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Reviews in Advanced Sciences and EngineeringVol. 3, pp. 1–18, 2014

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Mechanism and Applications of MetalNanoparticles Prepared by Bio-Mediated ProcessLaura Castro, Ma Luisa Blázquez∗, Jesús Ángel Muñoz,Felisa González González, and Antonio Ballester

Department of Materials Science and Metallurgical Engineering, Complutense University of Madrid,Av. Complutense s/n, 28040 Madrid, Spain

ABSTRACT

The increasing demand and limited natural resources of noble metals make its recovery from dilute industrialwastes attractive, especially when using environmentally friendly methods. Nowadays, the high impact thatnanotechnology is having in both science and society offers new research possibilities. Metal nanoparticleshave attracted a great scientific interest due to their unique optoelectronic and physicochemical properties.These properties strongly depend on size, shape, crystallinity and structure. Consequently, there are a widerange of potential applications in diverse areas such as molecular diagnostics, electronics, catalysis, drugdelivery or sensing. Different physical and chemical methods have been employed for the synthesis of metalnanoparticles. However, they use harmful chemicals that may represent a risk to the environment and publichealth. Hence the need to develop environmentally friendly procedures to recover precious metals. The use ofbiological organisms in synthesis and assembly of nanoparticles has received increasing attention. Biosynthesisof noble metal nanoparticles has attracted scientists’ attention as a new clean, cost-effective and efficientsynthesis technique. There are several organisms capable of synthesizing nanoparticles such as bacteria,yeasts, actinomycetes, fungi and plants. Although biological formation of nanoparticles has been broadly studiedin recent years, the mechanisms involved in this process have not been yet clearly elucidated. This reviewremarks the importance of bionanotechnology, considering new advances in biological synthesis of noble metalnanoparticles, its mechanisms and its potential applications.

KEYWORDS:

CONTENTS1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12. What is Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 53. Nanoparticles Biosynthesis in the Environment . . . . . . . . . . . 64. Synthesis of Metal Nanoparticles Using Biomass . . . . . . . . . 7

4.1. Bacteria in Nanoparticle Synthesis . . . . . . . . . . . . . . . 74.2. Yeasts in Nanoparticle Synthesis . . . . . . . . . . . . . . . . . 94.3. Fungi in Nanoparticle Synthesis . . . . . . . . . . . . . . . . . 94.4. Plants in Nanoparticle Synthesis . . . . . . . . . . . . . . . . . 94.5. Algae in Nanoparticle Synthesis . . . . . . . . . . . . . . . . . 11

5. Mechanisms of Biosynthesis . . . . . . . . . . . . . . . . . . . . . . 126. Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

6.1. Biomedical Applications . . . . . . . . . . . . . . . . . . . . . . 146.2. Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156.3. Catalysts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156.4. Bioremediation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

7. Concluding Remarks and Research Challenges for Biosynthesis 16References and Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

∗Author to whom correspondence should be addressed.Email: [email protected]: xx xx xxxxAccepted: xx xx xxxx

1. INTRODUCTIONDevelopment of novel functional materials lies at the heartof nanotechnology. In turn, nanotechnology-enabled pro-cesses require our constant innovation and improvementin many materials adopted in chemical engineering opera-tions including durable reaction vessels and piping, activecatalysts and adsorbents, multifunctional thin films andcoatings, and porous solids and membranes. With thefast development of nanoscience and nanotechnology overthe past decade, researchers now are able to gain addi-tional control over particle size, crystal shape, struc-tural phase, and chemical and physical properties (suchas catalytic, optical, electronic, magnetic, hydrophobicand hydrophilic) for these materials. As a whole, sig-nificant advancements have been made across varioustechnological sectors of chemical engineering, includingmany emerging applications in pharmaceuticals, consumerproducts, electronics, chemical sensing, catalysis, watertreatment, as well as fuels and renewable energy.Nanostructured materials often present properties which

are unusual in bulk materials of the same composition.

Rev. Adv. Sci. Eng. 2014, Vol. 3, No. 3 2157-9121/2014/3/001/018 doi:10.1166/rase.2014.1064 1

Mechanism and Applications of Metal Nanoparticles Prepared by Bio-Mediated Process Castro et al.

Laura Castro is a Ph.D. student in the Department of Materials Science and MetallurgicalEngineering in the Complutense University of Madrid. Her research is focused on biologicalsynthesis of metallic nanoparticles and bioleaching and bioremediation using iron reducingmicroorganisms.

Ma Luisa Blázquez is full Professor in the Department of Materials Science and Metallurgi-cal Engineering, Complutense University of Madrid (Spain) since 2009. She has participatedin more than thirty research projects and has published more than one hundred originalresearch papers. Her research background has been focused on different areas in the fieldof Biohydrometallurgy: Bioleaching, Biosorption, Acid mine drainages, Bioremediation andBiological synthesis of nanoparticles.

Jesús Ángel Muñoz is a full Professor in the Department of Materials Science and Met-allurgical Engineering, Complutense University of Madrid. His research activities havebeen focused on different areas in the field of Biohydrometallurgy, including: bioleaching,biosorption, bioremediation and biosynthesis.

Felisa González González is full Professor in the Department of Materials Science andMetallurgical Engineering, Complutense University of Madrid. Her research activity hasbeen focused mainly on the field of Biohydrometallurgy. She has directed six Ph.D. disser-tations and participated in more than thirty national and international research projects. Shehas published more than one hundred scientific articles and was awarded with the Nationalprize for Nobel Researchers by the Spanish Royal Society of Chemistry.

Antonio Ballester is a Ph.D. in Chemistry (Complutense University of Madrid, Spain/1978)and Professor of Materials Science and Metallurgical Engineering (Complutense Univer-sity of Madrid, Spain/1988 to date). Major fields of scientific research: Bacterial leaching;Biosorption; Acid rock drainage; Natural attenuation of soil contamination; Iron reduc-ing microorganisms; Bioproduction of nanoparticles; Hydrometallurgy; Extractive metal-lurgy. Scientific and/or professional institutions: Royal Spanish Society of Chemistry/Spain;CIM/Canada; ASM International/USA. TMS/USA. “Agustin Plana” Award (1st Edition,2008) granted by CENIM-CSIC Spain in recognition of his research merits.

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Castro et al. Mechanism and Applications of Metal Nanoparticles Prepared by Bio-Mediated Process

Noble metal nanoparticles exhibit unique electronic andoptical properties that are critically related to their sizeand shape. These effects are the result of changes in thesurface plasmon resonance, the frequency at which con-duction electrons oscillate in response to the alternatingelectric field of incident electromagnetic radiation. How-ever, only metals with free electrons (essentially Au, Ag,Cu, and the alkali metals) possess plasmon resonances inthe visible spectrum, which give rise to such intense col-ors. Elongated nanoparticles (ellipsoids and nanorods) dis-play two distinct plasmon bands related to transverse andlongitudinal electron oscillations. The longitudinal oscilla-tion is very sensitive to the aspect ratio of the particles,so that slight deviations from spherical geometry can leadto impressive color changes. Apart from single particleproperties, the environment in which the metal particlesare dispersed is also of relevance to the optical proper-ties. The refractive index of the surrounding medium, aswell as the average distance between neighboring metalnanoparticles, has been shown to influence the spectralfeatures.1

