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Insights into the molecular methods used to study gold nugget forming microbes—
outlook towards a promising future for these organisms in gold ore processing
1CRC LEME, Department of Earth and Marine Sciences, Australian National University, ACT, 0200 2CRC LEME, CSIRO Land and Water, PMB 2, Glen Osmond, SA, 5064
The residential microflorae of auriferous soils from the Tomakin Park Gold Mine in temperate south eastern
New South Wales and the Hit or Miss Mine in the tropical Palmer River gold fields in north eastern
Queensland have been shown to solubilize and precipitate gold (Reith & McPhail, in preparation). To
determine if microorganisms also contribute to the authigenic formation of gold nuggets, gold flakes were
obtained from soils overlying these deposits and from other prospects in the Palmer River gold fields.
Scanning electron microscopy revealed the presence of micrometer-sized 'bubbly' structures, which cover
large areas on the surface of these gold flakes. Based on their morphology, these bubbly structures were
described as microbial fossils, pseudomorphs or fossilised biofilms of microbial origin. They closely
resemble budding microbial cells of the genus Pedomicrobium and were detected on gold flakes from various
locations in Australia, Alaska and South America (Bischoff et al. 1992, Watterson 1992, Bischoff 1997).
However, proof of their microbial origin cannot be made based on describing their morphologies alone.
Scanning electron microscopy of gold flakes used in this study revealed regions that seemed to be covered by
recently active biofilms. This was verified by staining the gold flakes with 4',6'-Diamidino-2-phenylindole
(DAPI), a dye that forms fluorescent complexes with natural double-stranded DNA. Using DAPI-staining,
combined with confocal laser microscopy, we were able to create 3D-images of these biofilms on the gold
However, to understand which organisms are involved in the formation of gold flakes and nuggets the DNA
of these organisms has to be isolated and studied. We are using denaturing gradient gel electrophoresis
(DGGE) of certain variable areas of the 16S rDNA to obtain information relating to the microbial community
structure on the gold flakes, such as the organisms present, the number of species and the population
diversity. The ribosomal 16S rDNA gene is functionally conserved in all prokaryotic organisms (Amann et
1995). However, within the 16S rDNA gene are several variable regions where the nucleotide sequence
differs between different species, thus 16S rDNA can be used to identify groups and species of organisms
(Amann et al. 1995). 16S rDNA is today widely used to compare the microbial community structure and
diversity of environmental samples (Head et al. 1998).
In this paper we aim to explain the methodology we are currently using to obtain and study 16S rDNA from
the natural gold grains, describe our first results obtained by using these techniques and give an outlook on
how these findings might be used to construct biological accumulation plants for gold processing.
The Polymerase Chain Reaction (PCR)
The PCR amplifies a specific region of DNA as defined by two primer sequences, thus it can be used to
examine a particular region of the genome. Starting from a small amount of DNA in a sample a large number
of copies of this region of the DNA are created by using PCR-amplification. The PCR-amplification itself is
a three-stage process: (i), DNA is denatured (made to a single strand); (ii), the primers bind to their
complementary sequence; and (iii), primers are extended by addition of nucleotides complementary to that on
the template sequence. This process was repeated 25 times in our experiments. Depending on the focus of the
study the primers used can be universal (i.e., used for all bacteria or all fungi), or specific, so as to amplify
DNA from one species only. Since the aim of our study was to examine the microbial diversity, we used a
universal set of primers (27-GC and 534R), designed to anneal to all 16S rDNA. Because we expected the
amount of 16S rDNA on the gold flakes to be minimal, we did not use a conventional DNA extraction
method, but used 40 gold flakes directly as templates in our PCR-amplification. The product of this first
PCR-amplification was used as a template for a second PCR-amplification.
