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Biogeochemical cycling of gold: Transforming gold particles from arctic Finland
Accepted Manuscript Biogeochemical cycling of gold: Transforming gold particles from arctic Finland
Frank Reith, Maria Angelica D. Rea, Paige Sawley, Carla M. Zammit, Gert Nolze, Tina Reith, Kai Rantanen, Andrew Bissett PII: DOI: Reference:
S0009-2541(18)30133-5 doi:10.1016/j.chemgeo.2018.03.021 CHEMGE 18701
To appear in:
Chemical Geology
Received date: Revised date: Accepted date:
8 November 2017 9 March 2018 14 March 2018
Please cite this article as: Frank Reith, Maria Angelica D. Rea, Paige Sawley, Carla M. Zammit, Gert Nolze, Tina Reith, Kai Rantanen, Andrew Bissett , Biogeochemical cycling of gold: Transforming gold particles from arctic Finland. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Chemge(2017), doi:10.1016/j.chemgeo.2018.03.021
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Manuscript submitted to Chemical Geology
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Biogeochemical cycling of gold: Transforming gold particles from arctic Finland
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Frank Reith1,2*, Maria Angelica D. Rea1,2, Paige Sawley1, Carla M. Zammit3, Gert
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Nolze4, Tina Reith1, Kai Rantanen5, Andrew Bissett6
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Affiliations:
The University of Adelaide, School of Biological Sciences, Department of Molecular
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and Cellular Biology, Adelaide, South Australia 5005, Australia 2
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CSIRO Land and Water, Environmental Contaminant Mitigation and Technologies, PMB2, Glen Osmond, South Australia 5064, Australia
School of Earth Sciences, University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia
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Federal Institute for Materials Research and Testing, Berlin, Germany 5
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Gold Prospectors Association of Finnish Lapland, Inari, Finland
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CSIRO, Oceans and Atmosphere, Hobart, Tas, 7000, Australia
Running Title: Geomicrobial gold transformation in arctic Finland
*Corresponding author: Frank Reith
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ACCEPTED MANUSCRIPT A/Prof. Frank Reith Department of Molecular and Cellular Biology, School Biological Sciences, The University of Adelaide CURRENT ADDRESS:
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CSIRO Land and Water
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PMB 2
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Glen Osmond, 5064
FAX:
+61 8 8303 8550
[email protected]
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Number of Pages:
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E-MAIL:
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+61 8 8303 8469
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PHONE:
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South Australia, Australia
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Number of Tables
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Number of Figures:
Number of Supplementary Materials: 4
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ACCEPTED MANUSCRIPT Abstract (Bio)geochemical cycling of gold (Au) has been demonstrated in present-day (semi)arid, (sub)-tropical and temperate environment. Hereby biofilms on Au-bearing mineraland Au-particle surfaces drive Au dispersion and reconcentration, thereby (trans)forming
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the particles. However, it is unknown if biogeochemical cycling of Au occurs in polar
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environments, where air temperatures can reach -40°C and soils remain frozen for much
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of the year. Therefore, placer Au-particles, soils and waters were collected at two placer mining districts in arctic Finland, i.e., the Ivalojoki and Lemmenjoki goldfields. Sites were
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chosen based on contrasting settings ((glacio)-fluvial vs. glacial-till deposits) and depths
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(surface to 5 m below current surface). Gold particles were studied using a combination of tagged 16S rRNA gene next generation sequencing and electron microscopic / micro-
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analytical techniques. Across all sites a range of Au-particle morphologies were
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observed, including morphotypes indicative of Au dissolution and aggregation. Elevated Au concentrations indicative of Au mobility were detected in placer particle bearing soils
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at both districts. Typically Au-particles were coated by polymorphic biofilm layers
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composed of living and dead cells embedded in extracellular polymeric substances. Intermixed were biominerals, clays and iron-sulfides/oxides and abundant secondary Au
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morphotypes, i.e., nano-particles, micro-crystals, sheet-like Au, branched Au networks and overgrowths and secondary rims. Biofilms communities were composed of Acidobacteria (18.3%), Bacteroidetes (15.1%) and Proteobacteria (47.1%), with Proteobacteria (19.5%) being the most abundant proteobacterial group. Functionally, biofilms were composed of taxa contributing to biofilm establishment, exopolymer production and nutrient cycling, abundant taxa capable of Au mobilization, detoxification and biomineralization, among them Cupriavidus metallidurans, Acinetobacter spp. and
ACCEPTED MANUSCRIPT Pseudomonas spp, were detected. In conclusion, these results demonstrate that placer Au-particle transformation and Au dispersion occur in cold, arctic environments. This corroborates the existence of biogeochemical Au cycling in present-day cold
bacteria,
biogeochemistry,
mobility,
Finland,
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Keywords: Gold,
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environments.
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metallidurans, NGS
Cupriavidus
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Highlights:
Gold particles from Arctic placers are biogeochemically transformed.
Multispecies bacterial biofilms exists on Arctic placer gold particles.
Biofilm communities include bacterial taxa mediating gold cycling.
Elevated gold concentration were detected in soils and waters.
Biogeochemical gold-cycling occurs in modern day cold, arctic environments.
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ACCEPTED MANUSCRIPT 1. INTRODUCTION The cycle of gold (Au) is driven by physical, geochemical and geomicrobial processes mediating the dispersion and reconcentration of Au in Earth surface environments (Southam et al., 2009; Reith et al., 2013; Craw and Kerr, 2017). Thereby
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physical, chemical and biological processes co-occur and their interactions shape Au-
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particle morphologies and the distribution of Au in the landscape (Reith et al., 2007;
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Craw and Kerr, 2017). Physical erosion of host materials, e.g., quartz-Au veins, results in primary Au being deposited in placers, including eluvial, alluvial, glaciofluvial and
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glacial-till placers. Biological and chemical processes in combination with climatic and
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geological factors determine the physicochemical conditions at a site, and drive the solubilization and re-precipitation of Au (e.g., Reith et al., 2007; Craw and Kerr, 2017).
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Primary Au undergoes transformation through chemical de-alloying, biogeochemical
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Au/Ag dissolution as well as (bio)geochemical aggregation of Au (e.g., Southam et al., 2009; Reith et al., 2013; Craw and Kerr, 2017). This leads to the formation of secondary
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Au overgrowths, which are very pure (>99 wt. % Au) and can have a range of
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morphologies including triangle, spherical, hexagonal and octahedral nano-particles and µ-crystals, bacteriomorphic Au and sheet-like and wire Au (Reith et al., 2010; Reith et
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al., 2013; Shuster et al., 2017). Ultimately, high purity Au rims covering entire primary particles or parts thereof form. These can reach widths of several 100 µm and usually display a non-deformed matrix of nano- and microcrystals (Craw and Lilly, 2016; Stewart et al., 2017). Indeed most primary Au-particles recovered from placer environments display a Au-Ag core and have a partial or complete rim of high purity secondary Au (e.g., Groen et al., 1990, McCready et al., 2003, Fairbrother et al., 2012; Reith et al., 2012; Stewart et al., 2017; Shuster et al., 2017; Melchiorre et al., 2018). 3
ACCEPTED MANUSCRIPT Microorganisms contribute to the solubilization of Au via the formation of reactive Mn-oxide minerals, e.g., birnessite, capable of oxidizing Au(0) and Au(I) to Au(III)complexes (Ta et al., 2014 and 2015). Microbes can excrete Au-complexing ligands required for stabilization of Au-complexes in solution, e.g., soil- and groundwater, under
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Earth surface conditions (Reith et al., 2007; Fairbrother et al., 2009). Gold dissolution by
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bacteria has been observed in laboratory culture experiments and in soil microcosms
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(Fairbrother et al., 2009; Reith and McPhail, 2006 and 2007). In a recent study assessing supergene Au transformation in relation to groundwater geochemistry in New
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Zealand, thiosulfate was determined to be the dominant Au-complexing ligand (Craw
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and Kerr, 2017). While a range of microbiota have been shown to passively sorb Au(I/III)-complexes, some bacteria living in biofilm communities on placer Au-particle
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surfaces have developed active biochemical responses to deal with Au-toxicity and
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reductively precipitate Au-complexes leading to the formation of nanoparticulate Au (Nakajima, 2003; Reith et al., 2009; Kenney et al., 2012; Johnston et al. 2013; Checa et
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al., 2007). Dispersion of Au nanoparticles enhances Au mobility, aggregation of nano-
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and microphase Au concentrates Au in secondary enrichment zones (Craw and Kerr, 2017; Shuster et al., 2017).
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Natural Au-particles from a wide range of placer localities in (semi)-arid and (sub)-tropical Australia, arid North America, temperate New Zealand, and tropical South America (Brazil and Columbia) are coated by polymorphic layers consisting of cells, extracellular polymeric substances (EPS), as well as detrital and authigenic minerals, e.g., clays, Fe-oxides and sulfides (e.g., Reith et al., 2006; Rea et al., 2016; Shuster et al., 2016; Melchiorre et al., 2018). While species composition of resident biofilms varies between different sites, β- and γ-Proteobacteria dominated communities at sites in 4
ACCEPTED MANUSCRIPT Australia, New Zealand and Brazil (Reith et al., 2006; Reith et al., 2010; Rea et al., 2016). Functional assignment of detected operational taxonomic units (OTUs) suggests that communities living on Au-particle surfaces have highly diverse metabolic capabilities. OTUs can be functionally assigned to six groups linked to different stages of
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biofilm development (Rea et al., 2016): To group one, organisms capable of surface
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conditioning and attachment, e.g., Arthrobacter sp. and Staphylococcus sp., were
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assigned. In group two, OTUs mediating EPS production and autoaggregation, e.g., some Pseudomonas spp. and Rhodobacter spp., are found. To group three organisms
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largely involved in carbon and nitrogen cycling were assigned, these included
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Pelomonas spp., Herbaspirrillum spp., and Acidovorax spp.. To group four organisms capable of the metabolic turnover of complex and xenobiotic organics and toxins were
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assigned, including some Burkholderia spp., Sphingomonas spp. and Phenylobacterium
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include
putative
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spp.. In group five organisms capable of directly affecting Au cycling were identified. Au-mobilizers,
e.g.,
the
sulfur-oxidizing
bacterium
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Diaphorobacter sp., Sphingomonas spp. and Methylobacterium spp. Other species
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included have the ability to actively detoxify Au(I/III)-complexes, e.g., C. metallidurans, D. acidovorans, Stenotrophomonas sp., and Achromobacter spp.. The aurophillic
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bacterium C. metallidurans, which was detected on Au-particles from Australian and Brazilian sites, appears to be one of the key-species involved in Au cycling in these regions (Reith et al., 2006; Reith et al., 2010; Rea et al., 2016). It reduces toxic Au(I/III)complexes in the periplasm, which leads to the formation of Au nanoparticles (Reith et al., 2009; Wiesemann et al., 2013 and 2017). Another bacterium involved in active detoxification and precipitation of Au nanoparticles is D. acidovorans, which produces the metallophore delftibactin to extracellularly convert toxic Au(I/III)-complexes to Au 5
ACCEPTED MANUSCRIPT nanoparticles (Johnston et al., 2013). Species forming dispersal cells, such as Lysobacter spp., are found in group six. In Finnish Lapland Au has been mined from placer deposits for more than 150 years, making this region the last professional placer Au mining area in Western Europe
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and one of the northern most in the world (Saarnisto et al., 1991). Located
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approximately 350 km north of the Arctic Circle, average annual air temperatures lie
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below 0°C, with air temperature below -40°C common in winter. Soils and sediments are frozen for much of the year down to several meters. While grains from Lemmenjoki and
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Ivalojoki occur in a wide range of morphologies and links to physical, chemical and
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anthropogenic transformation have been described by Kinnunen (1996), the presence of biofilms and Au biominerals, e.g., nanoparticles, indicative of biogeochemical Au cycling
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have not been shown from these or any other localities in these climatic conditions.
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Hence, it is unknown if biogeochemical Au cycling occurs in low temperature environments. Therefore, the aims of this study are to assess: (i) the presence of
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secondary Au morphotypes and the dispersion of Au into the surrounding environment;
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(ii) the presence of biofilms on placer Au particles from Finnish Lapland; and (iii) the phylogenetic composition and putative functional capabilities of biofilm communities in
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relation to processes affecting Au mobility and particle transformation. To achieve this, Au-particles from ten individual sites in northern Finland were studied using a combination of Illumina Miseq next generation sequencing (NGS) of biofilm communities and advanced micro-analytical techniques, including field emission gun-scanning electron microscopy (FEG-SEM), focused ion beam-scanning electron microscopy (FIBSEM) coupled with energy dispersive x-ray spectroscopy (EDXS), electron microprobe
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ACCEPTED MANUSCRIPT analyses (EPMA). In addition, soil and water samples from key-sites were analyzed for more than 50 physicochemical properties, including Au concentrations.
2. MATERIALS AND METHODS
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2.1. Description of field sites and sampling
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Sample collection sites are located in the Ivalojoki and Lemmenjoki goldfields in northern
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Lapland, Finland (Fig.1, Table 1). Placer Au has been mined at these sites since the 19th century (Puustinen, 1991; Kojonen et al., 2005). The size of Au-particles mined ranges
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from very fine dust to rare large nuggets weighing several hundred grams (Puustinen,
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1991). Altogether more than 1000 kg of Au have been mined here during the past 120 years. Most of the Au fields are interpreted as (glacio)-fluvial and glacial-till placers; they
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vary in size and are generally composed of sorted to poorly sorted glacial sediments
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(Saarnisto et al., 1991; Kinnunen, 1996).
Geologically, the Ivalojoki and Lemmenjoki placer Au-fields are situated within the
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southern part of the granulite complex of Lapland, which extends from Norway in the
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west to the Russian border in the east (Saarnisto et al., 1991; Kojonen et al., 2005). The complex was metamorphosed during the Svecokarelian Orogeny (~1.9 Ga), under
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granulite conditions (Saarnisto et al., 1991; Tuisku et al., 2006). While primary bedrockhosted Au mineralized system occurs in the area (Sarnisto et al., 1991), large primary Au deposits have not been reported in the immediate vicinity of the sampling sites (Eilu et al., 2007; Eilu, 2015). During the Pleistocene the whole of northern Finland was glaciated (Stigzelius, 1977). Deglaciation began approximately 11600 years ago (Lunkka et al., 2004). During deglaciation, river valleys acted as meltwater channels. Today, interior Finnish Lapland 7
ACCEPTED MANUSCRIPT is characterized by gently undulating glacially formed terrain. The average annual temperature is -0.9 °C in nearby Ivalo, with an average minimum winter temperature of 18°C. Average rainfall is 400 to 500 mm (Finnish Meteorological Institute, 2014). The soil types range from typic haplocryod to skeletic podzol (Sutinen et al., 2007). Soil-pHs
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typically range from 3.0 to 5.4 in top soil horizons to 4.1 to >7 in the deeper soil and till
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horizons (Kähkönen, 1996; Närhi et al., 2011). Organic C and N in Ah-horizon soils can
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reach more than 10 and 0.5 wt.%, respectively; S, Fe and Ca concentration can reach more than 2000, 1400 and 2200 mg/kg in A and B horizons, respectively (Kähkönen,
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1996; Närhi et al., 2011).
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Placer Au-particles, soils and surface water samples from the Ivalojoki and Lemmenjoki placer deposits were collected in August 2013. Gold particles were
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collected from ten sites, all of which are currently mining claims (Fig. 1; Table 1). Three
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sites were within the Ivalojoki district and seven were within the Lemmenjoki district (Fig. 1; Table 1). Sites were chosen based on contrasting deposit styles (glavio)fluvial and till
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placers, at key locations soil and water samples were also collected (Table 1). Gold
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particle, soil and water samples were collected following a field sterile method to minimize contamination (Reith et al., 2010). Gold particles for electron microscopy (EM)
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were washed with sterile 0.9 wt. % NaCl solution and kept in EM fixative (4 wt.% paraformaldehyde, 1.25 wt.% glutaraldehyde in PBS, 4 wt.% sucrose, pH 7.2). Samples for DNA analysis were washed and stored in sterile saline solution. Samples were then transported on ice (Au particles for micro-analysis) or frozen (Au-particles for DNAanalysis, waters and soils) to the laboratory.
2.2. Electron microscopy and microanalyses of Au-particles 8
ACCEPTED MANUSCRIPT Gold particles for EM were processed and dehydrated using series of ethanol (70 vol.%, 90 vol.% and 100 vol.%; 2 x 10 min each) washes and placed in 100 wt.% hexamethyldisilazane (HDMS) to ensure the integrity of bacterial cells and biofilms (Fratesi et al., 2004). Samples were C coated and analyzed using FEG-SEM in
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secondary electron (SE) and backscatter imaging (BSE) modes at 5 kV and 20 kV,
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(Quanta™ 450 FEG Environmental SEM with EDS detectors, FEI, Netherlands).
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Selected Au-particles were analyzed further using a focus ion beam secondary electron microscope (FIB-SEM; Helios NanoLab DualBeam, FEI, Netherlands). Images of the
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surface features were collected at 2 to 20 kV and 86 pA, with sectioning and cleaning at
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20 kV and 9.7 pA. The instrument is equipped with energy dispersive X-ray spectroscopy (EDXA) used to collect element maps across the particles surfaces and
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milled sections; elements mapped were Au, Ag, Fe, C, N, O, Si, Ti, Al, Ga, Mg, Na, K
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and Pt. Quantitative maps of element compositions of particles were collected using an electron microprobe (EPMA; Cameca™ SXFive Electron Microprobe, France), with 23
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samples being mapped. Samples were set in epoxy resin and polished with 1 µm
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diamond paste. The Cameca™ SXFive Microprobe, equipped with five wavelength dispersive (WLD) X-Ray detectors, with PeakSite software for instrument control, and
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Probe for EPMA™ for data acquisition and processing to produce quantitative maps of the Au-particles. Mapping was conducted at 20 kV and 200 nA. The particles were analyzed for (detection limits in parenthesis in wt.%): Au (0.24). Ag (0.09), S (0.025), Fe (0.06) and Cu (0.11). All elements were calibrated using minerals and pure metal standards from Astimex and P&H. Cu, Fe and S was calibrated on chalcopyrite; Ag on telluride; and Au on a pure metal standard. EPMA software produces a full quantitative pixel by pixel calculation by using the mean Atomic number background correction 9
ACCEPTED MANUSCRIPT (Donovan and Tingle, 1996) in CalcImage, and false colorization and formatting in Surfer10™ to produce net intensity, detection limit map and totals image. Backscatter Diffraction (EBSD) analyses were conducted with a ZEISS Supra field emission (FE)SEM (Zeiss, Germany) with an e-Flash detector and data was analyzed using CrystAlign
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(Bruker AXS, Germany).
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2.3. Chemical analyses of soil and water samples
Soil and water samples were analyzed for a range of physicochemical parameters
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following methods published in Reith et al. (2012) and Brugger et al. (2013). Prior to
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analyses, soil samples were homogenized in a ring mill and water samples were filtered through 0.22 µm sterile syringe filters. Waters and soil (1:5 soil : H2O) were analyzed for
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pH, electrical conductivity and alkalinity. Total C and N in soil samples were determined
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by high temperature combustion in an atmosphere of oxygen using a Leco CNS-2000. Dissolved C in waters (DC) was determined by Skalar Formacs HT TOC/TN Analyzer.
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Inorganic carbon was acidified to form CO2 prior to detection. Dissolved organic carbon
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(DOC) was determined by the difference between total and inorganic C. Ammonianitrogen (NH4-N) was determined by flow analysis and spectrometric detection
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according to ISO 11732. Nitrate- and nitrite-N were determined using the automated segmented flow analyzer (Alpkem Flow Solution 3) at 540 nm. Anions in waters (F-, Cl-, NO2-, Br-, NO3-, SO42- and PO43-) were measured using a Dionex ICS-2500 ion chromatography system with 2 mm AS16 anion separation column. Soil samples were microwave digested in concentrated aqua regia. Element concentrations in digests and water samples were determined using inductively coupled plasma optical emission spectroscopy (ICP-OES) on a Spectro ARCOS (Spectro Analytical Instruments, Kleve, 10
ACCEPTED MANUSCRIPT Germany). Trace and ultratrace elements were analyzed by inductively coupled plasma mass spectrometry (ICP-MS; Agilent 7700, Japan). Concentrations of Au and Ag in soil samples were analyzed in pressed pellets using laser ablation (LA)-ICP-MS analyses with Resonetics M50 with 7700 ICP-MS (Resonetics, USA; Agilent, USA) calibrated with
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NIST standards. Elements analyzed and quantification limits are provided in Tables 2
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and 3.