The first synthesis of metallic gold nanoparticles mostprobably date back to the 5th or 4th century BC wheregold specimens were reported in China and Egypt.2 Theoptical properties of metal nanoparticles were exploitedfor coloration of glass, ceramics and pottery. One of themost fascinating examples of this technology is probablythe famous Lycurgus cup which can be seen at The BritishMuseum in London. This object, crafted by the Romansin the 4th century, features an amazing property of chang-ing color depending on the light shed on it. Reflected lightmakes it appear green, whereas in transmission a bright redcolor can be seen. Work of Barber evidenced the presenceof mixed Au–Ag particles of approximately 70 nm in theglass matrix of the vase, in fact, that alloy of gold and silverwas crucial for obtaining the scarlet red color.3 The opticalproperties of metal nanoparticles have long been of inter-est in physical chemistry, starting with Faraday’s investi-gations of colloidal gold.4 The optical properties of goldnanoparticles indeed filled Faraday with enthusiasm whenhe reported in 1857 the synthesis of colloidal solutions ofgold exhibiting colors ranging from ruby red to amethyst.He started to list the factors impacting the color of thosesolutions and evidenced that “the mere variation in the sizeof particles gave rise to a variety of resultant colors.” Manyapplications of this property have arisen recently.Nanomaterials are commonly synthesized using two

strategies: top–down and bottom–up (Fig. 1). In top–down methods, the bulk materials are gradually brokendown to nanosized materials. As a result, many top–downapproaches have been developed and applied for the syn-thesis of nanoparticles.Recently, mechanical milling has proved to be an

effective and simple technique without involving hightemperature treatment for the production of nanocrys-talline powders, with the possibility of obtaining large

quantities of materials with modified properties.5 In thistechnique, starting powder particles are trapped betweenhighly kinetic colliding balls and the inner surface ofthe vial, which causes repeated deformation, re-welding,and fragmentation of premixed powders resulting in theformation of fine, dispersed particles in the grain-refinedmatrix. During the milling operation, two essential pro-cesses affect the particle characteristics. First, the coldwelding process leads to an increase in average particlesize of the composite. In addition, the fragmentationprocess causes the breaking up of composite particles.Steady-state equilibrium is attained when a balance isachieved between these processes after a certain period ofmilling.The major use of the conventional ball milling is to

fracture the particles and to reduce the size, which is dif-ferent from the newly established high energy ball millingmethod. In this new method a magnet is placed close tothe cell to apply a strong magnetic pulling force on themagnetic milling balls, and therefore the impact energy ismuch higher than the conventional ball milling energy. Inaddition, different milling actions and intensities can beachieved by adjusting the cell rotation rate and the magnetposition.Etching can be used to generate complex particle

architectures by examining the opposite of the nanocrystalgrowth by using a chemical. A key strategy for controllingthe shape of single-crystalline nanoparticles is the abilityto distinguish between various crystallographic faces andgrowth directions.6

The electro-explosion is a process for the productionof nanoparticles whereby a wire is fed into a reactor,and subjected to a high-current, high-voltage microsecondpulse to cause it to explode. Sputtering is another processwhereby atoms are ejected from a solid target materialdue to bombardment of the target by energetic particles.7

Among the synthesis methods, sputtering represents themost economic and widely used physical depositionmethod.Laser ablation is a convenient way of generating

nanoparticles and their aggregates for laboratory studies.8

This method can generally only produce small amounts ofnanoparticles. However, laser ablation can vaporize mate-rials that cannot readily be evaporated. The purity of theaerosol product depends primarily on that of the carriergas and the target material. Since the laser beam is focusedon the target, contamination by materials released fromother parts of the apparatus is less likely. The handlingof the solid target is easier and often less hazardous thanusing metal organic compounds or compounds contain-ing chlorine or fluorine. Laser ablation allows control overthe ablated mass by varying pulse energy and frequency.The mass of material ablated can be measured by weighingthe collected aerosol and the target. Aerosols composed ofdifferent materials can be easily generated by changing thetarget material.

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SYNTHESIS OFMETAL NANOPARTICLES

TOP-DOWN METHODS

-Mechanical milling-Etching (chemical)

-Electro-explosion (thermal/chemical)-Sputtering (kinetic)

-Laser ablation (thermal)

BOTTOM-UP METHODS

-Supercritical fluid synthesis-Aerosol based processes

-Sol processes and sol-gel processes-Plasma or flames praying synthesis

-Green chemistry-Chemical vapour deposition

-Spinning-Pirolysis

-Atomic or molecular condensation

Fig. 1. Methods of nanoparticles production.

Some of the most popular methods to synthesizenanomaterials and the fabrication of nanostructures arebottom–up approaches.The sol–gel process is a wet-chemical technique, also

known as chemical solution deposition, widely usedrecently in the fields of materials science and ceramic engi-neering. Such methods are used primarily for the fabrica-tion of materials (typically a metal oxide) starting from achemical solution (“sol,” short for solution) which acts asthe precursor for an integrated network (or “gel”) of eitherdiscrete particles or network polymers.9

Supercritical fluid technology has become an importanttool of materials processing in the last two decades. Super-critical CO2 and H2O are extensively being used in thepreparation of a great variety of nanomaterials. Supercrit-ical carbon dioxide (scCO2) exhibits a hybrid of gas-likeand liquid-like properties. It can dissolve solutes like a liq-uid and yet possesses low viscosity, high diffusivity andzero surface tension like a gas. It is miscible with gases,and its solvation power can be tuned by changing tempera-ture and pressure. These properties make scCO2 an attrac-tive medium for delivering reactant molecules to the areas

with high aspect ratios, complicated surfaces and poorlywettable substrates, enabling deposition processes occur-ring in scCO2 to attain high uniformity and homogeneity.Unreacted materials and by-products from the scCO2 pro-cesses can be easily removed from the system by flush-ing the system with CO2 fluid, thus high purity becomesavailable. In addition, scCO2 is non-toxic, non-flammableand recyclable, therefore the liquid waste problem can beminimized.10

Inert gas condensation is frequently used to makenanoparticles from metals with low melting points.11 Themetal is vaporized in a vacuum chamber and then super-cooled with an inert gas stream. The supercooled metalvapor condenses into nanometer-sized particles, which canbe entrained in the inert gas stream and deposited on asubstrate or studied in situ.In chemical vapor deposition (CVD), vapor phase pre-

cursors favor nucleation of particles by deposition of afilm.12 In this approach, the precursors can be solid, liquidor gas at ambient conditions, but are delivered to the reactoras a vapor (from a bubbler or sublimation source, as neces-sary). CVD has some advantages over other methods: the

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process is relatively simple, it uses inexpensive equipment,and particle formation can be controlled by a variety ofprocess parameters like reactor temperature, precursor con-centration, or residence time in the reactor.In pyrolysis, a vaporous precursor (liquid or gas) is

forced through an orifice at high pressure and burned. Theresulting solid is air classified to recover oxide particlesfrom by-product gases. Pyrolysis often results in aggre-gates and agglomerates rather than singleton primary par-ticles. Spray pyrolysis use a nebulizer to use a nebulizerto directly inject very small droplets of precursor solution.Laser pyrolysis photothermal synthesis is an alternativethat uses the absorption of laser energy to heat the pre-cursors to induce reaction and homogeneous nucleation.13

Compared to heating the gases in a furnace, this allowshighly localized heating and rapid cooling, since only thegas (or a portion of the gas) is heated, and its heat capacityis small. Heating is generally done using an infrared (CO2)laser, whose energy is either absorbed by one of the pre-cursors or by an inert photosensitizer. Moreover, one candirectly spray liquid precursor into it. This process is gen-erally called flame spray pyrolysis. This method allows theuse of precursors that do not have sufficiently high vaporpressure to be delivered as a vapor.The injection of precursors in thermal plasma can also

deliver the energy necessary to cause evaporation of smallmicrometer size particles.14 This generally decomposes theprecursors fully into atoms, which can then react or con-dense to form particles when cooled by mixing with coolgas or expansion through a nozzle. The thermal plasmatemperatures are in the order of 10,000 K, so that solidpowder easily evaporates. Nanoparticles are formed uponcooling while exiting the plasma region.In spinning, the liquid spreads over a disk to form a

thin film due to the high centrifugal force. This methoduses a simpler type of equipment.15 The micromixing timeis short under rotation and this could provide uniformsupersaturation in the reactor and enhance homogeneousnucleation so that uniform distribution of the particle sizeis obtained. It was also shown that the spinning do notrequire as much power as a traditional continuous reactorto push the stirrer in the liquid. Furthermore, it is consid-ered a valid candidate for use in precipitation processes inindustrial applications with a very high production rate.Conventional physical and chemical methods are

extensively used to produce nanoparticles for a long time.Nevertheless, the high-energy consumption, the stabilityand the use of toxic chemicals is the subject of paramountconcern. These methods are unsuitable for the synthesisof ecofriendly and biocompatible metal nanoparticle. Theexploration of natural resources is a promising ecofrienlyalternative for physical and chemical methods.Green chemistry is defined as “utilization of a set of

principles that reduce or eliminates the use or generationof hazardous substances in the design, manufacture, and

application of chemical products.”16 Green nanoscienceand nanotechnology involves the application of theseprinciples.Green nanotechnology involves several goals:

(a) advancing the development of clean technologies thatuse nanotechnology,(b) minimizing potential environmental and human healthrisks associated with the manufacture and use of nanotech-nology products and(c) encouraging replacement of existing products withnew nanoproducts that are more environmentally friendlythroughout their life cycles.

