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1 Introduction

Gold is one of the rarest metals on earth, and its importance has been acknowledged since antiquity. Gold is not only used in jewelry industry but also in a diverse range of industrial applications covering the field of biology and medicine, environment, and technology (e.g., Oldenburg et al. 1998; Vieira and Volesky 2000; Salata 2004; Sperling et al. 2008; Cai et al. 2008; Chen et al. 2008; Mohanpuria et al. 2008). Because of the increased demand of gold in many industrial applications, there is a growing need for cost effectiveness as well as to implement green chemistry in the development of new nanoparticles. Although various chemical and physical synthesis methods successfully produce pure and well-defined nanoparticles, these methods remain expensive and involve the use of hazardous chemicals. In consequence, biological processes that lead to the formation of nanoscale inorganic materials are appealing as possible environmentally friendly nanofactories.

The synthesis of gold nanoparticles of different sizes, ranging from <1 to several hundred nanometers, and shapes has been conducted in both aqueous and nonpolar organic solutions (Brust et al. 1994; Handley 1989; Selvakannan et al. 2003; Daniel and Astruc 2004). The usual synthetic route to prepare gold nanoparticles involves the reduction of a gold salt (usually a halide) in solution by various reducing agents in the presence of a stabilizer (Brust et al. 1994; Handley 1989; Selvakannan et al. 2003; Daniel and Astruc 2004; Masala and Seshadri 2004). The use of rapid reductants (e.g., white phosphorus, tannic acid, formamide, o-anisidine) results in bigger and generally spherical nanoparticles, while weak reducing agents (e.g., citrate, tartarate) results in a slow reaction generating small nanoparticles (Handley 1989; Daniel and Astruc 2004). Sulfur-containing ligands (e.g., xanthates, disulfides, dithiols or trithiols), phosphanes, phosphines, amines, chalcogenides, carboxylates, and polymers have all been used to stabilize gold nanoparticles (Brust et al. 1994; Daniel and Astruc 2004).

Biological methods using organisms, such as bacteria and fungi (e.g., Southam and Beveridge 1994, 1996; Canizal et al. 2001; Kashefi et al. 2001; Mukherjee et al. 2001, 2002; Shankar et al. 2003; Keim and Farina 2005; Nair and Pradeep 2002; Ahmad et al. 2005; Lengke and Southam 2005, 2006, 2007; Lengke et al. 2006a, b, c, 2007), plants (e.g., Gardea-Torresdey et al. 2002), plant extracts (e.g., Shankar et al. 2003, 2004), and dead plant biomass (e.g., Gardea-Torresdey et al. 1999, 2000), for the synthesis of gold nanoparticles are relatively new areas, and are currently being explored. Although the biosynthesis of gold nanoparticles is relatively new, the interactions between bacteria and metals have been well documented (e.g., Heath 1981; Nash et al. 1981; Ehrlich 2002) and the ability of bacteria to accumulate metals has been recognized in the bioremediation field (e.g., Lloyd 2002; Dermont et al. 2008). Previous studies of the biosynthesis of gold nanoparticles have been focused on the use of gold (III)–chloride [AuCl −4 ] and gold(I)–thiosulfate [Au(S2O3) 3−2 ] complexes. Therefore, the objective of this chapter is to review the current state of knowledge concerning the biosynthesis of gold nanoparticles by bacteria, cyanobacteria, and algae using these two gold complexes, based on the existing literature.

2 Gold Chemistry

Gold can occur in one of the six oxidation states, from −1 to +5 (Puddephatt and Vittal 1994), which can be related to its relatively high electronegativity. The most common form of gold complexes is in aurous [Au(I)] and auric [Au(III)] oxidation states (Emery and Leddicotte 1961). The dissolution of gold in aqueous solution is a combination process of oxidation and complexation. In the presence of a complexing ligand, Au(I) and Au(III) can form stable complexes, otherwise they can be reduced in solution to metallic gold (Nicol et al. 1987). The stability of gold complexes is related not only to the properties of the complexing ligand, but also more specifically to the donor atom of the ligand that is bonded directly to the gold atom. According to Nicol et al. (1987), the first rule is that the stability of gold complexes tends to decrease when the electronegativity of the donor atom increases. For example, the stability of gold halide complexes in solution follows the order I− > Br− > Cl− > F−. The second rule is that Au(III) is generally favored over Au(I) with hard ligands and Au(I) over Au(III) with soft ligands. The soft ligands containing less electronegative donor atoms such as S, C, P, and I form more stable complexes with Au(I), whereas the hard electron donor ligands such as F, Cl, O, and N prefer Au(III). The preferred co-ordination number of Au(I) is 2, tending to form linear complexes, and that of Au(III) is 4, tending to form square planar complexes.

The review of gold complexes below is limited for the gold(III)–chloride and gold(I)–thiosulfate because the published biosynthesis of gold nanoparticles by bacteria, cyanobacteria, and algae mainly utilized these two gold complexes.

2.1 Gold(I)–Thiosulfate Complex

Gold(I)–thiosulfate complex is a predominant species at a wide range of pH (acidic to alkaline) and moderate oxidizing to reducing conditions (Senanayake et al. 2003). Sodium thiosulfate is one component of an alternative lixiviant to cyanide for extraction of gold (Aylmore and Muir 2001), and it forms a strong complex with Au(I). The advantage of this approach is that thiosulfate is essentially non-toxic, and ore types that are refractory to gold cyanidation (e.g., carbonaceous or Carlin type ores) can be leached by thiosulfate. Some problems with this alternative process include the high consumption of thiosulfate, and the lack of a suitable recovery technique, because gold(I)–thiosulfate does not adsorb to activated carbon, a standard technique used in gold cyanidation to separate the gold complex from the ore slurry.

