Soil sampling

We applied a randomized strategy49 to locate sampling sites in the gold anomaly over the shallow mineralization, as well as sites in an adjacent non-auriferous district 100 m away (serving as the reference) (Supplementary Fig. 1, Supplementary Table 1). Approximately 500 g of surface soil (0–10 cm) from each site were collected. Briefly, a 0.2 m × 0.2 m wide × 0.4 m deep bulk of soil was excavated using a sterilized spade. Soils were then carefully sampled with a sterilized scoop along the exposed profile. A total of 19 randomly collected soil samples were immediately stored in a −86 °C portable freezer (ULT25; Global Cooling, Inc., Athens, OH, USA) and a cooler with ice for DNA extraction and culture-dependent analysis, respectively.

Geochemical characterization

Soil physicochemical parameters, including temperature, moisture, and electrical conductivity were measured on site with a handheld Thermometer (DT-847U; OneTemp Pty Ltd, Adelaide, SA, Australia) and a HydroSense II Soil Moisture Measurement System (Campbell Scientific Pty Ltd, Garbutt, QND, Australia). Soil pH was determined in deionized water by a pH/EC meter (900-P; TPS Pty Ltd, Brisbane, QND, Australia). Element composition of the soils was analyzed by inductively coupled plasma mass spectrometry (ICP-MS) (Nexion 300Q; Perkin Elmer, Waltham, MA, USA) at LabWest Mineral Analysis Pty Ltd. in Perth, Australia. Detection limits for all elements were at the ng g−1 level. Gold concentrations in soil samples were determined by the aqua regia method23. Briefly, soil samples were ground to 200 μm, mixed with 100% aqua regia (HCl:HNO 3 = 3:1), and digested at room temperature for 24 h. The supernatant obtained by centrifugation was analyzed by ICP-MS. Total carbon and nitrogen were analyzed by CSBP Lab (CSBP Fertilizers, Kwinana, WA, Australia) using the Dumas high temperature combustion method50.

Soil microcosms

Six batch-type microcosms were set up under aerobic conditions with metallic gold particles (1.5–3.0 μm; Sigma-Aldrich; St Luis, MO, USA) supplementation. Conditions for all microcosms are listed in Table 1. Briefly, 20 g wet weight of fresh organic-rich soil was added to sterile 250-mL screw cap Erlenmeyer flasks (PYREX®, Sigma-Aldrich) with 200 mL of sterile Milli-Q water. For microcosms GA6F+cs and RA6F+cs, the mixture was supplemented with 0.2-μm filter-sterilized 30 μg mL−1 chloramphenicol (Fisher Scientific, Waltham, MA, USA) and 100 μg mL−1 streptomycin sulfate (Fisher Scientific) from ethanolic or aqueous stocks, respectively, to inhibit bacterial growth51. For microcosms GA8B+cyc and RA8B+cyc, the mixture was supplemented with 5% cycloheximide (0.2-μm filter-sterilized) to inhibit fungal growth26. For microcosms GA6I and RA6I, the mixture was sterilized by repeated autoclaving (121 °C, 20 min, twice over 24 h) to cease biological activities. We added sterilized gold particles to the microcosms to achieve an initial gold concentration of 40 μM. The microcosms were inoculated statically in the dark at 10 °C. Before each sampling, the microcosms were homogenized by gentle shaking. The microcosms GA6F+cs, RA6F+cs, GA6I, and RA6I were sampled at 0, 17, 45, 119, 191, 310, 453, and 693 h; whereas the microcosms GA8B+cyc and RA8B+cyc were sampled at 0, 47.5, 140.5, 185.5, 286.5, and 693 h. We pipetted 1 mL of slurry from each flask to colorimetrically determine the Au(III) concentration using TMB (Sigma-Aldrich) and measured the resulting absorbance at 654 nm24 with a DR 5000™ UV–Vis spectrophotometer (Hach, Dandenong South, VIC, Australia). The slurry was centrifuged at 3000 × g for 1 min and 0.5 mL supernatant was pipetted into a cuvette (BRAND® standard disposable cuvettes, Sigma-Aldrich). Then, 0.1 mL of TMB indicator was added to the cuvette and mixed with the supernatant by gentle pipetting. The mixture was incubated at 25 °C in the dark for 20 min before the colorimetric measurement. We prepared the TMB indicator by dissolving TMB in a mixture of ethanol (100%, Sigma-Aldrich) and 1.0 M sodium acetate/acetic acid buffer (pH 3.5) at a volume ratio of 4:1. The concentration of TMB in the solution was 2.0 mM. The standard curve was prepared using AuCl 3 (Sigma-Aldrich) in Milli-Q water solution at a final concentration of 2, 3, 10, 20, and 30 μM (Supplementary Table 8, Supplementary Fig. 6). The pH of the microcosms was also monitored at the sampling time using a pH meter (MC-80; TPS). The microcosm setup and all measurements were conducted in triplicate.