Researchers are already taking the first steps towardsthis green nano vision. Some aim to re-engineer andreduce the environmental impacts of products. Others aretrying to directly help the environment by creating newkinds of solar cells, remediation methods and water filters.However, this review focuses on green manufacturing pro-cesses that make existing processes more efficient and lesshazardous, and outline new green methods for producingnanomaterials and other nanoproducts. Biological entitiesand inorganic materials have been in constant touch witheach other ever since appearance of life on Earth. Due tothis regular interaction, life could sustain on this planetwith a well-organized deposit of minerals. Recently scien-tists have become more and more interested in the interac-tion between inorganic molecules and biological species.Studies have found that many microorganisms can pro-duce inorganic nanoparticles through either intracellular orextracellular routes.

2. WHAT IS BIOSYNTHESIS?Nanobiotechnology is one of the most promising areasin modern nanoscience and technology. This emergingarea of research interlaces various disciplines of sci-ence such as physics, chemistry, biology, and materialscience.Biotechnology and nanotechnology are two of the 21st

century’s most promising technologies. Nanotechnologyis defined as the design, development and applicationof materials and devices whose least functional makeup is on a nanometer scale. Generally, nanotechnologydeals with developing materials, devices, or other struc-tures possessing at least one dimension sized from 1to 100 nanometers. Meanwhile, biotechnology deals withmetabolic and other physiological processes of biologi-cal subjects. Nanobiotechnology is the association of thesetwo technologies and can play a vital role in developingand implementing many useful tools in the study of life.17

Nanotechnology is very diverse, ranging from exten-sions of conventional device physics to completelynew approaches based upon molecular self-assembly,from developing new materials with dimensions on thenanoscale to investigating whether we can directly con-trol matter at the atomic level. This idea entails the

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application of fields of science as diverse as surface sci-ence, organic chemistry, molecular biology, semiconductorphysics, microfabrication, etc.The organisms used in nanoparticle formation vary

widely, from simple prokaryotic bacterial cells to complexeukaryotes. Biosynthesis of gold, silver, gold–silver alloy,selenium, tellurium, platinum, palladium, silica, titania,zirconia, quantum dots, magnetite and uraninite nanopar-ticles by bacteria, actinomycetes, fungi, yeasts and viruseshave been reported.The use of organisms in nanotechnology is develop-

ing rapidly because of the ease of handling and forma-tion of nanoparticles.18 Furthermore, biosynthesis of metalnanoparticles is an environmentally friendly method with-out the use of harsh toxic and expensive chemicals.Although biological methods are regarded as safe,

cost-effective, sustainable and clean processes, they alsohave some drawbacks in culturing of microbes and usebiomasses, which is time-consuming and difficult in pro-viding better control over size distribution, shape andcrystallinity. Additionaly, biological nanoparticles are notmonodispersible and its production rate is slow. These arethe main problems that have delayed the biological syn-thesis approaches.The most relevant aspects to be considered in the

biological process to synthesize highly stable and well-characterized nanoparticles are the following:19

(1) Selection of the suitable organisms. Researches havebeen focused on the important intrinsic properties of theorganisms such as enzyme activity and biochemical path-ways with the aim of choosing the best candidates formetal nanoparticles production. There is also the possi-bility of producing genetically engineered microbes thatoverexpress specific reducing agents and thereby, can con-trol the size and shape of biological nanoparticles.(2) Optimal conditions for cell growth and enzyme activ-ity. The nutrients, inoculums size, light, temperature, pH,mixing speed, and buffer strength are very importantpoints that should be controlled. The presence of sub-strates or related compounds in subtoxic levels from thebeginning of the growth would increase the activity of theenzymes.(3) Optimal reaction conditions. The yield and the pro-duction rate are important points to be considered toimplement the use of organisms for the synthesis of metalnanoparticle at industrial scale. Bioreduction conditions inthe reaction mixture should be carefully optimize. In addi-tion, substrate concentration, biocatalyst concentration,electron donor and its concentration, exposure time, tem-perature, buffer strength, mixing speed, and light need tobe controlled. Moreover, some investigations have shownthat the use of some complementary factors such as visi-ble light or microwave irradiation, and boiling could affectthe size, morphology, and rate of reaction.

The optimization of these factors is required to imple-ment these approaches in large scale and for commercial

applications. It is particularly interesting to developnew low cost processes for the synthesis of metallicnanoparticles.

3. NANOPARTICLES BIOSYNTHESISIN THE ENVIRONMENT

Inspiration from nature, where living organisms produceinorganic materials through a biological guided processknown as biomineralization, is adopted as a superiorapproach to nanomaterials assembly.20 The biomineraliza-tion processes exploit biomolecular templates that inter-act with the inorganic material at nanoscale, resulting inextremely efficient and highly controlled syntheses.Typical examples of biomineralized products include

siliceous materials synthesized by diatoms and sponges.Diatoms are unicellular algae that have the extraordi-nary capability to produce an enormous variety of biosil-icates structures. Each diatom species is characterizedby a specific biosilicate cell wall that contains regularlyarranged slits or pores in the size range between 10 and1000 nm (nanopatterned biosilica). An important compo-nent of biomineralization is the protein or peptide templatethat controls the shape and crystal structure of biomineralsas well as the assembly procedure. Biosilica morphogene-sis takes place inside the diatom cell within a specializedmembrane-bound compartment termed the silica deposi-tion vesicle. Silica formation from a silicic acid solutionis catalyzed by biosilica-associated peptides (silaffins) andlong-chain polyamines in diatoms.21

Other examples of inorganic, nanostructured biosyn-thetic minerals include Fe3O4, CaSO4 or BaSO4, which aresynthesized by a variety of microorganisms. These mineralsoften have special uses such as magnetic sensors in magne-totactic bacteria (Fe3O4), gravity sensing devices (CaCO3,CaSO4, BaSO4) and iron storage and mobilization (Fe2O3 ·H2O in the protein ferritin). Magnetotactic bacteria (Mag-netospirillum magnetotacticum, Magnetobacterium bavar-icum, Magnetospirillum gryphiswaldense) have specialorganelles called magnetosomes that contain magneticcrystals, which enable the cells to orient themselves alongthe magnetic field lines of the Earth.22 Magnetotactic bac-teria synthesize magnetic particles comprising iron oxide(Fe3O4), iron sulfides (Fe3S4), or both in their intracellularcompartment. The magnetic particles are aligned in chainswithin the bacterium, and function as biological compassneedles. This enables the bacterium to migrate along oxy-gen gradients in aquatic environments under the influenceof the Earth’s geomagnetic field.In natural systems, trace elements (heavy metals and

metalloids) are important environmental pollutants, andare toxic even at very low concentrations. Bacteria play acrucial role in metal biogeochemical cycling and mineralformation in surface and subsurface environments.23�24 Inthe presence of high concentrations of toxic metal ions,bacteria develop numerous detoxification mechanisms like

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dissimilatory oxidation or reduction of metal ions, com-plexation and/or precipitation, impaired transport systemand efflux system.25 Microorganisms have been used witha certain success in metal bioremediation of contami-nated subsurface environments. Plants have also showngreat potential in heavy metal accumulation and detoxifi-cation. Researches have been focused on the use of plantbiomass for metal removal from aqueous solutions, knownas biosorption, a very promising method for the removal ofcontaminants from effluents in an eco-friendly approach.However, its role in nanoparticle synthesis has recentlybeen studied.The natural occurrence of inorganic nanomaterials in

several organisms encourages the use of prokaryotic andeukaryotic systems as possible eco-friendly nanofactoriesfor nanoparticle synthesis routes alternative to chemicalmethods.

