Biomineralization of Platinum by Escherichia coli

The widespread use of platinum in many industrial applications has led to its release into the environment at elevated concentrations with potential adverse effects on human and environmental health. However, the nature of interactions between mobile platinum complexes and the biotic components of the environment, which are increasingly being exposed to platinum, is poorly studied. The aim of this study was to assess the impact of Pt(IV)-chloride on the growth and activity of the well-characterized bacteria Escherichia coli. Bacterial survival and viability in the presence of different concentrations of Pt(IV)-chloride were assessed in liquid culture, while platinum retention was assessed using experimentation with sand-filled columns with the residual platinum concentration measured by atomic absorption spectroscopy. Bacterial biomineralization of platinum was studied with scanning electron microscopy. The results showed that E. coli tolerated PtCl4 at concentrations of up to 10,000 μM over 21 days and remained viable after 112 days of incubation with PtCl4 at 10,000 μM in sand columns. Overall, 74 wt.% and 50 wt.% of platinum was mineralized in E. coli and blank sand columns, respectively. The results of this study confirm that E. coli is capable of biomineralizing platinum. The results confirm that the interaction of platinum with bacteria is not limited to known metal-resistant bacterial species.


Introduction
Platinum (Pt) is one of the six platinum group elements.In recent years, platinum emissions into the environment have increased due to its use in the automobile [1], chemical, and medical industries [2][3][4][5].In the environment, platinum transformation is affected by many factors such as pH, redox potential, soil salinity, organic matter content, soil structure, the presence of complexing agents, and the activity of macro-and microbiota [5][6][7].In aqueous solutions, Pt ions are unstable and can be transported under extremely acidic and oxidizing conditions.Platinum can be transported in the environment as nanoparticles (NPs) and as complexes such as Pt-thiosulfate, -chloride, and complexes with low-and high-molecular-weight organic acids [5].
Microorganisms can influence the chemical and physical conditions within their environment, such as redox and pH, through the secretion of complex ligands, which can also affect Pt mobility in soils, sediments, surface-and groundwater [8][9][10].Hence, microbial activity may play a key role in the biogeochemical cycling of platinum in the environment.For example, some bacteria such as Pseudomonas maltophilia are known to be capable of metal removal in aqueous solutions [11,12].
There have been several studies over the last two decades investigating reactions between platinum and microorganisms.Bacteria can take up platinum through their cell walls [5,[13][14][15][16][17][18].They can also enable the mobilization of platinum, as observed in P. plecoglossicida, which mobilized platinum as Pt(IV)-cyanide after 10 days of incubation [19].In addition, halophilic bacteria have been used to biologically recover Pt(II) and Pt(IV) from dilute industrial process streams with flow cytometric membrane staining used to confirm cell viability during platinum recovery [20].
Metallophilic bacteria such as Cupriavidus metallidurans, which contain high numbers of heavy metal resistance genes [21], have been widely used in heavy metal biomineralization studies.C. metallidurans can actively reduce Pt toxicity when exposed to low concentrations of PtCl 4 [22].C. metallidurans can immobilize Pt complexes such as Pt(IV)-chloride (at concentrations of up to 5000 µM) [22].Transmission electron microscopy (TEM)-based investigations showed that Pt nanoparticles were present within the cell walls of C. metallidurans [22].However, few detailed studies have been carried out using non-heavy metal resistant bacteria.
Escherichia coli is among the most widely studied bacteria in terms of its genetics, physiology, biochemistry, and response to exposure to metals.This is due to the availability of the complete genomic sequence of E. coli, its low cost, and wide availability [23].E. coli can also be readily manipulated genetically [24].Most E. coli strains have been shown to be sensitive to heavy metals such as Zn, Cd, and Hg; studies have also shown that exposure of E. coli to Pt complexes (e.g.(NH 4 ) 2 [PtCl 6 ]) results in the inhibition of cell division [25][26][27][28][29].However, some E. coli strains have been shown to exhibit a range of tolerances to different concentrations of Zn(II), Cd(II), Co(II), Ni(II) [27].
An increasing concentration of Pt in the environment might be detrimental to ecosystem function and stability based on its toxicity to biota.Understanding bacterial interactions with platinum may offer considerable insights into the biochemical detoxification of Pt and potential mineralization and recovery using microbial agents.Therefore, the aim of the study was to examine the interaction of E. coli and platinum and to investigate toxic effects of platinum complexes on E. coli.