Geochemical modeling and thermodynamic calculation

The pH-Eh diagrams were constructed at 25 °C using the Geochemist’s Workbench (GWB), version 12 (Rockware Inc., Golden, CO, USA) to show the predicted gold solubility and predominant gold species. The systems contained 1 ppt to 1 ppm of Au+. HCO 3 − and S 2 O 3 2− with an activity of 0.01 were used to represent carbon-rich and sulfur-rich systems, respectively. In each diagram (Fig. 2d, e), the carbon or sulfur species were reacted at various pH-Eh conditions, and the predominant carbon or sulfur species in the sub-diagram were then reacted with gold.

Thermodynamic calculation (thermodynamic properties of substances shown in Supplementary Table 9): The standard enthalpy of reaction (3) is:

$$\Delta _{\mathrm{r}}H^\Theta = \hskip 2.5pt 2 \times \Delta _{\mathrm{f}}H_{{\mathrm{H}}_2{\mathrm{O}}\left( {\mathrm{l}} \right)}^\Theta + \Delta _{\mathrm{f}}H_{{\mathrm{Au}}^{3 + }}^\Theta - \Delta _{\mathrm{f}}H_{{\mathrm{Au}}^0\left( {\mathrm{s}} \right)}^\Theta - \Delta _{\mathrm{f}}H_{{\mathrm{O}}_2^ - \left( {\mathrm{g}} \right)}^\Theta \\ - 4 \times \Delta _{\mathrm{f}}H_{{\mathrm{H}}^ + }^\Theta = - 122.82\;{\mathrm{kJ}} {\cdot} {\mathrm{mol}}^{ - 1}$$

The standard entropy of reaction (3) is:

$$\Delta _{\mathrm{r}}S^\Theta = 2 \times S_{{\mathrm{H}}_2{\mathrm{O}}\left( {\mathrm{l}} \right)}^\Theta + S_{{\mathrm{Au}}^{3 + }}^\Theta - S_{{\mathrm{Au}}^0\left( {\mathrm{s}} \right)}^\Theta - S_{{\mathrm{O}}_2^ - \left( {\mathrm{g}} \right)}^\Theta - 4 \times S_{{\mathrm{H}}^ + }^\Theta = - 359.74\;{\mathrm{J}} {\cdot} {\mathrm{mol}}^{ - 1} \, {\mathrm{K}}^{ - 1}$$

The Gibbs free energy of reaction (3) is:

$$\Delta _{\mathrm{r}}G^\Theta = \Delta _{\mathrm{r}}H^\Theta - T \times \Delta _{\mathrm{r}}S^\Theta = - 15.62\;{\mathrm{kJ}} {\cdot} {\mathrm{mol}}^{ - 1}$$

as \(\Delta _{\mathrm{r}}G^\Theta = - 2.303\;RT{\mathrm{log}}K^\Theta\) (R is the gas constant, T is temperature in K)

The logKΘ of reaction (3) is 2.74.

