin > Pt(IV)-chloride > Pt(II)-cyanide. The aim was to investigate how Au/Pt-complex toxicity, in combination with the metabolic state of cells, affects Au/Pt accumulation, speciation and biomineralization. Overall, more Au(I)- than Pt-complexes were accumulated. Significantly more Au(I)-thiosulfate was taken up by metabolically active vs. inactive or dead cells. Toxicity of Au(I)-complexes was ‘managed’ via the formation of intermediate species, e.g., Au(I)-C mixed ligand complexes. Over time Au(I) associated with active cells was reduced to metallic particles, with higher rates of transformation being observed in experiments amended with Au(I)-thiosulfate- compared to Au(I)-cyanide complexes. In contrast, Pt uptake did not differ with respect to metabolic state. Pt(IV)-complexes were reduced to Pt(II) within 1 min of amendment; further reduction of the Pt(II) was not observed. In conclusion, toxicity of Au/Pt-complexes is linked to the ability of cells to take up and actively detoxify the complexes. Gold uptake was linked to the detoxification of the Au(I)-complexes via active reductive precipitation to Au(0). In contrast, metabolic activity/toxicity did not influence Pt accumulation and/or transformation. This indicates that the ability of bacteria to cycle Au via mobilization, accumulation and biomineralization provides a selective advantage for organisms able to detoxify highly mobile Au-complexes. Because Pt-complexes are not taken up as readily and are hence less toxic, they do not provide a similar selective advantage, and hence Pt is less readily cycled. This may explain the substantially higher environmental mobility of Au compared to Pt.

Introduction

Over the past decade, the role of microorganisms in driving the biogeochemical cycling of Au in near-surface environments has been widely recognized (e.g., Reith et al., 2006, Lengke and Southam, 2007, Southam et al., 2009, Reith et al., 2013, Shuster and Southam, 2015). In particular, biological pathways contributing to the dispersion and re-precipitation of Au have been characterized at the (bio)molecular level (Johnston et al., 2013, Pontel et al., 2007, Reith et al., 2009). Many of these studies were dedicated to understanding the formation of secondary Au deposits and the development of Au anomalies around buried hydrothermal ore deposits (e.g., Fairbrother et al., 2012, Reith et al., 2010, Reith et al., 2012). In addition, the fundamental understanding of geobiological Au cycling can support the development of biotechnologies, e.g., the use of the metallophillic bacterium C. metallidurans for: (i) the production of Au nano-particles, (ii) Au bio-accumulation in bio-hydrometallurgy and (iii) the development of biosensors for mineral exploration (Checa et al., 2007, Zammit et al., 2013). In contrast to Au, the role of microorganisms for the environmental mobility of Platinum-Group-Elements (PGEs) is poorly constrained (Reith et al., 2014, Reith et al., 2016).

Geochemical modeling of abiogenic systems indicated that there should be little difference between Au- and Pt-mobility in near-surface environments (Brugger et al., 2013). Due to the high redox-potentials of Au- and Pt-ions, which exceed that of water, free Au- or Pt-ions are not present in aqueous solution (e.g., Brugger et al., 2013, Liu et al., 2014). Gold(I)- and Pt(II)-complexes with chloride, cyanide, amines, thiosulfate, bisulfide are predicted to be the dominant Au/Pt species in surface fluids and groundwaters based on thermodynamic considerations (e.g., Brugger et al., 2013, Liu et al., 2014). In contrast, Au(III) and Pt(IV)-complexes are predicted to only be thermodynamically stable in highly oxidizing, acidic and chloride-rich environments (Usher et al., 2009). However, Ta et al. (2014) demonstrated that Au(III)-complexes exist under meta-stable conditions in the surface environments, e.g., in hypersaline, reducing and basic waters. The same study also confirmed that Au(I)-thiosulfate and Au(I)-cyanide dominate in other near-surface environments, e.g., groundwaters around buried ore-bodies (Ta et al., 2014, Reith et al., 2007). A recent study directly comparing Au and Pt mobility in groundwaters, soils, sediments and Pt/Au-grains from the Fifield Au/Pt-field (New South Wales, Australia) has shown that Au was far more mobile than Pt (Brugger et al., 2013). Elevated mobilization of Au compared to Pt was observed when biofilms of C. metallidurans or Chromobacterium violoaceum were growing on the surfaces of Au- or Pt-foils (Fairbrother et al., 2009, Brugger et al., 2013). This led Brugger et al. (2013) to suggest that differences in biotoxicity, passive uptake and biochemically active detoxification, result in high turnover rates for Au, and that this may cause the differing environmental mobility of Au vs. Pt.