Growth and Column Experiments
Pt(IV)-chloride (PtCl 4 , 99.99 wt.% (metal basis), Pt, 55-58%) was obtained from Sigma-Aldrich (St Louis, Missouri, USA).Sand was collected from Melbourne (Bundoora Sand and Building Supplies, VIC, Australia).The sand used in this experiment was a medium coarse sand with a particle size distribution of 0.5 to 3.0 mm with a median size of 1 mm.The E. coli strain W3110 used was obtained from the RMIT Bundoora Culture Collection.E. coli was grown in Tris Minimal Media (TMM, 30 mL) supplemented with sodium gluconate (4% w/v) for 3 days at 30 • C and 160 rpm.Bacteria were harvested by centrifugation at 4,000 g for 10 min, the pellet washed twice with deionized H 2 O and then resuspended in TMM (30 mL).The cell density was then measured spectrophotometrically at OD 600 .
Prior to setting up the column experiments, an initial short-term study was carried out to determine the tolerable concentrations of Pt(IV)-chloride for E. coli's survival and the time period for stabilization of bacterial growth.A stock solution (50 mM) of PtCl 4 was prepared (1.83 g of PtCl 4 dissolved in 100 mL of DDI water); 30 mL aliquots of TMM were then amended with a range of PtCl 4 concentrations (25, 50, 100, 150, 200, 250, 500, 1000, 5000, and 10,000 µM) together with bacterial cells.Samples were collected every 7 days over the 21 day incubation period.Experiments were carried out in triplicate at 25 • C.
Following the selection of the most appropriate Pt-complex concentration from the experiment described above, pre-sterilized glass columns of 10 mm diameter containing 10 g of acid-washed, sieved quartz sand were set up and incubated at 25 • C in a constant temperature room in the dark.Replicated sand columns (n = 6) amended with Pt(IV)-chloride and inoculated with E. coli were prepared as treatment columns along with control columns amended only with Pt(IV)-chloride and no bacterial inoculum.
Columns were inoculated with washed cell concentrate (1 mL) harvested from a pre-culture (10 mL grown in TMM) [30].The initial cell concentrations in the columns were 5.3 × 10 8 (± 2.3 × 10 8 ) CFU mL −1 for E. coli.Control columns were amended with sodium azide (0.15 mL, 1% w/v) to help maintain sterility.Columns were amended throughout the incubation by addition of TMM (10 mL) containing Pt (1000 µM) at time periods of 0, 1, 14, 28, 42, 56, 70, 84, 98, and 112 days.At each sampling time, columns were completely drained prior to amendment with a new medium and the bacterial viable count determined at each time point.

Analyses of the Outlet Solution and Column Sands
Following incubation, all samples were centrifuged at 4000 g for 10 min.The pellet was washed twice with water, resuspended in TMM (30 mL), and the OD 600 value recorded spectrophotometrically.Cell enumeration was carried out by plating aliquots (10 µL) of the outlet solution on agar plate counting media (Acumedia nutrient agar 7145).The assay was carried out based on the counting method previously described [31].The pH of the eluted solution was measured using a Hanna Instrument H11134 (Bedfordshire, UK) pH-electrode equipped with a CyberScan pH 310 meter.
At the end of the incubation period (after 112 days), the columns were sectioned into 20 mm segments and approximately 0.2 g of sand per segment (in triplicate) was taken and digested in aqua regia (1:3 HNO 3 :HCl).Following digestion, platinum concentrations were measured by AAS.The number of cells in the column were also enumerated.An aliquot (0.2 g per segment, in triplicate) of sand was taken and vortexed for two minutes in 1 mL NaCl (0.9 M).An aliquot (10 µL) was then plated on agar plate counting media [30].DNA was extracted from 0.25 g of sand from each segment using an UltraClean Microbial DNA kit (MO BIO Laboratories, Carlsbad, CA, USA) following the manufacturer's instructions.All columns including controls were subject to DNA extraction protocols to assess the sterility of the controls.