2.4. Biomolecular and statistical analyses
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Biofilm communities resident on Au-particles were assessed using nested 16S
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rRNA polymerase chain reaction (PCR) combined with next generation sequencing (NGS) using the Illumina MiSEQ platform – TruS eq SBS v.3 600 cycle using 300 bp
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paired end sequencing (Reith et al., 2010; Bissett et al., 2016). The universal primers
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27F (Lane, 1991) and 1492R (Osborn et al., 2000) were used for initial PCR amplification of 16s rRNA genes directly from the Au-particles (Reith et al., 2010), 58
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positive 1st round amplification were further amplified using primers 27F and 519R
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(Lane, 1991; Lane et al., 1985), and sequenced at the Australian Genome Research Facility (AGRF, Melbourne, Australia). DNA amplifications were performed in an Applied
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Biosystems Veriti™ Thermal Cycler. Amplicons were checked in 1.5% agarose gel with Gel Red (Biotium Inc., Hayward, CA, USA) 1:10,000 (v/v) and run at a constant voltage of 80 V for 1 h. Procedures for sequencing, open OTU picking and assignment are detailed in Bissett et al. (2016). Briefly, sequence read quality was visually assessed using FastQC (Andrews, 2010). Sequences were trimmed to remove poor quality bases and merged using FLASH (Magoc and Salzberg, 2011). Sequences < 400 bp or containing N or homopolymer runs of >8 bp were removed (MOTHUR v1.34.1; Schloss 11
ACCEPTED MANUSCRIPT et al., 2009). Remaining sequences were submitted to open reference (OTU) picking at 97 % sequence similarity using UPARSE (Edgar et al., 2013) and OTU abundance tables constructed by mapping all reads to these OTUs (usearch_global, 97 %). Maximum-likelihood (1000 bootstrap replicates) phylogenetic trees were constructed
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using GENEIOUS 7.0 (Biomatters Ltd., New Zealand); sequences used for tree
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construction were deposited in GenBank with accession numbers MG209801 to
conducted based on a study by Rea et al. (2016).
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MG209818. Functional assignment of OTUs to six stages of biofilm development was
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Multivariate statistical analyses were conducted using the PRIMER-6 software
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package with the PERMANOVA+ add-on (Clarke and Warwick, 2001; Anderson et al., 2008). Similarity matrices were established on square root transformed abundance data
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using the Bray–Curtis method (Bray and Curtis, 1957). Permutational multivariate
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analysis of variance (PERMANOVA) combined with canonical analysis of principal coordinates (CAP) and SIMPER analysis were used to assess the influence of deposit
3. RESULTS
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type, location and sampling depth on community composition (Anderson et al., 2008).
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3.1. Morphology and composition of Au particles and particle surfaces Placer Au-particles collected from the seven individual sites at the Lemmenjoki and three sites at the Ivalojoki goldfields ranged in grain size from 0.1 to 2.0 mm (Fig. 1). Across samples from all sites a wide range of particle morphologies were observed, including flattened, elongated, equant, irregular, branching and globular Au-particles (Fig. 1 D-H). Particles were slightly to highly rounded and showed signs of physical and (bio)geochemical transformations, which had occurred in Earth surface environments 12
ACCEPTED MANUSCRIPT during and post transport (Fig. 1 D-H). Signs of physical modification included rounded and (partially) folded edges, striations, randomly orientated scratches and pitted cavities indicative of glacial and alluvial transport (Fig. S1). Particles from glacial-till sites that had not undergone post-glacial fluvial redistribution by the Ivalojoki and Lemmenjoki
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river systems were irregular with partially folded edges, but fewer scratches and pits.
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With increasing fluvial transport Lemmenjoki particles transgressed from irregular to
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elongated and wire Au morphologies, and showed increasingly battered and roughened or rounded surface morphologies (Fig. 1 D-H; Fig. S1). Particles from Ivalojoki became
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more rounded and eventually flattened as sites changed from glacial-till to fluvial sites.
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In addition to showing signs of physical alteration, signs of biogeochemical transformations of the particles were common. These included dissolution features (Fig.
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2A), where progressive dissolution of Ag and Au has led to the irregular pitting of particle
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surfaces. Surfaces displaying dissolution features were commonly in close proximity to secondary-Au re-precipitation features (Fig. 2B). Particles from all sites were entirely or
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partially coated by a layer comprising C, N, O, S, Al, Si, Fe, Na, Mg, P, K, Ti and Au
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(Fig. 3A-C). This layer, which can reach a thickness of more than >20 µm in topographic lows, is composed of bacterial cells and EPS intermixed/underlain by Fe-oxides and -
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sulfides, clays, silicate minerals and secondary Au (Fig. 3D-H); indeed S and Fe containing mineral layers commonly coat particle surfaces (Fig. 4). In zones especially rich in organic material, indicative of active biofilms, the biofilm was also especially enriched in nano- and microphase Au (Figs. 5 and 6). Rod-shaped bacterial cells (2.5 to 3.5 µm in exopolymers) occurring individually or in clusters were observed in protected areas on many particles, where they were commonly attached by fimbriae to the Au surface and commonly associated with nano-particulate Au (Fig. 3E and 6). Aggregates 13
ACCEPTED MANUSCRIPT of nano and micro-sized, e.g., crystalline Au triangles, conglomerates of Au µ-crystals and bacterioform/budding Au, which can form bridging structures of the matrix, were also commonly observed and associated with polymorphic layers (Figs. 5 and 6). While abundant nano- and microphase secondary-Au was observed on particles from all sites,
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particles from sites with increasing recent fluvial influences displayed fewer Au nano-
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and micro-particles in the polymorphic layers. Similar results were also obtained from
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the Lemmenjoki sites, where more nanophase and micro-crystalline Au was observed on particles from glacial-till deposits. Overall, particles from IVK, IVH and LJ1 and LJ2
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showed the most extensive assembly of polymorphic layers and Au nanoparticles and
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aggregates (Table 1; Figs. 1, 5 and 6).
Elemental maps of a particle surface, in which a primary Au surface (composed of
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a Au-Ag alloy) was partially coated by polymorphic layer, showed that during the
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transformation of the primary Au-Ag-alloy Ag is lost, and the secondary Au aggregates, thereby forming sheet-like Au overgrowths, which are beginning to cover the particle
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(Fig. 7 A-C). This process was further developed on some particles, where below a 10
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µm deep polymorphic layer that contained nano- and microphase Au, a 1-2 µm wide rim of highly pure (99.9 wt.%) secondary Au coated the primary Au-Ag (Fig. 7 F,G). Note the
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transition between secondary Au and primary Au-Ag is sharp, not diffuse (Figs. 7 F,G and 8), suggesting that a new layer of secondary Au had formed over the primary Au-Ag surface, rather than that Ag had been progressively mobilized from the primary Au-Ag. This is supported by EDSD measurement of a polished particle, which showed finegrained, unstrained nano- and microcrystalline Au on the surface of the particles, whereas the core of the grain was composed of a strained large Au-mono-crystal (Fig. 7 H,I). 14
ACCEPTED MANUSCRIPT To further assess the grade of transformation across the sites particles from all sites were quantitatively mapped. Silver content in particles with a primary-Au-Ag core ranged from 4.0 to 18.0 wt.%, with Au concentrations being inverse to Ag concentrations (Fig. 8; Table S1). While 12 particles were partially or thinly (<1 µm) covered by a layer
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of secondary-Au, 11 grains displayed a thicker (>1 µm) layer of secondary-Au. Of these
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most showed layers of approximately 10 µm of secondary-Au, with some being
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progressive transformed along crevices and cracks and three particles being entirely
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3.2. Assessment of biofilm communities
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composed of 99.9 wt.% Au (Fig. 8; Table S1).
To assess the composition of the biofilm communities resident on the particles, the 16S
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rRNA gene was amplified directly from Au-particles. 16S rRNA gene sequences were
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obtained from >80 % of particles, suggesting that >80% of particles had biofilms on their surface at the time of sampling. Sequencing was conducted from six DNA-positive
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particles per site; four grains from LJ2 were amplified. After processing, >3.9 million
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reads were obtained from the 58 samples. Overall, 519 different OTUs were recorded (Table S2). Proteobacteria were the dominant phyla, comprising 46.7 % and 54.1 % of
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OTUs and total reads, respectively (Fig. 9; Table S2). Within the Proteobacteria, the βProteobacteria dominated, with 101 OTUs and 31.1 % of total reads. α- and γProteobacteria were numerous with 78 and 49 detected OTUs, which made up 13.1 and 8.3 % of total reads; - and ε-Proteobacteria made up 2.9 % of OTUs with 1.5 % of total reads (Fig. 9; Table S2). Also abundant were the phyla Acidobacteria and Actinobacteria, with 96 and 37 detected OTUs, which made up 15.1 and 6.6 % of total reads, respectively (Fig. 9; Table S2). Other phyla observed included Firmicutes (4.6 15
ACCEPTED MANUSCRIPT and 2.6 %, OTUs and reads) and Bacteriodetes (4.8 and 3.4 %), with 1-3 % of Cloroflexi,
Planctomycetes,
Cyanobacteria
Verrucomicrobia,
AD3,
Gemmantimonadetes, Nitrospira; with 16 other, rare phyla also detected (Table S2). The number of OTUs recorded in biofilms at individual sites ranged from 85 at
T
LJ2 to 215 at IVK; β-Proteobacteria were most numerous at eight and Acidobacteria at
IP
two sites (Table S2). Eighteen OTUs were detected on particles from all sites, of those
CR
eight were β-Proteobacteria, and included Gallionella spp., C. metallidurans and Ralstonia spp.; Arthobacter spp. and Pseudomonas spp. were also detected (Fig. 10A;
US
Table S3). Across all sites the top10/top20 most numerous OTUs (by number of reads)
AN
contributed between 59.2 to 99.6 and 66.6 to 99.8 % of total reads detected, respectively (Table 4). This suggests that communities are dominated by a few OTUs,
M
which make up the majority of the biofilm communities, with more rare species detected
ED
as a result of deeper NGS compared to PCR-DGGE combined with Sanger sequencing used in earlier studies (Reith et al., 2006, 2010 and 2016). PERMANOVA showed that
PT
community composition varied significantly with site (√CV = 3.36; P < 0.001), deposit
CE
type (√CV = 3.43; P < 0.001) and sampling depths, i.e. position of Au-particles sampled in the deposit (√CV = 2.16; P < 0.001). CAP and SIMPER analyses of community data
AC
suggested significant links of bacterial communities from Au-particles from different deposit type and depth were strongly related to differences in the diversity and abundance of α-, β- and γ- Proteobacteria, Acido- and Actinobacteria and Bacteriodetes (Fig. 10B).
3.3.
Assessment of water and soil geochemistry
16
ACCEPTED MANUSCRIPT To assess the mobility of Au in soils and till containing transported placer Au-particles, and thereby provide a framework of physicochemical conditions at which Au mobility occurs, three water and seven soil samples from key-sites were analyzed for >50 parameters (Table 2 and 3). The water sample from the Ivalojoki site, i.e., IVK,
T
contained 0.6 µg/L mobile Au as well as 0.17 µg/L of Ag, with S, Co, Cr, Ni, V, Ti and Zn
IP
also detected (Table 2). Surface waters had a pH of 5.1 and 5.4, groundwater pH at LK
CR
was 6.6 (Table 2). TOC in waters lay between 11 and 34 mg/L, and up to 2.1 mg/L of TN, 2.6 mg/L of chloride and 2.0 mg/L of sulfate were detected. All soil samples
US
contained elevated Au and Ag concentrations of up to 211 and 290 µg/kg of soil,
AN
respectively. The highest concentration of Au (i.e., 211 µg/kg) was detected in the Ahhorizon with concentrations decreasing with increasing sample depth to 24 µg/kg in the
M
till at LK (Table 3). Gold was not homogeneous distributed within each sample as
ED
evidenced by LA-ICP-MS lines across the pressed soil pellets (data not shown). In contrast Ag concentrations were more homogenous between (Table 3) and within
PT
samples (data not shown). While Au and Ag concentrations were strongly elevated
CE
compared to background concentration, concentrations of other typical Au pathfinder elements, including Cu, Cd, As, Mo, Pb, Co, Zn and Ni, were not elevated above
AC
background concentrations expected and homogenously distributed between samples (Salminen, 2009). Soil-pH was with 3.0 the lowest in the Ah-horizon at LJ1 and increased with depth to pH 6.8 in the till collected below 2 m depth at LK. Sulfur content in the Ah-horizon at LJ1 was 5957 mg/kg high, and much lower concentrations were detected in all deeper samples and ranged between 150 to below 40 mg/kg. Similarly TOC and TN ranged from 7.5 and 0.39 wt.%, respectively, in the Ah-horizon at LJ1 to 0.09 and 0.01 wt.% in the till (Table 3). 17
ACCEPTED MANUSCRIPT
4.
DISCUSSION
4.1
Gold particle transformations
The variety in chemical composition and morphology of the particles from the same sites
T
suggests that they have been affected by a range of physical and bio(geo)chemical
IP
processes since they have been exposed to surface conditions. Some particles from
CR
IVH, IVK and LTE are composed of almost pure 99.9 wt.% Au, while others contain cores with >10 wt.% Ag (Fig. 8). At the glacial-till site LJ1 some particles show limited
US
evidence of physical transport, such as slight rounding and pitting, scratches and
AN
striations, while other grains were highly transformed and spherical (Figs. 1 and S1). This suggests that Au-particle populations were derived from different primary sources.
M
These may have been exposed to surface conditions, including chemical and biological
ED
transformation, for different periods of time, and their co-occurrence is a result of glacial and (glacio)fluvial fluvial transport and deposition. Indeed earlier studies have shown
PT
that minor primary deposit types may be present throughout in region (Saarnisto et al.,
CE
1991). These have been described as quartz-carbonate, quartz-hematite and arsenopyrite-rich shear zone deposits (Saarnisto et al., 1991). They contain localized
AC
differences in Ag and trace metal contents in the Au, with an average Ag content of particles reported to be 6 wt.% (Saarnisto et al., 1991). Particle shapes and the extent of their rounding is commonly linked to the transport distance of the Au, and the purity of Au can be linked to the length of time/intensity the particles have undergone (bio)geochemical transformation and refining (Kinnunen, 1996). This is especially apparent with particles composed of 99.9 wt.% pure Au with little or no sign of a primaryAu-Ag core (Wilson, 1984). This suggests that a smaller part of the Au-particle 18
ACCEPTED MANUSCRIPT population had undergone pre-glacial weathering and transformation and/or subsequent glacial transport from more distant sources, confirming the earlier hypothesis of a redistribution of highly transformed particles from paleo-placer environments. The majority of particles, i.e., those with a full or partial layer of secondary-Au overlying a
T
primary core may have been eroded from regional sources by the progressing glaciers
IP
and deposited in glacial-till, which was reworked by fluvial processes.
CR
Pits on particles surfaces can be the result of physical damage, removal of crystals or (bio)geochemical dissolution (Kinnunen, 1996). Some of the pits observed on
US
particles from IVH are likely the result of (bio)geochemical Au dissolution, as dissolution
AN
pits generally produce irregular, mottled surfaces, as observed here (Fig. 2). Many of the pits found on the Finnish particles appear in zones of most intensive physical
M
transformation and are therefore have likely been obtained through physical scratching
ED
(Fig. S1). Extensive nano- and micro-particulate Au, dispersed through polymorphic layers, that are forming delicate aggregates of hexagonal or pseudohexagonal Au plates
PT
as well as spheroidal and bacterioform overgrowth have been observed on particles
CE
from all sites, but were most common on samples from glacial-till deposits. These are likely the result of Au dissolution and (bio)geochemical re-precipitation under surface
AC
conditions, as shown in numerous field- and experimental studies from sites around the world (e.g., Craw and Lilly, 2016; Falconer and Craw, 2009; Reith and McPhail, 2006; Reith et al., 2006, 2010 and 2012; Shuster et al., 2015 and 2017; Melchiorre et al., 2018). Processes affecting Ag also result in overall particle transformation, these include the de-alloying and mobilization of the Ag as well as re-crystallization of Ag-poor µm sized Au–crystals resulting from physical strain (Hough et al., 2009). There is evidence 19
ACCEPTED MANUSCRIPT to suggest Ag de-alloying has also contributed to the formation of the Au-rich rim on some grains. Particles with irregular core/rim boundaries that create deep embayments into the core, such as a particle from LKO (Fig. 7C), may be the result of Ag leaching (Youngson and Craw, 1993). When Ag is oxidized, its size is reduced, resulting in its
T
removal from its site in the alloy (Desborough (1970). Repetition of this process creates
IP
new Au/Ag alloy interfaces, resulting in porous/diffuse rims. Some authors suggest that
CR
the diffusion of Ag is too slow to create the sharp boundaries between the enriched Au rim and the core of the grain (Groen et al., 1990). However, a number of recent studies
US
of Au-particles collected at sites in New Zealand have suggested that the sharp
AN
boundary indicates re-crystallisation of Ag-poor Au in surface environments. In these samples, erosion and transport of Au-particles for short distances into Eocene paleo-
M
placer deposits caused internal deformation of particles, especially near the edges of the
ED
particle (Stewart et al., 2017). Rims of fine grained (1–20 µm) recrystallized Au formed around the edges of the particles as a result of stored strain energy, which was
PT
accompanied by expulsion of Ag from Au microcrystals leading to rims containing 0-3
CE
wt.% Ag.
The delicate nature of the secondary Au surface features observed on the
AC
particles especially from till placers, which includes the secondary Au aggregates, would not survive transport (Youngson and Craw, 1993), and is hence likely the result of postdeposition, biogeochemical transformations (Fig. 5). Therefore, estimates about the extent of supergene transformation experienced by particles can be made. The last deglaciation of northern Finland began 11600 years ago, with deglaciation came the deposition of grains in tills suggesting that post-glaciation, secondary Au transformation processes were beginning not long after particle deposition. This suggests that the scale 20
ACCEPTED MANUSCRIPT of transformation observed on the majority of grains, i.e., those derived from local sources, may be the result of 11,600 years of biogeochemical refining in cold arctic conditions. A recent study of particles from Kilkivan, Queensland, Australia, assessed similar surface morphologies on particles from assumed to be eluvial/fluvial placer
T
deposits, with the aim to assess environmental rates of these biogeochemical
IP
transformation processes leading to Au surface morphologies and transformations
CR
(Shuster et al., 2017). Gold particles from Queensland showed pronounced secondary Au structures, e.g., nanometer-sized Au colloids and micrometer-sized octahedral Au
US
platelets and aggregates thereof embedded in polymorphic layers, which covered 10-40
AN
% of particle surfaces (Shuster et al., 2017). A biogeochemical model developed based on these data, and environmental properties these particles were subjected to,
M
estimated 17.9–58.5 years of Au cycling and mobilization within this subtropical
ED
environment. This time estimate corresponds well with decadal time scales required for secondary Au enrichment to occur within subtropical placer deposits, as determined
PT
from early field observations (Freise, 1931).
