Microbial synthesis of gold nanoparticles: Current status and future prospects

Advances in Colloid and Interface Science 209 (2014) 40–48

Contents lists available at ScienceDirect

Advances in Colloid and Interface Science

j ourna l homepage: www.e lsev ie r .com/ locate /c i s

Microbial synthesis of gold nanoparticles: Current status andfuture prospects

Utkarsha Shedbalkar a, Richa Singh a, Sweety Wadhwani a, Sharvari Gaidhani b, B.A. Chopade a,⁎a Department of Microbiology, University of Pune, Pune 411007, Maharashtra, Indiab Institute of Bioinformatics and Biotechnology (IBB), University of Pune, Pune 411007, Maharashtra, India

⁎ Corresponding author at: Department of Microbio411007, Maharashtra, India. Tel.: +91 20 25690463.

E-mail addresses: [email protected], chopade@u

0001-8686/$ – see front matter © 2013 Elsevier B.V. All rihttp://dx.doi.org/10.1016/j.cis.2013.12.011

a b s t r a c t

Available online 2 January 2014

Keywords:Gold nanoparticlesMicrobial synthesisPropertiesTransmission electron microscopyMechanismApplications

Gold nanoparticles have been employed in biomedicine since the last decade because of their unique optical,electrical and photothermal properties. Present review discusses themicrobial synthesis, properties and biomedicalapplications of gold nanoparticles. Different microbial synthesis strategies used so far for obtaining better yield andstability have been described. It also includes different methods used for the characterization and analysis of goldnanoparticles, viz. UV–visible spectroscopy, Fourier transform infrared spectroscopy, X ray diffraction spectroscopy,scanning electronmicroscopy, ransmission electronmicroscopy, atomic forcemicroscopy, electron dispersive X ray,X ray photoelectron spectroscopy and cyclic voltametry. The different mechanisms involved in microbial synthesisof gold nanoparticles have been discussed. The information related to applications of microbially synthesized goldnanoparticles and patents on microbial synthesis of gold nanoparticles has been summarized.

© 2013 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402. Synthesis methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

2.1. Gold nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412.1.1. Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412.1.2. Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412.1.3. Microbial products for synthesis of gold nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

2.2. Gold nanoalloys . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422.3. Gold nanoconjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

3. Properties and characterization of gold nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 443.1. Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

3.1.1. Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 443.1.2. Optical and electronic properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

3.2. Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444. Mechanism of microbial synthesis of gold nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445. Applications of microbially synthesized gold nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 466. Patents on microbially synthesized gold nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 477. Future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

1. Introduction

Gold is one of thewell knownnoblemetals. It is used as heat insulatorin automobiles and reflective layer on some high-end CDs. It produces anintense red colorwhen used as a coloring agent in cranberry glass [1–3].

logy, University of Pune, Pune

nipune.ac.in (B.A. Chopade).

ghts reserved.

Gold, in variety of forms, has been used in medicine throughout thehistory of civilization [4,5]. Gold and gold compounds have been usedin treatment of rheumatic diseases and discoid lupus erythematosus[6,7], restorative dentistry [8–10] and various inflammatory skin disor-ders such as pemphigus, urticaria and psoriasis [11]. Gold eyelid im-plants are used in lagopthalmos patients and in patients sufferingfrom facial nerve palsy [12–15].

Nanomaterials are believed to display different properties than thatof bulk materials, and so is true in the case of gold nanoparticles

41U. Shedbalkar et al. / Advances in Colloid and Interface Science 209 (2014) 40–48

(AuNPs) [16–18]. In last few years, AuNPs have received importance inresearch because of their unique optical, electrical and photothermalproperties [19–21]. Moreover, they are highly stable to oxidation[22,23]. The phase and morphology variations in AuNPs can alter theirphysical and chemical properties [24]. The first review on the medicalapplications of gold and gold compounds was published by Fricker(1996) [5]. Since then, 33 reviews have been published till to date ongold, gold compounds, nanostructures and their importance in variousfields. However, up till now there has been no review on microbialsynthesis of AuNPs, this is the first review specifically about the micro-bial synthesis of AuNPs, its mechanism of synthesis, characterization,patents and applications.