Scanning Electron Microscopy (SEM)
Sand grains were fixed in electron microscope (EM) fixative (4% paraformaldehyde, 1.25% glutaraldehyde in phosphate buffer saline (PBS) and 4% sucrose at pH 7.2) for 30 min.Samples were then washed in buffer (PBS and 4% sucrose) for 5 min.Samples were dehydrated using a series of increasing ethanol concentrations 70%, 90%, 100%, twice for 10 min for each concentration.Samples were then fixed using hexamethyldisilazane (HMDS) 1:1 with ethanol for 10 min followed by 100% HMDS for 10 min, twice.HMDS was then removed and the samples dried.Grains were carbon coated and analyzed using focused ion beam-scanning electron microscopy (FIB-SEM; Helios NanoLab 600, FEI Inc., Eindhoven, The Netherlands).SEM-energy-dispersive X-ray elemental analysis (EDXA) operating at 200 kV (RMIT University) was used to detect Pt sections at a 90 • angle to the surfaces, with a Ga ion beam at 30 keV 21 nA −1 used to cut the section.Surfaces were cleaned by decreasing the power of the ion beam to 30 keV 2.8 nA −1 and 20 keV 0.34 nA −1 .Sections were element mapped by EDXA using a 10 mm 2 Sapphire Si(Li) EDX detector (FEI Inc., Eindhoven, The Netherlands) [32].

Optimization and Screening of Pt Concentration for Column Experiments
The optimum concentration of PtCl 4 that was used for the subsequent column experiment was determined by growing E. coli in varying concentrations of PtCl 4 .The concentration of platinum in the outlet solution from uninoculated columns remained constant and significantly higher (27% of the original platinum added), when compared with that in columns inoculated with E. coli (18% of the original platinum added), confirming the uptake of around 9% of the platinum added.The species survived up to the highest concentration of PtCl 4 (10,000 µM).However, the cell numbers of E. coli decreased with increasing metal concentrations of 100, 150, 1000 and 10,000 µM of Pt.The numbers of cells per milliliter of E. coli observed at the highest PtCl 4 concentration (10,000 µM) was 1.99 × 10 2 (± 3.0 × 10 1 ) (data not shown).This indicated that E. coli survived in columns containing PtCl 4 at 10,000 µM concentration.

Column Experiments
The impact of Pt concentration on the viability of E. coli W3110 was examined using a viable plate count method.At the start of the experiment, the E. coli viable cell counts (CFU mL −1 ) were 2.7 × 10 9 cells mL −1 .Once PtCl 4 was added, the viability of E. coli was largely maintained over the experimental period, although the cell numbers started decreasing from the third amendment (4.3 × 10 8 ± 2.7 × 10 8 mL −1 ) (Figure 1). the outlet solution from uninoculated columns remained constant and significantly higher (27% of the original platinum added), when compared with that in columns inoculated with E. coli (18% of the original platinum added), confirming the uptake of around 9% of the platinum added.The species survived up to the highest concentration of PtCl4 (10,000 µM).However, the cell numbers of E. coli decreased with increasing metal concentrations of 100, 150, 1000 and 10,000 µM of Pt.The numbers of cells per milliliter of E. coli observed at the highest PtCl4 concentration (10,000 µM) was 1.99 × 10 2 (± 3.0 × 10 1 ) (data not shown).This indicated that E. coli survived in columns containing PtCl4 at 10,000 µM concentration.

