Synthesis of extracellular gold nanoparticles using Cupriavidus metallidurans CH34 cells

Abstract

In this study, spontaneous synthesis of a gold (Au) colloid using cells of Cupriavidus metallidurans CH34 is reported, and compared with results obtained using cells of the model bacterium Escherichia coli MG1655. To investigate the synthesis mechanism, bacterial biomass and secretomes from both strains were incubated with Au(III) ions. Only CH34 cells were capable of producing extracellular dispersions of Au nanoparticles (NPs). Transmission electron microscopy images showed that AuNPs morphology was dominated by triangular and decahedral nanostructures. Energy dispersive X‐ray spectroscopy and Fourier transform infrared spectra showed the presence of sulphur and vibrations associated to proteins. Average AuNPs diameter was obtained by dynamic light‐scattering measurements (DLS), NP tracking analysis measurements and analysis of electron microscopy images. Moreover, DLS measurements showed that the biogenic colloid was stable after exposure to ultrasound, high ionic strength and extreme pH conditions. The biogenic AuNPs produced by strain CH34 did not show antibacterial activity, in contrast to biogenic silver NPs. Comparative bioinformatic analysis of genomes from strain CH34 and strain MG1655 showed potential CH34 proteins that may be electron donors during reduction of Au(III) ions. On the basis of these results, a mechanism for the extracellular Au reduction by strain CH34 is proposed.

1 Introduction

The need for cost–effective eco–friendly synthesis of metallic nanoparticles (NPs) has established new methods to replace chemical synthesis of colloids. Several bacterial strains are versatile biocatalysts for biotransformation of heavy metals and other toxic compounds [1, 2, 3, 4, 5, 6, 7, 8]. In this context, the biogenic synthesis of metallic NPs by microorganisms such as Pseudomonas, Shewanella and Streptomyces strains has been reported [9, 10, 11, 12, 13, 14, 15].

Cupriavidus metallidurans is a model heavy metal‐resistant bacterium that harbours gene clusters enabling detoxification of diverse heavy metal ions and complexes [1, 2, 16, 17]. Interestingly, C. metallidurans is also involved in the biogeochemical cycle of gold (Au). The presence of C. metallidurans cells in biofilms that covered the surface of Au grains has been reported [18, 19]. Transcriptomic analysis suggested that oxidative stress and heavy metal resistances genes including an Au‐specific operon were involved in Au reduction by C. metallidurans [19]. Recently, formation of AuNP and microparticle aggregates onto the surface C. metallidurans biofilms has also been reported [20].

The aim of this paper was to obtain a stable dispersion of AuNPs using C. metallidurans CH34 cells. For this, bacterial cultures were fractionated and exposed to an Au aqueous solution. Using this procedure, spontaneous extracellular dispersions of AuNPs were obtained. The biogenic colloid was characterised by surface plasmon spectroscopy, dynamic and electrophoretic light scattering and NP tracking analysis (NTA). Solid‐state biogenic AuNPs were characterised by powder X‐ray diffraction (XRD), Fourier transform infrared (FT‐IR) spectroscopy, energy dispersive X‐ray (EDX) spectroscopy and transmission electron microscopy (TEM). The stability of the colloid was characterised by electrophoretic and dynamic light scattering (DLS). Moreover, the antibacterial activity of the biogenic AuNPs on bacterial growth was evaluated. Finally, based on these results a protein‐mediated extracellular Au(III) reduction mechanism is proposed.

2 Materials and methods

2.1 Materials

Solid hydrated auric salt HAuCl₄ ·3 H₂ O (99.9% purity) was obtained from Sigma Aldrich (Saint Louis, MO, USA). Sodium chloride (NaCl), sodium hydroxide (NaOH) and potassium chloride (KCl) were obtained from Merck (Darmstadt, Germany). Beef extract and yeast extract were acquired from Becton Dickinson (Cockeysville, MD, USA) and agar solid medium was obtained from Merck (Darmstadt, Germany). Biogenic silver (Ag) NPs (40 nm average diameter) were recovered from Fusarium oxysporum permeates [13].