In alkaline solution34, Au3+ could complex to OH− forming Au(OH) 4 -:

$${\mathrm{Au}}^{3 + } + 4\;{\mathrm{OH}}^ - = {\mathrm{Au}}\left( {{\mathrm{OH}}} \right)_4^ - \quad {\mathrm{log}}K = 51.35$$ (4)

Together with water dissociation reaction:

$${\mathrm{H}}_2{\mathrm{O}}_{\left( {\mathrm{l}} \right)} = {\mathrm{H}}^ + + {\mathrm{OH}}^ - \quad {\mathrm{log}}K = -14.00$$ (5)

By combining reaction (3), (4), and (5), we get the log K of reaction (1)

For reaction of oxygen (O 2(aq) ) oxides Au (s) 0

$${\mathrm{Au}}_{\left( {\mathrm{s}} \right)} + 0.75\;{\mathrm{O}}_{2\left( {{\mathrm{aq}}} \right)} + 3{\mathrm{H}}^ + = {\mathrm{Au}}^{3 + } + 1.5\;{\mathrm{H}}_2{\mathrm{O}}_{\left( {\mathrm{l}} \right)}\quad {\mathrm{log}}K = -11.44$$ (6)

Considering the Au(OH) 4 − complexation, we get the log K of reaction (2) by combining reaction (4), (5), and (6)

To calculate the standard reduction potential of O 2 − (g) /H 2 O (l) redox couple, the reaction (3) can be divided into two half reactions

$${\mathrm{Au}}_{\left( {\mathrm{s}} \right)}-3\;{\mathrm{e}}^- = {\mathrm{Au}}^{3 + }$$ (7)

$${\mathrm{O}}_{2\left( {\mathrm{g}} \right)}^ - + 4{\mathrm{H}}^ + + 3\;{\mathrm{e}}^- = 2\;{\mathrm{H}}_2{\mathrm{O}}_{\left( {\mathrm{l}} \right)}$$ (8)

The change in free energy of each half reactions can be measured by Eq. (9)

$$\Delta G^{{0}} = -nFE^{{0}},$$ (9)

where n is number of moles of electrons (equivalents) involved in the reaction; F is the Faraday constant (96.49 kJ per volt gram equivalent), and E0 is the cell potential (V) at standard state. For reaction (7), E0 = –1.52V52. So, the ∆G0 for reaction (7) is:

$$\Delta G_{{\mathrm{Au}}^0\left( {\mathrm{s}} \right)/{\mathrm{Au}}^{3 + }}^0 = - nFE^0 = 440.00\;{\mathrm{kJ}} {\cdot} {\mathrm{mol}}^{ - 1}$$

So, for reaction (8):

$$\Delta G_{{\mathrm{O}}_2^ - /{\mathrm{H}}_2{\mathrm{O}}\left( {\mathrm{l}} \right)}^0 = \Delta _rG_{{\mathrm{reaction}}\left( 3 \right)}^{\mathrm{\Theta }} - \Delta G_{{\mathrm{Au}}^0\left( {\mathrm{s}} \right)/{\mathrm{Au}}^{3 + }}^0 = - 455.62\;{\mathrm{kJ}} {\cdot} {\mathrm{mol}}^{ - 1}$$

$${\mathrm{log}}K^0 = \frac{{ - \Delta G_{{\mathrm{O}}_2^ - /{\mathrm{H}}_2{\mathrm{O}}\left( {\mathrm{l}} \right)}^0}}{{2.303\;RT}} = 79.84$$

$$E_{{\mathrm{O}}_2^ - /{\mathrm{H}}_2{\mathrm{O}}\left( {\mathrm{l}} \right)}^0 = \frac{{ - \Delta G_{{\mathrm{O}}_2^ - /{\mathrm{H}}_2{\mathrm{O}}\left( {\mathrm{l}} \right)}^0}}{{nF}} = 1.57\,{\mathrm{V}}$$

$$K = \frac{{\alpha _{{\mathrm{H}}_2{\mathrm{O}}\left( {\mathrm{l}} \right)}^2}}{{\alpha _{{\mathrm{O}}_2^ - }\alpha _{{\mathrm{H}}^ + }^4\alpha _{{\mathrm{e}}^ - }^3}}$$ (10)

So \({\mathrm{log}}K = 4pH + 3pe\) as \(pe = \frac{F}{{2.303RT}}Eh\)

The correlation of pH-Eh for O 2 − (g) /H 2 O (l) redox couple is

$$79.84 = 4{\mathrm{pH}} + 50.7{\mathrm{Eh}}$$ (11)

As shown in Fig. 2, when pH = 0, Eh = 1.57; when pH = 14, Eh = 0.47.