CE
As particles from both climatic zones are at comparatively similar stages of transformation, this suggests that the biogeochemical transformation of particles
AC
appears to be two orders of magnitude faster in subtropical conditions compared to cold, arctic environments. Indeed temperature is an important factor affecting the kinetics of (bio)chemical reactions, the composition of microbial community and the microbial activity driving nutrient cycling (Paul and Clark, 1996; Männistö and Häggbloom, 2006). Studies have shown that bacterial growth rates and activity increase exponentially with temperature (Ratkowsky et al., 1982; Pietikäinen et al., 2005). A study of soils from Sweden showed that the respiration rate at 45°C was up to 120 times higher than at 0°C 21
ACCEPTED MANUSCRIPT (Pietikäinen et al., 2005). Bacterial activities at optimum temperatures were around 10 times above those at 0°C in a range of agricultural and humus soils, respectively. Other studies have shown that process rates of N and S cycling exponentially increase with temperature (e.g., Germinda and Janzen, 1993; Nicolardot et al., 1994). The key
T
organisms driving Au cycling at the site, e.g., C. metallidurans, are mesophilic, not
IP
psychrophilic, organisms (Rea et al., 2016), and may therefore only display limited Au
Environmental mobility of Au and Ag
US
4.2.
CR
cycling activity for most the year.
AN
Gold solubilization, dispersion and re-precipitation in Earth surface environments have been attributed to geochemical and biological processes (e.g., Reith et al., 2007;
M
Shuster et al., 2016; Anand et al., 2016; Craw and Kerr, 2017; Melchiorre et al., 2018).
ED
Analyses of the placer Au-particles combined with the characterization of soil and water samples suggest that a combination of direct and indirect geochemical and biological
PT
processes have contributed to the dissolution and precipitation of Au and the patterns of
CE
Au distribution in soils holding placer Au at the study sites in arctic Finland. Note physical transport and deposition of mm-sized Au-particles in glacial and (glacio)fluvial
AC
systems have been discussed in the section above. It is also important to note that Au and Ag are the only elements with significantly elevated concentrations in soils at the study sites. However, weathering of primary bedrock-hosted Au deposits commonly leads to the enrichment of other pathfinder elements in overlying soils and sediments (Boyle, 1979). The elements that are enriched depend on the specific type of primary deposit and can include As, Bi, Cu, Mo, S, Sb, Pb, Se, Te, Pb and U (Boyle, 1979). This indicates that Au and Ag detected in the soils have been mobilized from the placer 22
ACCEPTED MANUSCRIPT particles post deposition, rather than from potential underlying primary sources, which have not been reported in the immediate vicinity of the sampling sites (Eilu et al., 2007; Eilu, 2015). The highest concentrations of finely dispersed Au in soils were detected the top
T
soils, and were up to 10 fold higher at LJ1 and IVK compared the C-horizon till. Top soils
IP
also displayed the lowest pH, highest organic matter- and S contents (Table 3), and
CR
generally contain the highest number of metabolically active microorganisms (Paul and Clark, 1996). This suggests that this combination of factors enhances the mobility of Au,
US
contributes to the transformation of Au-particles and dispersion of Au into adjacent soils.
AN
Indeed placer Au-particles from IVK, and LJ1 and LJ2 showed the most extensive assembly of transformation of polymorphic layers and Au nanoparticles and aggregates,
M
with water at IVK, containing 0.6 and 0.17 µg/L of mobile Au and Ag, respectively (Table
ED
1; Figs. 5 and 6).
These data point to processes that may be important for Au dissolution from the
PT
placer Au in Finnish Lapland. These include the oxidation/complexation of Au by Mn-
CE
oxide biominerals, e.g., the birnessite, under localized acidic conditions through ligand coupled oxidation with inorganic or bioorganic ligands (Ta et al., 2014 and 2015).
AC
Resulting Au-complexes likely include Au-organic acid- or cyanide complexes as well as possible Au-chloride, -thiosulfate and -bisulfide complexes. Given the levels of Mn in soil samples, which range from 300 to 1000 mg/kg (Table 3), the presence of Mn-oxidizing bacteria on Au-particles capable of mediating the formation of Mn(III/IV)-oxides, the acidic pH of top soil, and the presence of sulfides on Au-particles, which upon oxidation produce localized acidic conditions, it is likely Mn-oxides play a role for the mobilization of Au. In addition, oxidation/complexation of metallic Au by bioorganic Mn-compounds, 23
ACCEPTED MANUSCRIPT similar to [(bpy)2MnIII(μ-O)2Mn-IV(bpy)2]3−, may be responsible for Au-complex formation through a ligand coupled oxidation (Ta et al., 2014 and 2015). Note, biologically mediated Au solubilization is assumed to occur as an indirect effect of the metabolic processes, e.g., in biofilms on the Au-particle surfaces, and include the production of
T
Mn-compounds, thiosulfate, organic acids and cyanide. In contrast Au detoxification and
IP
biomineralization of nanophase Au can be actively mediated by specific biochemical
CR
pathways aimed at reducing intracellular Au toxicity (e.g., Johnson et al., 2013; Rea et al., 2016; Wiesemann et al., 2017).
US
In the presence of high concentration of organic matter in soils, as observed in
AN
top soil horizons at the sampling sites (Table 3), complexation to organic ligands is likely, as Group IB elements strongly bind to organic matter (Boyle, 1979; Vlassopoulos
M
et al., 1990). A range of bacteria associated with the Au-particles are capable of
ED
producing large amounts of organic acids and some are known to excrete cyanide (Fig. 11). Earlier experimental studies have shown that amino acids, e.g., aspartic acid,
PT
alanine, histidine, serine, and glycine, excreted by heterotrophic microbiota, can facilitate
CE
Au solubilization leading the formation of Au-amino-acid complexes (e.g., Korobushkina et al., 1983). Korobushkina et al. (1976) have shown that during in vitro studies with
AC
heterotrophic bacteria in media containing metallic Au several mg/L of Au were solubilized. In microcosm studies with organic matter rich auriferous soils from Australia, experiments with a living resident microflora, resulted in up to 80 wt.% of Au being solubilized; here Au solubilization was linked to the microbial formation of amino acids and cyanide (Reith and McPhail, 2006 and 2007; Fairbrother et al., 2009). In soils amended with metallic Au pellets, Au was also liberated from the pellet; in contrast sterilized microcosm Au mobilization was negligible (Reith and McPhail, 2006). 24
ACCEPTED MANUSCRIPT Amino acids, especially glycine, are common metabolic precursors for cyanide production in the bacteria, e.g., Chromobacterium violaceum, Pseudomonas spp., which have been shown to solubilize of Au from metallic Au surfaces (Fairbrother et al., 2009). Bacteria with the ability to produce cyanide have been detected on the Finnish placer
T
Au-particles, suggesting that Au cyanidation contributes to Au mobilization. In some soil
IP
ecosystems cyanide producing microorganisms can make up to 50 % of the soil
CR
microbial community (Kremer and Souissi, 2001), which would further increases the mobility of Au. Humic and fulvic acids, which make up a large proportion of the total and
US
the dissolved organic matter in Arctic soil systems, may also contribute to the
AN
mobilization of Au (Boyle, 1979). They have been shown to contribute to the solubilization of Au by forming complexes with Au(I/III)-ions, in other studies the
M
reduction of Au-complexes and the formation of stabilized Au nanoparticles was
ED
observed, which may also contribute to Au mobility at the Finnish sites (e.g., Freise, 1931; Ong and Swanson, 1969).
PT
Choride has been detected in the surface water samples (Table 2), and provides
CE
a suitable inorganic ligand for Au complexation (Usher et al., 2009). Gold(III)-chloride complexes have been predicted only to exist under low pH and highly oxidizing
AC
conditions, but in a recent study have been shown to be present in saline surface water under circum-neutral reducing conditions (Ta et al., 2014). Particularly the Ah-horizon at LJ1 displayed strongly elevated concentrations of S, which in northern Finland is common and a result of atmospheric inputs linked to anthropogenic pollution (Forsius et al., 2010). This in combination with high organic matter contents and changing redox conditions suggests a highly active S cycle in these soils, waters and sediments, which can further increase heavy metal mobility. Secondary 25
ACCEPTED MANUSCRIPT sulfide minerals were abundant on Au-particles surfaces, especially on those collected from top soils (Fig. 3 and 4), and bacteria capable of S-oxidation and reduction were detected in biofilm communities. This suggests that mobilization as thiosulfate and bisulfide complexes may play a role for Au mobility in Finnish Lapland, similar to a
T
recently published study from New Zealand (Craw and Kerr, 2017). Craw and Kerr
IP
(2017) analyzed 655 waters from environments relevant to supergene processes in the
CR
Otago Schist goldfield of southern New Zealand, which they have used as proxies to deduct supergene Au transformation processes. They have found that Au was mobilized
US
as thiosulfate- and bisulfide complexes and re-precipitated as metallic Au near
AN
unconformities, when Au-complexes were destabilized as a result of changes to physicochemical conditions, e.g., redox status or pH conditions. They suggest that this
M
mechanism of Au mobilization can be very effective at circumneutral to slightly acid pHs
ED
in organic matter limited systems, which may apply to the till and sediment layers at the study sites.
PT
Thiosulfate also is considered an important ligand for the dissolution and
CE
dispersion of Ag (Zipperian et al., 1988). Generally, Ag is considered to more mobile than Au in surface environments as it readily forms complexes with Cl−, SO42− or NO3 –
AC
ligands (Shuster et al., 2017). Mobile Ag nano-particles can also form through a variety of process, including biogenic and abiogenic reduction and stabilization of Ag-complexes (Quang Huy et al., 2013). Silver nanoparticles are labile under a range of soil conditions, leading to their dissolution and the formation of mobile ionic complexes (Levard et al., 2012). Therefore, Ag is commonly more homogeneously distributed in soils, as was also observed at the study sites. In addition, Ag is incorporated in a range of secondary minerals, e.g., jarosite, as observed in another recent study, but formation 26
ACCEPTED MANUSCRIPT of mm-sized secondary Ag-particles formed in soils or sediments have not to the authors’ knowledge not been reported, suggesting that Ag becomes widely dispersed in the landscape (Mann, 1984; Shuster et al., 2017). Gold is also transported in the soil environment as mobile complexes and
T
(nano)-particles (; Hough et al., 2011; Craw and Kerr, 2017). Low molecular weight
IP
organic acids, e.g., citrate, acetate, oxalate, amino acids and high molecular weight
CR
organic acids, including humic and fulvic acids, peptides and proteins present in biofilms may can act as ligands for Au complexation as well as stabilize metallic Au
US
nanoparticles (Vlassopoulos et al., 1990; Hough et al., 2011; Reith and Cornelis, 2017).
AN
Gold nanoparticles are formed by the abiogenic or bioorganic reduction of Aucomplexes due to changing physicochemical conditions, reduction on mineral surfaces
M
or by organic acids, or during active or passive microbial biomineralization (Hough et
ED
al., 2011). Indeed recent studies point to biofilms on placer Au-particles as an important sources of mobile Au nanoparticles, because members the community actively form
PT
extracellular Au nanoparticles, organic compounds to stabilized them are plentiful, and
CE
biofilms are subject to cycles of formation and destruction via desiccation, freezing or grazing by soil fauna, leading to periodic release of nano-particulate Au and its
AC
dispersion in the environment via transport in soil solutions and groundwater (Reith et al., 2010; Hough et al., 2011; Johnston et al., 2013; Shuster et al., 2017). The release of nano-particulate Au may therefore contribute to the high mobility of Au at the study sites as well as the observed inhomogeneous distribution within samples. Complexes and nanoparticles are re-precipitated and thereby immobilized, when the complexing ligands or stabilizing agents are decomposed, and when they are sorbed by minerals, organics or microbiota and subsequently aggregate (Hough et al., 2011; 27
ACCEPTED MANUSCRIPT Craw and Kerr, 2017). Using selective sequential extractions on soils amended with mobile Au-complexes has shown that they are preferentially associated with organic matter, clays and Fe-oxides (Gray et al., 1998; Reith et al., 2005). The results of sequential extractions conducted with samples collected from an biologically active Ah-
T
horizon microcosms after 0, 10, 20, 30, 40, and 68 days of incubation indicated a
IP
continuous microscale solubilization and re-adsorption of mobile Au by the organic
CR
matter, which after 40 days of incubation contained more than 80 wt.% of the Au in the soil (Reith and McPhail, 2006). In the same soil collected after 10 days of amendment
US
with 100 µg/kg Au(III)-chloride, 95 wt.% of the Au was associated with the organic
AN
matter (Reith and McPhail, 2006). In another study ten Australian soils were amended with citrate-stabilized Au nanoparticles, of which 3- to >80 wt.% remained highly
M
mobile/dispersible in soil solutions after 28 days of incubation, with increasing clay, Corg
ED
and Fe/Mn-oxide contents decreasing and elevated sand contents and pH increasing their mobility (Reith and Cornelis, 2017). Results of selective extractions of soils after 28
PT
days of incubation showed that up to 80 wt.% of sorbed Au was associated with the
CE
organic matter in most soils, with Fe/Mn-oxides accounting for up to up to 42 wt.% of sorbed Au nanoparticles in some soils (Reith and Cornelis, 2017). Subsequently
AC
aggregation of Au nanoparticles can form µm-sized Au aggregates, which can grow to larger purely secondary Au particles (Fairbrother et al., 2013; Shuster and Southam 2015).
4.3.
Functional characterization of biofilm communities
Earlier studies have described biofilms on Au-particles from Australia, New Zealand and South America; commonly associated with these was abundant secondary Au resulting 28
ACCEPTED MANUSCRIPT from biogeochemical Au cycling (Fairbrother et al., 2012; Reith et al., 2006, 2010 and 2012; Craw et al., 2016; Rea et al., 2016; Shuster et al. 2016). These sites are located in (sub)-tropical,
(semi)-arid
and
temperate
environments,
with
average
annual
temperatures between 5 and >20°C, and limited, if any soil frost. Therefore, the present
T
study aimed to assess if biofilms occur on Au particles from cold sites with average
IP
annual temperatures approximating 0°C, how the communities compare to those on Au-
CR
particles from warmer climatic zones, and study the functional links between biofilms and processes affecting particle transformation and Au mobility. Results showed that
US
biofilms were highly abundant, with most particles being coated by polymorphic layers
AN
composed of cells, EPS and (bio)minerals, with >80 % of grains with detectable DNA (Figs. 3 and 5). Associated with these layers was nano- and microphase Au, which was
M
commonly underlain by a layer of secondary-Au that had formed on top the primary-Au
ED
(Figs. 4-6).
Earlier studies have shown no direct link of the community composition of Au
PT
particles and climatic settings under (sub)-tropical, (semi)-arid and temperate (Rea et al.,
CE
2016). This suggests that physicochemical factors other than climate, e.g., Au and associated heavy metal toxicity, may drive community composition and select for
AC
communities with the combined ability to detoxify these metals. Organisms living in Aurich environments are constantly exposed to harsh environmental conditions, e.g., heavy metal toxicity conferred from mobile Au(I/III)-complexes and Au-deposit associated pathfinder metals, e.g., Ag, Cu, Ni, Cr, As, Pb, Se, Te and W (Reith et al., 2012). At the site elevated concentration of mobile Au and Ag co-occur, and because of their mobility and high toxicity need to be detoxified, explaining the co-occurrence of a wide range of
29
ACCEPTED MANUSCRIPT heavy metal detoxifying organisms and abilities in the biofilms on Au particles required to confer protection to the entire biofilm (Reith et al., 2012; Wiesemann et al., 2017). Across the sites studied earlier, biofilm communities were dominated by Actinobacteria, Firmicutes and especially Proteobacteria, with β- and γ-Proteobacteria
T
being most abundant (Rea et al., 2016). Among these the Oxalobacteraceae and
IP
Comamonadaceae, which contain a range of known metal-resistant and metallophilic
CR
taxa, e.g., C. metallidurans and Ralstonia spp. as well as Delftia spp. and Variovorax spp. were highly abundant. Similar to these earlier studies, biofilm communities on
US
Finnish particles were dominated by β-Proteobacteria, especially Oxalobacteraceae and
AN
Comamonadaceae, which dominated communities and made up 8 of 18 OTUs detected on grains from all sites (Fig. 9). These results suggest that highly specialized biofilm
M
communities adapted to these Au-rich micro-environments perform key-roles in these
ED
biofilm communities, and thereby drive the biogeochemical cycling of Au and the biogeochemical transformation of Au particles.
PT
Biofilm communities on Finnish Au-particles include a range of phyla that can
CE
actively contribute to Au- and heavy metal cycling and detoxification (Fig., 11; Reith et al., 2010; Rea et al., 2016). C. metallidurans, which was detected on particles from all
AC
sites (Fig. 9B), is a non-spore forming aerobic β-Proteobacterium, able to tolerate high levels of heavy metals (Reith et al., 2009; Wiesemann et al., 2013). It is able to to oxidize molecular hydrogen allowing chemolithoautotrophic growth (Mergeay et al., 1985; Herzberg et al., 2015), and to degrade a variety of aromatic and unusual organic compounds (Mergeay, 2000; Springael et al., 1993; Miyake-Nakayama et al., 2006). During its evolution C. metallidurans has obtained the ability to resist high concentrations of transition metals, in particular the oxidative/metal stress induced by 30
ACCEPTED MANUSCRIPT toxic Au(I/III)-complexes, with toxicity levels similar to Hg(II)- and Ag(I)-compounds; note minimal inhibitory concentrations (MICs) for Au(I/III)-complexes, Ag and Hg-compounds are in the order of 1 to 5 µM in C. metallidurans and toxic effects to the organisms start at approximately 1/1000 of the MIC (Nies, 1999; Reith et al., 2009; Wiesemann et al.,
T
2013 and 2017; Etschmann et al., 2016). C. metallidurans reduces toxic Au(I/III)-
IP
complexes in the periplasm via synergistic co-utilization and regulation of Cu/Au
CR
resistance determinants (Wiesemann et al., 2013 and 2017; Zammit et al., 2016). This leads to the formation of ~10 nm sized Au nanoparticles in the periplasm (Reith et al.,
US
2009). The metal chaperone CupC and the periplasmic Cu-oxidase CopA have been
AN
shown to have key-functions for the detoxification of Au-complexes and the formation of Au nanoparticles (Wiesemann et al., 2013 and 2017; Zammit et al., 2016; Bütof et al.,
M
2018). Using these abilities C. metallidurans has been shown to survive up to 615 µM of
ED
Au(I)-complexes, retain 99.5 wt.% of Au(I)-thiosulfate percolating through columns, and actively biomineralize Au as nanoparticles and micro-crystalline aggregates (Fairbrother
PT
et al., 2013). Over time nanoparticle aggregates encapsulate and subsequently replace
CE
cells leading to the formation of mm-sized Au particles (Fairbrother et al., 2013; Shuster et al., 2015). By mediating Au biomineralization, C. metallidurans plays a key role in
AC
environmental cycling of Au in Earth surface environments. Other taxa, such as Gallionella sp., Variovorax boronicumulans, Paenibacillus sp., Rhodoferax sp., and Pseudomonas sp. are known to produce siderophores that may act as a metal-binding scaffold for the detoxification Au and various other metals, which can co-occur with Au (Fig. 11). Recent studies have shown that a siderophore, delftibactin, produced by Delftia acidovorans, another non-spore-forming, O2-utlizing aerobic β-Proteobacterium that occurred in biofilms from Australian Au particles, confers 31
ACCEPTED MANUSCRIPT Au-resistance and detoxification by extracellularly reducing mobile Au-complexes to metallic non-toxic nanoparticles (Johnston et al., 2013). Gallionella spp. have been shown to oxidize Fe(II) and mediate the biomineralization of insoluble micro-crystalline Fe(III)-oxides (Hallberg and Ferris, 2004). Fe-oxides have been commonly observed on
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the Finnish Au particles, and have been linked to co-precipitation and sorption of heavy
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metals during and after their formation (McKenzie, 1980 ; Manceau et al., 1992).