2. Synthesis methods

Synthesis of nanoscale gold with controlled phase and morphologyis one of the fundamental and technological interests. The synthesis ofgold colloids, recently named as AuNPs, was reported about 150 yearsago by Michael Faraday using phosphorous to reduce AuCl4− ions [25].In recent years, a wide range of AuNPs have been developed for applica-tions in biotechnology, industries, and electrical, pharmaceutical, medi-cal and agricultural fields [26–28] by variety of physical, chemical andbiological methods. Gold nanostructures of well defined compositionsare synthesized in the form of clusters, colloids, wires, rods, tubes, pow-ders, thin films, etc., by using these techniques [18,29–31].

2.1. Gold nanoparticles

Traditionally, AuNPs have been synthesized by physical and chemi-cal methods summarized in Fig. 1. AuNPs of size ranging from 1 to100 nm and different shapes have been obtained by using these tech-niques. Though these synthesis methods have been extensively studied,they have certain drawbacks such as use of harsh chemicals, stringentsynthesis conditions, energy and capital intensive [32–34] and less pro-ductivity [35,36]. Current synthetic methods result in mixed shapenanoparticles (NPs) that require expensive and low-yield purificationprocedures such as differential centrifugation [37]. Moreover, thesemethods impose environmental hazards due to toxic solvents or addi-tives and produce more sludge. Hence, there is an increasing need todevelop clean, nontoxic, environmentally benign and sustainable syn-thesis procedures. Development of high yield and low cost methodsfor NPs production is an important challenge [34]. Consequently, re-searchers in nanoparticle synthesis have turned to biological systemsdue to their rich diversity [38,39].

Biosynthesis can be successfully used for the production of smallerparticles in large scale [40,41]. It is important to note that biologicallysynthesized NPs exhibit enhanced stability [42] and better control over

Synthesiof AuNP

Physicalmethod

Chemicamethod

1. UV irradiation

2. Laser ablation

3. Plasma synthesis

1. Citrate synthesis

2. Turkevich method

3. Wet chemical synth

4. Chemical reduction

Fig. 1. Physicochemical metho

morphology [43]. Biological systems like bacteria, fungi, actinomycetesand plants have the ability to produce NPs [44–48]. NPs are producedby microbes, intra and/or extracellularly, due to their innate potential[46,49–51]. However, the extraction of NPs formed by intracellularbiosynthesis is generally difficult because of additional processingsteps such as ultrasonication and treatment with suitable detergents[52]. Thus, microorganisms giving extracellular biosynthesis of NPsmust be extensively screened [53–55]. Use ofmicroorganisms as poten-tial biofactories for synthesis of AuNPs is a relatively new area of re-search with considerable prospects. Further, it is environmentallyacceptable, economic, time saving and can be easily scaled up for largescale synthesis [41,56–59]. Subsequent sections give the detailed infor-mation about the various microbial synthetic strategies used for AuNPs.

2.1.1. BacteriaAmong microorganisms, prokaryotes have received the most atten-

tion in the area of AuNP synthesis [60]. For the first time microbialsynthesis of AuNPs was reported in Bacillus subtilis 168 which revealedthe presence of 5–25 nm octahedral NPs inside the cell wall [61]. InRhodopseudomonas capsulata, spherical AuNPs with 10–20 nm rangehave been observed [62] at lower concentration and nanowires withnetwork at higher concentration [63]. Six cyanobacteria have beenreported for production of AuNPs. Plectonema sp. [64,65], Anabaenasp., Calothrix sp., and Leptolyngbya sp. have been exploited for theAuNP synthesis [66]. Single-cell protein of Spirulina platensis was alsoshown to produce AuNPs and Au core–Ag shell NPs [67]. An overviewon bacterial synthesis of AuNPs is given in Table 1. If one tries togroup the AuNP producing bacteria according to 9th edition of Bergey'sManual of Systematic Bacteriology (2005) [68], themembers belongingto groups glidobacteria, and beta, epsilon and zeta proteobacteria havenot been reported so far.

2.1.2. FungiFungi appear to be more promising for large scale production of NPs

as they are simpler to grow both in the laboratory and at an industrialscale as well as secrete large amount of proteins [34,89,90]. Besides this,fungi synthesize NPs of defined dimensions with good monodispersity[34,89]. Different fungal species, e.g., Fusarium oxysporum, Verticilliumsp. have been reported [89,91,92] to synthesize NPs either intra or ex-tracellularly. Shankar et al. (2004) [44] demonstrated that goldnanoplates can be synthesized by using fungal extracts. Yeasts likePichia jadinii and Yarrowia lipolyticawere also shown to have a good po-tential for synthesis of AuNPs [93,114] and they are now under the spe-cialized exploration to engineer AuNPs. The yeast strains are havingmany advantages over bacteria for the bulk production of NPs as yeastsare easy to handle in laboratory conditions, synthesize high amount of

s

l Physicochemicalmethod

esis

method

1. Sonochemical

2. Sonoelectrochemical methods

ds for synthesis of AuNPs.