Isolation and identification of gold-oxidizing fungi

The auriferous soil samples were diluted (10−1) in sterilized 0.7% NaCl, and 10 μL of the dilution was plated onto gold-PYG agar without adjusting the pH. PYG medium contained (per liter) 20 g agar, 0.25 g peptone, 0.25 g yeast extract, 0.25 g glucose, 0.01 g CaCl 2 ·2H 2 O, and 0.5 g MgSO 4 ·7H 2 O26. We aseptically added AuCl to the medium to a final concentration of 10 μM. Colloidal gold formed in the medium overnight through AuCl disproportionation. All plates were incubated at 10 °C in the dark. Gold-oxidizing fungi were screened by the TMB plate-flooding method after 14 days of incubation. Briefly, 1 mg of TMB was dissolved in 1 mL of dimethyl sulfoxide (Sigma-Aldrich) and 9 mL of 0.05 M phosphate/citrate buffer (pH 5.0). A drop of TMB reagent was applied directly to a fungal colony and incubated for 20 min at room temperature in the dark prior to a visual inspection for color change. To eliminate false positive signals produced by peroxidase, a drop of 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS, Sigma-Aldrich) was applied to a spare area of the same fungal colony. The ABTS solution was prepared by dissolving ABTS in 0.2 M sodium acetate (Sigma-Aldrich) (pH 5.0). The final concentration of ABTS in the solution was 3.6 mM53. An additional spot of TMB reagent was placed on the agar as a reference for the abiotic oxidation of gold. TMB-positive (blue) and ABTS-negative (colorless) colonies were preliminarily identified as gold-oxidizing fungal isolates and were subjected to further analysis after purification by streaking on a plate five times.

Fungal genomic DNA was extracted using a plant/fungi DNA isolation kit (Norgen Biotek Corp., Thorold, ON, Canada) with one loop of fungal biomass, according to the user’s manual. Fungal ITS region primers comprising ITS1-F (CTTGGTCATTTAGAGGAAGTAA) (forward) and ITS4 (TCCTCCGCTTATTGATATGC) (reverse) were used for fungal rRNA gene ITS amplification54. Sequencing was conducted by the Australian Genome Research Facility (Melbourne, VIC, Australia). Sequences were assembled using Geneious Pro version 4.6.0. Fungal phylogeny was determined by performing a BLAST search of the obtained ITS sequences against the NCBI database.

X-ray photoelectron spectroscopy

The preliminarily identified gold-oxidizing fungal isolates were inoculated on gold-PYG agar at a final gold concentration of 400 μM. After 14 days of incubation at 10 °C in dark, a gold-depleted halo around the colony appeared, dividing the agar into a central zone, an oxidized zone, and an undisturbed zone. At the center of each zone, the agar was vertically cut to form a 5 mm wide × 10 mm long surface. The agar pieces were then carefully freeze-dried by lyophilization overnight (ALPHA 2–4 LD plus; John Morris Scientific, Sydney, NSW, Australia). All freeze-dried samples were adhered onto double-sided adhesive tape by exposing the cross-section upside prior to introducing them into the analysis chamber. XPS measurements were performed on an Axis Ultra DLD spectrometer (Kratos, Manchester, UK) using a monochromatic AlKα (1486.6 eV) irradiation source operated at 225 W in conjunction with a charge neutralizer. A hybrid lens system with a magnetic lens provided an analysis area constrained to a spot of 110 µm in diameter. The proportion between area of the beam and sample of each zone was 1:5000. The vacuum pressure of the analysis chamber of the spectrometer was maintained at ≤8 × 10–9 Torr throughout the duration of the analysis. The electron binding energy scale was referenced to the C 1s line of aliphatic carbon, which was set at 284.8 eV. XPS spectra were collected with a pass energy of 160 eV for survey spectra and 40 eV for high-resolution spectra. Data files were processed using CasaXPS (Casa Software Ltd, Teignmouth, UK). Shirley background subtraction was applied to all high-resolution spectra. Au 4f spectra were fitted using an asymmetric peak shape for metallic Au, whereas Gaussian-Lorentzian line shapes were used to fit the higher-oxidation state gold species. The area ratio for the Au 4f5/2:Au 4f7/2 doublets was set to 3:4, whereas the full width at half maximum was constrained to values considered reasonable for each chemical state.