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Paenibacillus spp. have been shown to release siderophores, exhibit significant biosorption capabilities and tolerance/resistance to a range of toxic heavy metals, e.g.,
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Cu, As, Pb, Cd, Co, Ni and Zn (Acosta et al., 2005 ; Grady et al., 2016). Rhodoferax
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spp., can reduce Fe(III) to Fe(II), which in the presence of sulfide may lead to the formation of Fe-sulfides, e.g., pyrite, possesses genes linked to As metabolism and uses
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heavy metal efflux pump to confer resistance to Cd, Zn, Co (Risso et al., 2009). Single
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euhedral pyrite particles, approximately 1.5 µm wide, were observed (Fig. 3E). Pyrites were undamaged, showing well defined edges and corners and smooth faces,
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suggesting post-deposition formation. Some Pseudomonas spp., are able to secrete
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siderophores during Fe-limiting conditions, and possess genes encoding resistance to Cu and Ag, and plasmid born resistance to Hg and As as well as other heavy metals
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(Visca et al., 1992; Schalk et al., 2011). A range of other taxa present on the particles have also been shown to be able to biomineralize Au nanoparticles, including Pseudomonas veronii, Escherichia coli and Arthrobacter spp. which, similar to Herbaspirillum sp., produces bioflocculants that bind to heavy metals and passively precipitates the metals in the EPS layer (Fig. 11; Bender et al., 1995). In addition Arthrobacter spp. are resistant to high levels of other heavy metals, including Ni and Cr (Margesin and Schinner, 1996; Abou-Shanab et al., 2007). Other metal-tolerant or 32
ACCEPTED MANUSCRIPT resistant
taxa
detected
include
Acinetobacter
woffii,
Lentzea
violacea,
and
Mesorhizobium sp. as well as Rahnella aquatilis that has been known to exhibit strong tolerance to As(III) and As(V) (Fig. 11; Valverde et al., 2011). Manganese-oxidizing bacteria were also present on the particles, e.g.,
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Hyphomicrobium sp. and Pedomicrobium australicum. Pedomicrobium spp. have the
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ability to adhere strongly to surfaces and take advantage of the nutrients and soluble
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Mn-ions attracted to the solid–liquid interface (Fig. 10; Sly et al., 1988a/b). They form intracytoplasmic membrane compartment within the cells to oxidize soluble Mn 2+ to
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insoluble Mn(III/IV)-oxides (Gebers, 1981; Gebers and Beese, 1988), which can
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contribute to the mobilization of Au (Ta et al., 2014). Mobilization of Au and Ag may be important to keep metal toxic loads in the biofilm micro-environment of the grains high,
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thereby eliminating outside competition and selecting for biofilm communities containing
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Au detoxification capabilities. This was confirmed in a study by Brugger et al. (2013), which has shown that Au-toxicity is a key-factor in driving environmental Au cycling.
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Interestingly, C. metallidurans has also been shown to mobilize Au from metallic Au
et al., 2013).
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surfaces, which suggests that it can form its own Au-cycling micro-environment (Brugger
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However, these metallophillic and metal-resistant organisms may not be able to survive without the help of other members of the biofilm community (Fig. 11; Rea et al., 2016). These auxiliary taxa appear to be dominantly involved in particle colonialization, biofilm establishment and nutrient cycling. These functions can be fulfilled by a wide variety of environmental taxa, provided they are protected from heavy metal toxicity, therefore their diversity may be linked to site-specific conditions. This may explain the overall link of community structures to different sites, conditions and deposit styles 33
ACCEPTED MANUSCRIPT observed for the Finnish particles as well as globally, in climatically different situations (Rea et al., 2016). Among these auxiliary taxa detected on Finnish particles are Gram-positive Streptococcus spp., which can colonize the surface during stage one of biofilm
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development (Fig. 10). Gram-positive bacteria can resist surface charge and form an
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attachment on the surface. Initial colonizers also need to produce EPS, resist surface
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charge like Massilia timonae (Harimawan et al., 2011) and form a strong attachment to the surface using eDNA and type IV pilli such as Acidovorax temperans (Heijstra et al.,
US
2009) and Curvibacter spp. (Dominiak et al., 2011). Another interesting taxum detected
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was Candidatus solibacter, which is equipped with a large number of anion : cation symporters, hypothesized to aid the community with its ability to deal with extreme
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variation in moisture, temperature and nutrient availability. Cellulose production
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exhibited by this species may contribute toward maintaining biofilm integrity by promoting the ability of a biofilm to adhere in environments (Flemming and Wingender,
PT
2010).
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After these initial colonizers have settled on the surface, other taxa easily grow on the preconditioned surfaces. In some environments, the lack of precursor nutrients may
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require recruitment of auto and phototrophs in the biofilm, such as Porchlorococcus sp., Rhodoplanes sp, and Polybucelobacter sp (Fig. 11). Some recruited bacteria can perform nitrate reduction and sulfide oxidation, which include Methylibium spp., Lutibacterium spp., Massilia spp. and Arcobacter spp. (Fig. 11). The increasingly taxarich community will produce metabolic by- and waste products, e.g., organic acids and external nutrient- and energy sources may also become available, e.g., fulvic and humic acids. Low molecular weight organic acids are the product of fermentation, common 34
ACCEPTED MANUSCRIPT metabolites of C-metabolism under anoxic conditions, part of the anoxic C-mineralization pathway and have been shown to form Au-complexes (Reith et al., 2008). OTUs able to utilize the C-sources are abundant in the biofilms and include Propionibacterium granulosum, Acinetobacter spp., and Corynebacterium spp. Other OTUs detected may
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employ proteolytic and saccharolytic activities such as Afifella spp., Pseudomonas
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viridiflava, Trichococcus spp., and Syntrophomonas spp. Sacccharolytic fermenting
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bacteria, Trichococcus spp. participates in decomposition of organic matter, utilize organic acids and produce electron donors for sulfate reduction (Fig. 11; Holzapfel and
US
Wood, 2014). Syntrophomonas spp, performs synthrophic degradation by feeding on
AN
other bacteria and is involved in acetogenesis (Zhang and Lu, 2016). Other OTUs are capable of degradation of hydrocarbon and xenobiotics as well as OTUs related to the
M
breakdown of polymers by enzymes and acids (Fig. 11). Important biodegradative
ED
processes for hydrocarbons and xenobiotics may be performed by Paucimonas lemoignei, Polaromonas spp., Spingomonas spp., Variovorax paradoxus, Variovorax
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spp., Herminiimonas spp., and Arthrobacter chlorophenolicus (Handrick et al., 2001;
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Mattes et al., 2008; Unell et al., 2009). Metabolic turnover of all of these energy sources may therefore fuel the entire biofilm community. When biofilms reach their maximum
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carrying capacity, some cells may be released back to the environment. These dispersal cells are capable of carrying associated Au nanoparticles and may thereby contribute to Au dispersion in the wider environment.
5. Conclusions Gold and Ag are mobilized, dispersed and re-precipitated at the study sites, confirming that active biogeochemical cycling of these metals occurs in cold, arctic settings. As a 35
ACCEPTED MANUSCRIPT result of this Au particles from the Ivalojoki and Lemmenjoki placer deposits have been transformed, confirming that biogeochemical process model responsible for supergene Au-particle transformation introduced by Fairbrother et al. (2012) and extended by an physical component by Stewart et al. (2017) is valid in cold environments. Given the
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mobility of both Au and Ag in the systems, the composition of bacterial biofilms on
aurophillic β-Proteobacterium C. metallidurans was detected on
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communities and
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particles is likely driven by metal toxicity. β-Proteobacteria dominate microbial
particles from all sites. Similar to other sites around the world C. metallidurans likely
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plays a key-role for Au detoxification and biomineralization. Overall, biogeochemical Au
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transformations lead to similar secondary morphotypes observed on particles in other environments, but it appears cycling occurs at slower rates compared to sites were
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higher temperatures and year-round available liquid water lead to higher (bio)chemical
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ED
reactivities and element cycling intensities.
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ACKNOWLEDGMENTS
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We dedicate this study to the memory of Vasant Mäkinen, gold prospector, engineer, test driver, great friend, may you rest in peace! We acknowledge the following individuals and institutions for their contributions: Australian Research Council (ARCFT100150200), The South Australian Museum and Adelaide Microscopy (University of Adelaide), especially Dr. Sarah Gilbert, Dr. Animesh Basak and Dr. Benjamin Wade for instrument support; the Gold Prospectors Association of Finnish Lapland for field support, Heikki Kallio-Mannila, Aila, Heikki and Antti Heikkilä, Risto and Maija Vehviläinen, Pekka Turkka, Raimo Kanamäki, Antti Kohtamäki, Sirkka and Kari 36
ACCEPTED MANUSCRIPT Merentuoto, Ami Telilä and Pekka Itkonen for their warm welcome, access to sites and the fantastic samples. Thanks to Manfred and Brigitte Roos for assistance with sampling, and Gregory Rinder for graphics support. We appreciate the comments by the two anonymous reviewers, which have improved the manuscript, as well as the handling
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and comments by the editor Prof. Johannesson.
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ACCEPTED MANUSCRIPT Tables
Mining District
Coordinates
Deposit style
Ivalojoki - Kalliomannila a,b (IVK) Ivalojoki – Heikkila (IVH) Ivalojoki – Vehviläinen (IVV) Lemmenjoki – a,b Jäkäläpää 1 (LJ1) Lemmenjoki – Jäkäläpää 1 (LJ2) Lemmenjoki – Turkka a (LT) Lemmenjoki – Telilä (LTE) Lemmenjoki – Itkonen a Salonen (LIS) Lemmenjoki – a,b Kanamäki (LK) Lemmenjoki – Kohtamäki (LKO)
Ivalojoki
N 68°27.460' E 26°56.823' N 68°27.527' E 26°56.580' N 68°25.200' E 26°54.833' N 68°41.899' E 25°43.701' N 68°41.877' E 25°43.632' N 68°42.518' E 25°42.020' N 68° 38.984' E 25° 37.778' N 68° 41.668' E 25° 41.147' N 68°39.915' E 25°36.305' N 68°39.100' E 25°37.756’
Glacial till
Sampling depth [m] 0.5 – 1.5
Glacial till
2–3
(Glacio)fluvial
0 – 0.5
Glacial till
0 – 1.5
Glacial till
0 – 1.5
(Glacio)fluvial
0–1
Glacial till
2–4
(Glacio)fluvial
0–1
(Glacio)fluvial
2–5
Glacial till
2–4
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LemmenjokiJäkäläpää LemmenjokiJäkäläpää LemmenjokiMiessi LemmenjokiMiessi LemmenjokiMiessi LemmenjokiMiessi LemmenjokiKaarreoja
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in addition to placer Au-particles, soil samples were obtained in addition to placer Au-particles, water samples were obtained
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b
Ivalojoki
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Ivalojoki
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Site
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Table 1 - Summary of relevant site properties.
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Parameter
Unit
Sample depth
n.a.
b
LJ1
LK
Surface water 5.4 1.6 34 1.6 0.067 0.021 0.16
Surface water 5.1 2.1 29 2.1 0.21 0.011 0.08
Groundwater 5m 6.6 1.2 11 1.2 0.03 0.10 0.14
Quantification Limit n.a.
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n.a. n.a. dS/m 0.01 mg/L 0.01 mg/L 0.01 mg/L 0.005 mg/L 0.005 meq/L 0.01 0.8 2.6 1.4 2 mg/L 0.05 0.03 0.33 2 mg/L 2.0 0.8 0.9 2 mg/L Al 0.12 0.08 <0.05 0.5 mg/L Ca 1.1 0.2 0.9 0.5 mg/L Fe 0.2 <0.1 <0.1 0.5 mg/L K 5.4 2.8 0.6 0.5 mg/L Mg 0.6 0.2 0.6 0.5 mg/L Mn <0.05 <0.05 0.08 0.5 mg/L Na 2.1 0.8 1.6 0.5 mg/L S 0.9 0.5 0.4 0.5 mg/L 0.17 <0.05 <0.05 0.5 Ag µg/L As µg/L 0.36 0.58 0.09 0.05 Au µg/L 0.6 <0.5 <0.5 0.5 Co µg/L 0.37 0.13 0.58 0.5 Cr µg/L 0.3 0.2 0.3 Ni µg/L 1.1 0.5 0.7 0.2 Pb µg/L <0.05 <0.05 0.06 0.05 V µg/L 1.03 1.00 0.06 0.05 Ti µg/L 0.8 0.7 <0.5 0.5 Zn µg/L 5.8 3.5 10.4 0.2 a Notes: The following elements were analyzed but found to be below detection limit: Sc, Sn, Se, Cd, Y, Pd, Ga, Ge, Ru, Re, Ta, La, Ce, Pr, Nd, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Re, Rh, Ir, Pt, U, Th -1 -1 < 0.05 < 0.2 µg L , Cu < 1 µg L . b n.a., not applicable
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pH Electrical conductivity TOC TN NH4-N NOx-N Total Alkalinity Cl NO3 = SO4
IVK
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ACCEPTED MANUSCRIPT Table 3 – Physicochemical parameters, major element contents and concentration of Au and its pathfinder elements in soil samples from Finnish Lapland. Parameter
a
Unit
IVK
LJ1 S1
LJ1 S2
LJ1 S3
LT
LIS
LK
Quantification Limit n.a.
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Soil horizon A2 Ah A2 B/C B C (Till) C(Till) Sample depth m 0.5-1.5 0-0.1 0.1-0.3 1 >0.5 >1 >2 pH n.a. 6.5 3.0 4.8 6.0 5.4 6.1 6.8 n.a. Electrical dS/m 7.96 2650 36.3 4.13 41.70 33.60 4.25 0.01 conductivity 0.01 Total C % 1.4 7.5 0.35 0.45 0.75 0.63 0.09 0.01 Total N % 0.07 0.39 0.03 0.03 0.02 0.01 0.02 Al mg/kg 39991 27103 24976 30210 36900 32052 23118 100 Ca mg/kg 1754 3621 2441 1937 319 1583 5041 100 Fe mg/kg 64193 48054 35775 45268 121458 61618 39315 100 K mg/kg 3221 1091 2463 2841 6003 5715 5460 100 Mg mg/kg 11502 12899 7498 6265 7577 7254 8003 100 Mn mg/kg 418 359 324 309 1000 852 754 50 Na mg/kg 255 146 272 259 214 242 341 50 P mg/kg 739 363 287 510 961 331 1386 100 S mg/kg 193 5957 89 77 150 <40 <40 40 Ag µg/kg 120 164 199 290 145 148 145 10 As mg/kg 0.9 2.0 1.1 0.9 1.3 1.5 1.1 0.1 Au µg/kg 100 211 98 83 61 48 24 10 Cd mg/kg 0.56 0.20 0.09 0.57 0.47 0.35 0.55 0.1 Co mg/kg 14.78 208 15.48 18.88 38.43 68.85 15.35 0.2 Cu mg/kg 28.9 26.5 17.2 40.1 56.6 43.1 25.9 0.2 Mo mg/kg 1.9 4.0 1.0 1.5 7.0 4.2 0.4 0.1 Pb mg/kg 7.3 2.5 5.8 5.6 10.4 8.6 4.9 0.1 Se mg/kg 0.5 1.0 <0.2 0.3 0.8 0.6 <0.2 0.2 Sn mg/kg 0.30 0.28 0.36 0.37 0.21 0.33 1.49 0.1 V mg/kg 97 58.0 56.5 83 114 100 59.5 0.1 U mg/kg 1.34 0.35 1.08 1.21 1.53 3.22 4.62 0.1 Zn mg/kg 106 77.0 65.5 80 184 112 80 0.2 a Notes: The following elements were analyzed and data is available on request: Sc, Sn, Se, Cd, Y, Pd, Ga, Ge, Ru, Re, Ta, La, Ce, Pr, Nd, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Re, Rh, Ir, Pt, Th. b n.a., not applicable
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ACCEPTED MANUSCRIPT Table 4 - Total reads, OTUs and percentage of total reads covered by the most
Percentage of total reads covered by the 20 most abundant OTUs 66.6
339599
283
67.8
89.2
IVV
306367
121
73.3
94.5
LJ1
222757
138
61.4
LJ2
208061
85
98.9
LT
1020754
136
66.1
LTE
416549
163
86.7
LIS
87554
98
98.6
LK
640476
108
99.6
LKO
254711
109
97.4
Sum
3915388
519 (different)
81.2
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99.7 89.4
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97.2
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IVH
99.1 99.8 99.5
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Number of OTUs
IVK
Total number of reads 418560
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Site
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Percentage of total reads covered by the 10 most abundant OTUs 59.2
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abundant OTUs for the ten sites in Finnish Lapland.
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ACCEPTED MANUSCRIPT Figure Captions Figure 1
(A) Sampling locations; (B) representative sample from one site; and (C-H) typical morphologies of Au particles from the Ivalojoki and Lemmenjoki goldfields in Finnish Lapland. Shown are backscatter electron (BSE)
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micrographs of particles that are slightly rounded (C, from LJ1; F from
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IVK), wiry (D from LT), spheroidal (E from LJ1), well-rounded (G from IVH)
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and flattened Au (H from IVV) grains (for site abbreviations please see
Figure 2
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Table 1).
BSE micrograph of a characteristic Au particle surface showing dissolution
Figure 3
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features (white arrows).
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pitting (A), and (B) showing dissolution pitting next to re-precipitation
Secondary electron (SE) micrographs of polymorphic layers on Au particle
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surfaces. (A) SE micrograph of a typical particle coated by a polymorphic
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layer; (B, D, E) SE micrographs and a representative EDS spectrum (C) of polymorphic layers composed of nano- and microcrystal minerals, cells
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and EPS; (G) SE micrograph and EDS spectrum (H) of a cell and a typical
Figure 4
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pyrite micro-crystal. Electron microprobe maps, Au (red) and S (blue) and Fe (green) and S (green), showing the association of S and Fe with transforming Au. A particle composed of Au–Ag alloy overlain by secondary Au (A) and a high purity Au-particle (B, C), overlin by a polymorphic layer rich in S- and Fecontaining minerals. Figure 5
BSE (A, B, D-F) and SE (C) micrographs of secondary-Au morphologies associated with polymorphic layers: (A) nano-phase Au; (B, C) triagonal 56
ACCEPTED MANUSCRIPT Au platelets; and E) vermiform (C, D) and triangular platy (F) aggregates of secondary-Au. Figure 6
BSE (A, D) and SE (B, C) micrographs showing the direct association of a rod-shaped bacterial cell with aggregates of Au nano and µ-crystals (A, B),
SE micrographs (A, F), EDS- (B-E, G) and EBSD- (H, I) maps of a Au
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Figure 7
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and (C, D, E (spectrum)) of nanophase Au in EPS (C, D from ).
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surface partially coated by a polymorphic layer (A-E) and a FIB-milled section (F, G) and polished block (H), respectively, showing progressive
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coating of primary-Au-Ag by secondary Au nano- and microcrystalline
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aggregates (B, C, white arrow), leading to the formation of a layer of highpurity (G, white arrow) microcrystalline (H, I) secondary-Au. Electron microprobe maps, Au (red) and Ag (green), showing the
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Figure 8
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progressive transformation of Au particles. (A) A particle composed of a homogenous Au–Ag alloy with a partial layer of secondary high purity Au;
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(B, C) increasingly transformed Au particles, and an almost entirely
Figure 9
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transformed particle (D). Composition of bacterial communities associated with Finnish Au particles.
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Distribution of dominant bacteria phyla/classes based on (A) number of OTUs (529) and (B) total number sequences (>3.9 million) for the 10 sites; note: classes are shown for Proteobacteria and Firmicutes.
Figure 10
(A) Neighbour-joining phylogenetic tree of representative 16S rRNA sequences of the 18 present on particles from all 10 sites. percentages of 1000 bootstrap values < 70% are not shown. Methanobrevibacter smithii was used as the out-group. (B) Ordination plot of the first two canonical 57
ACCEPTED MANUSCRIPT axes produced by canonical analyses of principal coordinates (CAP) of Miseq data analyzed for differences in community assemblages in relation to deposit type and depth; vectors of Pearson’s correlations of classes/phyla are overlain16S rRNA gene.
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Schematic model and putative functional classification of bacterial taxa
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constituting biofilms on Au particles (modified after Rea et al., 2016): (1)
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conditioning of surfaces to their attachment; (2) the recruitment of photoand heterotrophic Gram-negative bacteria; (3–5) the proliferation and
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growth of the biofilm community including heterotrophic and metallophillic
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species; (5) the mobilization, detoxification and re-precipitation of Au; and (6) the seeding of dispersal cells with release of nano-particle and Au
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complexes.