2.2 Gold(III)–Chloride Complex

Gold (III)–chloride complex is stable under oxidizing and acidic conditions (Ran et al. 2002). It decomposes above 160°C or in light. Many studies of biosynthesis gold nanoparticles had utilized the gold(III)–chloride acid solid as a starting material to form gold(III)–chloride complex (e.g., Nair and Pradeep 2002; Lengke et al. 2006a, b, c, 2007). Gold(III)–chloride acid solid (HAuC14⋅xH2O) can be produced by dissolving gold in aqua regia and then evaporating nitric acid (HNO3) with hydrochloric acid (HCl). This solid is also referred to as acid gold trichloride or gold(III)–chloride trihydrate (Emery and Leddicotte 1961; http://en.wikipedia.org/wiki/Gold(III)_chloride). Gold(III)–chloride acid – appearing as long, bright yellow, needle-like crystals – is very hygroscopic and highly soluble in water and ethanol.

3 Biosynthesis of Gold Nanoparticles

The interactions between bacteria/cyanobacteria/algae and gold have been well documented since 1986 for biosorption and biorecovery of gold. Our review includes 46 published papers, utilizing bacteria (24), cyanobacteria (8), and algae (14) to determine whether gold can be accumulated from gold(I)–thiosulfate and gold(III)–chloride solutions. Because of purposes other than the biosynthesis of gold particles, some studies of biosorption and biorecovery neither included details of the shape and size of gold particles (e.g., Kashefi et al. 2001; Nakajima 2003) nor described the experimental conditions such as pH, temperature, duration, initial gold concentrations, or bacterial population (e.g., Ting et al. 1995; Kashefi et al. 2001). In general, gold nanoparticles were precipitated intracellularly and/or extracellularly depending on the species (Fig. 3.1). The shape of gold particles precipitated by bacteria/cyanobacteria/algae includes spherical, oval, spongy clots, mushroom, irregular, triangular, tetragonal, hexagonal, octahedral, rod, cubic, docahedral, icosahedral, coil or wire, plate, and thin foil, with size ranging from 1 nm to several mm (Fig. 3.2), as discussed below.

Fig. 3.1
figure 1_3

(a) A TEM micrograph of a thin section of cyanobacteria cell with the gold nanoparticles inside the cell, (b) a SEM micrograph of gold nanoparticles on the surface of sulfate-reducing bacteria (Desulfovibrio sp). Scale bars in (a) and (b) are 0.5 and 1.5 μm, respectively

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Fig. 3.2
figure 2_3

TEM and SEM micrographs of selected gold nanoparticles formed by cyanobacterial interactions with gold(III)–chloride and gold(I)–thiosulfate complexes. Scale bars in (a), (b), (c), and (d) are 0.5, 2, 1, and 0.1 μm, respectively

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3.1 Gold(I)–Thiosulfate Complex

The interactions between bacteria and cyanobacteria with a gold–thiosulfate complex have been studied by Lengke and Southam (2005, 2006, 2007) and Lengke et al. (2006a), and are summarized in Table 3.1. Acidithiobacillus thiooxidans isolated from the Driefontein Consolidated gold mine in Witwatersrand Basin, Republic of South Africa was able to produce gold particles in the presence of up to 0.26 mmol/L gold at pH ~ 1.9–2.2 and 25°C within a period of 35–75 days (Lengke and Southam 2005). In the presence of dead bacterial cells and in the chemical abiotic experiments, gold was not precipitated under similar experimental conditions and duration. The bacterial system was able to precipitate 87–100% of the gold under diurnal light exposure, while only 11–69% of the gold was precipitated in the dark. The gold nanoparticles were precipitated inside the bacterial cells with the size <10 nm, forming a spherical shape. Outside the bacterial cells, the gold had formed coiled or wire, irregular, and rounded structures with an approximate size of 0.5–5 μm, and a crystalline octahedral shape.

Table 3.1 A summary of bacterial and cyanobacterial interactions with a gold(I)–thiosulfate complex
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Heterotrophic sulfate-reducing bacteria (SRB) isolated from the Driefontein Consolidated gold mine in Witwatersrand Basin, Republic of South Africa was able to produce gold particles in the presence of up to 2.6 mmol/L gold at pH ~ 7.4–7.9 and 23–25°C within a period of 6–53 days, in the presence or absence of iron (Lengke and Southam 2006). Only in the presence of live bacterial cells, gold was precipitated from solution. The gold nanoparticles were precipitated inside and at the bacterial cells with the size <10 nm, forming a spherical shape. In bulk solution, the gold had formed spherical, sub-octahedral to octahedral shape with size ranging from 0.5 to 6 μm (Lengke and Southam 2006). The role of SRB in the precipitation of gold was also investigated through column experiments in the presence of up to 2.6 mmol/L gold at pH ~ 7.4–8.5 and 23–25°C within a period of 148 days, in the presence or absence of iron (Lengke and Southam 2007). In the bacterial systems, SRB precipitated gold inside and at the cells, forming spherical nanoparticles (<10 nm). In bulk solution, the formation of octahedral gold crystals, framboid-like structures (~1.5 μm), and gold foil (mm-scale) was observed in the bacterial systems, while only a spherical shape with a size ~1 μm was observed in the chemical abiotic systems.