4. SYNTHESIS OF METAL NANOPARTICLESUSING BIOMASS

Even though many biotechnological applications, such asthe remediation of toxic metals,employ organisms such asbacteria26 and fungi,27 these organisms are recently discov-ered as possible eco-friendly nanofactories.28�29 Processesdevised by nature for the synthesis of inorganic materialson nano- and microlength scales have contributed to thedevelopment of a relatively new and largely unexploredarea of research based on the use of different organisms inthe biosynthesis of nanomaterials.30

The rate of intracellular particle formation and thereforethe size of the nanoparticles could, to an extent, be manip-ulated by controlling parameters such as pH, temperature,substrate concentration and exposure time to substrate.31

Efforts have also been made to manipulate the shapeand size of gold nanoparticles produced extracellularly bymicroorganisms through altering key growth parameters.32

This section provides an overview of the currentresearch worldwide on the use of biomass such as bacte-ria and actinomycetes (both prokaryotes), as well as algae,yeast, fungi, and plants (eukaryotes) in the biosynthesisof metal nanoparticles. Both unicellular and multicellularorganisms are known to produce inorganic materials eitherintra-or extracellularly (Table I).

4.1. Bacteria in Nanoparticle SynthesisInteractions between metals and microbes have beenexploited for various biological applications in thefields of bioremediation, biomineralization, bioleaching,and biocorrosion. The microbial synthesis of nanopar-ticles has emerged as a promising field of researchin nanobiotechnology interconnecting biotechnology andnanotechnology.Microbes are able to produce inorganic materials either

intra- or extracellularly in nanoscale dimensions. Micro-bial resistance to most toxic heavy metals is due to their

chemical detoxification as well as due to energy-dependention efflux from the cell by membrane proteins that func-tion either as ATPase or as chemiosmotic cation or protonanti-transporters. Alteration in solubility also plays a rolein microbial resistance. Therefore, microbial systems candetoxify the metal ions by either reduction and/or precipi-tation of soluble toxic inorganic ions to insoluble non-toxicmetal nanoclusters.33 Bacterial detoxification can be madeeither by extracellular biomineralization, biosorption, com-plexation or precipitation or intracellular bioaccumulation.Extracellular production of metal nanoparticles has morecommercial applications in various fields. Since the poly-dispersity is the major concern, it is important to optimizethe conditions for monodispersity in a biological processas it was exposed previously. In case of intracellular pro-duction, the accumulated particles are of particular dimen-sion and with less polydispersity.31

Early studies have revealed that Bacillus subtilis 168was able to reduce Au3+ ions to produce octahe-dral gold particles of nanoscale dimensions (5–25 nm)within bacterial cells by incubation of the cells withgold chloride under ambient temperature and pressureconditions.28�29

The first report on the biosynthesis of silver nanopar-ticles used Pseudomonas stutzeri AG259, isolated fromsilver mines, able to form single crystals with well-definedcompositions and shapes. These silver-containing crystalswere embedded in the organic matrix of the bacteria,growing up to 200 nm in size with equilateral trian-gle and hexagonal forms. The authors showed by TEMthat large quantities of silver accumulate when the bac-teria were cultured in a high concentration of AgNO3,and the majority was in the form of crystals. The EDXspectrum suggested the production of Ag2S and elementalAg.34

Nair and Pradeep reported that common Lactobacillusstrains found in buttermilk assisted the growth of micro-scopic gold, silver, and gold-silver alloy crystals of well-defined morphology.35

When growth-decoupled, resting cells of metal ionreducing bacteria, S. algae was incubated anaerobicallyin aqueous solution of H2PtCl6 at room temperature andneutral pH, it reduced PtCl−2

6 ions in the presence of lac-tate as the electron donor to metallic platinum chang-ing the color from pale yellow to black. It was foundplatinum nanoparticles deposited in the periplasmic spacebetween the inner and outer membranes of S. algae. Yetanother platinum group metal nanoparticle was producedby sulfate-reducing bacterium, Desulfovibrio desulfuri-cans NCIMB 8307. This bacterium anaerobically biore-duced and biocrystallized palladium (II) ions to palladiumnanoparticles on the surface of cells in the presence ofan exogenous electron donor as formate within minutes atneutral pH.36

De Windt and coworkers also showed that another iron-reducing bacterium, S. oneidensis MR-1, in the presence

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Table I. Metallic nanoparticles synthesized by different organism.

Organism Metal NP Location References

BacteriaBacillus subtilis 168 Au Intracellular [28, 29]Pseudomonas stutzeri AG259 Ag triangles and hexagons Intracellular [34]Lactobacillus Au, Ag, Au–Ag alloys Intracellular [35]Desulfovibrio desulfuricans Pd Intracellular [36]S. oneidensis Pd Intracellular [37]Rhodopseudomonas capsulata Au wires Extracellular [38, 39]Cupriavidus necator, Pseudomonas putida, Paracoccus denitrificans Pd (3–30 nm) Intracellular [41]Pseudomonas aeruginosa Au, Ag, Pd, Fe, Rh, Ni, Ru, Pt Extracellular [42, 43]Pyrobalaculum islandicum, G. sulfurreducens, Pyrococcus furiosus Au Extracellular [40]Morganella sp. Ag Intracellular [44]Bacillus licheniformis Ag Intracellular [45]

YeastYarrowia lipolytica NCIM 3589 Au Intracellular [46]Saccharomyces cerevisae Au, Ag Extracellular [47]Candida guilliermondii Au, Ag Extracellular [48]

FungiVerticillium Au, Ag Intracellular/extracellular [49, 50]Fusarium oxysporum Au, Ag, Pt Extracellular [51–53]Aspergillus flavus Ag Intracellular [54]Phoma Ag Extracellular [55]Trichoderma asperellum Ag Extracellular [56]Coriolus versicolor Ag Extracellular [57]Rhizopus oryzae Au Extracellular [58]

PlantsAlfalfa Au, Ag Intracellular [59, 60]Lemongrass (Cymbopogon flexuosus) Au triangular prisms Extracellular [61]C. annuum leaf extract Ag Extracellular [62]Aloe vera leaf extract Au triangles, Ag spheres Extracellular [63]Cinnamomum camphora leaf Au triangles, Ag spheres, Pd Extracellular [64]Jatropha latex Ag Extracellular [65]Sesbania drummondii Au Intracellular [66]Phragmites australis, Iris pseudacorus Cu Intracellular [67]Coffee and tea extract Ag, Pd Extracellular [68]Geranium (leaves, roots) Au, Ag Extracellular [69]Sugar beet pulp Au wires, triangles, rods Extracellular [70, 71]Gardenia jasminoides Ellis’ extract Pd Extracellular [72]Citrus limon leaf extract Ag Extracellular [73]Diopyros kaki leaf extract Pt Extracellular [74]Wood nanomaterials Pt Extracellular [75]Neem (A indica) leaf extract Au, Ag and bimetallic Au shell Extracellular [76]Psidium guajava leaf extract Au Extracellular [77]Citrullus colocynthis calli extract Ag Extracellular [78]

AlgaeChlorella vulgaris Au Intracellular [79]Spirulina platensis Au, Ag, Au/Ag bimetallic Extracellular [80]Sargassum wightii Au, Ag Extracellular [81, 82]Kappaphycus alvarezii Au Extracellular [83]Tetraselmis kochinensis Au Intracellular [84]

of formate as the electron donor reduced Pd(II) to Pd(0)nanoparticles on the cell wall and inside the periplasmicspace by changing the color of the biomass to black.37

Microbial synthesis of metal nanoparticles dependsupon the localization of the reductive components ofthe cell. When the cell wall reductive enzymes or solu-ble secreted enzymes are involved in the reductive pro-cess of metal ions then metal nanoparticles can be foundextracellularly.