Table 1Synthesis of gold nanoparticles by bacteria.

Bacteria Size (nm) Shape Localization Reference

CyanobacteriaPlectonema boryanum UTEX 485 – Cubic Membrane vesicles [64,65]Anabaena sp.Calothrix sp.Leptolyngbya sp.

– – Intracellular [66]

Spirulina platensis 6–10 – Extracellular [67]Lyngbya majuscula b20 nm Spherical Intracellular and extracellular [69]

Other glidobacteriaSulfate reducing bacteria b10 – Cell envelope [70]

Alpha proteobacteriaRhodobacter capsulatus – – Plasma membrane [71]Rhodopseudomonas capsulata 10–20

50–400a

50–60

SphericalTriangular nanoplatesSpherical nanowires

Extracellular [62][63]

Gamma proteobacteriaEscherichia coli 20 [72]Escherichia coli DH5α – Spherical Cell surface [73]Pseudomonas aeruginosa 15–30

10–20–

SphericalExtracellular––

[50][74]

Pseudomonas denitrificans 25–30 Face centered cubic Cell bound [75]Shewanella algae 10–20 – Periplasmic space, bacterial envelope [49]Shewanella oneidensis 12 ± 5 Spheres – [46]Stenotrophomonas maltophilia 40 – Intracellular [51]Marinobacter pelagius 10 Triangle, spherical, polygonal Cell wall bound [76]Stenotrophomonas sp. 10–50 Multi-shaped Extracellular [77]

Delta proteobacteriaDesulfovibrio desulfuricans 20–50 – Periplasmic space over the cell surface [78]

Gram positive bacteria

FirmicutesBacillus subtilis 168 5–25 Octahedral Inside cell wall [61]Bacillus subtilis 20 – – [79]Bacillus licheniformis 10–100 Cubes – [80]Bacillus licheniformis [43]Bacillus megaterium DO1 1.9 ± 0.8 Spherical Extracellular [81]Lactobacillus sp. 20–50 Hexagonal Intracellular [82]Geobacillus stearothermophilus 12 Polydispersed, circular Extracellular [83]Jeotgalibacillus sp. 5–35 Intracellular cell bound [84]

ActinobacteriaRhodococcus – – – [62]Brevibacterium casei 10–50 – – [85]Thermonospora sp. 7–12

8SphericalSpherical

Extracellular [56][86]

Rhodococcus sp. 5–15 – Intracellular on cytoplasmic membrane rather than cell wall. [87]Arthrobacter sp. 61BArthrobacter globiformis 151B

8–40 Spherical Cell wall bound [88]

– Not reported.a Sizes of these NPs are mentioned N100 nm as per previous reports.

42 U. Shedbalkar et al. / Advances in Colloid and Interface Science 209 (2014) 40–48

enzymes and grow rapidly employing simple nutrients [95]. Table 2gives the brief overview of different fungi used for synthesis of AuNPs.

2.1.3. Microbial products for synthesis of gold nanoparticlesSome other biological technologies are also available for ecofriendly

synthesis of AuNPs. Biological materials like DNA [120,121], biolipidcylinders [122,123], viroid capsules [124], bacterial rapidosomes [125],multicellular superstructures [126], bacterial enzymes [127], etc., havebeen used in template mediated synthesis of AuNPs.

2.2. Gold nanoalloys

Nanoalloys are the clusters formed of two or more metallic elements[128]. Gold nanoalloys exhibit unique and often enhanced electrical,optical, catalytic and magnetic properties relative to corresponding indi-vidual metal NPs [128,129]. Combinations of two metals occur either byself assembly [130] or artificially doneusing different fabricationmethods[131]. Formation of a single bimetallic nanoparticle leads to higher

physical stability and distinctive properties compared to monometallicparticles [129,132]. Synthesis of bimetallic NPs is receiving much atten-tion due to many exciting applications [132]. Gold nanoclusters exhibitstrong relativistic effect and hold a great promise for applications incatalysis, the medical field and sensors [129]. Extensive research is fo-cused on synthesis, characterization and applications of gold nanoalloysin various fields. The various physical and chemical methods have beenexploited for synthesis of gold nanoalloys with different metal ions(Fig. 2).