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Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10
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Figure 12
Frank Reith, Maria Angelica D. Rea, Paige Sawley, Carla M. Zammit, Gert Nolze, Tina Reith, Kai Rantanen, Andrew Bissett PII: DOI: Reference:
S0009-2541(18)30133-5 doi:10.1016/j.chemgeo.2018.03.021 CHEMGE 18701
To appear in:
Chemical Geology
Received date: Revised date: Accepted date:
8 November 2017 9 March 2018 14 March 2018
Please cite this article as: Frank Reith, Maria Angelica D. Rea, Paige Sawley, Carla M. Zammit, Gert Nolze, Tina Reith, Kai Rantanen, Andrew Bissett , Biogeochemical cycling of gold: Transforming gold particles from arctic Finland. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Chemge(2017), doi:10.1016/j.chemgeo.2018.03.021
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ACCEPTED MANUSCRIPT Confidential Information:
Manuscript submitted to Chemical Geology
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Biogeochemical cycling of gold: Transforming gold particles from arctic Finland
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Frank Reith1,2*, Maria Angelica D. Rea1,2, Paige Sawley1, Carla M. Zammit3, Gert
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Nolze4, Tina Reith1, Kai Rantanen5, Andrew Bissett6
1
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Affiliations:
The University of Adelaide, School of Biological Sciences, Department of Molecular
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and Cellular Biology, Adelaide, South Australia 5005, Australia 2
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CSIRO Land and Water, Environmental Contaminant Mitigation and Technologies, PMB2, Glen Osmond, South Australia 5064, Australia
School of Earth Sciences, University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia
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3
4
Federal Institute for Materials Research and Testing, Berlin, Germany 5
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Gold Prospectors Association of Finnish Lapland, Inari, Finland
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CSIRO, Oceans and Atmosphere, Hobart, Tas, 7000, Australia
Running Title: Geomicrobial gold transformation in arctic Finland
*Corresponding author: Frank Reith
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ACCEPTED MANUSCRIPT A/Prof. Frank Reith Department of Molecular and Cellular Biology, School Biological Sciences, The University of Adelaide CURRENT ADDRESS:
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CSIRO Land and Water
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PMB 2
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Glen Osmond, 5064
FAX:
+61 8 8303 8550
[email protected]
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Number of Pages:
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E-MAIL:
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+61 8 8303 8469
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PHONE:
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South Australia, Australia
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Number of Tables
4
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Number of Supplementary Materials: 4
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ACCEPTED MANUSCRIPT Abstract (Bio)geochemical cycling of gold (Au) has been demonstrated in present-day (semi)arid, (sub)-tropical and temperate environment. Hereby biofilms on Au-bearing mineraland Au-particle surfaces drive Au dispersion and reconcentration, thereby (trans)forming
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the particles. However, it is unknown if biogeochemical cycling of Au occurs in polar
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environments, where air temperatures can reach -40°C and soils remain frozen for much
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of the year. Therefore, placer Au-particles, soils and waters were collected at two placer mining districts in arctic Finland, i.e., the Ivalojoki and Lemmenjoki goldfields. Sites were
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chosen based on contrasting settings ((glacio)-fluvial vs. glacial-till deposits) and depths
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(surface to 5 m below current surface). Gold particles were studied using a combination of tagged 16S rRNA gene next generation sequencing and electron microscopic / micro-
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analytical techniques. Across all sites a range of Au-particle morphologies were
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observed, including morphotypes indicative of Au dissolution and aggregation. Elevated Au concentrations indicative of Au mobility were detected in placer particle bearing soils
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at both districts. Typically Au-particles were coated by polymorphic biofilm layers
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composed of living and dead cells embedded in extracellular polymeric substances. Intermixed were biominerals, clays and iron-sulfides/oxides and abundant secondary Au
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morphotypes, i.e., nano-particles, micro-crystals, sheet-like Au, branched Au networks and overgrowths and secondary rims. Biofilms communities were composed of Acidobacteria (18.3%), Bacteroidetes (15.1%) and Proteobacteria (47.1%), with Proteobacteria (19.5%) being the most abundant proteobacterial group. Functionally, biofilms were composed of taxa contributing to biofilm establishment, exopolymer production and nutrient cycling, abundant taxa capable of Au mobilization, detoxification and biomineralization, among them Cupriavidus metallidurans, Acinetobacter spp. and
ACCEPTED MANUSCRIPT Pseudomonas spp, were detected. In conclusion, these results demonstrate that placer Au-particle transformation and Au dispersion occur in cold, arctic environments. This corroborates the existence of biogeochemical Au cycling in present-day cold
bacteria,
biogeochemistry,
mobility,
Finland,
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Keywords: Gold,
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environments.
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metallidurans, NGS
Cupriavidus
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Highlights:
Gold particles from Arctic placers are biogeochemically transformed.
Multispecies bacterial biofilms exists on Arctic placer gold particles.
Biofilm communities include bacterial taxa mediating gold cycling.
Elevated gold concentration were detected in soils and waters.
Biogeochemical gold-cycling occurs in modern day cold, arctic environments.
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ACCEPTED MANUSCRIPT 1. INTRODUCTION The cycle of gold (Au) is driven by physical, geochemical and geomicrobial processes mediating the dispersion and reconcentration of Au in Earth surface environments (Southam et al., 2009; Reith et al., 2013; Craw and Kerr, 2017). Thereby
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physical, chemical and biological processes co-occur and their interactions shape Au-
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particle morphologies and the distribution of Au in the landscape (Reith et al., 2007;
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Craw and Kerr, 2017). Physical erosion of host materials, e.g., quartz-Au veins, results in primary Au being deposited in placers, including eluvial, alluvial, glaciofluvial and
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glacial-till placers. Biological and chemical processes in combination with climatic and
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geological factors determine the physicochemical conditions at a site, and drive the solubilization and re-precipitation of Au (e.g., Reith et al., 2007; Craw and Kerr, 2017).
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Primary Au undergoes transformation through chemical de-alloying, biogeochemical
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Au/Ag dissolution as well as (bio)geochemical aggregation of Au (e.g., Southam et al., 2009; Reith et al., 2013; Craw and Kerr, 2017). This leads to the formation of secondary
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Au overgrowths, which are very pure (>99 wt. % Au) and can have a range of
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morphologies including triangle, spherical, hexagonal and octahedral nano-particles and µ-crystals, bacteriomorphic Au and sheet-like and wire Au (Reith et al., 2010; Reith et
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al., 2013; Shuster et al., 2017). Ultimately, high purity Au rims covering entire primary particles or parts thereof form. These can reach widths of several 100 µm and usually display a non-deformed matrix of nano- and microcrystals (Craw and Lilly, 2016; Stewart et al., 2017). Indeed most primary Au-particles recovered from placer environments display a Au-Ag core and have a partial or complete rim of high purity secondary Au (e.g., Groen et al., 1990, McCready et al., 2003, Fairbrother et al., 2012; Reith et al., 2012; Stewart et al., 2017; Shuster et al., 2017; Melchiorre et al., 2018). 3
ACCEPTED MANUSCRIPT Microorganisms contribute to the solubilization of Au via the formation of reactive Mn-oxide minerals, e.g., birnessite, capable of oxidizing Au(0) and Au(I) to Au(III)complexes (Ta et al., 2014 and 2015). Microbes can excrete Au-complexing ligands required for stabilization of Au-complexes in solution, e.g., soil- and groundwater, under
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Earth surface conditions (Reith et al., 2007; Fairbrother et al., 2009). Gold dissolution by
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bacteria has been observed in laboratory culture experiments and in soil microcosms
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(Fairbrother et al., 2009; Reith and McPhail, 2006 and 2007). In a recent study assessing supergene Au transformation in relation to groundwater geochemistry in New
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Zealand, thiosulfate was determined to be the dominant Au-complexing ligand (Craw
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and Kerr, 2017). While a range of microbiota have been shown to passively sorb Au(I/III)-complexes, some bacteria living in biofilm communities on placer Au-particle
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surfaces have developed active biochemical responses to deal with Au-toxicity and
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reductively precipitate Au-complexes leading to the formation of nanoparticulate Au (Nakajima, 2003; Reith et al., 2009; Kenney et al., 2012; Johnston et al. 2013; Checa et
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al., 2007). Dispersion of Au nanoparticles enhances Au mobility, aggregation of nano-
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and microphase Au concentrates Au in secondary enrichment zones (Craw and Kerr, 2017; Shuster et al., 2017).
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Natural Au-particles from a wide range of placer localities in (semi)-arid and (sub)-tropical Australia, arid North America, temperate New Zealand, and tropical South America (Brazil and Columbia) are coated by polymorphic layers consisting of cells, extracellular polymeric substances (EPS), as well as detrital and authigenic minerals, e.g., clays, Fe-oxides and sulfides (e.g., Reith et al., 2006; Rea et al., 2016; Shuster et al., 2016; Melchiorre et al., 2018). While species composition of resident biofilms varies between different sites, β- and γ-Proteobacteria dominated communities at sites in 4
ACCEPTED MANUSCRIPT Australia, New Zealand and Brazil (Reith et al., 2006; Reith et al., 2010; Rea et al., 2016). Functional assignment of detected operational taxonomic units (OTUs) suggests that communities living on Au-particle surfaces have highly diverse metabolic capabilities. OTUs can be functionally assigned to six groups linked to different stages of
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biofilm development (Rea et al., 2016): To group one, organisms capable of surface
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conditioning and attachment, e.g., Arthrobacter sp. and Staphylococcus sp., were
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assigned. In group two, OTUs mediating EPS production and autoaggregation, e.g., some Pseudomonas spp. and Rhodobacter spp., are found. To group three organisms
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largely involved in carbon and nitrogen cycling were assigned, these included
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Pelomonas spp., Herbaspirrillum spp., and Acidovorax spp.. To group four organisms capable of the metabolic turnover of complex and xenobiotic organics and toxins were
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assigned, including some Burkholderia spp., Sphingomonas spp. and Phenylobacterium
These
include
putative
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spp.. In group five organisms capable of directly affecting Au cycling were identified. Au-mobilizers,
e.g.,
the
sulfur-oxidizing
bacterium
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Diaphorobacter sp., Sphingomonas spp. and Methylobacterium spp. Other species
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included have the ability to actively detoxify Au(I/III)-complexes, e.g., C. metallidurans, D. acidovorans, Stenotrophomonas sp., and Achromobacter spp.. The aurophillic
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bacterium C. metallidurans, which was detected on Au-particles from Australian and Brazilian sites, appears to be one of the key-species involved in Au cycling in these regions (Reith et al., 2006; Reith et al., 2010; Rea et al., 2016). It reduces toxic Au(I/III)complexes in the periplasm, which leads to the formation of Au nanoparticles (Reith et al., 2009; Wiesemann et al., 2013 and 2017). Another bacterium involved in active detoxification and precipitation of Au nanoparticles is D. acidovorans, which produces the metallophore delftibactin to extracellularly convert toxic Au(I/III)-complexes to Au 5
ACCEPTED MANUSCRIPT nanoparticles (Johnston et al., 2013). Species forming dispersal cells, such as Lysobacter spp., are found in group six. In Finnish Lapland Au has been mined from placer deposits for more than 150 years, making this region the last professional placer Au mining area in Western Europe
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and one of the northern most in the world (Saarnisto et al., 1991). Located
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approximately 350 km north of the Arctic Circle, average annual air temperatures lie
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below 0°C, with air temperature below -40°C common in winter. Soils and sediments are frozen for much of the year down to several meters. While grains from Lemmenjoki and
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Ivalojoki occur in a wide range of morphologies and links to physical, chemical and
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anthropogenic transformation have been described by Kinnunen (1996), the presence of biofilms and Au biominerals, e.g., nanoparticles, indicative of biogeochemical Au cycling
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have not been shown from these or any other localities in these climatic conditions.
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Hence, it is unknown if biogeochemical Au cycling occurs in low temperature environments. Therefore, the aims of this study are to assess: (i) the presence of
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secondary Au morphotypes and the dispersion of Au into the surrounding environment;
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(ii) the presence of biofilms on placer Au particles from Finnish Lapland; and (iii) the phylogenetic composition and putative functional capabilities of biofilm communities in
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relation to processes affecting Au mobility and particle transformation. To achieve this, Au-particles from ten individual sites in northern Finland were studied using a combination of Illumina Miseq next generation sequencing (NGS) of biofilm communities and advanced micro-analytical techniques, including field emission gun-scanning electron microscopy (FEG-SEM), focused ion beam-scanning electron microscopy (FIBSEM) coupled with energy dispersive x-ray spectroscopy (EDXS), electron microprobe
6
ACCEPTED MANUSCRIPT analyses (EPMA). In addition, soil and water samples from key-sites were analyzed for more than 50 physicochemical properties, including Au concentrations.
2. MATERIALS AND METHODS
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2.1. Description of field sites and sampling
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Sample collection sites are located in the Ivalojoki and Lemmenjoki goldfields in northern
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Lapland, Finland (Fig.1, Table 1). Placer Au has been mined at these sites since the 19th century (Puustinen, 1991; Kojonen et al., 2005). The size of Au-particles mined ranges
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from very fine dust to rare large nuggets weighing several hundred grams (Puustinen,
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1991). Altogether more than 1000 kg of Au have been mined here during the past 120 years. Most of the Au fields are interpreted as (glacio)-fluvial and glacial-till placers; they
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vary in size and are generally composed of sorted to poorly sorted glacial sediments
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(Saarnisto et al., 1991; Kinnunen, 1996).
Geologically, the Ivalojoki and Lemmenjoki placer Au-fields are situated within the
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southern part of the granulite complex of Lapland, which extends from Norway in the
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west to the Russian border in the east (Saarnisto et al., 1991; Kojonen et al., 2005). The complex was metamorphosed during the Svecokarelian Orogeny (~1.9 Ga), under
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granulite conditions (Saarnisto et al., 1991; Tuisku et al., 2006). While primary bedrockhosted Au mineralized system occurs in the area (Sarnisto et al., 1991), large primary Au deposits have not been reported in the immediate vicinity of the sampling sites (Eilu et al., 2007; Eilu, 2015). During the Pleistocene the whole of northern Finland was glaciated (Stigzelius, 1977). Deglaciation began approximately 11600 years ago (Lunkka et al., 2004). During deglaciation, river valleys acted as meltwater channels. Today, interior Finnish Lapland 7
ACCEPTED MANUSCRIPT is characterized by gently undulating glacially formed terrain. The average annual temperature is -0.9 °C in nearby Ivalo, with an average minimum winter temperature of 18°C. Average rainfall is 400 to 500 mm (Finnish Meteorological Institute, 2014). The soil types range from typic haplocryod to skeletic podzol (Sutinen et al., 2007). Soil-pHs
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typically range from 3.0 to 5.4 in top soil horizons to 4.1 to >7 in the deeper soil and till
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horizons (Kähkönen, 1996; Närhi et al., 2011). Organic C and N in Ah-horizon soils can
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reach more than 10 and 0.5 wt.%, respectively; S, Fe and Ca concentration can reach more than 2000, 1400 and 2200 mg/kg in A and B horizons, respectively (Kähkönen,
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1996; Närhi et al., 2011).
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Placer Au-particles, soils and surface water samples from the Ivalojoki and Lemmenjoki placer deposits were collected in August 2013. Gold particles were
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collected from ten sites, all of which are currently mining claims (Fig. 1; Table 1). Three
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sites were within the Ivalojoki district and seven were within the Lemmenjoki district (Fig. 1; Table 1). Sites were chosen based on contrasting deposit styles (glavio)fluvial and till
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placers, at key locations soil and water samples were also collected (Table 1). Gold
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particle, soil and water samples were collected following a field sterile method to minimize contamination (Reith et al., 2010). Gold particles for electron microscopy (EM)
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were washed with sterile 0.9 wt. % NaCl solution and kept in EM fixative (4 wt.% paraformaldehyde, 1.25 wt.% glutaraldehyde in PBS, 4 wt.% sucrose, pH 7.2). Samples for DNA analysis were washed and stored in sterile saline solution. Samples were then transported on ice (Au particles for micro-analysis) or frozen (Au-particles for DNAanalysis, waters and soils) to the laboratory.
2.2. Electron microscopy and microanalyses of Au-particles 8
ACCEPTED MANUSCRIPT Gold particles for EM were processed and dehydrated using series of ethanol (70 vol.%, 90 vol.% and 100 vol.%; 2 x 10 min each) washes and placed in 100 wt.% hexamethyldisilazane (HDMS) to ensure the integrity of bacterial cells and biofilms (Fratesi et al., 2004). Samples were C coated and analyzed using FEG-SEM in
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secondary electron (SE) and backscatter imaging (BSE) modes at 5 kV and 20 kV,
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(Quanta™ 450 FEG Environmental SEM with EDS detectors, FEI, Netherlands).
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Selected Au-particles were analyzed further using a focus ion beam secondary electron microscope (FIB-SEM; Helios NanoLab DualBeam, FEI, Netherlands). Images of the
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surface features were collected at 2 to 20 kV and 86 pA, with sectioning and cleaning at
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20 kV and 9.7 pA. The instrument is equipped with energy dispersive X-ray spectroscopy (EDXA) used to collect element maps across the particles surfaces and
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milled sections; elements mapped were Au, Ag, Fe, C, N, O, Si, Ti, Al, Ga, Mg, Na, K
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and Pt. Quantitative maps of element compositions of particles were collected using an electron microprobe (EPMA; Cameca™ SXFive Electron Microprobe, France), with 23
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samples being mapped. Samples were set in epoxy resin and polished with 1 µm
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diamond paste. The Cameca™ SXFive Microprobe, equipped with five wavelength dispersive (WLD) X-Ray detectors, with PeakSite software for instrument control, and
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Probe for EPMA™ for data acquisition and processing to produce quantitative maps of the Au-particles. Mapping was conducted at 20 kV and 200 nA. The particles were analyzed for (detection limits in parenthesis in wt.%): Au (0.24). Ag (0.09), S (0.025), Fe (0.06) and Cu (0.11). All elements were calibrated using minerals and pure metal standards from Astimex and P&H. Cu, Fe and S was calibrated on chalcopyrite; Ag on telluride; and Au on a pure metal standard. EPMA software produces a full quantitative pixel by pixel calculation by using the mean Atomic number background correction 9
ACCEPTED MANUSCRIPT (Donovan and Tingle, 1996) in CalcImage, and false colorization and formatting in Surfer10™ to produce net intensity, detection limit map and totals image. Backscatter Diffraction (EBSD) analyses were conducted with a ZEISS Supra field emission (FE)SEM (Zeiss, Germany) with an e-Flash detector and data was analyzed using CrystAlign
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(Bruker AXS, Germany).
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2.3. Chemical analyses of soil and water samples
Soil and water samples were analyzed for a range of physicochemical parameters
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following methods published in Reith et al. (2012) and Brugger et al. (2013). Prior to
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analyses, soil samples were homogenized in a ring mill and water samples were filtered through 0.22 µm sterile syringe filters. Waters and soil (1:5 soil : H2O) were analyzed for
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pH, electrical conductivity and alkalinity. Total C and N in soil samples were determined
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by high temperature combustion in an atmosphere of oxygen using a Leco CNS-2000. Dissolved C in waters (DC) was determined by Skalar Formacs HT TOC/TN Analyzer.
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Inorganic carbon was acidified to form CO2 prior to detection. Dissolved organic carbon
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(DOC) was determined by the difference between total and inorganic C. Ammonianitrogen (NH4-N) was determined by flow analysis and spectrometric detection
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according to ISO 11732. Nitrate- and nitrite-N were determined using the automated segmented flow analyzer (Alpkem Flow Solution 3) at 540 nm. Anions in waters (F-, Cl-, NO2-, Br-, NO3-, SO42- and PO43-) were measured using a Dionex ICS-2500 ion chromatography system with 2 mm AS16 anion separation column. Soil samples were microwave digested in concentrated aqua regia. Element concentrations in digests and water samples were determined using inductively coupled plasma optical emission spectroscopy (ICP-OES) on a Spectro ARCOS (Spectro Analytical Instruments, Kleve, 10
ACCEPTED MANUSCRIPT Germany). Trace and ultratrace elements were analyzed by inductively coupled plasma mass spectrometry (ICP-MS; Agilent 7700, Japan). Concentrations of Au and Ag in soil samples were analyzed in pressed pellets using laser ablation (LA)-ICP-MS analyses with Resonetics M50 with 7700 ICP-MS (Resonetics, USA; Agilent, USA) calibrated with
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NIST standards. Elements analyzed and quantification limits are provided in Tables 2
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and 3.