Plectonema boryanum UTEX 485, a filamentous cyanobacterium, had been reacted with a gold–thiosulfate complex (up to 2.8 mmol/L gold) at 25–100°C for up to 1 month and at 200°C for 1 day (Lengke et al. 2006a). The interaction of cyanobacteria with a gold–thiosulfate complex caused the separation of filaments into their constituent cells and released membrane vesicles after 14 days at 25°C. The precipitation of irregular gold particles on the surface of membrane vesicles was observed. At 60–200°C, nanoparticles of gold and gold sulfide were deposited inside the cells (<10 nm) and on the outer surface of the sheaths (~10–25 nm). The larger nanoparticles of gold (~20–25 nm) were distinctly cubic in shape. In the chemical abiotic systems, the gold–thiosulfate were stable for 1 month at 25°C, whereas gold nanoparticles (~25 nm) with a cubohedral habit were precipitated at 60–200°C.

3.2 Gold(III)–Chloride Complex

The interactions between bacteria, cyanobacteria, and algae with a gold(III)–chloride complex have been studied using different species, and the results of these studies are summarized in Tables 3.2, 3.3, and 3.4. The first published laboratory study to understand the interaction of a mixed bacteria (Bacillus mesentericus niger, Bacillus oligonitrophilus, and Bacterium nitrificans) with initial elemental gold was conducted by Korobushkina and Korobuskhin (1986). The objective of their study was to determine whether gold can be biologically re-deposited. The elemental gold was dissolved into solution, presumably as gold(III)–chloride or gold–organic complexes, and a reduction process occurred simultaneously, forming spherical/oval (1–2 μm), spongy clots/clusters/nodules (0.1–20 μm), and mushroom (0.1–0.5 μm) shapes. Southam and Beveridge (1994, 1996) employed Bacillus subtilis 168 in their experiments at 4, 23–25, 60, and 90°C within 1 week to 8 months. Gold nanoparticles were initially precipitated inside the bacterial cells (5–50 nm) and were released into solution, forming spherical (20 nm) and hexagonal–octahedral (4–20 μm) gold. The formation of crystalline octahedral gold was enhanced by an increase in the experimental duration and temperature. The potential of 14 hyperthermophilic and mesophilic dissimilatory iron(III) reducing bacteria to precipitate elemental gold from solutions was investigated by Kashefi et al. (2001) using hydrogen (H2), lactate, or acetate electron donor at neutral pH. Of 14 species, 6 were able to precipitate elemental gold only in the presence of H2 electron donor at 100°C. Most of the six bacteria precipitated gold at the cell surfaces (nm scale), except for Geovibrio ferrireducens (ATCC 51996). However, a closer inspection of the thin sections of several species [e.g., Geobacter sulfurreducens (ATCC 51573), Pyrobaculum islandicum (DSM 4184), Thermotoga maritima (DSM 3109)] indicated that gold was also precipitated intracellularly. The interaction of Pseudomonas aeruginosa PAO1 biofilm and a gold(III)–chloride complex resulted in the formation of both extracellular and intracellular gold (Karthikeyan and Beveridge 2002). Most gold particles were precipitated at the cell surfaces with lesser amounts inside the cells and little throughout the exopolymeric substances (EPS). Nair and Pradeep (2002) used Lactobacillus sp. to assist the precipitation of gold, silver, and gold–silver alloy. Gold particles were precipitated both within and outside the cells in the range of 20–50 nm and >100 nm forming spherical, triangular, and hexagonal shapes. In the system containing both silver and gold, gold–silver alloy precipitated inside the bacterial cells, forming 100–300 nm crystals within 3 days. Nakajima (2003) and Tsuruta (2004) conducted gold biosorption to determine maximum gold accumulation using 8 and 25 bacterial species, respectively. However, the size and shape of gold accumulation were not described in their studies. Escherichia coli and Pseudomonas maltophilia have extremely high ability to accumulate gold from a gold(III)–chloride solution at pH 3 and 30°C (Nakajima 2003). Tsuruta (2004) found that some gram-negative bacterial species (e.g., Acinetobacter calcoaceticus, Erwinia herbicola, Pseudomonas aeruginosa, and Pseudomonas maltophilia) have high ability to sorb gold at pH 3 and 25°C. Uncultured magnetotactic cocci bacteria, collected from Itaipu lagoon, Rio de Janeiro, Brazil were able to precipitate gold in the capsule and granules (Keim and Farina 2005). 16S ribosomal DNA clones from bacterial biofilms of the secondary gold grains from two sites in Australia (Tomakin Park Gold Mine, New South Wales and the Hit and Miss Gold Mine, Queensland) displayed 99% similarity to Ralstonia metallidurans (Reith et al. 2006). The laboratory experiments using the same bacteria confirmed that elemental gold was precipitated during the interaction with a gold(III)–chloride solution with both viable and dead cells. Gericke and Pinches (2006a, b) conducted laboratory experiments using three bacterial species (Pseudomonas stutzeri NCIMB 13420, Bacillus subtilis DSM 10, and Pseudomonas putida DSM 291) to determine the effect of pH (3–9), temperature (25–50°C), and gold concentrations (1.3–13 mol/L) on the size and shape of gold particles. In all cases, gold was precipitated uniformly inside the cells as spherical shapes (<10 nm) at all pH values, but larger particles with irregular, undefined shapes formed at pH 7 and 9. The size and rate of gold precipitation increased with increasing temperature and gold concentrations. Konishi et al. (2006) investigated the reduction and precipitation of gold using Shewanella algae ATCC 51181. The results showed that elemental gold was only formed in the presence of bacteria with H2 electron donor, forming 10–20 nm irregular shape. The biosynthesis of gold nanoparticles was investigated using Escherichia coli DH5α by Du et al. (2007). The gold nanoparticles formed on the bacterial surfaces, mostly as spherical with triangular and quasi-hexagonal shapes. Deplanche and Macaskie (2007) achieved gold precipitation intracellularly (<10 nm) and extracellularly (20–50 nm) using both Escherichia coli and Desulfovibrio desulfuricans with H2 as the electron donor. Gold particles were not precipitated in the abiotic conditions or the presence of heat-killed cells, and the presence of copper in solution inhibited gold precipitation. At pH 2, red suspension was observed, indicating the formation of smaller gold particles, and at pH 9, dark blue suspension was formed, presumably containing larger gold particles. Rhodopseudomonas capsulata was able to precipitate gold from a gold(III)–chloride solution at pH 4–7, forming spherical, plate, triangular, and hexagonal gold with size ranging from 10 to 400 nm (He et al. 2007). Biosynthesis of gold nanoparticles using Pseudomonas aeruginosa was achieved and formed extracellular spherical gold with size ranging from 15 to 40 nm (Husseiny et al. 2007). Feng et al. (2008) discovered that Rhodobacter capsulatus were able to precipitate gold nanoparticles within and outside the bacterial cells, forming spherical and irregular gold with average size ranging from 10 to 48 nm. Bacillus licheniformis precipitated gold nanocubes (10–100 nm) within 2 days at 25°C (Kalishwaralal et al. 2009). Monodispersed gold nanoparticles capped with dodecanethiol were biosynthesized extracellularly by Bacillus megatherium D01 at 26°C (Wen et al. 2009). The presence of thiol capping ligand induced the formation of spherical gold nanoparticles with size <2.5 nm. Nangia et al. (2009) reported the biosynthesis of gold nanoparticles by Stenotrophomonas maltophilia forming ~40 nm spherical and irregular shapes at 25°C for 8 h.