Column Experiments
The impact of Pt concentration on the viability of E. coli W3110 was examined using a viable plate count method.At the start of the experiment, the E. coli viable cell counts (CFU mL −1 ) were 2.7 × 10 9 cells mL −1 .Once PtCl4 was added, the viability of E. coli was largely maintained over the experimental period, although the cell numbers started decreasing from the third amendment (4.3 × 10 8 ± 2.7 × 10 8 mL −1 ) (Figure 1).The calculated retention percentages of Pt in the column were 75% and 50% for E. coli and the control, respectively.This indicated that E. coli was able to take up platinum from the supplied Pt complex.SEM was utilized to observe the appearance of bacterial cells in the presence and absence of Pt (Figure 2A).E. coli, when treated with PtCl4, grew as single cells within the biofilm.Energydispersive X-ray analysis (EDXA) carried out for platinum, as well as carbon and silicon, showed the presence of platinum (shown in green in Figure 2B) associated with most E. coli cells, while silicon (blue) can be seen lying outside of the cells.Carbon is present in abundance outside the E. coli cells although a number of cells can be seen to be stained red (Figure 2B).Higher magnification images of E. coli cells containing Pt revealed the presence of nanoparticulate Pt formed within the cells.The EDXA map (C, Pt, Si) shown as an insert in Figure 2D reveals the presence of conglomerates of Pt, which have replaced bacterial cells in the biofilm (C, D) of replaced cells (D).In addition, conglomerates of platinum can be seen replacing E. coli cells (Figure 2D). Figure 2D shows a FIB cut made through the biofilm, exposing the porous nature of the secondary platinum.The calculated retention percentages of Pt in the column were 75% and 50% for E. coli and the control, respectively.This indicated that E. coli was able to take up platinum from the supplied Pt complex.SEM was utilized to observe the appearance of bacterial cells in the presence and absence of Pt (Figure 2A).E. coli, when treated with PtCl 4 , grew as single cells within the biofilm.Energy-dispersive X-ray analysis (EDXA) carried out for platinum, as well as carbon and silicon, showed the presence of platinum (shown in green in Figure 2B) associated with most E. coli cells, while silicon (blue) can be seen lying outside of the cells.Carbon is present in abundance outside the E. coli cells although a number of cells can be seen to be stained red (Figure 2B).Higher magnification images of E. coli cells containing Pt revealed the presence of nanoparticulate Pt formed within the cells.The EDXA map (C, Pt, Si) shown as an insert in Figure 2D reveals the presence of conglomerates of Pt, which have replaced bacterial cells in the biofilm (C, D) of replaced cells (D).In addition, conglomerates of platinum can be seen replacing E. coli cells (Figure 2D). Figure 2D shows a FIB cut made through the biofilm, exposing the porous nature of the secondary platinum.To assess the location preference of E. coli within the sand column together with the dispersion of platinum through the column, bacterial cells and Pt concentrations were determined throughout the column from 20 mm segments cut at different depths, and with 100 mm representing the top of the column and 20 mm the bottom.Viable cell numbers were found to be 2.2 × 10 4 (± 2.52 × 10 3 ) CFU mL −1 and zero for the recovery of cells from columns amended with E. coli for the top and bottom, respectively.Examination of the column for platinum revealed that most platinum was also found at the top of the column (1.38 ± 0.07 µmol) in columns amended with E. coli (Table 1).To assess the location preference of E. coli within the sand column together with the dispersion of platinum through the column, bacterial cells and Pt concentrations were determined throughout the column from 20 mm segments cut at different depths, and with 100 mm representing the top of the column and 20 mm the bottom.Viable cell numbers were found to be 2.2 × 10 4 (± 2.52 × 10 3 ) CFU mL −1 and zero for the recovery of cells from columns amended with E. coli for the top and bottom, respectively.Examination of the column for platinum revealed that most platinum was also found at the top of the column (1.38 ± 0.07 µmol) in columns amended with E. coli (Table 1).