Related to improve the control of shape and size inbiological methods, Rhodopseudomonas capsulata showedthe ability to produce gold nanoparticles in differentsizes, and the shape of gold nanoparticles was controlledby pH.38 R. capsulata, was capable of producing goldnanoparticles extracellularly and the gold nanoparticleswere quite stable in the solution. The aqueous chloroaurateions were reduced during exposure to the biomass and thecolor of the reaction solution turned from pale yellow to

8 Rev. Adv. Sci. Eng., 3, 1–18, 2014


purple. They have shown that when the pH was adjusted to4, a number of nanoplates were observed in addition to thespherical gold nanoparticles in the reaction solution. Sizeand morphology of gold nanoparticles might be affected bythe concentration of AuCl−4 ions. At lower concentration ofAuCl−4 ions as the substrate, spherical gold nanoparticles(10–20 nm) were synthesized exclusively, while at higherconcentrations, networked gold nanowires were producedin the aqueous solution.39

Among hyperthermophilic and mesophilic dissimilatoryFe(III) reducing bacteria and archea like Pyrobalaculumislandicum, Thermotoga maritime, S. algae, G. sulfurre-ducens and Pyrococcus furiosus gold was precipitated byreducing gold(III) to metallic gold in the presence ofhydrogen as electron donor. The precipitation occurredextracellularly due to the presence of Au(III) reductasesnear the outer cell surfaces of Fe(III) reducers.40

4.2. Yeasts in Nanoparticle SynthesisAmong the eukaryotic microorganism, yeasts have beenexploited mainly for the synthesis of semiconductors.In a classical study,85 demonstrated that yeasts such asS. pombe and C. glabrata produced intracellular CdSnanoparticles when challenged with cadmium salt in solu-tion. These biological quantum dots were capped bypeptides.On the other hand, Agnihotri et al. reported the bio-

logical formation of gold nanoparticles in the cell wall oftropical marine yeast, Yarrowia lipolytica NCIM 3589.46

Recently, the biosynthesis of gold and silver nanoparti-cles was investigated using the culture supernatant brothof the yeast Saccharomyces cerevisae47 and Candidaguilliermondii.48 Metal nanoparticles were formed aftergold and silver ions came in contact with the culture super-natant broth.

4.3. Fungi in Nanoparticle SynthesisFungi are eukaryotic, spore-producing, achlorophyl-lous organisms usually containing filamentous, branchedsomatic structures with hyphae surrounded by cell walls.Fungi can accumulate metals by physicochemical and bio-logical mechanisms including extracellular binding thoughmetabolites and polymers, such as specific polypeptides,and metabolism-dependent accumulation. Fungi are moreadvantageous compared to other microorganisms in manyways. Fungal mycelia can withstand flow pressure andagitation and other conditions in bioreactors compared tobacteria. The extracellular secretions of reductive proteinsare abundant and can be easily handled in downstreamprocessing.The nanoparticles formed inside the organism are usu-

ally smaller than extracellularly reduced nanoparticles. Thesize limit could be related to the particles nucleatinginside the organisms. First reported examples of biosyn-thesis of nanoparticles using fungi are the appearance of

gold and silver nanoparticles by reduction of metal ionsintracellularly. A distinctive purple color in the biomassof Verticillium after exposure to HAuCl4 solution indicatesthe formation of gold nanoparticles intracellularly.49

Mukherjee et al. reported the intracellular synthesis ofgold nanoparticles within Verticillium with the deposits ofthe metal clearly bound to the surface of the cytoplasmicmembrane. Verticillium spp. fungal biomass on exposureto aqueous silver nitrate solution also resulted in the accu-mulation of silver nanoparticles beneath the fungal cellsurface with a negligible amount on the solution.50

From the application point of view, it would be imper-ative to harvest the metal nanoparticles formed within thefungal biomass. It is possible to release the intracellu-lar metallic nanoparticles via ultrasound treatment of thebiomass-nanoparticles composite or via reaction with suit-able detergents. Nonetheless, it would be far more practicalif the metal ions exposed to the fungus could be reducedoutside the fungal biomass, leading to the formation ofmetal nanoparticle in the solution.An extracellular synthesis of silver nanoparticles has

been achieved with the fungus Phoma when the fungal cellfiltrate was exposed to an aqueous silver nitrate solutionat room temperature.55

The plant pathogenic fungal strain Fusarium oxysporumhas been widely studied. Certain fungal species such asFusarium oxysporum can synthesize metal nanoparticlesextracellularly by secretion of high quantities of proteinsand/or enzymes. Then, the rapid formation of highly stablegold52 and silver51 nanoparticles has been reported. Apartfrom individual metal nanoparticles, the bimetallic Au–Agalloy can be synthesized by F. oxysporum. Moreover, ithas been shown that when the biomass of F. oxysporumis exposed to equimolar solutions of HAuCl4 and AgNO3,highly stable Au–Ag alloy nanoparticles of varying molefractions can be achieved.86

Fusarium oxysporum fungal strain was screened andfound to be successful for inter-and extracellular produc-tion of platinum nanoparticles.87 Nanoparticles of varyingsize (10–100 nm) and shape (hexagons, pentagons, circles,squares, rectangles) were produced at both extracellularand intercellular levels by Fusarium oxysporum.Recently, the fungus Aspergillus flavus also resulted

in the accumulation of silver nanoparticles on the cellwall surface when incubated with silver nitrate solution.54

They showed the nanoparticles with variable shapes wereobtained but predominantly spherical and triangular.

4.4. Plants in Nanoparticle SynthesisThe use of plants in the recovery of noble metals from oremines and runoffs is known as phytomining. It is a cost-effective environmentally compatible method compared toconventional chemical methods.79 The use of plants andplant products as sustainable and renewable resources inthe synthesis of nanoparticles is more advantageous over

Rev. Adv. Sci. Eng., 3, 1–18, 2014 9


prokaryotic microbes, which needs expensive methodolo-gies for maintaining microbial cultures and downstreamprocessing.The natural phenomenon of heavy metal tolerance of

plants has interested researchers to investigate the relatedbiological mechanisms as well as physiology and geneticsof metal tolerance in hyperaccumulator plants. Gardea–Torresdey et al. were the first to report the formation ofgold and silver nanoparticles inside living plants.59�60 Theysynthesized gold and silver nanoparticles within live alfalfaplants by metal ion uptake from solid media. Differentmorphologies of gold particles were obtained by reactinggold(III) with alfalfa biomass including irregular shaped,fcc tetrahedral and hexagonal platelet particles which werelarger than decahedral and icosahedral multiple twinnedparticles.88

Armendariz et al. reported for the first time the bio-logical formation of rod-shaped nanoparticles.89 Theycharacterized the gold nanoparticles formed by wheatbiomass exposed to potassium tetracholoaurate solutionat different pH values at room temperature. It was con-cluded that wheat biomass was able to reduce Au(III)to Au(0) forming fcc tetrahedral, hexagonal, decahe-dral, icosahedral multitwinned, irregular, and rod-shapednanoparticles.Sharma and coworkers also observed the intracellu-

lar accumulation of stable gold nanoparticles in rootsand shoots of Sesbania drummondii seedlings. The reduc-tion process was presumed to be mediated by the pres-ence of secondary metabolites present in the cells. Thus,biomatrixed nanomaterial containing gold nanoparticleswas used as stable heterogeneous catalyst in the reductionof industrially toxic pollutant, 4-nitrophenol.66