2.2 Biosynthesis of AuNPs

C. metallidurans CH34 [21] and E. coli MG1655 [22] were grown in a low ionic strength medium with meat extract (20 g/l) and yeast extract (20 g/l) [23]. Biogenic synthesis of AuNPs was done according to methods A and B [13], with modifications (Fig. 1). Method A evaluates the effect of bacterial biomass on the Au(III) reduction process and method B evaluates the effect of bacterial extracellular medium on the Au(III) reduction process. In method A, biomass (40 ml) was directly exposed to an Au solution achieving a final concentration of 2 mM. In method B, bacterial biomass was used to obtain bacterial filtrates that were exposed to an Au(III) solution (2 mM). In both methods, cell cultures grown until stationary phase were used as starting bacterial biomass. For method A, cells were collected by centrifugation and washed three times with doubly deionised water. Cell pellet was suspended in doubly deionised water and diluted up to a concentration of 3.3 × 10⁹ CFU/ml. The bacterial suspension was exposed to an Au(III) solution (2 mM) and then incubated during 96 h in darkness without shaking at 30°C. The stock solution of an Au(III) solution (40 mM) was previously adjusted to pH 7 with NaOH. To recover the colloid of biogenic AuNPs, the bacterial suspension was centrifuged at 5000 g during 10 min and the supernatant was filtered using a 0.45 μm pore size nitrocellulose membrane. The dispersion of biogenic AuNPs was recovered in the permeate fraction. For method B, cells from a stationary phase bacterial culture were collected by centrifugation and washed three times with doubly deionised water. Cell pellet was suspended in doubly deionised water and diluted up to a concentration of 6 × 10⁹ CFU/ml. To obtain the bacterial secretome, the biomass was incubated during three days in flasks with shaking (150 rpm) at 30°C. Then, cells were centrifuged at 5,000g during 10 min and the supernatant was recovered and filtered using a 0.2 μm pore size nitrocellulose membrane. Bacterial secretomes were recovered [24] and incubated with an Au(III) solution (2 mM) during 96 h in darkness without shaking at 30°C.

Fig. 1.

Workflow for method A (left) and method B (right)

2.3 Characterisation of NPs in colloidal state

The ultraviolet–visible absorbance spectra of Au colloid were obtained at room temperature with an Agilent 8453 spectrophotometer equipped with a diode array (Palo Alto, CA, USA). Hydrodynamic diameter (Z‐Ave) and zeta‐potential (ζ‐ potential) were determined by light‐scattering measurements using a ZetaSizer Nano ZS instrument. Additional particle size distributions and concentration were determined by NTA using a NanoSight LM20 device. In all cases, temperature was fixed at 25°C and 1 mM KCl solution was used as dissolvent. Concentration of metal in the colloidal dispersions was determined by inductively coupled plasma using a Perkin Elmer Optima 3000 DV ICP‐OES spectrometer.

2.4 Characterisation of NPs in solid state

Morphology and size distribution of the biogenic AuNPs were observed by TEM. Images were obtained using a Carl Zeiss (Libra) TE microscope operating at 120 keV and analysed using the ImageJ 1.47v software. To obtain the images, one drop of biogenic AuNPs was deposited on a carbon‐coated parlodion film supported in 300 mesh copper (Cu) grids (Ted Pella, Redding, CA, USA). For EDX analysis, biogenic AuNPs were deposited on a carbon grid and spectra were acquired with a Carl Zeiss, EVO MA‐10 device. Additional solid‐state characterisation was done to powder samples of biogenic AuNPs. To obtain the powder samples, colloidal dispersions of biogenic AuNPs were centrifuged at 20,000g during 20 min and then washed three times with MilliQ water. The sediment containing biogenic AuNPs was lyophilised and then analysed by powder XRD and FT‐IR spectroscopy. Powder XRD spectra were obtained in a Shimadzu XRD 6000 diffractometer using Cu Kα radiation (1.5406 Å) operating at 30 mA and 40 kV. Scan speed was 0.02°/min and the time constant was 2 s. FT‐IR spectra were recorded using a Bomem MB spectrometer in 4000–400 cm−1 frequency range using an attenuated total reflectance mode. A total of 250 scans and a resolution of 4 cm−1 were employed to obtain each spectrum.