Surprisingly, very few reports are available on themicrobial synthe-sis of gold nanoalloys. F. oxysporum was found to synthesize an Au–Agnanoalloy of 8–14 nm and its synthesis was controlled by the NADHdependent protein released by the fungus [57]. It was also suggestedthat by controlling the amount of cofactor, synthesis of stable Au–Agalloy particles is possible [57]. S. platensis, a microscopic alga, has beenshown to form the Ag–Au alloy of size 17–25 nm [67]. Filamentousfungus Neurospora crassa produced Au–Ag bimetallic alloy type NPsconcentrated on the outer cell wall when exposed to ionic solutions of

Table 2Synthesis of gold nanoparticles by fungi.

Fungus Size (nm) Shape Localization Reference

ZygomycetesRhizopus oryzae 10 – Cell free extract

Cell surface[96]

Rhizopus stolonifer 1–5 – – [97]

AscomycetesAspergillus niger 10–20 Polydispersed Extracellular [98]Aspergillus niger 12.79 ± 5.61 – Extracellular [99]Aureobasidium pullulans 29 ± 6 Spherical Intracellular [100]Colletotrichum sp. 20–40 Decahedral and icosahedral – [101]

[102]Fusarium semitectum 25 – – [103]Fusarium oxysporum 2–50 – – [104]

[92][86][91]

Fusarium oxysporum 128 ± 70a Aggregates Intracellular [100]Helminthosporum solani 2–70 Polydispersed Extracellular [105]Neurospora crassa 32 Spherical – [106]Penicillium brevicompactum 10 ∼ 50 Spherical – [107]Trichoderma koningii 30–40 Small spheres to polygons – [108]Trichoderma koningii 10–14 Spheres Cell free filtrate [109]Verticillium sp. 20 ± 8 Spherical

QuasihexagonalCell wallCytoplsmic membrane

[89]

Verticillium luteoalbum b10 SphericalSpheres and rods

Intracellular [94][93]

Cylindrocladium floridanum 19.05 Spherical Extracellular [110]

BasidiomycetesPhanerochaete chrysosporium 10–100 Spherical Extracellular [111]Volvariella volvacea 20–150a Spherical, – [42]Sclerotium rolfsii 25 Triangles, decahedrals, hexagonals and rods Extracellular [112]

YeastsSchizosaccharomyces cerevisiae N100a – Cell wall [113]Pichia jadinii b100 Spherical Cytoplasm [93]Yarrowia lipolytica NCIM 3589 15 Hexagonal, triangular Associated with cell wall [114]Yarrowia lipolytica 9–27 Spherical Cell associated [115]Hansenula anomala 14 – – [116]Candida albicans 20–40 Spherical – [117]Candida albicans 5 Monodispersed spherical Cell free extract [118]Extremophilic yeasts (isolated from acid mine drainage) 30–100 – On cell surface, extracellular [119]

– Not reported.a Sizes of these NPs are mentioned N100 nm as per previous reports.

43U. Shedbalkar et al. / Advances in Colloid and Interface Science 209 (2014) 40–48

silver and gold [133]. Further, authors have also reported change in thesize of alloy Au–Ag bimetallic NPs from 3 to 90 nm with change in theionic concentration of solution [133]. Escherichia coli cells were able to

Gold Nanoalloys

Physical and Chemical methods

Brust methodGalvanic substitutionMicrowave assistedLaser ablationReduction by sodium boSputter depositionPhotochemical reduction

Biological methods

Fusarium oxysporumLactobacillus spp.Spirulina platensisE. coliCupriavidus necator

Fig. 2. Synthesis, properties and a

precipitate palladium (Pd) ions leading to reduction of Au (III) to formAu–Pd core shells [134]. The formation of the Au–Pd alloy on the cellsurface of Cupriavidus necator cells was also reported [135]. Lactobacillus

rohydride

Au-AgAu-PdAu-PtAu-RuAu-T

Au-AgAu-Pd

Properties

Physical stabilityUnique optical,

magnetic, catalytic

properties

Applications

MedicineCatalysisSensors

pplications of Au nanoalloys.

44 U. Shedbalkar et al. / Advances in Colloid and Interface Science 209 (2014) 40–48

strains,which are found in buttermilk,were able to synthesize the gold–silver alloy crystals of well defined morphology [82].

