Geomicrobiology Journal, 23:591597, 2006 Copyright c Taylor & Francis Group, LLC ISSN: 0149-0451 print / 1521-0529 online

DOI: 10.1080/01490450600964326

Bioaccumulation of Gold by Filamentous Cyanobacteria Between 25 and 200 C

Maggy F. Lengke, Michael E. Fleet, and Gordon Southam
Department of Earth Sciences, University of Western Ontario, London, Ontario, N6A 5B7, Canada

Plectonema boryanum UTEX 485 was reacted with aqueous AuCl solutions (2 mM Au) at 25 to 100 C for 1 month, and 200 C 4 for one day. Addition of AuCl to cyanobacteria killed the cultures 4 instantly, and Au was precipitated throughout the cells as nanoparticles. Precipitation of octahedral crystal platelets of Au occurred in the aqueous uid, with particle size increasing with increase in temperature from about 1.5 m at 25 C to 10 m at 100 C. Addition of AuCl to suspensions of the dead, autoclaved cyanobacteria 4 also precipitated Au from solution, suggesting that the presence of cell degradation products caused instability of AuCl . 4 Keywords biomineralization, cyanobacteria, gold, octahedral gold, transmission electron microscopy (TEM)

INTRODUCTION Gold is a precious metal with economic importance since antiquity. Although the pathways of Au cycling in natural environments is the subject of considerable research, much still remains to be explored. During weathering of rocks, Au dissolution is generally attributed to thiosulfate (Au(S2 O3 )3 ) or 2 possibly sulte (Au(SO3 )3 ) complexes in near neutral solution 2 (Pitulko 1976; Plyusnin et al. 1981; Webster 1986; Benedetti and Boulegue 1991). Under acidic and oxic environments, gold is mobilized as complexes with Cl (AuCl or AuCl ) in areas 2 4 with high concentrations of Cl such as in arid lateritic terrains and in hydrothermal environments (Mann 1984; Webster 1986; Benedetti and Boulegue 1991; Ran et al. 2002). The accumulation of Au in natural environments from Australia, Venezuela, Alaska, and South Africa has been

attributed to cyanobacteria and other bacteria (i.e., Hallbauer 1986; Bischoff et al. 1992; Watterson 1992; Bischoff 1994, 1997; Mossman et al. 1999), based largely on the similar morphology of secondary Au grains or Au nuggets and cyanobacteria or bacteria. The carbonaceous matter, including solidied bitumen, associated with much of the Au in the Witwatersrand basin is thought to be of cyanobacteria origin (Mossman and Dyer 1985; Dyer et al. 1988; Mossman et al. 1999). The carbon seams in the Witwatersrand basin have been hypothesized to be the remains of lamentous cyanobacteria because they exhibit the size and shape of modern lamentous cyanobacteria (Dyer et al. 1988). Therefore, our study on bioaccumulation of gold was conducted using lamentous cyanobacteria. Several laboratory experiments have been conducted to understand the interaction of Au with cyanobacteria using gold(III)-chloride (AuCl ) (e.g. Darnall et al. 1986; Greene 4 et al. 1986; Watkins et al. 1987; Dyer et al. 1994). Previous studies demonstrated that cyanobacteria were able to accumulate Au from AuCl by a biosorption mechanism; however, the 4 morphology of Au particles resulting from this mechanism has not been investigated. Therefore, the goals of this study were to investigate the role of the lamentous cyanobacterium Plectonema boryanum strain UTEX 485 in the mobility of Au and secondary Au deposition using aqueous AuCl solution, and to 4 understand the effect of complex organic/inorganic reactions on the morphology of Au particles. MATERIALS AND EXPERIMENTAL PROCEDURES Chemicals Aqueous AuCl solution used in this study was prepared 4 from gold(III)-chloride, HAuCl4 .3H2 O (Alfa Aesar Company, Ward Hill, Massachusetts, USA), dissolved in distilled, deionized water at 18.2 M cm1 obtained from a Millipore system. The Au solutions were lter-sterilized using a 0.45 m membrane ltration before being added to both cyanobacterial and abiotic experiments. Cyanobacteria Conditions and Experiments The lamentous cyanobacterium, Plectonema boryanum strain UTEX 485 (obtained from the culture collection at the