Discussion
This study examined the potential of E. coli to biomineralize Pt.In preliminary experiments aimed at determining the optimum concentration for the subsequent column experiment, a Pt(IV)-chloride concentration of 10,000 µM was selected for the long-term column experiment.This concentration was selected based on the observed survival and maintenance of cell viability by E. coli.In the subsequent column experiments, the growth characteristics of E. coli was investigated to determine the response of E. coli to the presence of Pt.E. coli cells were found to grow more as single cells when exposed to PtCl 4 compared to the growth pattern in the absence of platinum (data not shown) indicating that platinum could be inhibiting cell division of E. coli cells [25,33].However, E. coli showed decreased cellular viability from the fourth amendment probably in response to platinum toxicity.This is not unusual as a previous study had shown a reduction in E. coli growth when treated with several toxic metals including Cu, Cd, Zn, and Hg (1-5 ppm) [26].In this study, E. coli was found to be capable platinum biomineralization.This is in agreement with previous studies where an accumulation of PtCl 4 and subsequent formation of platinum nanoparticles was observed in a range of bacteria (Pseudomonas spp., E. coli, and sulfate-reducing bacteria) and fungi (Neurospora crassa and Fusarium oxysporum) [34][35][36][37].The concentrations of platinum detected in the outlet solutions of E. coli were very low compared to the blank or negative control; this might be because Pt was absorbed or complexed into the bacterial biofilm.This is in agreement with a previous study on the biomineralization of toxic metals such as gold [28,35].These studies found that biomineralization of gold occurred in the presence of viable biofilms, forming intra-and extra-cellular nanoparticles.Nano and micro-particles were observed through SEM around the cells.Platinum nanoparticles were found within and around the E. coli cells.The platinum micro-crystal aggregates (>5 µm) that formed in the biofilm or around cells may provide nucleation sites for the accretion and growth of grains due to biomineralization [38].Bacteria are capable of accumulating metals in amounts greater than their own weight due to their high surface area to volume ratio [39,40].FIB-SEM showed nanoparticles in E. coli through sectioning of single cells.In order to assess the distribution of E. coli in the columns, sectioning of the column in 20-mm segments was carried out.This resulted in the observation of dense cells at the top and none at the bottom of the columns with E. coli.Crucially, with E. coli, 74% of platinum (mineralized from the PtCl 4 ) was detected in the sand column.This was higher than the 50% of platinum retained in the blank control, suggesting the ability of E. coli to facilitate platinum solubilization, transport, and precipitation, thus affecting the dispersion and re-concentration of Pt in surface environments.Microbes can also influence redox and pH conditions and are involved in the formation and secretion of ligands in soils and surface-and groundwaters [8][9][10]41].In this experiment, the pH of the column outlet solution varied from 5.5 to 6.6 (average of 6.5) throughout the experiment, and the pH in the control column did not significantly change.

Figure 1 .
Figure 1.Cell numbers of Escherichia coli during the amendment with PtCl4 over 112 days.The results represent the mean of triplicate values with standard deviations.

Figure 1 .
Figure 1.Cell numbers of Escherichia coli during the amendment with PtCl 4 over 112 days.The results represent the mean of triplicate values with standard deviations.

Figure 2 .
Figure 2. SEM micrographs of the E. coli biofilm from column experiments showing overall morphology (A); energy-dispersive X-ray analysis (EDXA) element map (C, Pt, Si) showing the distribution of platinum in the biofilm (B); detailed view of individual active cells and conglomerates of platinum replacing bacteria cells in the biofilm (C,D), with EDXA map (C, Pt, Si) of replaced cells (D).A FIB cut in (D) exposes the porous nature of the secondary platinum.

Figure 2 .
Figure 2. SEM micrographs of the E. coli biofilm from column experiments showing overall morphology (A); energy-dispersive X-ray analysis (EDXA) element map (C, Pt, Si) showing the distribution of platinum in the biofilm (B); detailed view of individual active cells and conglomerates of platinum replacing bacteria cells in the biofilm (C,D), with EDXA map (C, Pt, Si) of replaced cells (D).A FIB cut in (D) exposes the porous nature of the secondary platinum.

Table 1 .
Number of cells extracted from each 20 mm segment cut at different depths; 100 mm is the top of the column (n = 3).

Table 1 .
Number of cells extracted from each 20 mm segment cut at different depths; 100 mm is the top of the column (n = 3).