Laser ablation inductively coupled plasma mass spectrometry

Colloidal gold distributions within colonies on the freeze-dried PYG agar plates were measured using LA-ICP-MS. The freeze-dried agar was vertically cut to a 5 mm wide × 80 mm long piece, which was then pressed against a polystyrene plate that had a 2-mm vertical slit to form an even surface for LA-ICP-MS analysis. Profiles (length, ca. 8 cm) covering biomass and agar alone were ablated with a New Wave 193 nm ArF excimer laser coupled to an Agilent 7700 ICP-MS instrument (Agilent Technology, Santa Clara, CA, USA). The line scan ablation was performed using a 30-µm square spot, a laser repetition rate of 20 Hz, and 60 µm s−1 stage translation speed. The monitored isotopes were 25Mg, 39K, and 197Au; the dwell times used for these isotopes were 0.01, 0.01, and 0.2 s, respectively. Absolute quantifications of gold were not performed because of the absence of a standard reference. Gold signals from the line scan ablation were presented as counts. Signal intensity was normalized to 25Mg and 39K in the agar.

Cyclic voltammetry

Cyclic voltammetry was applied to examine gold oxidation by TA_pink1. The cyclic voltammetry experiment was carried out in a 30 mL three-electrode reactor equipped with a graphite rod (diameter 5 mm) working electrode, a Ag/AgCl (3 M KCl) reference electrode, and a platinum wire counter electrode. The electrodes were connected to a potentiostat (SP-150; Bio-Logic, Seyssinet-Pariset, France) via copper wires. Twenty milliliter of PYG medium containing the fungal biomass with or without 400 μM colloidal gold were introduced to the reactor. Cyclic voltammetry was performed at 0 and 17 h at a scan rate of 10 mV/s55.

Scanning electron microscopy

TA_pink1 cultured in 10 mL liquid PYG medium was harvested by centrifugation at 3000 × g for 5 min. Sterilized cotton fiber was used as a non-biological control. Briefly, 200 mL of liquid PYG medium spiked with 400 μM colloidal gold was aseptically mixed with either the TA_pink1 biomass or the sterilized cotton fiber. The mixtures were incubated without shaking at 10 °C in the dark for 14 days. The fungal biomass and cotton fiber were then harvested by centrifugation and freeze-dried by lyophilization overnight (ALPHA 2–4 LD plus). The surface of dried fungal biomass and cotton fiber was coated with carbon and examined with a scanning electron microscope (XL-40 FEG; Philips, Eindhoven, Netherlands) fitted with a semi-quantitative energy dispersive X-ray spectrometer.

Gold-oxidizing fungus hyphal extension assay

Czapek Dox agar31 was sterilized by autoclaving at 121 °C for 20 min prior to addition of 50 µM AuCl 3 once the agar had cooled to 50 °C56. Then, 30 g of sucrose and 8 g of lignin were added to 1 L of medium, respectively. TA_pink1 was centrally inoculated from actively growing stock cultures maintained on PYG medium and the diameter of the fungal colony was measured manually with a ruler to calculate radial expansion rates.