Received 15 September 2005; accepted 21 July 2006. Address correspondence to Maggy F. Lengke, Department of Earth Sciences, University of Western Ontario, London, Ontario N6A 5B7, Canada. E-mail: mlengke@uwo.ca or maggylengke@yahoo.com The authors thank Dr. Marianna Krol, Ronald Smith, and Elizabeth Myscich for their assistance with the cyanobacteria cultures. We thank Dr. William C. Ghiorse and two anonymous reviewers for their constructive comments, and Patti Butler for her assistance during the manuscript correspondence. This research was supported in part by the National Science Foundation LExEn Program (EAR-9714214) and the Natural Sciences and Engineering Research Council of Canada Discovery Grants to G. Southam and M. E. Fleet.

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University of Texas at Austin, Texas, USA), was grown axenically in batch cultures in BG-11 medium (Rippka et al. 1979) buffered with 10 mM HEPES at a control temperature of 29 C under ambient CO2 conditions. The pH of the medium was adjusted to pH 8.0 using 1 M NaOH solution. Before experimentation, the culture was transferred (20% [vol/vol] of inoculum), and grown to a stationary growth phase, approximately 68 weeks, and washed three times with distilled, deionized water to remove salts and trace metals from the medium using centrifugation. Dead cyanobacteria were prepared by autoclaving the washed cultures for 1 hour at 121 C. Two types of cyanobacterial experiments were conducted to examine the role of cyanobacteria on the accumulation of Au from aqueous solutions of AuCl : Type 1, in which Au solu4 tions were added to the cultures and incubated at a temperature range from 25 to 200 C; and Type 2, in which Au solutions were added to dead, autoclaved cultures. Relatively high Au concentrations (up to 5 mM) were used so that the effects of the biogeochemical processes could be observed within a reasonable laboratory time frame. While these Au concentrations are well beyond the levels encountered in natural waters (Bowell et al. 1993), they are nevertheless below the concentrations of Au in natural hydrothermal environments. Type 1 Cyanobacterial Experiments. Experiments at 25, 60, and 100 C were performed in sealed borosilicate serum vials. To initiate each experiment, 5 mL of aqueous AuCl solution 4

(5 mM) were added to 5 mL of cyanobacteria culture (8 mg dry weight) and incubated for 1 h at 25 C. The cyanobacteria with incipient immobilized Au were separated from the soluble AuCl by centrifugation (14,000 g), and a 5 mL aliquot of the 4 supernatant liquid was removed for analysis and replaced with distilled, deionized water. For experiments at 60 and 100 C, the cyanobacteria and Au solutions were reacted for up to one month by placing the sealed borosilicate vials in temperature regulated laboratory ovens (Blue M Electric Company and Fisher Isotemp Oven, respectively). In addition, experiments were conducted at 200 C for 1 day in an acid digestion bomb (Parr 4749) and heated in a box furnace (Thermolyne 1500), after the initial incubation period. Initial Au concentrations of 2 mM and starting pH and Eh were established from the supernatant liquid removed after 1 hour incubation (Figure 1). pH, Eh, total Au, and cyanobacteria population were measured with time. All experiments were conducted in either duplicate or triplicate. Type 2 Cyanobacterial Experiments. Procedures were similar to those of the Type 1 experiments except that the cyanobacteria were killed by autoclaving for one hour at 121 C after growth of the cyanobacteria culture to stationary phase. Experiments were made at 25 C only. Abiotic Experiments Chemical control experiments consisting of 2 mM Au were conducted using AuCl solutions and no cyanobacteria present. 4

FIG. 1.