There are not many reports about the biological synthe-sis of copper nanoparticles. Copper, which is an essentialelement in electron transport chain of cellular respirationcan be toxic in large amounts. Certain plants can pre-vent copper toxicity by hyperaccumulating cationic copperto organic ligands in tissues. In plants such as Phrag-mites australis and Iris pseudacorus, the copper toxicitywas tackled by the transformation of copper into metal-lic nanoparticles (Cu0) in and near roots with the assis-tance of arbuscular endomycorrhizal fungi in oxygenatedenvironment.67 These nanoparticles were spherical, cuboc-tahedron and hemispherical.The extraction of metal nanoparticles from either liv-

ing or non-living plants for further applications is not wellinvestigated, but studies are moving towards solving suchthese problems and finding best ways for extraction andpurification of the produced nanoparticles.19 Armendarizhave extracted gold nanoparticles from inactivated tissuesof wheat and oat biomass. The extraction process involvedthe use of cetyltrimethylammonium bromide (CTAB) andcitrate combined with sonication to transfer the nanopar-ticles from the biomass into aqueous solution. Moreover,

TEM analysis of the extraction solutions indicated the goldnanoparticles extracted at first had a smaller radius thanthose being extracted later. Researchers’ aim of extract thenanoparticles from the cells was achieved using physico-chemical methods including freeze-thawing, heating pro-cesses, and osmotic shock. It seems that these processesmay interfere with the structure of nanoparticles, and someevents such as aggregation, precipitation, and sedimenta-tion could happen. These may change the shape and sizeof nanoparticles and interfere with the suitable propertiesof nanomaterials. On the other hand, enzymatic lysis of theplant cells (e.g., cocktail of enzymes isolated from Tricho-derma longibrachiatum) containing intracellular nanopar-ticles could be used, but this method is expensive andcould not be used in up-scalable and industrial produc-tion of nanoparticles. In addition, a rigid cell wall aroundthe plant cells makes nanoparticle extraction difficult. Toavoid these problems, researchers make efforts to developthe extracellular production of nanoparticles.Triangular or spherical shaped gold nanoparticles could

be easily modulated by reaction of the sundried biomass ofCinnamomum camphora leaf with aqueous gold precursorsat ambient temperature.64 Silver nanoparticles and triangu-lar or spherical nanoparticles of gold could be easily con-trolled by simply adjusting the amount of dried biomass.In other words, the size dispersion of quasi-spherical sil-ver nanoparticles as well as triangular or spherical shapesof gold nanoparticles could be easily controlled by simplevariation of the amount of biomass reacting with aqueoussolution of metal salts.Therefore, the utilization of biomass wastes in the syn-

thesis of nanoparticles is interesting. Castro and coworkersdemonstrated that biomolecules present in the sugar beetpulp, a waste from the sugar industry, were involved in thereduction of gold ions to anisotropic gold nanostructuressuch as rods, triangular and hexagonal by changing thepH70 (Fig. 2). In addition, these researchers developed amethod to synthesize gold nanowires using sugar beet pulpcontrolling the concentration of precursor and the pH ofthe solution.71

Another dimension was added to the ‘green chemistry’approach for pure metal synthesis with the use of plantbroths. Biosynthesis of nanoparticles by plant extractsis currently under exploitation. Synthesis of noble metalnanoparticles using plant extracts is very cost effective,and therefore can be used as an economic and valuablealternative for the large-scale fabrication of metal nanopar-ticles. Extracts from plants may act both as reducing andcapping agents in nanoparticle synthesis. The bioreductionof metal nanoparticles by combinations of biomoleculesfound in plant extracts (enzymes, proteins, amino acids,vitamins, polysaccharides, and organic acids such as cit-rates) is environmentally benign, despite of the chemicalcomplexity. There are a huge amount of reports publishedin the last times in order to obtain nanoparticles and con-trol shape and size using plant extract. Some of the most

10 Rev. Adv. Sci. Eng., 3, 1–18, 2014


(a) (b)

Fig. 2. Biosynthesized nanostructures using plants: (a) nanotriangles and (b) nanowire.

remarkable and recent studies are going to be related inthese chapter.Extracts from different parts of the geranium (leaves,

roots) were used to produce extracellular gold and silvernanoparticles.69 In other study, lemongrass (Cymbopogonflexuosus) plant extract was used to obtain triangular goldnanoprisms complexed by aldehydes or ketones present inthe plant.61

Chandran and coworkers reported the biogenic goldnanotriangles and spherical silver nanoparticles were syn-thesized by a simple procedure using Aloe vera leaf extractas the reducing agent.63 This procedure offers control overthe size of the gold nanotriangle and thereby a way totune their optical properties, particularly the position ofthe longitudinal surface plasmon resonance.Apart from these, there are many recent examples

such us the biosynthesis of nanoparticles using gumolibanum (Boswellia serrata),90 piper betle91 or Rumexhymenosepalus92 extracts as an evidence of the growingnumber of the researchers’ attempts in this direction.Gold and silver nanoparticles are the most common

ones synthesized and used for many important applica-tions in emerging interdisplinary field of nanobiotechnol-ogy. For instance, most of the publications are referredto these metal nanoparticles. Nevertheless, the relevanceof the recovery and applications of other metals inducesthe development of new biological alternatives for theirnanoparticle production.The first report on platinum nanoparticle synthesis using

a plant extract is relatively new.74 The leaf extract ofDiopyros kaki was used as a reducing agent in aque-ous H2PtCl6 · 6H2O solution. Very recently, platinumnanoparticles with controlled shapes and sizes were syn-thesized using wood nanomaterials in aqueous phase with-out employing any other reductants, capping or dispersingagents.75 The obtained spherical and cubic Pt nanoparticlesand spherical Pt nanoclusters exhibit high activities in thecatalytic reduction of p-nitrophenol as a model reaction.

At present, the advances in fabrication of ultrafine pal-ladium nanoparticles have gained great importance due totheir application both in heterogeneous and homogeneouscatalysis, due to their high surface-to-volume ratio andtheir high surface energy. However, most of traditional pro-cesses were performed in the presence of various stabiliz-ers to prevent the formation of undesired agglomerates oraggregates of Pd nanoparticles. Additionally, there are fewreports concerning biological production of Pd nanopar-ticles by plant extract or biomass, where the biomasswas found to act as both reducing agent and stabilizer.Nadagouda and Varma showed production of Pd nanopar-ticles using coffee and tea extract.68 Aqueous extract of thefruit of Gardenia (Gardenia jasminoides Ellis) was usedin the bioreduction of palladium chloride to metallic pal-ladium nanoparticles.72 The biogenic fabrication of pal-ladium nanoparticles has also been reported by a simpleprocedure using broth of Cinnamomum camphora leaf.93

4.5. Algae in Nanoparticle SynthesisAlgae are eukaryotic aquatic oxygenic photoautotrophs,and some of them are able to accumulate various heavymetals. However, there are very few reports about biolog-ical synthesis of noble metal nanoparticles using algae.The dried alga Chlorella vulgaris, a single-celled green

alga, was found to have strong binding ability towardstetrachloroaurate ions to form algal-bound gold, which wassubsequently reduced to Au(0). Approximately 88% ofalgal-bound gold attained metallic state and the crystalsof gold were accumulated in the interior and exterior ofcell surfaces with tetrahedral, decahedral and icosahedralstructures.Spirulina platensis is an edible blue–green alga and the

dried alga was used for the extracellular synthesis of gold,silver and Au/Ag bimetallic nanoparticles.80 Recently, ithas been reported the synthesis of extracellular metal bio-nanoparticles using Sargassum wightii82 and Kappaphy-cus alvarezii.83 Also, Senapati and coworkers reported