Table 3.2 A summary of bacterial interactions with a gold(III)–chloride complex
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Table 3.3 A summary of cyanobacterial interactions with a gold(III)–chloride complex
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Table 3.4 A summary of algae interactions with a gold(III)–chloride complex
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In the cyanobacterial system, Karamushka et al. (1991a, b) investigated gold biosorption using nine different species, and all of them were able to accumulate gold. Dyer et al. (1994) found that live and dead cells of Plectonema terebrans were able to precipitate and accumulate elemental gold. The studies by Karamushka et al. (1991a, b) and Dyer et al. (1994) did not describe the size and shape of the gold formed. Plectonema boryanum UTEX 485, a filamentous cyanobacterium, had been reacted with a gold(III)–chloride complex (up to 2.8 mmol/L gold) at 25–100°C for up to ~1 month and at 200°C for 1 day (Lengke et al. 2006a, b, c, 2007). On the addition of a gold(III)–chloride solution to the cyanobacteria, all cyanobacteria were killed within several minutes at all temperatures investigated. A significant increase in the precipitation of gold occurred with increasing temperature from 25 to 60–200°C. Gold nanoparticles were precipitated inside the cells (<10 nm) and also formed octahedral shape in the bulk solution from 1 μm at 25°C to 10 μm at 100°C. For abiotic experiments using a gold(III)–chloride solution, total soluble gold was stable in solution at 25–80°C, but it decreased with time at 100 and 200°C, forming irregular or cubic gold (~25 nm).

In the algae system, most of the published literature focused on the biosorption and biorecovery of gold from solution (e.g., Darnall et al. 1986; Hosea et al. 1986; Watkins et al. 1987); therefore, the size and shape of gold particles were not described in their published papers. Darnall et al. (1986) conducted the biosorption and biorecovery experiments of Au, Hg, Pb, Al, Zn, and Cu on Chlorella vulgaris biomass at pH 2–7. Between pH 5 and 7, these metals bound strongly to the cell surfaces, but most of these algae-bound metals were desorbed back into solution by lowering the pH to 2, except for Au, Hg, and Ag. Hosea et al. (1986) also conducted the biosorption and biorecovery experiments of gold using Chlorella vulgaris biomass. Elemental gold was precipitated inside and outside the cells forming nanometer-scale of irregular and spherical gold. Biosorption of gold by Chlorella vulgaris biomass reached maximum binding at pH 3 (Greene et al. 1986). Inhibition and reversal of gold binding by strong ligands were pH dependent, with the order of effectiveness: thiourea > cyanide > mercaptoethanol > bromide. Watkins et al. (1987) confirmed that Chlorella vulgaris biomass has high affinity for a gold(III)–chloride solution. Eleven species of brown, green, and red algae were tested for their ability to sorb gold from a gold(III)–chloride solution, and the maximum gold sorption was shown by Sargassum natans at pH 2.5 (Kuyucak and Volesky 1989a, b). The rates of gold biosorption increased at higher temperature from 4 to 60°C. The presence of other cations such as U, Fe, Zn, Ni, Ca, Ag, and Co did not affect the gold biosorption of Sargassum natans biomass at pH 2.5; however, anions such as sulfate, nitrate, and phosphate affected the biosorption ability. Gardea-Torresdey et al. (1990) showed that the gold binding capacity of five alga biomass increased as the algal carboxyl groups were esterified. Karamushka et al. (1991a, b) investigated gold biosorption using nine different species of algae and all of them were able to accumulate gold. The uptake of gold (III)–chloride solution was enhanced by several treatments such as acid, alkali, heat, and formaldehyde over the live/pristine cells (Ting et al. 1995). Gold precipitation by dealginated seaweed waste has been studied to elucidate the mechanisms of gold uptake from solution at pH 2–9 by Romero-Gonzãlez et al. (2003). In this study, gold was precipitated as hexagonal, tetrahedral, rod, irregular, and decahedral shapes with size ranging from ~20 nm to 5 μm. Lin et al. (2005) conducted a study of gold biosorption by Saccharomyces cerevisiae and determined that the gold bound on the cell wall had been reduced to elemental gold. Biosynthesis of gold nanoparticles was performed using Sargassum wightii biomass, collected from Mandapam Camp, Tamil Nadu, India (Singaravelu et al. 2007). The gold particles were precipitated as monodisperse spherical gold with size ranging from 8 to 12 nm. Mata et al. (2009) investigated gold biosorption and bioreduction using Fucus vesiculosus and reported that the maximum gold uptake occurred between pH 4 and 9, with optimum uptake at pH 7. The precipitated gold formed irregular or spherical shapes.