2.3. Gold nanoconjugates

Gold nanoconjugates are prepared by combining AuNPs with differ-ent biomolecules such as antibody, proteins, peptides, DNA, drugs, etc.The resulting conjugate exhibits the thermal, optical, imaging andcarrier properties of NPs along with the selectivity and specificity of abiomolecule [136]. AuNPs of smaller size are attractive materials forprotein and enzyme immobilization and these nanoconjugates haveseveral advantages in the fields of biocatalysis, biosensing andmedicine[137,138]. Because of its high surface area to volume ratio, NPs exhibithigh enzyme loading capacity. Further, the nanoconjugates possessless or no internal diffusion in solution and Brownian motion whencompared to the conventional immobilization techniques and porousmacroscopic supports [138]. AuNPs conjugated with antibodies, antibi-otics and various drugs have been extensively used for cancer diagnosis,imaging and treatment [139,140]. However, no reports are available ongold nanoconjugates synthesized by microbial methods.

3. Properties and characterization of gold nanoparticles

3.1. Properties

AuNPs exhibit intriguing properties than metallic gold which implytheir tremendous applications in medicine. These properties include(i) excellent chemical and mechanochemical stability [141], (ii) excel-lent optical andelectronic properties, (iii) easy surface functionalization,(iv) surface plasmon resonance effect, (v) unique catalytic activity[19,142–144], and (vi) biocompatibility [145]. Further, the AuNPs areresistant to oxidation, are crystallizable, have a controllablemorphologyand good size dispersion properties [19]. In the recent five years, theproperties of AuNPs are under extensive investigation and researchersare trying to device newer methods to improve their properties.AuNPs exhibit different shades of color from yellow (large particles) tored (small particles) and even mauve based on their size, shape andmonodispersity when suspended in a fluid usually water [3]. Changein color of the solutions of AuNPs is attributed to the nature of plasmonbands (One for spherical and two for nanorods) and anisotropic NPshave different reactivity for different crystal faces [3]. The variousshapes exhibited by AuNPs are shown in Fig. 3. Completely differentphysiochemical/thermodynamic properties are exhibited by AuNPsof diameters less than 10 nm because of their higher surface-area tovolume ratio [146]. Stability and optical and electronic properties areexceptionally important, as these impart the rest of the other propertiesto AuNPs. Alteration in these properties is possible because of slightchange in size, shape, storage, crystallinity, surfacemorphology, surfacemodification and conjugation with biomolecules [144,147].

3.1.1. StabilityThough AuNPs exhibit excellent mechanical and chemical stability,

aggregation of nanostructures in a surfactant-free reaction is a commonphenomenon leading to difficulty in studies of properties and applica-tions [148]. It has been demonstrated that AuNPs can be reduced andstabilized by chemical agents like ascorbic acid, citrate, etc. [149]. Thestable AuNPs can also be formedby surfacemodificationwith thiol com-pounds [150], cysteine [151] and coating of AuNPs with polyvinyl alco-hol [152]. The storage of AuNPs in the dark at 4 °C has been shown toprolong the stability of AuNPs [153]. Studies have also shown that thepresence of a nonionic fluorosurfactant during the chemical reductionleads to production of more stable AuNPs in colloidal solution. Theseparticles showed stability up to 1 month in the presence of high saltconcentration and over a pH range of 2–11 [154].

3.1.2. Optical and electronic propertiesAuNPs produce vibrant colors by interactionwith visible light. AuNP

interaction with light is strongly dictated by their environment, sizeand physical dimensions [155–157]. Recently, these unique optical–electronic properties have been utilized in high technology applicationssuch as electronic conductors, catalysis, organic photovoltaics, sensoryprobes, therapeutics and drug delivery [19,155]. The optical and elec-tronic properties of AuNPs are changed by changing the size, shape,surface chemistry or aggregation state and hence, can be customizedbased on applications like targeted cancer imaging and anticancerdrug delivery [158–160].