Denaturing gradient gel electrophoresis (DGGE)
The PCR-products, i.e., the amplified 16S rDNA, from the gold flakes were separated by denaturing gradient
In: Roach I.C. ed. 2004. Regolith 2004. CRC LEME, pp. 292-294. gel electrophoresis (DGGE). This technique is used to split up small DNA fragments (200-1000 base pairs—
bp) according to their melting behavior. The small fragments of genomic DNA, which are run on a low-to-
high denaturing gradient acrylamide gel, initially move according to molecular weight. As they progress into
regions of higher denaturing conditions the DNA begins to melt; the point at which this melting occurs
depends on its individual nucleotide composition. A shift in mobility of the DNA in the gel occurs after the
melting and the DNA gets 'stuck' and forms a characteristic band, which can be stained. In our study we used
an 8 % acrylamide gel made up with 40 to 65 urea/formamide denaturing solutions, which was run for 20 h at
60ºC and 60 V in 1 % Tris-acetate-EDTA-electrophoresis-buffer (TAE). The gels were stained for band
analysis using a silver staining technique (Bio-Rad Laboratories, California, USA). SYBR-gold staining was
used on gels used for band excision for sequencing (Invitrogen®, Oregon, USA). SYBR-gold stained gels
were digitalized using a UV transluminator system equipped with Diversity database software (Bio-Rad
Laboratories, California, USA). Silver stained gels were digitalized using a conventional scanner and scanner
Cloning and sequencing
To obtain information on which species are present on these gold flakes individual bands from the SYBR-
gold stained gels were excised by inserting a small pipette tip into the gel at each band of interest. The pipette
tips were placed into individual microcentrifuge tubes containing 40 µl H2O and held at 4ºC for 18 h to elute
the DNA. The pipette tip was then removed and PCR-amplification was used to multiply the particular bands
of 16S rDNA using the 27 GC and 534R primers. The product was run on another DGGE gel, to insure that
just single bands had been amplified. These individual bands were excised, amplified by PDR reaction and
the PCR-products were run on 2% agarose gels. These were stained for 1 h with ethidium bromide and the
16S rDNA bands were excised using a sterile scalpel and tweezers on a UV-transluminatior. These excised
bands were gel purified using Eppendorf Perfectprep® Gel Cleanup kit (Eppendorf AG, Hamburg,
Germany). Purified fragments were cloned into E. coli cells using a Promega pGEM-T SysII (Promega
Bioscience, California, USA). Cells were plated out and white colonies selected. Colonies were transferred
into LB-broth containing ampicillin and incubated at 37ºC overnight. The DNA was extracted using Mobio
UltracleanTM Mini Plasmid Prep kit (Mobio Laboratories Inc., California, USA). Extracted DNA was
sequenced using Beckman-Coulter-dye™ Terminator Cycle Sequencing with Quick-Start kit (Beckman-
Coulter Inc., California, USA). Samples were run on the Beckman Coulter™ CEQ 8000 capillary sequencer
(Beckman-Coulter Inc., California, USA). Base calling was checked by eye.
Band and sequence analysis
To analyze the DGGE gels the Diversity database software is used. The program is designed to identify and
group bands and compare band set from different gels. Two reference lanes of E. coli 16S rDNA extracts
were used as standards in our gels. A dissimilarity matrix will be calculated, based on band presence or
absence to compare the community structure of the different gold flakes. These can be analyzed using cluster
The sequence data was aligned using Bioedit software. The GenBank database was searched for matching
sequences using the BLAST software. Furthermore a phylogentic tree will be assembled from the 16S rDNA
sequences obtain from the gold flakes to show their relation to other groups of organisms.
DAPI staining has shown the presence of biofilms on the gold flakes. By successfully extracting 16S rDNA
from gold flakes we have now obtained further proof for their existence and are now able to identify their
nature. We were able to obtain 16S rDNA from 37 of 40 of gold flakes, indicating that bacteria are
commonly associated with gold flakes. Examination of the scanned images of the DGGE gels revealed the
presence of 2 to 25 (on average 6 to 8) clearly visible bands in every positive lane, indicating that up to 25
different species of prokaryotes were associated with the gold flakes. A widespread variation within the
samples was generally visible. Several bands were only detected on individual flakes or on flakes from one
particular site, while some bands occur in almost all samples from one site or in some case can be detected on
several samples from both sides. Generally, biofilms work like a well-structured factory where every group
of organisms has their specific tasks; while some tasks can be fulfilled by different groups of organisms,
others can only be fulfilled by a specific group (Little et al. 1997). The 16S rDNA shows that the biofilms
consist of a variety of species, but that one or two species seem to occur in most samples, indicating that
these organisms might have such a special task, such as the selective precipitation of the gold from solution
to form the actual gold flake.