2.5 Antibacterial activity of metallic NPs

The antibacterial activity of biogenic Au and AgNPs was evaluated by minimal inhibition concentration (MIC) measurements. The MIC of NPs dispersions was determined by microdilution method [25] and was defined as the minimum concentration of metal that inhibits E. coli MG1655 growth in liquid medium after an incubation period of 16 h at 37°C. The liquid medium used in the assays contained meat extract (5 g/l) and yeast extract (5 g/l) and was inoculated to a final bacterial concentration of 5 × 10⁵ CFU/ml. Aliquots of bacterial cultures were exposed to equivalent concentrations of Au and Ag obtained from serial dilutions of the colloids. The initial concentration of both Au and Ag NPs was 14.5 μg/ml. Also, the effect of Au and AgNPs on bacterial growth was visually analysed on agar plates. Solid medium contained meat extract (5 g/l), yeast extract (5 g/l) and agar (15 g/l). The agar plates (85 mm diameter) were seeded with an aliquot (100 μl) of 1 × 10⁸ CFU/ml of MG1655 cells and exposed to AuNPs and AgNPs. For this, NP dispersions were serially diluted and drops containing 20 μl of each dilution were poured onto the previously seeded solid medium and incubated during 16 h at 37°C.

3 Results

3.1 Biogenic synthesis of Au colloid

Using method A, supernatant of strain CH34 spontaneously developed an evident purple colour characteristic of Au colloids [Fig. 2 a (left)], whilst the supernatant of strain MG1655 remained translucid with no evident development of purple colour as determined by visual inspection [Fig. 2 a (right)]. Also, accumulation of a purple sediment was observed in both samples. No viable cells were detected after this treatment.

Fig. 2.

Optical properties of AuNPs synthesised by bacteria

(a) Reduction of Au(III) by C. metallidurans CH34 (left) and E. coli MG1655 cells (right), (b) Visible spectra of the supernatants obtained from C. metallidurans CH34 and E. coli MG1655 cultures

Using method B, bacterial secretomes incubated with Au ions did not develop purple colour, and appearance of an insoluble yellow Au complex was observed (not shown).

3.2 Characterisation of biogenic AuNPs in colloidal state

The visible spectrum of supernatants of CH34 cultures showed a maximum absorbance peak at a wavelength of 536 nm (Fig. 2 b). This maximum absorbance peak value is characteristic of the presence of dispersed AuNPs. On the other hand, the absorbance peak was neither evident in supernatants of E. coli MG1655 cells (Fig. 2 b), nor the secretomes obtained from both strains (not shown).

Light‐scattering measurements indicated that Z‐Ave of the biogenic AuNPs was 126.4 nm (Fig. 3 a), with an associated zeta‐potential of −0.25 ± 3 mV. Additional NTA measurements indicated that mean diameter of the colloid was 124 nm, with a mode of 78 nm and the presence of minor populations of 302 and 405 nm (Fig. 3 b). Concentration of four times diluted AuNPs was 4.19 × 10⁹ particles/ml. Therefore, original concentration of biogenic AuNPs was estimated as 1.68 × 10¹⁰ particles/ml.

Fig. 3.

Size distributions of biogenic AuNPs obtained by light‐scattering measurements

(a) Size distribution obtained by DLS measurements. Reported Z‐Ave of 126.4 nm and PdI of 0.284, (b) Size distribution and particles concentration obtained by NTA. Reported mean diameter of 124 nm, mode of 78 nm and SD of 88 nm

Afterwards, hydrodynamic diameter was evaluated after exposure to ultrasound, extreme pH conditions and elevated ionic strength to determine the stability of the colloid (Table 1). Z‐Ave of control samples was 126.4 nm, with an associated PdI of 0.284. Ultrasound treatment did not affect size distribution of the colloid. The presence of an acidic environment reduced Z‐Ave from 126.4 to 114.6 nm, whereas an alkaline environment increased Z‐Ave to 135.3 nm. In both cases, PdI of the colloid increased to 0.33. Finally, the presence of elevated ionic strength (500 mM NaCl) did not show effect on size distribution.

Table 1.

DLS measurements of biogenic AuNPs dispersions produced by C. metallidurans CH34 cells after exposure to different experimental conditions (average of three measurements)

Condition	Z‐Ave, d nm	PdI
control	126.4	0.284
ultrasound	124.9	0.279
pH 1	114.6	0.332
pH 10	135.3	0.334
NaCl (500 mM)	127.5	0.286

3.3 Characterisation of biogenic AuNPs in solid state

The XRD spectrum of powder AuNPs showed Bragg's reflection peaks at positions 2θ  = 38.1°, 44.3°, 64.6°, 77.5° and 81.7° (Fig. 4 a). Position of reflection peaks can be associated to diffraction planes (111), (200), (220), (31) and (222) of the crystalline unit cells of elemental Au as seen in the 04–0784 file of the JCPDS database. The all odd or all even Miller index values of the diffraction planes indicate that biogenic AuNPs have a face‐centred cubic crystalline structure. Calculated lattice constant (a) using the distance value (d) associated to {220} diffraction planes was estimated to be 4.1 Å. This value is in agreement with the standard report value from JCPDS (a  = 4.1 Å), thus confirming that biogenic AuNPs synthesised by strain CH34 are composed of elemental Au.