3.2. Characterization

Microbially synthesized AuNPs and gold nanoalloys have been char-acterized by many techniques. However, not a single technique is suffi-cient to characterize but combinations of methods are needed forcharacterization. UV–vis spectroscopy is reported for the primary detec-tion of synthesis of AuNPs in all the microbial AuNP syntheses. Few ofthe examples include R. capsulata, Sclerotium rolfsii, Thermomonosporaand extremophilic yeasts where peak in the range of 500–550 nm is ev-ident of the presence of AuNPs. [86,112,119]. Fourier Transform InfraredSpectroscopy analysis [86,116] is used in Thermomonospora to detectthe functional groups involved in the reduction of Au ions. Crystal struc-ture, phase composition as well as mean size of the AuNPs are analyzedby X ray diffraction spectroscopy (XRD) by Bhambure et al. (2009) andBinupriya et al. (2010) [97,99]. The elemental analysis, electronic stateand chemical characterization can be carried out by energy dispersivespectrum (EDS) [114], energy dispersive X ray (EDX) [62,78,96] andX ray photoelectron spectral (XPS) techniques [62]. EDS, EDX andXPS have been used in characterizing AuNPs synthesized by E. coli,Y. lipolytica, Rhizopus oryzae and R. capsulata [62,78,96,114]. The imag-ing andmappingof NPs are carried out by transmission electronmicros-copy (TEM) to elucidate its morphology [97,116]. Under high resonancetransmission electron microscopy (HRTEM) one can observe latticefringes of AuNPs to confirm its crystalline nature [96]. HRTEM analysiswas used by Das et al. (2009) to characterize the AuNPs produced byR. oryzae. Other microscopy techniques used for characterization ofAuNPs include scanning electron microscopy (SEM) [73,80], field emis-sion scanning electronmicroscopy (FESEM) and atomic forcemicroscopy(AFM) [96,116].

For characterization of gold nanoconjugates, besides the above-mentioned techniques, few other additional methods such as cyclicvoltametry and resonance Raman scattering [161] have been used.Thermogravimetric analysis (TGA) and X-ray electron spectroscopyare also used for characterization of gold nanoconjugates [162]. Atomicforce microscopy for detection of surface characters of AuNPs conjugat-ed with glucose oxidase have also been carried out to detect surfacestructures [163].

4. Mechanism of microbial synthesis of gold nanoparticles

AuNPs are formed by variety of microorganisms. Though the com-mon underlying mechanism involved in synthesis is the reduction ofAu+3 ions to form AuNPs. It has been postulated that the enzymessecreted by microorganisms play an important role in the reduction ofmetal ions, leading to NP nucleation and growth [62,86,164]. Thesemechanisms and processes involved in AuNP synthesis are depicted inFig. 4. In spite of large number of reports on microbial mediated AuNPsynthesis, the mechanistic aspects have not been established and needto be studied in depth [34]. Such studies have been performed previous-ly to explain the mechanism underlying silver NP synthesis, where theproteinmediated but nitrate reductase independent [165], hydrogenaseand nitrate reductase mediated synthesis [166] has been explained. Arational approach for AuNP synthesis can be developed by elucidationof biochemical pathways leading to gold biomineralization [34].

d e

j k

cba

f

l

g h i

Fig. 3. Electron microscopic images of AuNPs. TEM images of (a) Au nanoplates by Aspergillus niger [98], (b) spherical, triangular and hexagonal-shaped AuNPs by Penicilliumbrevicompactum [107], (c) spherical AuNPs by Rhodopseudomonas capsulata [63], (d) AuNPs by Verticillium luteoalbum [94], (e) oval shaped AuNPs by Stenotrophomonas maltophilia[51], (f) AuNPs by Sclerotium rolfsii [112], (g) intracellular synthesis of AuNPs by Pichia jadiniiafter reaction with AuCl4 [94], (h) AuNPs in the presence of a live cell filtrate ofP. brevicompactum [107], (i) biosynthesis of AuNPs using the bacteria Rhodopseudomonas capsulata [63]. SEM images of (j) Au nanocubes from Bacillus licheniformis [80], (k) hexagonaland triangular Au crystals by marine yeast Yarrowia lipolytica NCIM 3589 [114], (l) membrane bound AuNPs by Escherichia coli [73].

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The physicochemical parameters such as temperature, pH and sub-strate concentrationwere found to influence the intracellular AuNP syn-thesis [93,94]. Morphology of AuNPswas reported to bemanipulated byaltering these parameters and growth conditions of microorganisms[93,94]. Further, monodispersity can be achieved by optimizing theseparameters. However, the extensive studies on optimization have notbeen done yet. Proteins and amino acid residues in proteins such ascysteine, tyrosine and tryptophan are reported to play an importantrole in biosynthesis and stabilization of AuNPs [56,167–169]. Freeamino or cysteine groups in proteins can bind to the AuNPs and thus,

surface bound proteins can stabilize them [56,167]. In some cases, tyro-sine can bind to gold surface via amine groups and reduce silver ions athigh pH, thereby, producing Au core–Ag shell nanostructures [168].Tryptophan is also shown to produce metal NPs at basic pH. A metalion forms NPs by accepting an electron from a transient tryptophyl rad-ical formed because of conversion of tryptophan residue present in pep-tide [169]. It was also reported that the capping and stabilization ofAuNPs are affected by different proteins [56].