Rev. Adv. Sci. Eng., 3, 1–18, 2014 11


the intracellular production of gold nanoparticles usingTetraselmis kochinensis.84

5. MECHANISMS OF BIOSYNTHESISWhile a large number of microbial species and eukary-otes are capable of producing metals, the mechanism ofnanoparticle biosynthesis has not been well established.The metabolic complexity of the organisms complicatesthe analysis and identification of active species in thenucleation and growth of metal nanoparticles.The elucidation of the biochemical pathways is nec-

essary to develop a rational approach to nanoparticlesbiosynthesis. A number of issues need to be addressedfrom the nanotechnology and microbiological points ofview before such biosynthetic procedures can competewith the conventional protocols.Researchers have focused their attention on under-

standing the biological mechanisms and enzymatic pro-cesses of nanoparticle biosynthesis as well as detectionand characterization of biomolecules involved in the syn-thesis of metal nanoparticles.33 Many biomolecules suchas proteins/enzymes, amino acids, polysaccharides, alka-loids, alcoholic compounds, and vitamins can be involvedin bioreduction, formation, and stabilization of metalnanoparticles.The majority of metals are toxic to most microorgan-

isms, especially silver ions. Despite of this fact, prokary-otic bacteria have been extensively used for the synthesisof metallic nanoparticles. Some bacteria are resistant andmetals do not cause any effect on them. This was associatedto the accumulation of nanoparticles in intracellular spacesin order to reduce the toxicity. Klaus et al. suggested thatthe formation and accumulation of silver outside the cellu-lar membrane of bacteria, after reacting with H2S gas pro-duced by Pseudomonas, as a mechanism to avoid its toxiceffect.34 A step forward to understand the mechanism ofsilver nanoparticles formation involves the study of silverresistance by Morganella spp. at the phenotypic and geno-typic level. Parikh identified three major gene homologuesfrom Morganella spp. The gene homologue of silE wasfound to be highly similar to reported silE, which encodes asilver-binding protein. silP and silS gene homologues alsoshowed a high percentage similarity with reported silver-resistance genes. It indicated that silver-resistance machin-ery might have a similar role to play inMorganella spp. andcould be associated with silver nanoparticles synthesis.44

Moreover, the absence of such activity in closely relatedbacterial genera reinforced the uniqueness and specificityof this bacterium to silver cations.Some studies have indicated that NADH and NADH

dependent nitrate reductase enzymes are important factorsin the biosynthesis of metal nanoparticles. Gold nanoparti-cles with different sizes and shapes have been synthesizedusing Rhodopseudomonas capsulate. In this case, the bac-teria show a resistance to gold ion and gold nanoparticles.

The AuCl−4 ions could bind to the biomass through themain groups of secreted enzymes. Bacterium R. capsulateis known to secrete cofactor NADH- and NADH depen-dent enzymes. The bioreduction of gold ions was foundto be initiated by the electron transfer from NADH byNADH-dependent reductase as electron carrier, as alreadydescribed for other microorganisms.94�95

Similarly, Bacillus licheniformis is known to secretethe cofactor NADH and NADH-dependent enzymes, espe-cially nitrate reductase, which might be responsible for thebioreduction of silver ions to silver nanoparticles.45 Theauthors also reported the optimization of production ofnitrate reductase from B. licheniformis. The particles syn-thesized using the optimized enzyme activity ranged from10 to 80 nm.Fungi can accumulate metals by physicochemical and

biological mechanisms including extracellular binding bymetabolites and polymers, binding to specific polypep-tides, and metabolism-dependent accumulation. In somecases, as it have been described for extracellular reductionof metallic ions by bacteria, an enzymatic process seems tobe responsible for nanoparticle production. Protein assaysindicate that an NADH-dependent reductase, is also themain responsible factor of biosynthesis processes in fungi.This reductase gains electrons from NADH and oxidizesit to NAD+. The enzyme is then oxidized by the simulta-neous reduction of metal ions.Kumar et al. have demonstrated enzymatic synthesis

of silver nanoparticles using �-NADPH-dependent nitratereductase purified from F . oxysporum.95 Protein assaysindicate that an NADH-dependent reductase, is the mainresponsible factor of biosynthesis processes. The enzy-matic route of in vitro synthesis of silver nanoparticlesby NADPH-dependent nitrate reductase from F. oxyspo-rum with capping peptide, phytochelatin was demonstratedrecently and the mechanistic aspect was explained.94 Apartfrom enzymes, quinine derivates, such as naphthoquinonesand anthraquinones, also act as redox centers in the reduc-tion of silver nanoparticles. A similar finding was alsoreported in the reduction of gold(III) chloride to metal-lic gold by NADPH-dependent sulfite reductase and phy-tochelatin.In addition, one other important enzyme that is respon-

sible for this reduction in some microorganisms is nitratedependent reductase. In Fusarium oxysporum, this enzymeis conjugated with an electron donor (quinine), reducesthe metal ion, and changes it to elemental form. In thecase of rapid extracellular synthesis, because the reduc-tion happens in very few minutes, complex electron shuttlematerials may be involved in the biosynthesis process.The fungus Aspergillus flavus also accumulate silver

nanoparticles on the surface of its cell wall when incu-bated with silver nitrate solution.54 Extracellularly pro-duced nanoparticles were stabilized by the proteins andreducing agents secreted by the fungus. A minimum offour high molecularweight proteins released by the fungal

12 Rev. Adv. Sci. Eng., 3, 1–18, 2014


biomass have been found in association with nanoparti-cles. One of these was strain specific NADH-dependentreductase.Extracellular synthesis of gold nanoparticles using Rhi-

zopus oryzae has been reported recently and the authorsconcluded that the gold nanoparticles are produced bysurface-bound protein molecules that act as reducing andstabilizing agents.58 Biosynthesis of nanocrystalline sil-ver particles by Trichoderma asperellum56 and Coriolusversicolor57 were reported and the result confirms sil-ver nanoparticle stabilization by fungal protein. This bondcan be due to electrostatic attraction between free aminegroups or cysteine residues in the proteins with carboxy-late groups in enzymes present in the cell wall of myceliabecause of the evolution of S H stretching band.

In the case of intracellular formation of gold nanopar-ticles by algal biomass, Mandal and coworkers have sug-gested that metal ions are trapped on the surface of thecells via electrostatic interaction between the ions and neg-atively charged carboxylate groups present in the cell sur-face. It was also mentioned that enzymatic reduction ofthese ions leads to the formation of nuclei which subse-quently grow through further reduction of metal ions andaccumulation of these nuclei.96

The presence of various secondary metabolites,enzymes, proteins and/or other reducing agents withelectron-shuttling compounds is usually involved in thesynthesis of metal nanoparticles by plant components. Inbioaccumulation, the localization of nanoparticles is basedon the presence of particular enzymes or proteins involvedin it. The recovery of these nanoparticles from plant tissuesis tedious and expensive and needs enzymes to degradethe cellulosic materials which surrounds it. Thus, the syn-thesis of various metal nanoparticles using plant extractsis easy in downstream processing and in scaling up ofnanoparticles. Biosynthesis of metal nanoparticles usingdead biomass such as plant extracts or industrial wastesproduce extracellular nanoparticles and the process is notcontrolled enzymatically. Biosynthesis of nanoparticles byplant extracts is under exhaustive research; however, anexact mechanism for the synthesis of nanoparticles usingplants has not yet been elucidated although several hypo-thetical mechanisms have been proposed.17�97