Fig. 4.

Physical properties of biogenic AuNPs synthesised using C. metallidurans CH34 cells

(a) Powder XRD spectrum, (b) Size frequency distribution and morphology of the AuNPs determined by analysis of TEM images (n  = 142). Mean of 37.1 with SD of 15.5 nm. Inset: representative field of biogenic AuNPs

TEM images showed that diameter of the metallic core was ≥10 nm, with 85% of the particles showing a diameter between 20 and 60 nm. Few truncated triangular nanoplates with diameter ≥70 nm were also observed. Mean of the frequency distribution was 37.1 ± 15.5 nm (Fig. 4 b). Morphology was dominated by decahedral and triangular nanostructures [Fig. 4 b (inset)]. Also the perimeter of all NPs was surrounded by a layer of non‐metallic material with a width of ∼4.5 nm.

Chemical groups associated to AuNPs were investigated using FT‐IR spectroscopy. Spectrum showed several peaks that correlate with molecular vibrations of functional groups from proteins (Fig. 5 a); therefore, biogenic AuNPs spectrum was analysed based on the FT‐IR spectrum of bovine serum albumin [26]. The broadband between 3700 and 3000 cm−1 corresponds to vibrations of water molecules and protein functional groups. The higher peak at 3450 cm−1 corresponds to OH··· stretching vibrations of residual H₂ O (ν_O–H···). The following absorption bands at 3290 and 3100 cm−1 correspond with amide A group of vibrations. The absorption band between 3000 and 2800 cm−1 corresponds with stretching vibrations of C–H groups. The broadband between 1600 and 1700 cm−1 corresponds with molecular vibrations of amide I group. The absorption band between 1600 and 1500 cm−1 corresponds with molecular vibrations of amide II group. A similar absorbance pattern has been described for albumin adsorbed onto chemically synthesised AuNPs [27, 28]. In the latter case, the absorption band between 1400 and 1360 cm−1 corresponded to symmetric stretch (ν^S _COO−) of carboxylate groups of aspartic acid and glutamic acid residues. Finally, EDX spectrum showed signals at 2.309 and 2.465 keV, indicating the presence of sulphur atoms associated to biogenic AuNPs (Fig. 5 b).

Fig. 5.

Chemical characterisation of biogenic AuNPs synthesised using C. metallidurans CH34 cells

(a) FT‐IR spectrum of biogenic NPs (straight line) and bovine serum albumin adapted from [26] (dotted line), (b) EDX spectrum of biogenic AuNPs

3.4 Antibacterial activity of biogenic Au and AgNPs

The effect of biogenic Au and AgNPs on bacterial cell growth was compared. Microdilution method indicated that biogenic AuNPs did not affect the growth of E. coli MG1655, whereas biogenic AgNPs showed an MIC of 0.45 μg/ml (Fig. 6). This value is in agreement with previous reports [10, 29, 30]. Similar results were observed with biogenic Au and AgNPs on solid growth medium. These results indicate that under these experimental conditions, and in contrast to biogenic AgNPs, biogenic AuNPs synthesised by C. metallidurans CH34 do not show antibacterial activity against E. coli cells.

Fig. 6.

Minimal inhibitory concentration of biogenic NPs on planktonic cultures of E. coli MG1655

(a) MIC of AuNPs produced by C. metallidurans CH34, (b) MIC of AgNPs produced by F. oxysporum. nd: No growth detected. Inset: growth of E. coli MG1655 cells on agar plates after exposure to equivalent amounts of NPs

4 Discussion

Exposure of intact C. metallidurans CH34 cells to Au(III) ions produced evident extracellular dispersions of AuNPs, whereas exposure of MG1655 cells produced a purple sediment and a translucid supernatant (Figs. 2 a and b). Similar to our results, exposure of intact E. coli DH5α cells to Au(III) ions produced purple precipitates and a colourless extracellular solution [31]. In this case, reduction of Au ions and accumulative growth of AuNPs on cells surface was attributed to sugars or enzymes located in bacterial surface. Further experiments confirmed that E. coli cell membrane is capable of reducing Au(III) ions to produce AuNPs [32].