2.4. Biomolecular and statistical analyses
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Biofilm communities resident on Au-particles were assessed using nested 16S
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rRNA polymerase chain reaction (PCR) combined with next generation sequencing (NGS) using the Illumina MiSEQ platform – TruS eq SBS v.3 600 cycle using 300 bp
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paired end sequencing (Reith et al., 2010; Bissett et al., 2016). The universal primers
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27F (Lane, 1991) and 1492R (Osborn et al., 2000) were used for initial PCR amplification of 16s rRNA genes directly from the Au-particles (Reith et al., 2010), 58
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positive 1st round amplification were further amplified using primers 27F and 519R
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(Lane, 1991; Lane et al., 1985), and sequenced at the Australian Genome Research Facility (AGRF, Melbourne, Australia). DNA amplifications were performed in an Applied
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Biosystems Veriti™ Thermal Cycler. Amplicons were checked in 1.5% agarose gel with Gel Red (Biotium Inc., Hayward, CA, USA) 1:10,000 (v/v) and run at a constant voltage of 80 V for 1 h. Procedures for sequencing, open OTU picking and assignment are detailed in Bissett et al. (2016). Briefly, sequence read quality was visually assessed using FastQC (Andrews, 2010). Sequences were trimmed to remove poor quality bases and merged using FLASH (Magoc and Salzberg, 2011). Sequences < 400 bp or containing N or homopolymer runs of >8 bp were removed (MOTHUR v1.34.1; Schloss 11
ACCEPTED MANUSCRIPT et al., 2009). Remaining sequences were submitted to open reference (OTU) picking at 97 % sequence similarity using UPARSE (Edgar et al., 2013) and OTU abundance tables constructed by mapping all reads to these OTUs (usearch_global, 97 %). Maximum-likelihood (1000 bootstrap replicates) phylogenetic trees were constructed
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using GENEIOUS 7.0 (Biomatters Ltd., New Zealand); sequences used for tree
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construction were deposited in GenBank with accession numbers MG209801 to
conducted based on a study by Rea et al. (2016).
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MG209818. Functional assignment of OTUs to six stages of biofilm development was
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Multivariate statistical analyses were conducted using the PRIMER-6 software
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package with the PERMANOVA+ add-on (Clarke and Warwick, 2001; Anderson et al., 2008). Similarity matrices were established on square root transformed abundance data
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using the Bray–Curtis method (Bray and Curtis, 1957). Permutational multivariate
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analysis of variance (PERMANOVA) combined with canonical analysis of principal coordinates (CAP) and SIMPER analysis were used to assess the influence of deposit
3. RESULTS
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type, location and sampling depth on community composition (Anderson et al., 2008).
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3.1. Morphology and composition of Au particles and particle surfaces Placer Au-particles collected from the seven individual sites at the Lemmenjoki and three sites at the Ivalojoki goldfields ranged in grain size from 0.1 to 2.0 mm (Fig. 1). Across samples from all sites a wide range of particle morphologies were observed, including flattened, elongated, equant, irregular, branching and globular Au-particles (Fig. 1 D-H). Particles were slightly to highly rounded and showed signs of physical and (bio)geochemical transformations, which had occurred in Earth surface environments 12
ACCEPTED MANUSCRIPT during and post transport (Fig. 1 D-H). Signs of physical modification included rounded and (partially) folded edges, striations, randomly orientated scratches and pitted cavities indicative of glacial and alluvial transport (Fig. S1). Particles from glacial-till sites that had not undergone post-glacial fluvial redistribution by the Ivalojoki and Lemmenjoki
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river systems were irregular with partially folded edges, but fewer scratches and pits.
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With increasing fluvial transport Lemmenjoki particles transgressed from irregular to
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elongated and wire Au morphologies, and showed increasingly battered and roughened or rounded surface morphologies (Fig. 1 D-H; Fig. S1). Particles from Ivalojoki became
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more rounded and eventually flattened as sites changed from glacial-till to fluvial sites.
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In addition to showing signs of physical alteration, signs of biogeochemical transformations of the particles were common. These included dissolution features (Fig.
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2A), where progressive dissolution of Ag and Au has led to the irregular pitting of particle
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surfaces. Surfaces displaying dissolution features were commonly in close proximity to secondary-Au re-precipitation features (Fig. 2B). Particles from all sites were entirely or
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partially coated by a layer comprising C, N, O, S, Al, Si, Fe, Na, Mg, P, K, Ti and Au
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(Fig. 3A-C). This layer, which can reach a thickness of more than >20 µm in topographic lows, is composed of bacterial cells and EPS intermixed/underlain by Fe-oxides and -
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sulfides, clays, silicate minerals and secondary Au (Fig. 3D-H); indeed S and Fe containing mineral layers commonly coat particle surfaces (Fig. 4). In zones especially rich in organic material, indicative of active biofilms, the biofilm was also especially enriched in nano- and microphase Au (Figs. 5 and 6). Rod-shaped bacterial cells (2.5 to 3.5 µm in exopolymers) occurring individually or in clusters were observed in protected areas on many particles, where they were commonly attached by fimbriae to the Au surface and commonly associated with nano-particulate Au (Fig. 3E and 6). Aggregates 13
ACCEPTED MANUSCRIPT of nano and micro-sized, e.g., crystalline Au triangles, conglomerates of Au µ-crystals and bacterioform/budding Au, which can form bridging structures of the matrix, were also commonly observed and associated with polymorphic layers (Figs. 5 and 6). While abundant nano- and microphase secondary-Au was observed on particles from all sites,
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particles from sites with increasing recent fluvial influences displayed fewer Au nano-
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and micro-particles in the polymorphic layers. Similar results were also obtained from
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the Lemmenjoki sites, where more nanophase and micro-crystalline Au was observed on particles from glacial-till deposits. Overall, particles from IVK, IVH and LJ1 and LJ2
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showed the most extensive assembly of polymorphic layers and Au nanoparticles and
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aggregates (Table 1; Figs. 1, 5 and 6).
Elemental maps of a particle surface, in which a primary Au surface (composed of
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a Au-Ag alloy) was partially coated by polymorphic layer, showed that during the
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transformation of the primary Au-Ag-alloy Ag is lost, and the secondary Au aggregates, thereby forming sheet-like Au overgrowths, which are beginning to cover the particle
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(Fig. 7 A-C). This process was further developed on some particles, where below a 10
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µm deep polymorphic layer that contained nano- and microphase Au, a 1-2 µm wide rim of highly pure (99.9 wt.%) secondary Au coated the primary Au-Ag (Fig. 7 F,G). Note the
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transition between secondary Au and primary Au-Ag is sharp, not diffuse (Figs. 7 F,G and 8), suggesting that a new layer of secondary Au had formed over the primary Au-Ag surface, rather than that Ag had been progressively mobilized from the primary Au-Ag. This is supported by EDSD measurement of a polished particle, which showed finegrained, unstrained nano- and microcrystalline Au on the surface of the particles, whereas the core of the grain was composed of a strained large Au-mono-crystal (Fig. 7 H,I). 14
ACCEPTED MANUSCRIPT To further assess the grade of transformation across the sites particles from all sites were quantitatively mapped. Silver content in particles with a primary-Au-Ag core ranged from 4.0 to 18.0 wt.%, with Au concentrations being inverse to Ag concentrations (Fig. 8; Table S1). While 12 particles were partially or thinly (<1 µm) covered by a layer
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of secondary-Au, 11 grains displayed a thicker (>1 µm) layer of secondary-Au. Of these
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most showed layers of approximately 10 µm of secondary-Au, with some being
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progressive transformed along crevices and cracks and three particles being entirely
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3.2. Assessment of biofilm communities
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composed of 99.9 wt.% Au (Fig. 8; Table S1).
To assess the composition of the biofilm communities resident on the particles, the 16S
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rRNA gene was amplified directly from Au-particles. 16S rRNA gene sequences were
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obtained from >80 % of particles, suggesting that >80% of particles had biofilms on their surface at the time of sampling. Sequencing was conducted from six DNA-positive
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particles per site; four grains from LJ2 were amplified. After processing, >3.9 million
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reads were obtained from the 58 samples. Overall, 519 different OTUs were recorded (Table S2). Proteobacteria were the dominant phyla, comprising 46.7 % and 54.1 % of
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OTUs and total reads, respectively (Fig. 9; Table S2). Within the Proteobacteria, the βProteobacteria dominated, with 101 OTUs and 31.1 % of total reads. α- and γProteobacteria were numerous with 78 and 49 detected OTUs, which made up 13.1 and 8.3 % of total reads; - and ε-Proteobacteria made up 2.9 % of OTUs with 1.5 % of total reads (Fig. 9; Table S2). Also abundant were the phyla Acidobacteria and Actinobacteria, with 96 and 37 detected OTUs, which made up 15.1 and 6.6 % of total reads, respectively (Fig. 9; Table S2). Other phyla observed included Firmicutes (4.6 15
ACCEPTED MANUSCRIPT and 2.6 %, OTUs and reads) and Bacteriodetes (4.8 and 3.4 %), with 1-3 % of Cloroflexi,
Planctomycetes,
Cyanobacteria
Verrucomicrobia,
AD3,
Gemmantimonadetes, Nitrospira; with 16 other, rare phyla also detected (Table S2). The number of OTUs recorded in biofilms at individual sites ranged from 85 at
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LJ2 to 215 at IVK; β-Proteobacteria were most numerous at eight and Acidobacteria at
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two sites (Table S2). Eighteen OTUs were detected on particles from all sites, of those
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eight were β-Proteobacteria, and included Gallionella spp., C. metallidurans and Ralstonia spp.; Arthobacter spp. and Pseudomonas spp. were also detected (Fig. 10A;
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Table S3). Across all sites the top10/top20 most numerous OTUs (by number of reads)
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contributed between 59.2 to 99.6 and 66.6 to 99.8 % of total reads detected, respectively (Table 4). This suggests that communities are dominated by a few OTUs,
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which make up the majority of the biofilm communities, with more rare species detected
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as a result of deeper NGS compared to PCR-DGGE combined with Sanger sequencing used in earlier studies (Reith et al., 2006, 2010 and 2016). PERMANOVA showed that
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community composition varied significantly with site (√CV = 3.36; P < 0.001), deposit
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type (√CV = 3.43; P < 0.001) and sampling depths, i.e. position of Au-particles sampled in the deposit (√CV = 2.16; P < 0.001). CAP and SIMPER analyses of community data
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suggested significant links of bacterial communities from Au-particles from different deposit type and depth were strongly related to differences in the diversity and abundance of α-, β- and γ- Proteobacteria, Acido- and Actinobacteria and Bacteriodetes (Fig. 10B).
3.3.
Assessment of water and soil geochemistry
16
ACCEPTED MANUSCRIPT To assess the mobility of Au in soils and till containing transported placer Au-particles, and thereby provide a framework of physicochemical conditions at which Au mobility occurs, three water and seven soil samples from key-sites were analyzed for >50 parameters (Table 2 and 3). The water sample from the Ivalojoki site, i.e., IVK,
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contained 0.6 µg/L mobile Au as well as 0.17 µg/L of Ag, with S, Co, Cr, Ni, V, Ti and Zn
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also detected (Table 2). Surface waters had a pH of 5.1 and 5.4, groundwater pH at LK
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was 6.6 (Table 2). TOC in waters lay between 11 and 34 mg/L, and up to 2.1 mg/L of TN, 2.6 mg/L of chloride and 2.0 mg/L of sulfate were detected. All soil samples
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contained elevated Au and Ag concentrations of up to 211 and 290 µg/kg of soil,
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respectively. The highest concentration of Au (i.e., 211 µg/kg) was detected in the Ahhorizon with concentrations decreasing with increasing sample depth to 24 µg/kg in the
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till at LK (Table 3). Gold was not homogeneous distributed within each sample as
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evidenced by LA-ICP-MS lines across the pressed soil pellets (data not shown). In contrast Ag concentrations were more homogenous between (Table 3) and within
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samples (data not shown). While Au and Ag concentrations were strongly elevated
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compared to background concentration, concentrations of other typical Au pathfinder elements, including Cu, Cd, As, Mo, Pb, Co, Zn and Ni, were not elevated above
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background concentrations expected and homogenously distributed between samples (Salminen, 2009). Soil-pH was with 3.0 the lowest in the Ah-horizon at LJ1 and increased with depth to pH 6.8 in the till collected below 2 m depth at LK. Sulfur content in the Ah-horizon at LJ1 was 5957 mg/kg high, and much lower concentrations were detected in all deeper samples and ranged between 150 to below 40 mg/kg. Similarly TOC and TN ranged from 7.5 and 0.39 wt.%, respectively, in the Ah-horizon at LJ1 to 0.09 and 0.01 wt.% in the till (Table 3). 17
ACCEPTED MANUSCRIPT
4.
DISCUSSION
4.1
Gold particle transformations
The variety in chemical composition and morphology of the particles from the same sites
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suggests that they have been affected by a range of physical and bio(geo)chemical
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processes since they have been exposed to surface conditions. Some particles from
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IVH, IVK and LTE are composed of almost pure 99.9 wt.% Au, while others contain cores with >10 wt.% Ag (Fig. 8). At the glacial-till site LJ1 some particles show limited
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evidence of physical transport, such as slight rounding and pitting, scratches and
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striations, while other grains were highly transformed and spherical (Figs. 1 and S1). This suggests that Au-particle populations were derived from different primary sources.
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These may have been exposed to surface conditions, including chemical and biological
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transformation, for different periods of time, and their co-occurrence is a result of glacial and (glacio)fluvial fluvial transport and deposition. Indeed earlier studies have shown
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that minor primary deposit types may be present throughout in region (Saarnisto et al.,
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1991). These have been described as quartz-carbonate, quartz-hematite and arsenopyrite-rich shear zone deposits (Saarnisto et al., 1991). They contain localized
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differences in Ag and trace metal contents in the Au, with an average Ag content of particles reported to be 6 wt.% (Saarnisto et al., 1991). Particle shapes and the extent of their rounding is commonly linked to the transport distance of the Au, and the purity of Au can be linked to the length of time/intensity the particles have undergone (bio)geochemical transformation and refining (Kinnunen, 1996). This is especially apparent with particles composed of 99.9 wt.% pure Au with little or no sign of a primaryAu-Ag core (Wilson, 1984). This suggests that a smaller part of the Au-particle 18
ACCEPTED MANUSCRIPT population had undergone pre-glacial weathering and transformation and/or subsequent glacial transport from more distant sources, confirming the earlier hypothesis of a redistribution of highly transformed particles from paleo-placer environments. The majority of particles, i.e., those with a full or partial layer of secondary-Au overlying a
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primary core may have been eroded from regional sources by the progressing glaciers
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and deposited in glacial-till, which was reworked by fluvial processes.
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Pits on particles surfaces can be the result of physical damage, removal of crystals or (bio)geochemical dissolution (Kinnunen, 1996). Some of the pits observed on
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particles from IVH are likely the result of (bio)geochemical Au dissolution, as dissolution
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pits generally produce irregular, mottled surfaces, as observed here (Fig. 2). Many of the pits found on the Finnish particles appear in zones of most intensive physical
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transformation and are therefore have likely been obtained through physical scratching
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(Fig. S1). Extensive nano- and micro-particulate Au, dispersed through polymorphic layers, that are forming delicate aggregates of hexagonal or pseudohexagonal Au plates
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as well as spheroidal and bacterioform overgrowth have been observed on particles
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from all sites, but were most common on samples from glacial-till deposits. These are likely the result of Au dissolution and (bio)geochemical re-precipitation under surface
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conditions, as shown in numerous field- and experimental studies from sites around the world (e.g., Craw and Lilly, 2016; Falconer and Craw, 2009; Reith and McPhail, 2006; Reith et al., 2006, 2010 and 2012; Shuster et al., 2015 and 2017; Melchiorre et al., 2018). Processes affecting Ag also result in overall particle transformation, these include the de-alloying and mobilization of the Ag as well as re-crystallization of Ag-poor µm sized Au–crystals resulting from physical strain (Hough et al., 2009). There is evidence 19
ACCEPTED MANUSCRIPT to suggest Ag de-alloying has also contributed to the formation of the Au-rich rim on some grains. Particles with irregular core/rim boundaries that create deep embayments into the core, such as a particle from LKO (Fig. 7C), may be the result of Ag leaching (Youngson and Craw, 1993). When Ag is oxidized, its size is reduced, resulting in its
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removal from its site in the alloy (Desborough (1970). Repetition of this process creates
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new Au/Ag alloy interfaces, resulting in porous/diffuse rims. Some authors suggest that
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the diffusion of Ag is too slow to create the sharp boundaries between the enriched Au rim and the core of the grain (Groen et al., 1990). However, a number of recent studies
US
of Au-particles collected at sites in New Zealand have suggested that the sharp
AN
boundary indicates re-crystallisation of Ag-poor Au in surface environments. In these samples, erosion and transport of Au-particles for short distances into Eocene paleo-
M
placer deposits caused internal deformation of particles, especially near the edges of the
ED
particle (Stewart et al., 2017). Rims of fine grained (1–20 µm) recrystallized Au formed around the edges of the particles as a result of stored strain energy, which was
PT
accompanied by expulsion of Ag from Au microcrystals leading to rims containing 0-3
CE
wt.% Ag.
The delicate nature of the secondary Au surface features observed on the
AC
particles especially from till placers, which includes the secondary Au aggregates, would not survive transport (Youngson and Craw, 1993), and is hence likely the result of postdeposition, biogeochemical transformations (Fig. 5). Therefore, estimates about the extent of supergene transformation experienced by particles can be made. The last deglaciation of northern Finland began 11600 years ago, with deglaciation came the deposition of grains in tills suggesting that post-glaciation, secondary Au transformation processes were beginning not long after particle deposition. This suggests that the scale 20
ACCEPTED MANUSCRIPT of transformation observed on the majority of grains, i.e., those derived from local sources, may be the result of 11,600 years of biogeochemical refining in cold arctic conditions. A recent study of particles from Kilkivan, Queensland, Australia, assessed similar surface morphologies on particles from assumed to be eluvial/fluvial placer
T
deposits, with the aim to assess environmental rates of these biogeochemical
IP
transformation processes leading to Au surface morphologies and transformations
CR
(Shuster et al., 2017). Gold particles from Queensland showed pronounced secondary Au structures, e.g., nanometer-sized Au colloids and micrometer-sized octahedral Au
US
platelets and aggregates thereof embedded in polymorphic layers, which covered 10-40
AN
% of particle surfaces (Shuster et al., 2017). A biogeochemical model developed based on these data, and environmental properties these particles were subjected to,
M
estimated 17.9–58.5 years of Au cycling and mobilization within this subtropical
ED
environment. This time estimate corresponds well with decadal time scales required for secondary Au enrichment to occur within subtropical placer deposits, as determined
PT
from early field observations (Freise, 1931).
CE
As particles from both climatic zones are at comparatively similar stages of transformation, this suggests that the biogeochemical transformation of particles
AC
appears to be two orders of magnitude faster in subtropical conditions compared to cold, arctic environments. Indeed temperature is an important factor affecting the kinetics of (bio)chemical reactions, the composition of microbial community and the microbial activity driving nutrient cycling (Paul and Clark, 1996; Männistö and Häggbloom, 2006). Studies have shown that bacterial growth rates and activity increase exponentially with temperature (Ratkowsky et al., 1982; Pietikäinen et al., 2005). A study of soils from Sweden showed that the respiration rate at 45°C was up to 120 times higher than at 0°C 21
ACCEPTED MANUSCRIPT (Pietikäinen et al., 2005). Bacterial activities at optimum temperatures were around 10 times above those at 0°C in a range of agricultural and humus soils, respectively. Other studies have shown that process rates of N and S cycling exponentially increase with temperature (e.g., Germinda and Janzen, 1993; Nicolardot et al., 1994). The key
T
organisms driving Au cycling at the site, e.g., C. metallidurans, are mesophilic, not
IP
psychrophilic, organisms (Rea et al., 2016), and may therefore only display limited Au
Environmental mobility of Au and Ag
US
4.2.