4 Mechanisms of Biosynthesis of Gold Nanoparticles

The mechanisms for the biosynthesis of gold particles by bacteria, cyanobacteria, and algae from two gold complexes have been tested using several methods including laboratory-based experiments, microscopy technique [transmission electron microcopy (TEM) or scanning electron microscopy (SEM)], Fourier transform infrared spectroscopy (FT-IR), and/or X-ray absorption spectroscopy (XAS), as discussed below.

4.1 Gold(I)–Thiosulfate Complex

The mechanisms of gold precipitation from a gold(I)–thiosulfate complex by Acidithiobacillus thiooxidans, SRB, and Plectonema boryanum UTEX 485 are summarized here. Gold(I)–thiosulfate was entered into the cell of Acidithiobacillus thiooxidans as part of a metabolic process (Lengke and Southam 2005). This gold complex was initially decomplexed to Au(I) and thiosulfate (S2O 2−3 ) ions. Thiosulfate was used as energy source and Au(I) was presumably reduced to elemental gold within the bacterial cells. During the late stationary growth phase, the gold nanoparticles that were initially precipitated inside the cells were released from the cells, resulting in the formation of gold particles at the cell surface. Finally, the gold particles in the bulk solution were grown into micrometer-scale wire and octahedral gold.

The precipitation of gold(I)–thiosulfate complex by SRB was caused by three possible mechanisms: (1) iron sulfide formation, (2) localized reducing conditions, and (3) a metabolic process (Lengke and Southam 2006, 2007). In the iron system, the formation of iron sulfide generated by SRB could have adsorbed a gold(I)–thiosulfate complex onto freshly forming surfaces, leading to the precipitation of elemental gold. The localized reducing conditions generated by SRB were associated with metabolism. Thiosulfate ion from a gold(I)–thiosulfate complex was initially reduced to hydrogen sulfide (HS−), as the end product of metabolism. The release of hydrogen sulfide through the outer membrane pores decreased redox conditions around the cells and caused the precipitation of elemental gold. The precipitation of elemental gold by SRB through metabolic process was initiated when a gold(I)–thiosulfate entered the bacterial cells and was decomplexed to Au(I) and thiosulfate (S2O 2−3 ) ions. Thiosulfate was used as energy source and Au(I) was presumably reduced to elemental gold within the bacterial cells. During the late stationary growth phase or death phase, the gold nanoparticles that were initially precipitated inside the cells were released into the bulk solution and formed sub-octahedral to octahedral, sub-spherical to spherical aggregates resembling framboids, and ultimately millimeter-thick gold foil at longer experimental duration.

In the cyanobacterial system (Plectonema boryanum UTEX 485) with the addition of gold(I)–thiosulfate complex, gold was precipitated as thin nanoparticles associated with the surfaces of membrane vesicles at 25°C (Lengke et al. 2006a). Plectonema boryanum UTEX 485 exhibits the characteristics of gram-negative bacteria which can produce membrane vesicles. When the cyanobacteria interacted with relatively high concentrations of gold(I)–thiosulfate complex, the cells released membrane vesicle as a protective shield to prevent the uptake of gold(I)–thiosulfate from solutions and to keep gold(I)–thiosulfate away from sensitive cellular components. The interaction of gold(I)–thiosulfate complex with membrane vesicle caused the precipitation of elemental gold, possibly through the reaction with phosphorus, sulfur, or nitrogen ligands of outer membrane proteins, lipopolysaccharide, periplasmic proteins, phospholipids, DNA, and RNA. In the experiments at 60–200°C, gold nanoparticles were precipitated both intracellularly (<10 nm) and extracellularly (~10–25 nm), the latter with a distinct cubic shape and admixed with gold sulfide from 60 to 100°C. The stress on cyanobacteria caused by increasing temperature presumably decreased the cell sensitivity to receiving gold(I)–thiosulfate, and this gold complex was then reduced to gold sulfide and gold inside the cells. During the death of cyanobacteria, nanoparticles of gold sulfide and gold produced inside the cells were released through the cell membrane into solutions, as indicated by the precipitation of gold nanoparticles around the cells.