Thefirstmechanistic approach for bacterial synthesis of AuNPs statedprecipitation of AuNPs in B. subtilis 168 cells under ambient temperature

HAuCl4Uptake by Microbes

Reductases(NADH/NADPH

dependent/ independent)

Amino acids (Tyrosine,Tryptophan,

cysteine)

Tyrosine containing peptides

Ligninases / Laccases

Biomineralization

H2 gas as electron donor (Anaerobic condition)

Intracellular synthesis

Extracellular synthesis

Aqueous Environment

Synthesis and Stabilization

Capping

Proteins, Polysaccharides,

organic acids

Factors affecting synthesis and monodispersity:

TemperaturepH

Substrate concentrationShaking and static condition

Organic phosphate compounds

AuNPs

Au+++

Fig. 4.Mechanism of synthesis of gold nanoparticles.

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and pressure, during incubation of the cells with Au+3 ions [61]. Organicphosphate compounds were shown to play an important role, possiblyas bacteria–Au complexing agents, leading to in vitro development ofoctahedral AuNPs of 5–25 nm size in the cell wall [61]. Shewanella algaecan reduce Au+3 ions in anaerobic environments. In the presence ofS. algae and hydrogen gas, the Au ions are completely reduced forming10–20 nm AuNPs. [49]. In E. coli and Desulfovibrio desulfuricans, hydro-gen gas (H2) acts as an electron donor and periplasmic hydrogenasesare suggested to participate in the reduction of Au3+ ions and bioaccu-mulation of AuNPs [78]. In E. coli, the introduction of additional cysteinederived thiol residues with in FLiC protein is found to enhance the goldabsorption as well as diminution on the surface of flagellar filamentresulted in fabrication of AuNPs of 20–25 nm [78]. In filamentouscyanobacterium, Plectonema boryanum UTEX 485, the precipitation ofcubic AuNPs is observed at 25–100 °C up to one month and at 200 °Cin one day. This could be due to the interaction of cyanobacteria withaqueous gold chloride which initially promote the precipitation of NPsof amorphous gold (I) sulfide at the cell walls and deposit metallicgold near cell surface and in solutions [64].

In Trichothecium sp., the synthesis of AuNPs was controlled byvarying growth condition such as static and shaking. Extracellular andintracellular syntheses of AuNPs were achieved at static and shakingconditions respectively [170]. Further, fungi secrete proteins and reduc-ing agents which help in the stabilization of extracellularly synthesizedNPs [104]. Controlled synthesis of AuNPs is achieved by controllingactivities and cellular growth conditions in yeast strains [57,93,94].In F. oxysporum and R. capsulata, species specific NADH dependentreductases are suggested to be involved for bioreduction of Au3+ ionsto carry out the synthesis of AuNPs [62,100]. However, the exact mech-anism underlying the NADH dependent reduction of Au3+ ions is notknown. This has first time opened out a novel fungal enzyme based invitro approach for nanomaterial biosynthesis [90,104]. In Verticillum

electrostatic interactions with positively charged groups such as lysineresidues in enzymes, present in the cell wall of mycelia, caused trappingof Au3+ ions on the surface of fungal cells. Hence, the reduction ofAu3+ ions by enzymes led to aggregation of metal atoms and formationof AuNPs. However, the exact mechanism for formation of AuNPs wasnot elucidated inVerticillium [89,93,94]. In Phanerochaete chrysosporium,the lignolytic enzymes such as laccase and ligninase were reported tosynthesize extracellular and intracellular AuNPs respectively [111]. Atnormal pH (2–3.5) the cell surface proteins and enzymes trap andreduce gold ions to form nuclei which finally undergo crystal growthto form aggregates (size 128 ± 70 nm) of AuNPs. Binding and reduc-tion of gold ions are mediated by positive amino and sulfhydryl groupsand negative carboxylic groups in proteins [111]. Extracellular glycosyl-ated derivative of laccasewas stable and found to be involved in synthe-sis of extracellular AuNPs in growth medium [111]. In actinomyceteThermomonospora, enzymes were shown to play an important role inthe reduction of metal ions as well as stability of NPs; resulting in theefficient production of monodispersed AuNPs [86]. It has been hypoth-esized that proteins, polysaccharides and organic acids released by thefungi are able to differentiate crystal shapes and direct their growth into extended spherical crystals [171].