CR
cycling activity for most the year.
AN
Gold solubilization, dispersion and re-precipitation in Earth surface environments have been attributed to geochemical and biological processes (e.g., Reith et al., 2007;
M
Shuster et al., 2016; Anand et al., 2016; Craw and Kerr, 2017; Melchiorre et al., 2018).
ED
Analyses of the placer Au-particles combined with the characterization of soil and water samples suggest that a combination of direct and indirect geochemical and biological
PT
processes have contributed to the dissolution and precipitation of Au and the patterns of
CE
Au distribution in soils holding placer Au at the study sites in arctic Finland. Note physical transport and deposition of mm-sized Au-particles in glacial and (glacio)fluvial
AC
systems have been discussed in the section above. It is also important to note that Au and Ag are the only elements with significantly elevated concentrations in soils at the study sites. However, weathering of primary bedrock-hosted Au deposits commonly leads to the enrichment of other pathfinder elements in overlying soils and sediments (Boyle, 1979). The elements that are enriched depend on the specific type of primary deposit and can include As, Bi, Cu, Mo, S, Sb, Pb, Se, Te, Pb and U (Boyle, 1979). This indicates that Au and Ag detected in the soils have been mobilized from the placer 22
ACCEPTED MANUSCRIPT particles post deposition, rather than from potential underlying primary sources, which have not been reported in the immediate vicinity of the sampling sites (Eilu et al., 2007; Eilu, 2015). The highest concentrations of finely dispersed Au in soils were detected the top
T
soils, and were up to 10 fold higher at LJ1 and IVK compared the C-horizon till. Top soils
IP
also displayed the lowest pH, highest organic matter- and S contents (Table 3), and
CR
generally contain the highest number of metabolically active microorganisms (Paul and Clark, 1996). This suggests that this combination of factors enhances the mobility of Au,
US
contributes to the transformation of Au-particles and dispersion of Au into adjacent soils.
AN
Indeed placer Au-particles from IVK, and LJ1 and LJ2 showed the most extensive assembly of transformation of polymorphic layers and Au nanoparticles and aggregates,
M
with water at IVK, containing 0.6 and 0.17 µg/L of mobile Au and Ag, respectively (Table
ED
1; Figs. 5 and 6).
These data point to processes that may be important for Au dissolution from the
PT
placer Au in Finnish Lapland. These include the oxidation/complexation of Au by Mn-
CE
oxide biominerals, e.g., the birnessite, under localized acidic conditions through ligand coupled oxidation with inorganic or bioorganic ligands (Ta et al., 2014 and 2015).
AC
Resulting Au-complexes likely include Au-organic acid- or cyanide complexes as well as possible Au-chloride, -thiosulfate and -bisulfide complexes. Given the levels of Mn in soil samples, which range from 300 to 1000 mg/kg (Table 3), the presence of Mn-oxidizing bacteria on Au-particles capable of mediating the formation of Mn(III/IV)-oxides, the acidic pH of top soil, and the presence of sulfides on Au-particles, which upon oxidation produce localized acidic conditions, it is likely Mn-oxides play a role for the mobilization of Au. In addition, oxidation/complexation of metallic Au by bioorganic Mn-compounds, 23
ACCEPTED MANUSCRIPT similar to [(bpy)2MnIII(μ-O)2Mn-IV(bpy)2]3−, may be responsible for Au-complex formation through a ligand coupled oxidation (Ta et al., 2014 and 2015). Note, biologically mediated Au solubilization is assumed to occur as an indirect effect of the metabolic processes, e.g., in biofilms on the Au-particle surfaces, and include the production of
T
Mn-compounds, thiosulfate, organic acids and cyanide. In contrast Au detoxification and
IP
biomineralization of nanophase Au can be actively mediated by specific biochemical
CR
pathways aimed at reducing intracellular Au toxicity (e.g., Johnson et al., 2013; Rea et al., 2016; Wiesemann et al., 2017).
US
In the presence of high concentration of organic matter in soils, as observed in
AN
top soil horizons at the sampling sites (Table 3), complexation to organic ligands is likely, as Group IB elements strongly bind to organic matter (Boyle, 1979; Vlassopoulos
M
et al., 1990). A range of bacteria associated with the Au-particles are capable of
ED
producing large amounts of organic acids and some are known to excrete cyanide (Fig. 11). Earlier experimental studies have shown that amino acids, e.g., aspartic acid,
PT
alanine, histidine, serine, and glycine, excreted by heterotrophic microbiota, can facilitate
CE
Au solubilization leading the formation of Au-amino-acid complexes (e.g., Korobushkina et al., 1983). Korobushkina et al. (1976) have shown that during in vitro studies with
AC
heterotrophic bacteria in media containing metallic Au several mg/L of Au were solubilized. In microcosm studies with organic matter rich auriferous soils from Australia, experiments with a living resident microflora, resulted in up to 80 wt.% of Au being solubilized; here Au solubilization was linked to the microbial formation of amino acids and cyanide (Reith and McPhail, 2006 and 2007; Fairbrother et al., 2009). In soils amended with metallic Au pellets, Au was also liberated from the pellet; in contrast sterilized microcosm Au mobilization was negligible (Reith and McPhail, 2006). 24
ACCEPTED MANUSCRIPT Amino acids, especially glycine, are common metabolic precursors for cyanide production in the bacteria, e.g., Chromobacterium violaceum, Pseudomonas spp., which have been shown to solubilize of Au from metallic Au surfaces (Fairbrother et al., 2009). Bacteria with the ability to produce cyanide have been detected on the Finnish placer
T
Au-particles, suggesting that Au cyanidation contributes to Au mobilization. In some soil
IP
ecosystems cyanide producing microorganisms can make up to 50 % of the soil
CR
microbial community (Kremer and Souissi, 2001), which would further increases the mobility of Au. Humic and fulvic acids, which make up a large proportion of the total and
US
the dissolved organic matter in Arctic soil systems, may also contribute to the
AN
mobilization of Au (Boyle, 1979). They have been shown to contribute to the solubilization of Au by forming complexes with Au(I/III)-ions, in other studies the
M
reduction of Au-complexes and the formation of stabilized Au nanoparticles was
ED
observed, which may also contribute to Au mobility at the Finnish sites (e.g., Freise, 1931; Ong and Swanson, 1969).
PT
Choride has been detected in the surface water samples (Table 2), and provides
CE
a suitable inorganic ligand for Au complexation (Usher et al., 2009). Gold(III)-chloride complexes have been predicted only to exist under low pH and highly oxidizing
AC
conditions, but in a recent study have been shown to be present in saline surface water under circum-neutral reducing conditions (Ta et al., 2014). Particularly the Ah-horizon at LJ1 displayed strongly elevated concentrations of S, which in northern Finland is common and a result of atmospheric inputs linked to anthropogenic pollution (Forsius et al., 2010). This in combination with high organic matter contents and changing redox conditions suggests a highly active S cycle in these soils, waters and sediments, which can further increase heavy metal mobility. Secondary 25
ACCEPTED MANUSCRIPT sulfide minerals were abundant on Au-particles surfaces, especially on those collected from top soils (Fig. 3 and 4), and bacteria capable of S-oxidation and reduction were detected in biofilm communities. This suggests that mobilization as thiosulfate and bisulfide complexes may play a role for Au mobility in Finnish Lapland, similar to a
T
recently published study from New Zealand (Craw and Kerr, 2017). Craw and Kerr
IP
(2017) analyzed 655 waters from environments relevant to supergene processes in the
CR
Otago Schist goldfield of southern New Zealand, which they have used as proxies to deduct supergene Au transformation processes. They have found that Au was mobilized
US
as thiosulfate- and bisulfide complexes and re-precipitated as metallic Au near
AN
unconformities, when Au-complexes were destabilized as a result of changes to physicochemical conditions, e.g., redox status or pH conditions. They suggest that this
M
mechanism of Au mobilization can be very effective at circumneutral to slightly acid pHs
ED
in organic matter limited systems, which may apply to the till and sediment layers at the study sites.
PT
Thiosulfate also is considered an important ligand for the dissolution and
CE
dispersion of Ag (Zipperian et al., 1988). Generally, Ag is considered to more mobile than Au in surface environments as it readily forms complexes with Cl−, SO42− or NO3 –
AC
ligands (Shuster et al., 2017). Mobile Ag nano-particles can also form through a variety of process, including biogenic and abiogenic reduction and stabilization of Ag-complexes (Quang Huy et al., 2013). Silver nanoparticles are labile under a range of soil conditions, leading to their dissolution and the formation of mobile ionic complexes (Levard et al., 2012). Therefore, Ag is commonly more homogeneously distributed in soils, as was also observed at the study sites. In addition, Ag is incorporated in a range of secondary minerals, e.g., jarosite, as observed in another recent study, but formation 26
ACCEPTED MANUSCRIPT of mm-sized secondary Ag-particles formed in soils or sediments have not to the authors’ knowledge not been reported, suggesting that Ag becomes widely dispersed in the landscape (Mann, 1984; Shuster et al., 2017). Gold is also transported in the soil environment as mobile complexes and
T
(nano)-particles (; Hough et al., 2011; Craw and Kerr, 2017). Low molecular weight
IP
organic acids, e.g., citrate, acetate, oxalate, amino acids and high molecular weight
CR
organic acids, including humic and fulvic acids, peptides and proteins present in biofilms may can act as ligands for Au complexation as well as stabilize metallic Au
US
nanoparticles (Vlassopoulos et al., 1990; Hough et al., 2011; Reith and Cornelis, 2017).
AN
Gold nanoparticles are formed by the abiogenic or bioorganic reduction of Aucomplexes due to changing physicochemical conditions, reduction on mineral surfaces
M
or by organic acids, or during active or passive microbial biomineralization (Hough et
ED
al., 2011). Indeed recent studies point to biofilms on placer Au-particles as an important sources of mobile Au nanoparticles, because members the community actively form
PT
extracellular Au nanoparticles, organic compounds to stabilized them are plentiful, and
CE
biofilms are subject to cycles of formation and destruction via desiccation, freezing or grazing by soil fauna, leading to periodic release of nano-particulate Au and its
AC
dispersion in the environment via transport in soil solutions and groundwater (Reith et al., 2010; Hough et al., 2011; Johnston et al., 2013; Shuster et al., 2017). The release of nano-particulate Au may therefore contribute to the high mobility of Au at the study sites as well as the observed inhomogeneous distribution within samples. Complexes and nanoparticles are re-precipitated and thereby immobilized, when the complexing ligands or stabilizing agents are decomposed, and when they are sorbed by minerals, organics or microbiota and subsequently aggregate (Hough et al., 2011; 27
ACCEPTED MANUSCRIPT Craw and Kerr, 2017). Using selective sequential extractions on soils amended with mobile Au-complexes has shown that they are preferentially associated with organic matter, clays and Fe-oxides (Gray et al., 1998; Reith et al., 2005). The results of sequential extractions conducted with samples collected from an biologically active Ah-
T
horizon microcosms after 0, 10, 20, 30, 40, and 68 days of incubation indicated a
IP
continuous microscale solubilization and re-adsorption of mobile Au by the organic
CR
matter, which after 40 days of incubation contained more than 80 wt.% of the Au in the soil (Reith and McPhail, 2006). In the same soil collected after 10 days of amendment
US
with 100 µg/kg Au(III)-chloride, 95 wt.% of the Au was associated with the organic
AN
matter (Reith and McPhail, 2006). In another study ten Australian soils were amended with citrate-stabilized Au nanoparticles, of which 3- to >80 wt.% remained highly
M
mobile/dispersible in soil solutions after 28 days of incubation, with increasing clay, Corg
ED
and Fe/Mn-oxide contents decreasing and elevated sand contents and pH increasing their mobility (Reith and Cornelis, 2017). Results of selective extractions of soils after 28
PT
days of incubation showed that up to 80 wt.% of sorbed Au was associated with the
CE
organic matter in most soils, with Fe/Mn-oxides accounting for up to up to 42 wt.% of sorbed Au nanoparticles in some soils (Reith and Cornelis, 2017). Subsequently
AC
aggregation of Au nanoparticles can form µm-sized Au aggregates, which can grow to larger purely secondary Au particles (Fairbrother et al., 2013; Shuster and Southam 2015).
4.3.
Functional characterization of biofilm communities
Earlier studies have described biofilms on Au-particles from Australia, New Zealand and South America; commonly associated with these was abundant secondary Au resulting 28
ACCEPTED MANUSCRIPT from biogeochemical Au cycling (Fairbrother et al., 2012; Reith et al., 2006, 2010 and 2012; Craw et al., 2016; Rea et al., 2016; Shuster et al. 2016). These sites are located in (sub)-tropical,
(semi)-arid
and
temperate
environments,
with
average
annual
temperatures between 5 and >20°C, and limited, if any soil frost. Therefore, the present
T
study aimed to assess if biofilms occur on Au particles from cold sites with average
IP
annual temperatures approximating 0°C, how the communities compare to those on Au-
CR
particles from warmer climatic zones, and study the functional links between biofilms and processes affecting particle transformation and Au mobility. Results showed that
US
biofilms were highly abundant, with most particles being coated by polymorphic layers
AN
composed of cells, EPS and (bio)minerals, with >80 % of grains with detectable DNA (Figs. 3 and 5). Associated with these layers was nano- and microphase Au, which was
M
commonly underlain by a layer of secondary-Au that had formed on top the primary-Au
ED
(Figs. 4-6).
Earlier studies have shown no direct link of the community composition of Au
PT
particles and climatic settings under (sub)-tropical, (semi)-arid and temperate (Rea et al.,
CE
2016). This suggests that physicochemical factors other than climate, e.g., Au and associated heavy metal toxicity, may drive community composition and select for
AC
communities with the combined ability to detoxify these metals. Organisms living in Aurich environments are constantly exposed to harsh environmental conditions, e.g., heavy metal toxicity conferred from mobile Au(I/III)-complexes and Au-deposit associated pathfinder metals, e.g., Ag, Cu, Ni, Cr, As, Pb, Se, Te and W (Reith et al., 2012). At the site elevated concentration of mobile Au and Ag co-occur, and because of their mobility and high toxicity need to be detoxified, explaining the co-occurrence of a wide range of
29
ACCEPTED MANUSCRIPT heavy metal detoxifying organisms and abilities in the biofilms on Au particles required to confer protection to the entire biofilm (Reith et al., 2012; Wiesemann et al., 2017). Across the sites studied earlier, biofilm communities were dominated by Actinobacteria, Firmicutes and especially Proteobacteria, with β- and γ-Proteobacteria
T
being most abundant (Rea et al., 2016). Among these the Oxalobacteraceae and
IP
Comamonadaceae, which contain a range of known metal-resistant and metallophilic
CR
taxa, e.g., C. metallidurans and Ralstonia spp. as well as Delftia spp. and Variovorax spp. were highly abundant. Similar to these earlier studies, biofilm communities on
US
Finnish particles were dominated by β-Proteobacteria, especially Oxalobacteraceae and
AN
Comamonadaceae, which dominated communities and made up 8 of 18 OTUs detected on grains from all sites (Fig. 9). These results suggest that highly specialized biofilm
M
communities adapted to these Au-rich micro-environments perform key-roles in these
ED
biofilm communities, and thereby drive the biogeochemical cycling of Au and the biogeochemical transformation of Au particles.
PT
Biofilm communities on Finnish Au-particles include a range of phyla that can
CE
actively contribute to Au- and heavy metal cycling and detoxification (Fig., 11; Reith et al., 2010; Rea et al., 2016). C. metallidurans, which was detected on particles from all
AC
sites (Fig. 9B), is a non-spore forming aerobic β-Proteobacterium, able to tolerate high levels of heavy metals (Reith et al., 2009; Wiesemann et al., 2013). It is able to to oxidize molecular hydrogen allowing chemolithoautotrophic growth (Mergeay et al., 1985; Herzberg et al., 2015), and to degrade a variety of aromatic and unusual organic compounds (Mergeay, 2000; Springael et al., 1993; Miyake-Nakayama et al., 2006). During its evolution C. metallidurans has obtained the ability to resist high concentrations of transition metals, in particular the oxidative/metal stress induced by 30
ACCEPTED MANUSCRIPT toxic Au(I/III)-complexes, with toxicity levels similar to Hg(II)- and Ag(I)-compounds; note minimal inhibitory concentrations (MICs) for Au(I/III)-complexes, Ag and Hg-compounds are in the order of 1 to 5 µM in C. metallidurans and toxic effects to the organisms start at approximately 1/1000 of the MIC (Nies, 1999; Reith et al., 2009; Wiesemann et al.,
T
2013 and 2017; Etschmann et al., 2016). C. metallidurans reduces toxic Au(I/III)-
IP
complexes in the periplasm via synergistic co-utilization and regulation of Cu/Au
CR
resistance determinants (Wiesemann et al., 2013 and 2017; Zammit et al., 2016). This leads to the formation of ~10 nm sized Au nanoparticles in the periplasm (Reith et al.,
US
2009). The metal chaperone CupC and the periplasmic Cu-oxidase CopA have been
AN
shown to have key-functions for the detoxification of Au-complexes and the formation of Au nanoparticles (Wiesemann et al., 2013 and 2017; Zammit et al., 2016; Bütof et al.,
M
2018). Using these abilities C. metallidurans has been shown to survive up to 615 µM of
ED
Au(I)-complexes, retain 99.5 wt.% of Au(I)-thiosulfate percolating through columns, and actively biomineralize Au as nanoparticles and micro-crystalline aggregates (Fairbrother
PT
et al., 2013). Over time nanoparticle aggregates encapsulate and subsequently replace
CE
cells leading to the formation of mm-sized Au particles (Fairbrother et al., 2013; Shuster et al., 2015). By mediating Au biomineralization, C. metallidurans plays a key role in
AC
environmental cycling of Au in Earth surface environments. Other taxa, such as Gallionella sp., Variovorax boronicumulans, Paenibacillus sp., Rhodoferax sp., and Pseudomonas sp. are known to produce siderophores that may act as a metal-binding scaffold for the detoxification Au and various other metals, which can co-occur with Au (Fig. 11). Recent studies have shown that a siderophore, delftibactin, produced by Delftia acidovorans, another non-spore-forming, O2-utlizing aerobic β-Proteobacterium that occurred in biofilms from Australian Au particles, confers 31
ACCEPTED MANUSCRIPT Au-resistance and detoxification by extracellularly reducing mobile Au-complexes to metallic non-toxic nanoparticles (Johnston et al., 2013). Gallionella spp. have been shown to oxidize Fe(II) and mediate the biomineralization of insoluble micro-crystalline Fe(III)-oxides (Hallberg and Ferris, 2004). Fe-oxides have been commonly observed on
T
the Finnish Au particles, and have been linked to co-precipitation and sorption of heavy
IP
metals during and after their formation (McKenzie, 1980 ; Manceau et al., 1992).