4.2 Gold(III)–Chloride Complex

In the bacterial system using a gold(III)–chloride solution, gold was precipitated intracellularly (<10 nm) and extracellularly (~1 to 10 μm) (e.g., Southam and Beveridge 1994, 1996; Karthikeyan and Beveridge 2002; Nair and Pradeep 2002; Reith et al. 2006; Gericke and Pinches 2006a, b; Deplanche and Macaskie 2007; Feng et al. 2008). The presence of intracellular gold nanoparticles suggests that gold entered the cells as a gold(III)–chloride complex and was then reduced to elemental gold. These bacteria were killed and the resulting release of organics caused further precipitation of gold (Southam and Beveridge 1996). A role of sulfur and phosphorus has been linked to the precipitation of octahedral gold at acidic pH by Bacillus subtilis 168 (Southam and Beveridge 1996). The precipitation of gold by magnetotactic cocci bacteria occurred in the Phosphorus–Sulfur–Iron (PSFe) granules which suggests the association of gold with these elements (Keim and Farina 2005). The reduction and precipitation of gold by dissimilatory iron(III) reducing bacteria were catalyzed by H2 electron donor and not by lactate, indicating the involvement of hydrogenase (Kashefi et al. 2001). Deplanche and Macaskie (2007) further investigated the hydrogenase enzyme on the role of gold precipitation by Escherichia coli and Desulfovibrio desulfuricans ATCC 29577. They suggested that the periplasmic hydrogenases were involved in the gold precipitation but were not essential; however, the cytoplasmic hydrogenases and/or cytochrome c3 could be a complementary mechanism of gold reduction.

In the cyanobacterial system, the mechanisms of gold reduction by Plectonema boryanum UTEX 485 from gold(III)–chloride solutions have been studied at several gold concentrations (0.8–7.6 mmol/L) and at 25–80°C, using both fixed-time laboratory and real-time synchrotron radiation XAS experiments (Lengke et al. 2006c, 2007). The XAS results showed that Au(III) was reduced to Au(I) in a very fast reaction (within minutes), and Au(I) was immediately coordinated with sulfur atoms from cyanobacteria forming gold(I)–sulfide for all gold concentrations and temperatures. Within the cyanobacteria, sulfur presumably was present as cysteine or methionine groups of the outer membrane or periplasmic proteins. Sulfur from the cysteine or methionine groups was released into the bulk solutions through the outer membrane and immediately bound with the reduced gold forming gold(I)–sulfide on the cells or at the cell walls. The fast reduction of Au(III) to Au(I) and the fast release of organic sulfur from the dying and dead cyanobacteria at the higher temperatures investigated (60 and 80°C) resulted in the presence of gold(I)–sulfide as a major phase at the initial stage in the reaction. The reduction of gold(I)–sulfide to elemental gold was slower at 25°C than at 60 and 80°C. The cysteine or methionine within the outer membrane or periplasmic proteins also contained carbon and nitrogen, and this may explain indirect relationship between nitrogen and gold. The steps of mechanism of gold reduction and precipitation by cyanobacteria are deduced:

$$ {\hbox{gold}}\left( {\hbox{III}} \right) - {\hbox{chloride}}\left( {{\hbox{AuC}}{{\hbox{l}}_4}^{-} } \right) \to {\hbox{gold}}\left( {\hbox{I}} \right) - {\hbox{sulfide}}\left( {{\hbox{A}}{{\hbox{u}}_2}{\hbox{S}}} \right) \to {\hbox{gold}}\left( {\hbox{Au}} \right) $$
(3.1)

In the algae system, the mechanisms of gold reduction by Chlorella vulgaris biomass from gold(III)–chloride solutions have been studied using XAS (Watkins et al. 1987). The XAS results showed that Au(III) was partly reduced to Au(I) and Au(I) was coordinated with sulfur atoms from free sulfhydryl residues and also to a light-atom element, probably nitrogen. Kuyucak and Volesky (1989b) showed that elemental gold was mostly precipitated on the cell wall of Sargassum natans biomass and suggested that the carbonyl (C ≡ O) groups of the cellulosic materials were the main functional group in the gold binding with N-containing groups involved in a lesser degree. Lin et al. (2005) suggested that the hydroxyl group of saccharides, the carboxylate anion of amino-acid residues, from the peptidoglycan layer on the cell wall appeared to be the sites for gold binding. However, the gold uptake by algal biomass increased after esterification, suggesting that carboxyl groups played a minor role in gold binding (Gardea-Torresdey et al. 1990). Romero-Gonzãlez et al. (2003) studied the mechanisms of gold biosorption by dealginated seaweed biomass using FT-IR and XAS. Although FT-IR showed the presence of carboxylate groups on the surface of the biomass, XAS showed that the reduction of gold species occurred on the biomass surfaces to form gold nanoparticles and was followed by retention of Au(I) at the sulfur containing sites. Therefore, the steps of mechanism of gold reduction and precipitation by algae are similar to cyanobacteria (Reaction 3.1).

5 Application of Gold Nanoparticles

The properties of gold nanoparticles remarkably differ from the bulk gold because of quantum size confinement imposed by nano-size regimen (Link et al. 1999; Reddy 2006). The electronic, magnetic, and catalytic properties of gold nanoparticles depend mainly on their size and shape (Link et al. 1999). For example, spherical gold nanoparticles show a strong absorption band in the visible region of electromagnetic field (~520 nm) but is absent for very small particles (≤2 nm) as well as in the bulk gold. With a variety of unique properties, when gold nanoparticles are manipulated effectively, it can be applied to many different applications across the field of biology and medicine, environment, and technology (e.g., Oldenburg et al. 1998; Vieira and Volesky 2000; Salata 2004; Sperling et al. 2008; Chen et al. 2008; Cai et al. 2008).