5. Applications of microbially synthesized gold nanoparticles

There are only two reports available which state that AuNPsproduced by microbial source can be used for applications in medi-cine. AuNPs produced by Candida albicans have been studied fortheir potential in detection of liver cancer [116]. The AuNPs synthe-sized by Penicillium brevicompactum have been exploited to studytheir cytotoxic effects against mouse mayo blast cancer C2C12 cells[107].

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6. Patents on microbially synthesized gold nanoparticles

More than 100 patents are available on biogenic synthesis of metalNPs and their applications. In all these patents, only three patents specif-ically claim AuNP synthesis employing microorganisms. Endophytic mi-croorganisms such as Pseudomonas aeruginosa, Trichoderma atrovirideand Streptomyces sp. isolated from tea leaves have been demonstratedto synthesize AuNPs. These particles have been used to stabilize antifun-gal activity andhave been claimed as biocontrol agents and biofertilizers.Further it has also been claimed that the conjugation of these NPs withantimicrobials/biocontrol agent/plant growth promoter leads to in-creased stability of antimicrobials and enhanced activity of a biocontrolagent [74]. The synthesis of colloidal AuNPs by using probiotic bacteriahas also been claimed [172]. The green synthesis of monodisperseAuNPs and the process for direct embedment/integration of AuNPs inbiological system such as E. coli have been claimed previously [173].

7. Future prospects

• Understanding the large number of microorganisms in environment,it is necessary to screen the various groups of microorganisms forsynthesis of AuNPs. As there are no reports on NP synthesis byglidobacteria, and beta, epsilon and zeta proteobacteria, themembersof these groups should be studied for production of AuNPs.

• Future research goals on microbially synthesized AuNPs shouldaddress the development of reliable experimental protocols for thesynthesis of monodispersed AuNPs of defined composition.

• The research should also be focused on optimization of growth condi-tions of microorganisms and physicochemical parameters which havean effect on synthesis such as pH, temperature, age of the culture,amount of the biomass, salt concentration, etc. The large scalemicrobialsynthesis ofmonodispersedAuNPsneeds further detailed investigation.

• Oneof the challenging issues innanotechnology is the elucidationof themechanism of microbially synthesized AuNPs. But the exact role of re-ductases and biomolecules involved in gold ion reduction should bestudied. Purification and physicochemical characterization of the pro-teins/biomolecules involved in microbial synthesis of AuNPs need tobe investigated. A proteomic approach should also be used to explorethe exactmechanismof synthesis of AuNPs. Differential proteome anal-ysis involved in AuNP synthesis needs to be carried out. Researchshould also be focused on the synthesis of protein and biomoleculeme-diated synthesis and characterization of AuNPs.

• Studies should also be focused on stability of AuNPs and means to pre-vent aggregation of AuNPs. Many a times these proteins involved insynthesis are reported to stabilize the NPs, besides their role in synthe-sis. The discovery of newer stabilizing agents for NPs is required fortheir applications in various fields.

• Gold nanoalloys and nanoconjugates synthesized from microbial ori-gin; study of their characteristics and properties is another interestingarea of research. Emphasis should also be given on the synthesis of bi-metallic and trimetallic gold alloys by using microbial sources and theconjugation of microbially synthesized AuNPs should also be carriedout using different antibiotics, proteins, antibodies, peptides and otherbiomolecules. The microbially synthesized alloys and conjugate parti-cles will be more ecofriendly and open up new avenues of research innanomedicine.

Acknowledgment

US is thankful to the University Grants Commission (UGC), NewDelhi, India for awarding the Dr. D. S. Kothari Post Doctoral Fellowship.RS and SW acknowledge UGC, New Delhi for awarding the Junior Re-search Fellowship (JRF). BAC acknowledges the financial support fromthe Board of College and University Development (BCUD), Universityof Pune (2011–2013). The authors also acknowledge the University ofPune for UPE Phase II: Focus Area: Biotechnology (2012–2017).

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