CR
Paenibacillus spp. have been shown to release siderophores, exhibit significant biosorption capabilities and tolerance/resistance to a range of toxic heavy metals, e.g.,
US
Cu, As, Pb, Cd, Co, Ni and Zn (Acosta et al., 2005 ; Grady et al., 2016). Rhodoferax
AN
spp., can reduce Fe(III) to Fe(II), which in the presence of sulfide may lead to the formation of Fe-sulfides, e.g., pyrite, possesses genes linked to As metabolism and uses
M
heavy metal efflux pump to confer resistance to Cd, Zn, Co (Risso et al., 2009). Single
ED
euhedral pyrite particles, approximately 1.5 µm wide, were observed (Fig. 3E). Pyrites were undamaged, showing well defined edges and corners and smooth faces,
PT
suggesting post-deposition formation. Some Pseudomonas spp., are able to secrete
CE
siderophores during Fe-limiting conditions, and possess genes encoding resistance to Cu and Ag, and plasmid born resistance to Hg and As as well as other heavy metals
AC
(Visca et al., 1992; Schalk et al., 2011). A range of other taxa present on the particles have also been shown to be able to biomineralize Au nanoparticles, including Pseudomonas veronii, Escherichia coli and Arthrobacter spp. which, similar to Herbaspirillum sp., produces bioflocculants that bind to heavy metals and passively precipitates the metals in the EPS layer (Fig. 11; Bender et al., 1995). In addition Arthrobacter spp. are resistant to high levels of other heavy metals, including Ni and Cr (Margesin and Schinner, 1996; Abou-Shanab et al., 2007). Other metal-tolerant or 32
ACCEPTED MANUSCRIPT resistant
taxa
detected
include
Acinetobacter
woffii,
Lentzea
violacea,
and
Mesorhizobium sp. as well as Rahnella aquatilis that has been known to exhibit strong tolerance to As(III) and As(V) (Fig. 11; Valverde et al., 2011). Manganese-oxidizing bacteria were also present on the particles, e.g.,
T
Hyphomicrobium sp. and Pedomicrobium australicum. Pedomicrobium spp. have the
IP
ability to adhere strongly to surfaces and take advantage of the nutrients and soluble
CR
Mn-ions attracted to the solid–liquid interface (Fig. 10; Sly et al., 1988a/b). They form intracytoplasmic membrane compartment within the cells to oxidize soluble Mn 2+ to
US
insoluble Mn(III/IV)-oxides (Gebers, 1981; Gebers and Beese, 1988), which can
AN
contribute to the mobilization of Au (Ta et al., 2014). Mobilization of Au and Ag may be important to keep metal toxic loads in the biofilm micro-environment of the grains high,
M
thereby eliminating outside competition and selecting for biofilm communities containing
ED
Au detoxification capabilities. This was confirmed in a study by Brugger et al. (2013), which has shown that Au-toxicity is a key-factor in driving environmental Au cycling.
PT
Interestingly, C. metallidurans has also been shown to mobilize Au from metallic Au
et al., 2013).
CE
surfaces, which suggests that it can form its own Au-cycling micro-environment (Brugger
AC
However, these metallophillic and metal-resistant organisms may not be able to survive without the help of other members of the biofilm community (Fig. 11; Rea et al., 2016). These auxiliary taxa appear to be dominantly involved in particle colonialization, biofilm establishment and nutrient cycling. These functions can be fulfilled by a wide variety of environmental taxa, provided they are protected from heavy metal toxicity, therefore their diversity may be linked to site-specific conditions. This may explain the overall link of community structures to different sites, conditions and deposit styles 33
ACCEPTED MANUSCRIPT observed for the Finnish particles as well as globally, in climatically different situations (Rea et al., 2016). Among these auxiliary taxa detected on Finnish particles are Gram-positive Streptococcus spp., which can colonize the surface during stage one of biofilm
T
development (Fig. 10). Gram-positive bacteria can resist surface charge and form an
IP
attachment on the surface. Initial colonizers also need to produce EPS, resist surface
CR
charge like Massilia timonae (Harimawan et al., 2011) and form a strong attachment to the surface using eDNA and type IV pilli such as Acidovorax temperans (Heijstra et al.,
US
2009) and Curvibacter spp. (Dominiak et al., 2011). Another interesting taxum detected
AN
was Candidatus solibacter, which is equipped with a large number of anion : cation symporters, hypothesized to aid the community with its ability to deal with extreme
M
variation in moisture, temperature and nutrient availability. Cellulose production
ED
exhibited by this species may contribute toward maintaining biofilm integrity by promoting the ability of a biofilm to adhere in environments (Flemming and Wingender,
PT
2010).
CE
After these initial colonizers have settled on the surface, other taxa easily grow on the preconditioned surfaces. In some environments, the lack of precursor nutrients may
AC
require recruitment of auto and phototrophs in the biofilm, such as Porchlorococcus sp., Rhodoplanes sp, and Polybucelobacter sp (Fig. 11). Some recruited bacteria can perform nitrate reduction and sulfide oxidation, which include Methylibium spp., Lutibacterium spp., Massilia spp. and Arcobacter spp. (Fig. 11). The increasingly taxarich community will produce metabolic by- and waste products, e.g., organic acids and external nutrient- and energy sources may also become available, e.g., fulvic and humic acids. Low molecular weight organic acids are the product of fermentation, common 34
ACCEPTED MANUSCRIPT metabolites of C-metabolism under anoxic conditions, part of the anoxic C-mineralization pathway and have been shown to form Au-complexes (Reith et al., 2008). OTUs able to utilize the C-sources are abundant in the biofilms and include Propionibacterium granulosum, Acinetobacter spp., and Corynebacterium spp. Other OTUs detected may
T
employ proteolytic and saccharolytic activities such as Afifella spp., Pseudomonas
IP
viridiflava, Trichococcus spp., and Syntrophomonas spp. Sacccharolytic fermenting
CR
bacteria, Trichococcus spp. participates in decomposition of organic matter, utilize organic acids and produce electron donors for sulfate reduction (Fig. 11; Holzapfel and
US
Wood, 2014). Syntrophomonas spp, performs synthrophic degradation by feeding on
AN
other bacteria and is involved in acetogenesis (Zhang and Lu, 2016). Other OTUs are capable of degradation of hydrocarbon and xenobiotics as well as OTUs related to the
M
breakdown of polymers by enzymes and acids (Fig. 11). Important biodegradative
ED
processes for hydrocarbons and xenobiotics may be performed by Paucimonas lemoignei, Polaromonas spp., Spingomonas spp., Variovorax paradoxus, Variovorax
PT
spp., Herminiimonas spp., and Arthrobacter chlorophenolicus (Handrick et al., 2001;
CE
Mattes et al., 2008; Unell et al., 2009). Metabolic turnover of all of these energy sources may therefore fuel the entire biofilm community. When biofilms reach their maximum
AC
carrying capacity, some cells may be released back to the environment. These dispersal cells are capable of carrying associated Au nanoparticles and may thereby contribute to Au dispersion in the wider environment.
5. Conclusions Gold and Ag are mobilized, dispersed and re-precipitated at the study sites, confirming that active biogeochemical cycling of these metals occurs in cold, arctic settings. As a 35
ACCEPTED MANUSCRIPT result of this Au particles from the Ivalojoki and Lemmenjoki placer deposits have been transformed, confirming that biogeochemical process model responsible for supergene Au-particle transformation introduced by Fairbrother et al. (2012) and extended by an physical component by Stewart et al. (2017) is valid in cold environments. Given the
T
mobility of both Au and Ag in the systems, the composition of bacterial biofilms on
aurophillic β-Proteobacterium C. metallidurans was detected on
CR
communities and
IP
particles is likely driven by metal toxicity. β-Proteobacteria dominate microbial
particles from all sites. Similar to other sites around the world C. metallidurans likely
US
plays a key-role for Au detoxification and biomineralization. Overall, biogeochemical Au
AN
transformations lead to similar secondary morphotypes observed on particles in other environments, but it appears cycling occurs at slower rates compared to sites were
M
higher temperatures and year-round available liquid water lead to higher (bio)chemical
PT
ED
reactivities and element cycling intensities.
CE
ACKNOWLEDGMENTS
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We dedicate this study to the memory of Vasant Mäkinen, gold prospector, engineer, test driver, great friend, may you rest in peace! We acknowledge the following individuals and institutions for their contributions: Australian Research Council (ARCFT100150200), The South Australian Museum and Adelaide Microscopy (University of Adelaide), especially Dr. Sarah Gilbert, Dr. Animesh Basak and Dr. Benjamin Wade for instrument support; the Gold Prospectors Association of Finnish Lapland for field support, Heikki Kallio-Mannila, Aila, Heikki and Antti Heikkilä, Risto and Maija Vehviläinen, Pekka Turkka, Raimo Kanamäki, Antti Kohtamäki, Sirkka and Kari 36
ACCEPTED MANUSCRIPT Merentuoto, Ami Telilä and Pekka Itkonen for their warm welcome, access to sites and the fantastic samples. Thanks to Manfred and Brigitte Roos for assistance with sampling, and Gregory Rinder for graphics support. We appreciate the comments by the two anonymous reviewers, which have improved the manuscript, as well as the handling
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and comments by the editor Prof. Johannesson.
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ACCEPTED MANUSCRIPT Tables
Mining District
Coordinates
Deposit style
Ivalojoki - Kalliomannila a,b (IVK) Ivalojoki – Heikkila (IVH) Ivalojoki – Vehviläinen (IVV) Lemmenjoki – a,b Jäkäläpää 1 (LJ1) Lemmenjoki – Jäkäläpää 1 (LJ2) Lemmenjoki – Turkka a (LT) Lemmenjoki – Telilä (LTE) Lemmenjoki – Itkonen a Salonen (LIS) Lemmenjoki – a,b Kanamäki (LK) Lemmenjoki – Kohtamäki (LKO)
Ivalojoki
N 68°27.460' E 26°56.823' N 68°27.527' E 26°56.580' N 68°25.200' E 26°54.833' N 68°41.899' E 25°43.701' N 68°41.877' E 25°43.632' N 68°42.518' E 25°42.020' N 68° 38.984' E 25° 37.778' N 68° 41.668' E 25° 41.147' N 68°39.915' E 25°36.305' N 68°39.100' E 25°37.756’
Glacial till
Sampling depth [m] 0.5 – 1.5
Glacial till
2–3
(Glacio)fluvial
0 – 0.5
Glacial till
0 – 1.5
Glacial till
0 – 1.5
(Glacio)fluvial
0–1
Glacial till
2–4
(Glacio)fluvial
0–1
(Glacio)fluvial
2–5
Glacial till
2–4
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LemmenjokiJäkäläpää LemmenjokiJäkäläpää LemmenjokiMiessi LemmenjokiMiessi LemmenjokiMiessi LemmenjokiMiessi LemmenjokiKaarreoja
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in addition to placer Au-particles, soil samples were obtained in addition to placer Au-particles, water samples were obtained
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b
Ivalojoki
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a
Ivalojoki
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Site
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Table 1 - Summary of relevant site properties.
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ACCEPTED MANUSCRIPT Table 2 - Physicochemical conditions and element concentrations in water samples a
Parameter
Unit
Sample depth
n.a.
b
LJ1
LK
Surface water 5.4 1.6 34 1.6 0.067 0.021 0.16
Surface water 5.1 2.1 29 2.1 0.21 0.011 0.08
Groundwater 5m 6.6 1.2 11 1.2 0.03 0.10 0.14
Quantification Limit n.a.
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n.a. n.a. dS/m 0.01 mg/L 0.01 mg/L 0.01 mg/L 0.005 mg/L 0.005 meq/L 0.01 0.8 2.6 1.4 2 mg/L 0.05 0.03 0.33 2 mg/L 2.0 0.8 0.9 2 mg/L Al 0.12 0.08 <0.05 0.5 mg/L Ca 1.1 0.2 0.9 0.5 mg/L Fe 0.2 <0.1 <0.1 0.5 mg/L K 5.4 2.8 0.6 0.5 mg/L Mg 0.6 0.2 0.6 0.5 mg/L Mn <0.05 <0.05 0.08 0.5 mg/L Na 2.1 0.8 1.6 0.5 mg/L S 0.9 0.5 0.4 0.5 mg/L 0.17 <0.05 <0.05 0.5 Ag µg/L As µg/L 0.36 0.58 0.09 0.05 Au µg/L 0.6 <0.5 <0.5 0.5 Co µg/L 0.37 0.13 0.58 0.5 Cr µg/L 0.3 0.2 0.3 Ni µg/L 1.1 0.5 0.7 0.2 Pb µg/L <0.05 <0.05 0.06 0.05 V µg/L 1.03 1.00 0.06 0.05 Ti µg/L 0.8 0.7 <0.5 0.5 Zn µg/L 5.8 3.5 10.4 0.2 a Notes: The following elements were analyzed but found to be below detection limit: Sc, Sn, Se, Cd, Y, Pd, Ga, Ge, Ru, Re, Ta, La, Ce, Pr, Nd, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Re, Rh, Ir, Pt, U, Th -1 -1 < 0.05 < 0.2 µg L , Cu < 1 µg L . b n.a., not applicable
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pH Electrical conductivity TOC TN NH4-N NOx-N Total Alkalinity Cl NO3 = SO4
IVK
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ACCEPTED MANUSCRIPT Table 3 – Physicochemical parameters, major element contents and concentration of Au and its pathfinder elements in soil samples from Finnish Lapland. Parameter
a
Unit
IVK
LJ1 S1
LJ1 S2
LJ1 S3
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LIS
LK
Quantification Limit n.a.
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Soil horizon A2 Ah A2 B/C B C (Till) C(Till) Sample depth m 0.5-1.5 0-0.1 0.1-0.3 1 >0.5 >1 >2 pH n.a. 6.5 3.0 4.8 6.0 5.4 6.1 6.8 n.a. Electrical dS/m 7.96 2650 36.3 4.13 41.70 33.60 4.25 0.01 conductivity 0.01 Total C % 1.4 7.5 0.35 0.45 0.75 0.63 0.09 0.01 Total N % 0.07 0.39 0.03 0.03 0.02 0.01 0.02 Al mg/kg 39991 27103 24976 30210 36900 32052 23118 100 Ca mg/kg 1754 3621 2441 1937 319 1583 5041 100 Fe mg/kg 64193 48054 35775 45268 121458 61618 39315 100 K mg/kg 3221 1091 2463 2841 6003 5715 5460 100 Mg mg/kg 11502 12899 7498 6265 7577 7254 8003 100 Mn mg/kg 418 359 324 309 1000 852 754 50 Na mg/kg 255 146 272 259 214 242 341 50 P mg/kg 739 363 287 510 961 331 1386 100 S mg/kg 193 5957 89 77 150 <40 <40 40 Ag µg/kg 120 164 199 290 145 148 145 10 As mg/kg 0.9 2.0 1.1 0.9 1.3 1.5 1.1 0.1 Au µg/kg 100 211 98 83 61 48 24 10 Cd mg/kg 0.56 0.20 0.09 0.57 0.47 0.35 0.55 0.1 Co mg/kg 14.78 208 15.48 18.88 38.43 68.85 15.35 0.2 Cu mg/kg 28.9 26.5 17.2 40.1 56.6 43.1 25.9 0.2 Mo mg/kg 1.9 4.0 1.0 1.5 7.0 4.2 0.4 0.1 Pb mg/kg 7.3 2.5 5.8 5.6 10.4 8.6 4.9 0.1 Se mg/kg 0.5 1.0 <0.2 0.3 0.8 0.6 <0.2 0.2 Sn mg/kg 0.30 0.28 0.36 0.37 0.21 0.33 1.49 0.1 V mg/kg 97 58.0 56.5 83 114 100 59.5 0.1 U mg/kg 1.34 0.35 1.08 1.21 1.53 3.22 4.62 0.1 Zn mg/kg 106 77.0 65.5 80 184 112 80 0.2 a Notes: The following elements were analyzed and data is available on request: Sc, Sn, Se, Cd, Y, Pd, Ga, Ge, Ru, Re, Ta, La, Ce, Pr, Nd, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Re, Rh, Ir, Pt, Th. b n.a., not applicable
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ACCEPTED MANUSCRIPT Table 4 - Total reads, OTUs and percentage of total reads covered by the most
Percentage of total reads covered by the 20 most abundant OTUs 66.6
339599
283
67.8
89.2
IVV
306367
121
73.3
94.5
LJ1
222757
138
61.4
LJ2
208061
85
98.9
LT
1020754
136
66.1
LTE
416549
163
86.7
LIS
87554
98
98.6
LK
640476
108
99.6
LKO
254711
109
97.4
Sum
3915388
519 (different)
81.2
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99.7 89.4
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97.2
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IVH
99.1 99.8 99.5
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Number of OTUs
IVK
Total number of reads 418560
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Site
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210
Percentage of total reads covered by the 10 most abundant OTUs 59.2
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abundant OTUs for the ten sites in Finnish Lapland.
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ACCEPTED MANUSCRIPT Figure Captions Figure 1
(A) Sampling locations; (B) representative sample from one site; and (C-H) typical morphologies of Au particles from the Ivalojoki and Lemmenjoki goldfields in Finnish Lapland. Shown are backscatter electron (BSE)
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micrographs of particles that are slightly rounded (C, from LJ1; F from
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IVK), wiry (D from LT), spheroidal (E from LJ1), well-rounded (G from IVH)
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and flattened Au (H from IVV) grains (for site abbreviations please see
Figure 2
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Table 1).
BSE micrograph of a characteristic Au particle surface showing dissolution
Figure 3
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features (white arrows).
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pitting (A), and (B) showing dissolution pitting next to re-precipitation
Secondary electron (SE) micrographs of polymorphic layers on Au particle
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surfaces. (A) SE micrograph of a typical particle coated by a polymorphic
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layer; (B, D, E) SE micrographs and a representative EDS spectrum (C) of polymorphic layers composed of nano- and microcrystal minerals, cells
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and EPS; (G) SE micrograph and EDS spectrum (H) of a cell and a typical
Figure 4
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pyrite micro-crystal. Electron microprobe maps, Au (red) and S (blue) and Fe (green) and S (green), showing the association of S and Fe with transforming Au. A particle composed of Au–Ag alloy overlain by secondary Au (A) and a high purity Au-particle (B, C), overlin by a polymorphic layer rich in S- and Fecontaining minerals. Figure 5
BSE (A, B, D-F) and SE (C) micrographs of secondary-Au morphologies associated with polymorphic layers: (A) nano-phase Au; (B, C) triagonal 56
ACCEPTED MANUSCRIPT Au platelets; and E) vermiform (C, D) and triangular platy (F) aggregates of secondary-Au. Figure 6
BSE (A, D) and SE (B, C) micrographs showing the direct association of a rod-shaped bacterial cell with aggregates of Au nano and µ-crystals (A, B),
SE micrographs (A, F), EDS- (B-E, G) and EBSD- (H, I) maps of a Au
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Figure 7
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and (C, D, E (spectrum)) of nanophase Au in EPS (C, D from ).
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surface partially coated by a polymorphic layer (A-E) and a FIB-milled section (F, G) and polished block (H), respectively, showing progressive
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coating of primary-Au-Ag by secondary Au nano- and microcrystalline
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aggregates (B, C, white arrow), leading to the formation of a layer of highpurity (G, white arrow) microcrystalline (H, I) secondary-Au. Electron microprobe maps, Au (red) and Ag (green), showing the
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Figure 8
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progressive transformation of Au particles. (A) A particle composed of a homogenous Au–Ag alloy with a partial layer of secondary high purity Au;
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(B, C) increasingly transformed Au particles, and an almost entirely
Figure 9
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transformed particle (D). Composition of bacterial communities associated with Finnish Au particles.
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Distribution of dominant bacteria phyla/classes based on (A) number of OTUs (529) and (B) total number sequences (>3.9 million) for the 10 sites; note: classes are shown for Proteobacteria and Firmicutes.
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(A) Neighbour-joining phylogenetic tree of representative 16S rRNA sequences of the 18 present on particles from all 10 sites. percentages of 1000 bootstrap values < 70% are not shown. Methanobrevibacter smithii was used as the out-group. (B) Ordination plot of the first two canonical 57
ACCEPTED MANUSCRIPT axes produced by canonical analyses of principal coordinates (CAP) of Miseq data analyzed for differences in community assemblages in relation to deposit type and depth; vectors of Pearson’s correlations of classes/phyla are overlain16S rRNA gene.
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Schematic model and putative functional classification of bacterial taxa
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constituting biofilms on Au particles (modified after Rea et al., 2016): (1)
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conditioning of surfaces to their attachment; (2) the recruitment of photoand heterotrophic Gram-negative bacteria; (3–5) the proliferation and
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growth of the biofilm community including heterotrophic and metallophillic
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species; (5) the mobilization, detoxification and re-precipitation of Au; and (6) the seeding of dispersal cells with release of nano-particle and Au
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complexes.
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Figure 11
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Figure 1
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Figure 4
Figure 5
Figure 6
Figure 7
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Figure 12