Ancient cultures in Egypt, India, and China have used gold to treat diseases such as smallpox, skin ulcers, syphilis, and measles (Huaizhi and Yuantao 2001; Richards et al. 2002; Gielen and Tiekink 2005; Kumar 2007; Chen et al. 2008). Gold is currently used for medical devices including pacemakers and gold plated stents (Edelman et al. 2001; Svedman et al. 2005), for the management of heart disease, middle ear gold implants, and gold alloys in dental restoration (Svedman et al. 2006). Organogold compounds are widely used for the treatment of rheumatoid arthritis but at high doses, side effects such as proteinuria and skin reactions have been observed (Sun et al. 2007; Chen et al. 2008).

5.1 Biology and Medicine

Gold nanoparticles have been primarily used for labeling and bioimaging applications (Sperling et al. 2008; Chen et al. 2008). The gold nanoparticles are directed and enriched at the region of interest, providing contrast for observation and visualization. With the characteristic of strongly absorption and scattering visible light, the light energy excites the free electrons in the gold nanoparticles to a collective oscillation, known as surface plasmon. The excited electron gas relaxes thermally by transferring the energy to the gold lattice, and finally the light absorption leads to heating of the gold nanoparticles. The interaction of gold nanoparticles with light can be used for the visualization of particles using optical microscopy, fluorescence microscopy, photothermal, and photoacoustic imaging. In addition, the interaction of gold nanoparticles with both electron waves and X-rays can also be used for visualization, e.g., using TEM. Other noninvasive diagnostic tools such as magnetic resonance imaging (MRI) and X-ray computed tomography (X-ray CT) have also utilized gold nanoparticles as contrasting agent due to the ease of surface modification and higher X-ray absorption coefficient, respectively (Debouttiére et al. 2006; Kim et al. 2007).

Gold nanoparticles have been used for a long time for delivery of molecules into cells (Sperling et al. 2008; Chen et al. 2008). The molecules are adsorbed on the surface of gold nanoparticles and then are introduced into the cells using gene guns or particle ingestion. Inside the cells, these molecules will eventually detach themselves from the gold nanoparticles. Gene guns have been used for the introduction of plasmic DNA into plant cells and animal cells, which result in expression of the corresponding proteins inside the cells.

Gold nanoparticles can also be used as a heat source (Sperling et al. 2008; Cai et al. 2008). When gold particles absorb light, the free electrons in the gold particles are excited. Excitation at the plasmon resonance frequency causes a collective oscillation of the free electrons. During the interaction between the electrons and the crystal lattice of the gold nanoparticles, the electrons relax and the thermal energy is transferred to the lattice. The heat from the gold particles can be used for manipulating the surrounding tissues, e.g., hyperthermia, optically triggered opening of bonds, and opening of containers. Cells are sensitive to small increases in temperature and an increase of a few degrees can lead to cell death. If gold nanoparticles can be directed to the localized cancer tissue and is then heated by external stimuli, the cells in the vicinity of the gold particles can be selectively killed. Photo-induced heating of gold nanoparticles can also be used for the opening of chemical bonds (e.g., melting double stranded DNA and disassembling protein aggregates). Finally, photo-induced heating of gold nanoparticles can also be used for remotely controlled release of cargo molecules from capsules inside living cells. A newly functionalized gold nanoparticle (dendrimers) has been designed for not only targeting and killing tumors but also to fight cancer (Nam et al. 2009; Escosura-Muńiz et al. 2009). Gold nanoparticle is engineered not only to identify, target, and kill tumors but also to carry the additional drug to slow down the growth of cancer cells or kill the cancer cells. Dendrimers acts as an arm to the gold nanoparticles so that different molecules are attached to the arms. Once the cancer cells are surrounded by gold nanoparticles, lasers or infrared light heats the gold particles and the dendrimers release the various molecules to kill the tumors.

Gold nanoparticles can also be used for active sensor applications (Sperling et al. 2008). The objectives for sensor applications are to determine the presence of analyte and to provide its concentration. The plasmon resonance frequency is a reliable feature of gold nanoparticles that can be used for sensing. The binding of molecules to the particle surface can change the plasmon frequency directly. On the other hand, the plasmon resonance frequency is changed when the average distance among gold nanoparticles is reduced by forming small aggregates. The effect of plasmon coupling can be used for colorimetric detection of the analyte, known as a gold-based sensor. In the presence of target sequence, the gold nanoparticles bind to the target by hybridization of complementary strands of DNA which lead to a change in the plasmon resonance and the solution appears to be a violet/blue color. When the sample is heated, single sequence mismatches result in a different melting temperature of the aggregates and cause color change. The same concept applied for DNA assays can also be used for RNA- and peptide-based sequences and enzyme. The color changes can also be used to measure lengths, known as “rulers on the nanometer scale”. The fluorescence quenching occurs when many fluorophores are in close proximity to gold surfaces and this effect can be used for several sensor strategies. Raman scattering is enhanced if the analyte is close to a gold surface, called as surface-enhanced Raman scattering. Gold nanoparticles modified with Raman-active reporter molecules have been used for the detection of DNA (Krug et al. 1999), protein (Ni et al. 1999), and two-photon excitation (Kneipp et al. 2006). Gold nanoparticles can also be used for the transfer of electrons in redox reactions (Willner et al. 2006). The enzyme is conjugated to the surface of the gold particles and is immobilized on the surface of an electrode (Xiao et al. 2003). An electrode covered with a layer of gold nanoparticles has a much higher surface roughness and larger surface area which lead to higher currents. In addition, due to the presence of small gold particles, the contact of the gold nanoparticles with the enzyme can be more intimate, i.e., located in a close proximity to the reactive center, which can facilitate the electron transport.