Variation of pH, Eh, and total Au with time for the cyanobacterial and abiotic experiments with AuCl solution. 4

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The experiments were conducted at 25, 60, and 100 C for up to 1 month, and 200 C for 1 day after the addition of Au solution. Bacterial Viability and Total Bacterial Counts The effects of AuCl on the cyanobacteria were monitored 4 during the course of the cyanobacterial experiments by determining bacterial viability using the LIVE/DEAD BacLightTM bacterial viability kit (L-7007, Molecular Probes, Inc., Eugene, Oregon, USA). When viewed by a uorescence microscope, viable bacteria stained with the reagents appear green whereas dead bacteria appear red. The total number of bacteria in the cultures was determined by the direct counting method using a Petroff-Hauser counting chamber and a phase contrast light microscope (Nikon Labophot microscope). Chemical Analyses The pH and Eh were monitored using a Denver Instrument Basic pH/ORP/temperature-meter. The pH electrode was calibrated using buffer solutions 4, 7, and 10 with analytical uncertainties in measurements of pH of 0.05 pH unit. The Eh was measured using an ORP electrode and calibrated using ZoBells solution (Nordstrom 1977). During the cyanobacterial experiments, the pH was measured using pH indicator non-bleeding strips from EM Science. Total Au concentrations were measured over the course of the experiments with a Perkin-Elmer 3300-DV Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) instrument. The solutions were centrifuged (14,000 g) prior to ICP analyses. The uncertainty in measured Au is 5%, with detection limits of 0.25 M. Transmission Electron Microscopy (TEM) and Scanning Electron Microscope (SEM) Unstained whole sample mounts and thin sections of cyanobacteria and Au particles from the experiments were examined with a Phillips CM-10 transmission electron microscope (TEM) operated at 80 kV and a Phillips EM400T transmission electron microscope (TEM) with energy dispersive X-ray spectrometer (EDS). The whole mounts were prepared by oating Formvar carbon-coated 200 mesh copper grids on a drop of culture for several minutes to allow the bacteria and any negrained minerals to adsorb to the grid. The mounts were then washed with distilled, deionized water and allowed to air dry. Selected Area Electron Diffraction (SAED) patterns of the precipitated solids were obtained by TEM from unstained whole mounts to ascertain the crystallinity of solids (Southam and Beveridge 1996). The spacings of the reections on the diffraction patterns were calculated using the formula Rd = L, where R is the distance from a reection to the origin, d is interplanar spacing, is wavelength of the incident electron beam, and L is camera length. Samples for ultrathin sections were xed in 2% glutaraldehyde, enrobed in Noble Agar, dehydrated with a 25, 50, 75 and

100%(aq) ethanol series and embedded in LR White resin. The embedded samples were ultrathin sectioned on a Reichert-Jung Ultracut E ultramicrotome using a diamond knife to a thickness of 70 nm and collected on Formvar-carbon coated 200 mesh copper grids. The precipitated Au was also examined using a LEO 1540 XB Focus ion beam scanning electron microscope equipped with an energy dispersive spectrometer (FIB-SEM-EDS). All samples for FIB-SEM-EDS were carbon-coated before the analyses. RESULTS Cyanobacterial and Abiotic Experiments The results of cyanobacterial experiments using AuCl are 4 shown in Figure 1. In the Type 1 cyanobacterial experiments, Au solution was added to the stationary phase cultures and incubated at 25 to 200 C, while in Type 2 cyanobacterial experiments, Au solution was added to dead, autoclaved cultures and incubated at 25 C. On addition of AuCl to the cyanobacteria in the Type 1 4 cyanobacterial experiments, all cyanobacteria were killed within several minutes, with concomitant change in color of the solution from green to purple. Soluble Au concentrations decreased with increase reaction time indicating the precipitation of Au (Figure 1). Signicant increase in precipitation of Au occurred with increase in temperature from 25 C to 60200 C (Figure 1). The Eh values were relatively constant at 1.0 volts during the course of experiments at 25 C, while at 60 to 200 C, Eh decreased with time (Figure 1). The pH increased slightly from 1.6 to 1.9 or 2.2 from 25 to 200 C. After the addition of AuCl to the dead, autoclaved cyanobac4 teria in the Type 2 cyanobacterial experiments, the soluble Au concentrations decreased signicantly with time at 25 C (Figure 1). The rate of Au precipitation was faster by approximately 3 times in the Type 2 experiments than that in the Type 1 experiments at similar temperature. pH and Eh values remained relatively constant during the course of experiments. Total soluble Au concentrations and pH values were relatively constant during the abiotic experiments using AuCl at 4 25 and 60 C, and these concentrations and values decreased with time at 100 and 200 C (Figure 1). Macroscopic evidence of Au precipitation was indicated by change in color in the reaction systems to gold. Eh values were relatively constant at 25, 60, and 200 C but decreased to minimum values at about 15 days at 100 C. TEM and SEM TEM observations of the Type 1 cyanobacterial experiments with the addition of AuCl at 25200 C are presented in 4 Figure 2. At 25 C, the addition of AuCl to the cyanobacteria 4 killed the cells instantly, but the laments remained intact. Gold particles of irregular to octahedral habit were precipitated on the bacteria cells (Figure 2A). Gold particles were also dispersed