On the other hand, exposure of CH34 and MG1655 secretomes to Au(III) ions did not produce AuNPs dispersions. Therefore, it is concluded that under these experimental conditions both strains do not secrete enzymes or compounds to the extracellular medium that allow production of biogenic AuNPs.

These results indicate that the mechanism of Au(III) reduction during the synthesis of extracellular AuNPs dispersions implies oxidation of molecules that are present in C. metallidurans CH34 cells, and are absent in MG1655 cells. Therefore, it is proposed that reduction of Au(III) ions with concomitant production of extracellular dispersions is mediated by oxidation of macromolecules that are located in the cell membrane or periplasm of CH34 cells, and are absent in the cell membrane or periplasm MG1655 cells.

To study this possibility, a comparative bioinformatic analysis of both genomes indicated that, in contrast to strain MG1655, C. metallidurans CH34 possesses a cop gene cluster that encodes periplasmic and outer membrane sulphur rich proteins that contain a significant number of methionine (Met) and cysteine (Cys) residues (CopA 36 Met/1 Cys, CopB 52 Met, CopC 7 Met, CopK 9 Met, CopJ 4 Met/2 Cys) (Fig. 7). Complementary proteomic studies of strain CH34 demonstrated constitutive expression of the periplasmic protein CopK and the outer membrane bound protein CopB [33].

Fig. 7.

Proposed mechanism for the biosynthesis of extracellular dispersions of AuNPs by C. metallidurans CH34 based on genomic and proteomic data. Extracellular Au ions are reduced by the sulphur atom of Met and Cys residues of Cop proteins located in the cell membrane and periplasm. Au atoms evolve into small tetrahedral NPs. Small tetrahedral NPs evolve into small decahedra. Small decahedra evolve into the final biogenic colloid

Thus, during the biogenic synthesis experiments, the presence of Cop sulphur rich proteins located at the membrane and periplasm of CH34 cells might allow the direct interaction between sulphur atoms of Met and Cys residues and the diffusive Au(III) ions dissolved in the extracellular medium. In this manner, it is proposed that extracellular Au(III) ions oxidised the sulphur atom of Met residues into Met sulphoxide. This event has been described for Met residues from ribonuclease A and glycyl‐D,L‐Met dipeptide, with concomitant production of Au(0) [34, 35, 36]. In addition, Au(III) ions might oxidise Cys to cystine, and subsequently to sulphonic acid [37, 38]. After interaction, the reduced Au atoms might return to the extracellular medium and generate nucleation centres that finally evolve into extracellular AuNPs dispersions (Fig. 7). Moreover, under physiological conditions, redox cycling enzymes such as Met sulphoxide reductases might reduce and recover the functionality of oxidised sulphur containing residues. Therefore, this analysis suggests that Met residues of CopA and CopB are especially involved in the reduction of Au ions to produce biogenic extracellular AuNPs; and that a redox cycling process of these residues, probably mediated by Met sulphoxide reductases, is involved in the biochemical synthesis of Au nuggets in nature [19].

Further analysis of electron microscopy images showed that NPs morphology was dominated by decahedral and triangular structures [Fig. 4 b (inset)]. The origin of these structures can be attributed to interaction of ultra‐small tetrahedral NPs [39, 40, 41]. Tetrahedral morphology becomes unstable when NPs reach diameters close to 10 nm. Then, interaction of five tetrahedral units lowers down the surface area allowing formation of decahedral structures [39] (Figs. 4 b and 7). On the other hand, formation of major triangular nanoplates has been attributed to a slow kinetic process during NPs formation [42]. After long periods, the interaction of decahedra‐like such as structures serve as nucleation centres that finally evolve into triangular and truncated triangular structures. So, it is proposed that the biosynthesis mechanism of these AuNPs involves an initial step where small tetrahedral NPs are generated. The interaction of small tetrahedral units evolves into decahedral structures. As observed by TEM, one population of these decahedral structures may serve as nucleation centres for subsequent biosynthesis of triangular nanoplates, and other population might evolve up to reach diameters close to 50 nm (Fig. 7).

Additionally, TEM images also showed that AuNPs were covered by a ∼4.5 nm layer of non‐metallic material [Fig. 4 b (inset)]. FT‐IR analysis showed the presence of protein chemical groups associated to the biogenic NPs (Fig. 5 a), and EDX spectrum showed the presence of sulphur atoms (Fig. 5 b). In this manner, this data indicates that biogenic AuNPs synthesised by strain CH34 are covered by a layer of proteins, probably containing Cys and Met residues.