5.2 Environment

Gold nanoparticle based technologies are currently being developed for the environmental applications to pollution control and water purification (Das et al. 2009). Bimetallic gold–palladium nanoparticles provide an active catalyst to degrade trichloroethene (TCE), one of the major pollutants in groundwater to a non-toxic form (Wong et al. 2009). Palladium catalyst has been shown to degrade TCE in water but the cost is significant to be widely adopted. Although gold is more expensive than palladium, a combination of gold–palladium nanoparticles is much more reactive to remove TCE, resulting in more cost-effective application. Gold nanoparticles incorporated in a water purification device can effectively capture and remove halocarbon-based pesticides from drinking water (Das et al. 2009) and can also enhance the oxidation of mercury generated from coal power plants (Pradeep and Anshup 2009). A mixture of gold nanoparticles as well as the traditional platinum and palladium has been developed for automotive catalysts in diesel engines to increase hydrocarbon oxidation activity by U.S. Nanostellar (http://www.nanostellar.com). The use of gold nanoparticles as a catalyst has a major role to play in green chemistry (Dahl et al. 2007; Herzing et al. 2008). Most industrial oxidation processes tend to use chlorine or organic peroxides which result in large amounts of chloride salts and chlorinated organic byproducts. Gold nanoparticles supported on carbon active molecular oxygen in air are able to convert alkenes to partial oxidation products such as epoxides at atmospheric pressure and at 60–80°C (Hughes et al. 2005). Gold nanoparticles have been developed for selective oxidation of the biomass-derived chemicals, furfural and hydroxymethylfurfural, to form methyl esters. These chemicals are used for flavor and fragrance applications, in plastics and industrial solvents (Taarning et al. 2008).

5.3 Technology

Gold nanoparticles have been designed to improve computer memory (Lee et al. 2007). A three-dimensional computer memory device composed of layers of gold nanoparticles has been developed to increase the memory capacity of a single chip. The device is built on a base of silicon that is coated with hafnium oxide. Then, alternating layers of 16-nm sized gold nanoparticles and insulating polymers are formed on top. Another development of computer memory using gold nanoparticles is an organic nonvolatile bistable memory, which is a mixture of plastic and gold (Lin et al. 2007). Organic nonvolatile memories have been developed using plastic and other carbon-based chemicals but the organic memory devices tend to break down in air and under the stress of many read–write cycles. Therefore, a new memory consisting of gold nanoparticles mixed into a polymer and sandwiched between two aluminum electrodes is considered essential to be implemented in flexible electronics, such as radio-frequency identification, smart cards, e-paper, and flexible displays.

6 Future Directions

During the past two decades, the interaction of a variety of bacteria and two gold solutions [gold(I)–thiosulfate and gold(III)–chloride] have been well investigated, although the focus of these studies is not necessarily on the biosynthesis of gold nanoparticles. Living and dead bacteria/cyanobacteria/algae have huge potential for the production of gold nanoparticles; however, the previous studies are still largely in the discovery phase. Given the anticipated wide application of gold nanoparticles for commercial applications, consideration of the material design, processes, and applications using bacteria/cyanobacteria/algae that minimize hazard and waste will be essential for the transition of nanoscience discoveries to commercial products of nanotechnology. Future work should implement systematic experiments which include development of gold nanoparticles of well-defined size and shape. Better understanding of the mechanisms of gold biosynthesis will enable us to achieve better control over size, shape, and monodispersivity which will lead to the development of high precision in the production level and the application of nanoparticles for commercial scale applications.

7 Conclusions

We provide a comprehensive review of research conducted on the biosynthesis of gold nanoparticles by bacteria, cyanobacteria, and algae using gold(I)–thiosulfate [Au(S2O3) 3−2 ] and gold(III)–chloride [AuCl −4 ] complexes. In general, many bacteria, cyanobacteria, and algae have the ability to produce gold nanoparticles with properties similar to chemically synthesised materials. Intracellular synthesis of gold nanoparticles, as well as extracellular formation of nanoparticles in the presence of these microorganisms has been successfully demonstrated. The shape of gold particles precipitated by bacteria/cyanobacteria/algae includes spherical, oval, spongy clots, mushroom, irregular, triangular, tetragonal, hexagonal, octahedral, rod, cubic, docahedral, icosahedral, coil or wire, plate, and thin foil, with size ranging from 1 nm to several millimeters. The mechanisms of gold precipitation from a gold(I)–thiosulfate complex by Acidithiobacillus thiooxidans and SRB are directly or indirectly associated with metabolism. For the dissimilatory iron(III) reducing bacteria using gold(III)–chloride complex, the involvement of cytoplasmic hydrogenases and/or cytochrome c3 could be a complementary mechanism of gold reduction. For the cyanobacteria and algae using gold(III)–chloride complex, Au(III) was reduced to Au(I), and Au(I) was immediately coordinated with sulfur atoms from the cysteine or methionine groups, forming gold(I)–sulfide. The reduction of gold(I)–sulfide to elemental gold was a slower step mechanism than the formation of gold(I)–sulfide.

Previous work on the biosynthesis of gold nanoparticles is still largely in the discovery phase. Given the anticipated wide application of gold nanoparticles for commercial applications, continuing work is recommended to focus on the mechanisms of the biosynthesis of gold nanoparticles and the development of gold nanoparticles of well-defined size and shape.