Nucleic acid extraction and MiSeq sequencing

Soil DNA was extracted from 1 g wet weight of soil with a PowerSoil Total DNA Isolation kit (MO BIO Laboratories, Carlsbad, CA, USA) according to the manufacturer’s instructions. The extracted DNA was quantified using real-time quantitative polymerase chain reaction (qPCR) to assess template copy number and identify whether PCR inhibitors were present. qPCR was conducted for each sample at three dilutions (undiluted, 10–1, and 10–2) using the primers ITS2_ITS7F (GTGAGTCATCGAATCTTTG)57 and ITS2_ITS4R (TCCTCCGCTTATTGATATGC)58 to sequence the fungal rRNA ITS region, and the primers Bact_16S_F515 (GTGCCAGCMGCCGCGGTAA)59 and Bact_16S_R806 (GGACTACHVGGGTWTCTAAT)60 to sequence part of the bacterial 16S rRNA gene.

All PCRs were conducted in a 25-μL volume that included 2.5 mM MgCl 2 (Applied Biosystems, Waltham, MA, USA), 1× PCR Gold Buffer (Applied Biosystems), 0.25 mM deoxynucleotide triphosphates (dNTPs) (Astral Scientific, Taren Point, NSW, Australia), 0.4 mg mL−1 bovine serum albumin (Fisher Biotec, Wembley, WA, Australia), 0.4 μM of each primer, 0.2 μL AmpliTaq Gold DNA polymerase (Applied Biosystems), and 0.6 μL SYBR-Green dye. The qPCR reaction included denaturation at 95 °C for 5 min, 45 cycles at 95 °C for 30 s, annealing at 54 °C for 30 s, and extension at 72 °C for 30 s; plus a final extension at 72 °C for 10 min. The optimal dilution point for each sample was selected and used as the template for multiplex identifier (MID)-tagged PCR reactions.

All MID-tagged PCRs were performed in a 25-μL volume using the same PCR and thermocycler conditions, as stated above. All amplicons were generated in duplicate and assigned unique forward and reverse MID tags to ensure that any contamination from previously generated amplicons could be excluded post-sequencing. The resulting amplicons were pooled together to reduce PCR stochasticity. They were quantified on a Lab Chip and subsequently pooled in equimolar ratios into an amplicon library. The resulting library was run on a Pippin Prep (Sage Science, Beverly, MA, USA) to size-select for fragments in the range of 250–600 bp to reduce the amount of primer dimers within the library. The resulting eluate was purified using a QIAquick PCR purification kit (Qiagen, Chadstone Center, VIC, Australia) per the manufacturer’s protocol. Illumina MiSeq sequencing was performed using the MiSeq Reagent Kit v2 (500 cycles; Illumina, San Diego, CA, USA) 250 bp paired-end protocol following the manufacturer’s instructions.

Statistics and bioinformatics analysis

Differences and correlation between two sets of data were determined to be significant using an unpaired two-tailed t-test and Pearson correlation coefficient (GraphPad Prism Version 7; GraphPad Software, La Jolla, CA, USA).

MiSeq sequencing data were denoised and analyzed using Mothur v1.37.6 on the EC2 cloud service of the Amazon Web Server61. According to the MiSeq standard operating procedure (https://www.mothur.org/wiki/MiSeq_SOP), sequences were filtered based on the quality score using the make.contigs command. Sequences were then trimmed to a length of 250 bp, screened for chimeras (UCHIME), and grouped into OTUs at 0.03 distance cut-offs. Statistical analyses and taxonomic classification against the UNITE (fungi)62 and the Silva (bacteria)63 reference databases were also performed in Mothur, including the coverage, inverse Simpson index, and Berger-Parker index64. Relationships between geochemical variables and fungal community structures were analyzed using CCA in Canoco 565.

We constructed MENs based on Illumina MiSeq sequencing data of fungal ITS sequences through random matrix theory66. The sequence abundance table of the fungal community after Mothur analysis was split into two datasets: auriferous and reference. For each of them, only the OTUs that appeared in seven or more replicates were used for correlation calculations. The calculations of the global network properties, centrality of individual nodes, and module separation and modularity were performed using Pipeline online (http://ieg2.ou.edu/MENA/main.cgi). Data were visualized by Cytoscape v 2.8.367.