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FIG. 2. TEM micrographs of whole mounts of cyanobacterial cells cultured in the presence of AuCl at incubation temperatures of (A) 25, (B) 60, (C) 100 and 4 day 30, and (D) 200 C and day 1. Note that increase in temperature caused distortion of cyanobacteria laments and structure. (E) A TEM micrograph of a thin C. (F) TEM-EDS indicates the presence of Au and C; from a probe location within E. Scale bars in A, B, C, D, and E are 3, 2, section of cyanobacteria cells at 25 5, 2, and 0.5 m, respectively.

throughout the interior of the cells (Figure 2E). At 60 C, the cell structure was distorted and rounded, and the laments were separated into their constituent cells (Figure 2B). At 100 C, the laments were distorted and encrusted by Au particles (Figure 2C). At 200 C, the cyanobacteria cells exhibited rounded form, and Au particles were deposited on the individual cells (Figure

2D). TEM-EDS revealed that the cyanobacteria cells consist of C, Au and minor Cl (Figure 2F). Addition of AuCl to cyanobacteria ultimately resulted in 4 quantitative precipitation of octahedral crystal platelets of Au (Figures 3AC and 3F). The size of Au particles increased with increase in temperature from an approximate diameter of

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FIG. 3. (A, B, and C) TEM micrographs of whole mounts of octahedral Au platelets in the cyanobacterial system with the presence of AuCl at incubation 4 temperature of 25 C.; and (F) a SEM micrograph of octahedral gold at 100 C. (D) Reections in the TEM-SAED diffraction pattern are consistent with crystalline Au. (E) TEM-EDS indicates the presence of Au and C. Scale bars in A, B, C, and F are 0.5, 1, 0.5, and 10 m, respectively.

1.5 m at 25 C (Figures 3AC) to 10 m at 100 C (Figure 3F) with a nanometre scale thickness. TEM-SAED revealed a crystalline diffraction pattern consistent with crystalline Au and TEM-EDS showed that octahedral platelets consisted only of Au (Figure 3E). TEM observation of the abiotic AuCl experiments at 100 C 4 are presented in Figure 4. The solutions of AuCl were stable for 4

1 month at 25 C and 60 C, while at 100200 C, cubic nanoparticles of gold (25 nm) were precipitated from the reagent solution and tended to agglomerate as framboid-like clusters. TEM-SAED of cubic nanoparticles of Au revealed powder ring diffraction patterns (data not shown), consistent with crystalline Au. TEM-EDS of Au nanoparticles from the abiotic AuCl sys4 tem contained only Au (Figure 4B).