Bands were excised based on the band analysis results of the DGGE gels and to date 8 of these bands have
F. Reith and S.L. Rogers. Gold flakes and the art of molecular bioscience. been sequenced. Six of these sequences are not described in the GenBank database, which could indicate the
presence of formerly unknown or undescribed organisms on the gold flakes. Two of our sequences matched a
known extremophile. This organism seems to appear on most of the samples from both sampling sites. This
organism has been described as a metallophile, i.e., an organism that thrives in the presence of mM
concentrations of several heavy metals. It has been shown to harbor a large variety of heavy metal
resistances, such as resistance to Zn, Co, Cr, Cd, Co, Ni, Hg on two plasmids. It is commonly found in
natural or industrial heavy metal contaminated soils. It has also been shown to accumulate and precipitate
heavy metals, such as Cd, and form structures similar to those described on our gold flakes. Commonly it is
used in ecotoxicology testing of soils, but has not been tested for its gold resistance or for its use in gold
Organisms such as Thiobacillus ferrooxidans are commonly used in mineral processing plants to break down
sulfide minerals and liberate the metals bound within (Bosecker 1997). However, to then precipitate the
liberated gold the gold-containing solution, the leachate is run over activated carbon, which adsorbs the gold.
The adsorbed gold has to then be leached again, usually with concentrated cyanide solutions, before it can be
recovered by electro-winning. The process is highly energy consuming and also produces considerable
amounts of environmentally dangerous waste. Metal accumulating organisms, especially those showing high
metal resistance, could be successfully used to precipitate gold from solution. Apparently, the organisms on
our gold flakes selectively adsorb gold from the soil solution and form gold flakes of more than 97% purity.
Used in mineral processing, especially in conjunction with a bio-oxidation process using Thiobacilli, a highly
efficient environmentally friendly gold bio-oxidation/bio-accumulation could be constructed. Further
research should be undertaken to secure progress towards an environmentally sustainable and efficient future
for the mining- and minerals-processing industries, which make up a large proportion of Australia's GDP.

Acknowledgements: The authors express sincere appreciation to: the Cooperative Research Centre for
Landscape Environment and Mineral Exploration (CRC LEME) for funding and support of this project; Peter
and Kinuyo Wyatt at Tomakin Park Gold Mine for the access to the property and help during my stay; and,
Diana Harley at CSIRO Entomology for sequencing of the 16S rDNA fragments.

AMANN R.I., LUDWIG W. & SCHLEIFER K.H. 1995. Phylogenetic identification and in situ detection of
individual microbial cells without cultivation. Microbiological Reviews 59, 143-169.
BISCHOFF G.C.O. 1997. The biological origin of bacterioform gold from Australia. N. Jb. Geol. Paläont. Abh. H6, 329-338.
BISCHOFF G.C.O., COENRAADS R.R. & LUSK J. 1992. Microbial accumulation of gold: an example from Venezuela. N. Jb. Geol. Paläont. Abh. 194, 187-209.
BOSECKER K. 1997. Bioleaching: metal solubilization by microorganisms. FEMS Microbiol. Rev. 20, 591-
HEAD I. SAUNDERS J.R. & PICKUP R.W. 1998. Microbial evolution, diversity, and ecology: a decade of ribiosomal RNA analysis of uncultivated microorganisms. Microbial Ecology 35, 1-21.
LITTLE B.J., WAGNER P.A. & LEWANDROWSKI Z. 1997. Spatial relationships between bacteria and mineral surfaces. In: BANFIELD J.F. & NEALSON K.H. eds. Geomicrobiology: Interactions Between
Microbes and Minerals. Reviews in Mineralogy 35 Mineralogical Society of America, Washington
D.C., USA, 123-155.
REITH F. & MCPHAIL D.C. in preparation. Effect of the complex natural microflora on the solubilization of gold in soils from the Tomakin Park Gold Mine, New South Wales, Australia. To be submitted to Geochimica and Cosmochimica Acta. WATTERSON J.R. 1992. Preliminary evidence for the involvement of budding bacteria in the origin of Alaskan placer gold. Geology 26, 1147-1151.
F. Reith and S.L. Rogers. Gold flakes and the art of molecular bioscience.


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