Many naturally metal-rich environments are nutrient-poor (Ehrlich, 1996, Kenney et al., 2012). Here, bacteria are metabolically inactive for extended periods of time. Consequently, passive adsorption to cell walls in addition to active detoxification mechanisms as well as combinations of both may become important factors determining metal toxicity and resistance (e.g., Reith et al., 2009, Brugger et al., 2010). In this context passive processes are defined as being mediated by the cell matrix or the external organic framework of an organism, i.e., it will happen equally in dead, metabolically inactive or active organisms. Previous studies of passive sorption of Au(I) and Au(III) by bacterial cells have shown that removal of Au from solution was most extensive at low pHs (Niu and Volesky, 1999, Ran et al., 2002, Nakajima, 2003, Tsuruta, 2004, Kenney et al., 2012). Independent of bacterial species, cell wall ligands are protonated to a higher degree at lower pH conditions, which results in an overall more positive surface charge, increasing electrostatically driven passive sorption of negatively charged Au-complexes onto cells (Nakajima, 2003, Mack et al., 2007). Previous studies have also shown that in several Gram-positive and Gram-negative bacteria, passive sorption of Au(III)-complexes was associated with their reduction to Au(I) (Song et al., 2012, Kenney et al., 2012, Reith et al., 2009), the formation of complexes with sulfhydryl-, amine- (Kenney et al., 2012), or C- (possibly carboxyl group) sites (Legatzki et al., 2003, Reith et al., 2009). Kenney et al. (2012) demonstrated that these intermediate Au(I)-complexes may bind to cells more strongly than Au(III)-complexes.

Further reduction to metallic Au particles was not observed in dead or inactive cells, but active reduction to metallic Au from these cell-bound complexes may further improve the survival rates of those species capable of Au detoxification. Gold-specific, active detoxification pathways have been described for Plectonema boryanum, Salmonella enterica, Delftia acidovorans and C. metallidurans (Johnston et al., 2013, Lengke and Southam, 2006b, Pontel et al., 2007, Reith et al., 2009, Wiesemann et al., 2013). Jian et al. (2009) and Reith et al. (2009) studied the removal of Au(III)-complexes from solution by C. metallidurans. C. metallidurans reduced Au(III)-complexes to metallic Au via the rapid (within minutes), passive accumulation and formation of Au(I)-S and Au(I)-C intermediates on the cell wall. This passive uptake was followed by a slow (within days), biochemical (active) reduction, and the intra- and extracellular deposition of metallic Au nanoparticles (Reith et al., 2009, Wiesemann et al., 2013).

In contrast, few studies have assessed the uptake and reduction (active or passive) of Pt-complexes by bacteria. Early studies using the Gram-negative bacterium Escherichia coli amended with cisplatin showed that cisplatin inhibits cell division, causing filamentous growth, suggesting that these Pt-complexes entered the cells (Rosenberg et al., 1965). Lengke et al. (2006a) have shown that cyanobacteria can reduce Pt(IV) to Pt(II) under ambient conditions. At higher temperature (60–180 °C), the bacteria died, releasing organics that contributed to the further reduction of Pt(II) to Pt(0). Other studies (e.g., Rashamuse and Whiteley, 2007, Riddin et al., 2009) have found that sulfate-reducing bacteria (SRB) reduce Pt(IV) rapidly to Pt(II) using a cytoplasmic hydrogenase redox system. Once all the Pt(IV) had been reduced, the Pt(II) diffused to the periplasm, where it was further reduced via a slower and different mechanism to Pt(0). Konishi et al. (2007) showed that, using lactate as an electron donor, Shewanella algae was able to reduce PtCl₆^2 − complexes to Pt(0) within 60 min at room temperature and neutral pH.

In this study, we hypothesize that differing toxicity levels of Au(I)- and Pt(II/IV)-complexes are a result of the cells ability to sorb and actively biotransform them, causing the observed differences in environmental mobility. To assess if the uptake/reduction of Au(I)-and Pt(II/IV)-complexes by C. metallidurans CH34 is primarily a biologically active mechanism affected by complex toxicity, or if it is a function of passive adsorption, the effect of metabolic state of cells (viable, i.e., metabolically active; inactive, i.e., intact but non-functioning; or sterile, i.e., dead), and the length of exposure (1 min to 18 days) of cells to Au/Pt-complexes was tested. The distribution and speciation of Au and Pt in cells were measured using Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) and synchrotron-based X-ray Fluorescence (SXRF) and X-ray Absorption Near Edge Structure (XANES) spectroscopy.