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The amount of gold precipitation and ultimately the formation of octahedral Au platelets was directly related to the incubation temperature (Figures 1 and 3). Increase in temperature increased the amount of cell damage. The precipitation of Au for experiments with autoclaved bacteria was about 3 times faster than with initially live cyanobacteria at the same temperature (Figure 1). This indicates that Au precipitation and formation of octahedral platelets from AuCl were directly related to degra4 dation of cyanobacteria. Possible Mechanisms of Au Precipitation by Cyanobacteria In cyanobacterial experiments using AuCl , both extracel4 lular and intracellular precipitation of Au was observed. The presence of intracellular Au nanoparticles suggests that Au entered the cells complexed as AuCl , and then Au3+ was re4 duced to Au . The cyanobacteria were probably killed by either the gold(III)-chloride or the acidic pH; this resulted in the degradation of cells that caused further precipitation of Au. The octahedral form of the Au particles is a striking feature of the AuCl experiments. Evidently the interaction of the cyanobac4 teria (or their degradation products) with the AuCl promoted 4 the growth of {111} Au faces. Clearly, crystal growth of {111} faces was much more efcient than nucleation of new Au crystals. Crystal growth was controlled by deposition of Au metal atoms along cubic [100] directions of the Au crystals. In the abiotic AuCl experiments at 100200 C, the Au was precipitated 4 in the form of nanoparticles, and octahedral Au platelets were not observed. This demonstrates that interaction of cyanobacteria with the chemical environment is an important factor controlling the morphology of Au particles. Our recent research based on X-ray absorption spectroscopy (XAS) shows that the precipitation of Au from aqueous AuCl solutions by cyanobacteria 4 does indeed involve reaction with organic compounds (work in progress). The reduction of Au(111) is actually two-step, involving an intermediate Au(I)-S phase, with the sulfur being of organic origin. Implications for the Formation of Secondary Au This study shows that the interaction of cyanobacteria with AuCl solutions results in distinctive and characteristic mor4 phology for Au particles at 25200 C. The abiotic experiments yielded only nanoparticles of Au, whereas the experiments with cyanobacteria yielded extracellular octahedral Au platelets and intracellular Au nanoparticles. The interaction between Bacillus subtilis with AuCl also precipitated octahedral platelets of Au 4 (Southam and Beveridge 1994, 1996). This evidence points to an important role for cyanobacteria and presumably heterotrophic bacteria in Au mineralization through bacterially derived organic material. The occurrence of nanoparticles and octahedral platelets of Au have been observed in natural environments. Nanoparticles of Au are a signicant component of invisible Au, and could be

FIG. 4. (A) TEM micrographs of whole mounts of nanoparticles of Au in the abiotic system at 100 C; TEM-SAED diffraction patterns were consistent with crystalline nanoparticles of Au metal. (B) TEM-EDS analysis shows the presence of Au and C. Scale bar in A is 0.5 m.

DISCUSSION Comparison of Cyanobacterial Experimental Systems and Abiotic Experimental Systems In the cyanobacterial experiments with addition of AuCl , 4 Au was precipitated in the form of octahedral crystal platelets (Figures 2 and 3), while under abiotic experiments, the precipitation of Au was observed only at 100200 C and in the form of nanoparticles (Figure 4). The abiotic precipitation reaction of Au was likely promoted by heat by the following reaction: HAuCl4 + 1.5H2 O Au + 4H+ + 4Cl + 0.75O2 [1]

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incorporated in ore assemblages by sorption on organic matter and sulde surfaces (i.e., Gregoire 1985; Wood 1996; Fleet and Mumin 1997; Maddox et al. 1998; Ran et al. 2002). More generally, the association of organic C with Au deposits has been reported in Carlin-type deposits in Nevada (i.e., Arehart 1996; Dobra 1997; Hofstra and Cline 2000) and China (i.e., Cunningham et al. 1988; Rhui-Zhong 2002), in Australia (Kucha and Plimer 1999), and in the Witwatersrand Au deposit, South Africa (i.e., Mossman and Dyer 1985; Dyer et al. 1988; Mossman et al. 1999). Although chemi-sorption is important in the deposition of Au, this study shows that cyanobacteria could contribute to the direct precipitation of secondary Au through reduction processes. Octahedral Au grains have been identied as an important characteristic of secondary Au and observed in Mother Lode, California (Leicht 1982), weathered Archean metamorphic rocks in Western Australia (Wilson 1984), and in the Witwatersrand Au deposit, South Africa (Frimmel et al. 1993; Minter et al. 1993). This study demonstrates that biogeochemical precipitation of Au from AuCl solutions could contribute to the 4 formation of Au in placer environments and may also contribute to the growth of Au nuggets in ne-grained placer deposits by destabilizing AuCl resulting in the formation of secondary 4 Au. REFERENCES
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