Analysis of size distributions obtained by DLS and NTA indicates that results obtained with these technologies are comparable (Fig. 3). For example, DLS measurements showed that Z‐Ave of the particles was 126.4 nm, whereas NTA indicated that mean diameter of the distribution was 124 nm. Nevertheless, these results do not correlate with size distribution deduced from TEM images (average diameter of 37.1 nm) (Fig. 4 b). In this case, the difference between diameters determined by TEM and light‐scattering technologies can be explained by the characteristics of each technique. Size distributions obtained from TEM images are proportional to the length of the NPs and do not include the width of capping ligands adsorbed onto the AuNPs surface. In addition, size distributions obtained from TEM images reflect results from the analysis of AuNPs samples that are in a solid static state. In contrast, size distributions obtained from DLS and NTA measurements are proportional to the volume of the NPs and represent the accumulation of multiple measurements obtained from NPs population in dynamic state. Moreover, size distributions obtained by these light‐scattering technologies reveal intermolecular interactions between dispersed AuNPs that lead to multiple equilibriums, and are finally detected as an average of major‐sized NPs complexes [28, 43, 44, 45]. Therefore, size distributions of colloids obtained by DLS and NTA contain information about interacting and non‐interacting NPs, and detect small amounts of larger‐sized NPs that are outside the normal distribution that are not captured in TEM image analyses [46].

Also, stability of the biogenic AuNPs colloid was determined by DLS measurements after exposure to ultrasound, extreme pH conditions and elevated ionic strength (Table 1). Previous reports show that ultrasound fuses non‐functionalised AuNPs into worm‐like units with concomitant increment in Z‐Ave and PdI [47]. In our case, exposure to prolonged sonication periods did neither change Z‐Ave nor PdI values, indicating that the biogenic colloid is not sensitive to ultrasound perturbation probably by the protective effect of the layer of capping ligands that cover the surface of the biogenic particles. Additional Z‐Ave determinations after exposure to extreme pH conditions indicated that the biogenic colloid endures the chemical perturbance of acidic and alkaline environments, and suggests the dominance of basic functional groups associated to NPs [48]. Exposure to an acidic environment reduced the hydrodynamic diameter and increased the PdI of the colloid. The opposite was observed in the presence of alkaline environment. So, it is proposed that low pH conditions generate a re‐distribution of the interacting NPs population. Protonation of basic groups of capping ligands might increase the electrostatic repulsion of at least one fraction of the former interacting NPs. This process probably generated a new group of non‐interacting NPs that split the population distribution and is detected as a reduction in Z‐Ave and an increment in the PdI of the colloid. On the other hand, alkaline environment increased both Z‐Ave and PdI of the colloid. In this case, deprotonation of basic groups might reduce the electrostatic repulsion of at least one fraction of the interacting NPs. This process would generate a group of highly interacting NPs that split the former population distribution and is detected as an increment in both Z‐Ave and PdI of the biogenic colloid. Finally, the presence of high concentrations of NaCl did not alter the size distribution of the colloid. This result demonstrates that biogenic AuNPs do not flocculate nor aggregate in the presence of elevated ionic strength conditions, indicating that biogenic AuNPs colloids synthesised with C. metallidurans CH34 cells are not stabilised by electrostatic repulsion forces. This result also indicates that the stability mechanism is based on steric repulsion between the capping ligands of NPs [48], and that these biogenic NPs would remain stable even under ionic physiological conditions (150 mM NaCl).

5 Conclusions

Stable colloidal dispersions of AuNPs were obtained after incubation of C. metallidurans CH34 cells with Au(III). Potential CH34 proteins such as CopaA, CopB or CopK may act as electron donors for the reduction of Au(III) ions during the biosynthesis process, and a mechanism for the synthesis of extracellular AuNPs by strain CH34 was proposed.

Average diameter of the biogenic AuNPs in colloidal state obtained from light‐scattering measurements (DLS and NTA) was four times higher than diameter of the biogenic AuNPs in solid state obtained from analysis of TEM images. These results indicate that size distributions obtained from analyses of NPs in colloidal and solid state are not comparable.

The biogenic colloid is stable under chemical and physical perturbations, which are useful for potential applications. Clinical applications include the topic delivery of bioactive compounds that may interact with the protein layer of the NPs and the development of colorimetric biosensors that require colloidal probes with high stability and absorptivity.