In the extracellular synthesis of silver and gold nanopar-ticles using plants, biomolecules act as reducing agents,and the heterocyclic compounds as capping agents for thenanoparticles. Different studies point out that polysaccha-rides and other organic and water-soluble compounds areinvolved in the reduction of the metallic ions and the sta-bilization of nanoparticles. Complimentary investigationsin the biosynthesis of nanoparticles have explained thatC. annuum extract contains proteins with amine groups,which play reducing and controlling role during the for-mation of silver nanoparticles in the solutions.62 Recently,focusing on the role of peptides for the synthesis of

nanoparticles, Jatropha latex was used as reducing andcapping agent to form silver nanoparticles. The data basedon this plant reveal that the major peptide constituents ofthe latex are curcacycline A (cyclic octapeptide) and cur-cacycline B (cyclic nonapeptide) and these molecules wereinvolved in the reduction of silver ions to silver nanopar-ticles and in their stabilization.65 The authors suggested apossible mechanism to form silver nanoparticles. Firstly,the silver ions are entrapped in the core structure of thecyclic peptide. After that, the ions are reduced and stabi-lized in situ by the amide group of the host peptide andadditionally, the enzyme curcain (from the latex) stabilizessilver nanoparticles.Moreover, it is possible to stabilize the nanoparticles by

reducing sugars and/or terpenoids present in the neem (A.indica) leaf broth.76 In the same direction, the use of leafextract from Psidium guajava gave stable polyshaped goldnanoparticles. It was demonstrated that flavonoids in theextracellular solution from the leaves were responsible forthe biosynthesis of the gold nanoparticles.77

Despite most of the biological methods performed withorganisms lead to gold and silver nanoparticles production,the increasing demand and limited natural resources forindustrially important platinum-group metal catalysts ren-der the recovery from secondary sources such as industrialwaste economically interesting.Govender et al.98 studied the bioreduction of different

platinum salts using hydrogenase isolated from cultures ofF. oxysporum. It was suggested that H2PtCl6 may act as anelectron acceptor during the redox mechanism of a hydro-genase from sulfate reducing bacteria.99 It was also sug-gested a direct electron transfer between metal/metal ionsand the enzyme.100 A network of hydrophobic channelsexisted between the active site and the molecular surfacein the Ni-Fe hydrogenase of Desulfovibrio.101 These chan-nels served as a passage for metal ions, and even thoughthe diameter of these channels was about 0.45–0.60 nm,which was too small for H2PtCl6, it was not too small forPtCl2. It is believed, as shown in Figure 6, that H2PtCl6is rapidly reduced by a passive two-electron transfer pro-cess into a Pt(II) species at a remote site on the molecularsurface of the enzyme. Then, Pt(II) migrates to the activeregion through the network of channels and is reduced, bya second, slower two-electron reduction into Pt(0).Moreover, Bunge et al.41 showed that the ability of bac-

teria to support the formation of Pd(0) catalysts is notrestricted to a limited number of dissimilatory metal reduc-ing bacteria but is extended to a broader spectrum ofGram-negative bacteria, which can likewise act as tem-plates for the immobilization and subsequent reduction ofPd(II). This work strengthen the concept of cellular inter-faces acting as nucleation sites of Pd(0) metal depositionand as scaffolds for the growth of Pd(0) crystals. ThePd(0) nanoparticle formation was not restricted to livingcells, or to cells with active hydrogenases. It occurred bothon pasteurizedcells and on autoclaved cells with inactive

Rev. Adv. Sci. Eng., 3, 1–18, 2014 13


hydrogenases. In consequence, there was no evidence forthe involvement of active hydrogenases or the participationof other active enzymes in nucleating Pd(II) reduction andit was postulated a hydrogenase-independent mechanism,unlike the findings with Desulfovibrio spp.102 Althoughactive enzymes appear not to be required, it is possible thatthe coordination of Pd(II) to chemical groups on the cellsurface contributes to nucleating the reduction process.

6. APPLICATIONSDiscoveries in the past decade about inorganic nanopar-ticles have demonstrated that the electromagnetic, opticaland catalytic properties of noble-metal nanoparticles arestrongly influenced by shape and size. This has motivatedan upsurge in research on the synthesis routes that allowbetter control of shape and size for various nanotechno-logical applications. In this section we show some of thenovel properties and applications of the most widely usedmetal.Living and dead organisms have huge potential for the

production of nanoparticles/nanodevices with wide appli-cations. By using the organisms from simple bacteria tohighly complex eukaryotes in the reaction mixture, theproduction of nanoparticles/nanodevices with controlledshape and size can be achieved. Nano-biotechnology isat its first steps but various examples of this technologyand their use would attract the attention of people towardsits applications. In the ever expanding field of nanomate-rial research, metal nanoparticle received particular atten-tion due to their wide application in catalysis, electronics,sensing, photonics, environmental clean-up, imaging, bio-labelling, and drug delivery.

6.1. Biomedical ApplicationsBiomedical applications of metal nanoparticles have beendominated by the use of nanobioconjugates. Develop-ment of nanodevices using biological materials and theiruse in wide array of applications on living organismshas recently attracted the attention of biologists towardsnanobiotechnology.The applications of biogenic nanoparticle in

biomedicine are interesting because of the biocompati-bility. While the field of biosynthesized nanoparticles isrelatively new, researchers have already started explor-ing their use in applications such as probes for electronmicroscopy to visualize cellular components, targeted drugdelivery (vehicle for delivering drugs, proteins, peptides,plasmids, DNAs, etc.), cancer treatment, gene therapy andDNA analysis, antibacterial agents, biosensors, enhancingreaction rates, separation science, and magnetic resonanceimages. Here, we provide some examples to illustratethese applications.Since the 1970s, researchers have been trying to develop

systems capable of transporting active substances to spe-cific regions of the body for the treatment of many

diseases and the small size of the nanoparticle make themsuitable candidates. Nanoparticles were for targeted oraldrug delivery to the inflamed gut tissue in inflamma-tory bowel disease and found that this strategy of localdrug delivery showed improvement compared with exist-ing delivery devices.Gold and its compounds have long been used as medic-

inal agents throughout the history of civilization with itsearliest record dating back to 5000 years ago. The surfacesof gold nanoparticles can be readily modified with ligandscontaining functional groups such as thiols, phosphines,and amines, which exhibit affinity for gold surfaces. Goldnanoparticles have emerged as a promising scaffold fordrug and gene delivery that provide a useful complementto more traditional delivery vehicles. The combination oflow inherent toxicity, high surface area, stability, and func-tion tunability provides them with unique attributes thatshould enable new delivery strategies.103 Although thereis still a long way to go in this application, the anti-angiogenic properties of gold nanoparticles, as well as theeffects of gold nanopaticles in ovarian cancer, in multiplemyeloma, B-chronic lymphocytic leukemia, and the appli-cation in arthritis have been reported.104

Moreover, triangular gold nanoparticles can be used forhyperthermia cancer treatment by controlling the lengthof nanotriangles edges produced using Aloe vera leafextract.63 Hyperthermia cancer treatment involves admin-istering nanoparticles into the body, specifically at cancertissue sites. Local heating at specific sites is enabled bymeans of an external electromagnetic field.Silver nanoparticles have been widely used as a

novel therapeutic agent extending its use as antibacte-rial, antifungal, antiviral and antiinflammatory agent. Theantimicrobial activity of silver compounds is well known.Colloidal silver, however, was only discovered at thebeginning of the 20th century and with the growing resis-tance of microorganisms to antibiotics has renewed theinterest in biological effects of this form of silver.With the prevalence and increase of microorganisms

resistant to multiple antibiotics, silver based antisepticshave been emphasized in recent years. Silver nanoparticleswere biosynthesized using a great number of organisms.The nanoparticles were also evaluated for their increasedantimicrobial activities with various antibiotics againstGram-positive and Gram-negative bacteria. The antibacte-rial activities of ampicillin, kanamycin, erythromycin, andchloramphenicol were increased in the presence of silvernanoparticles against test strains. The highest enhancingeffect was observed for ampicillin against test strains. Thecombination of antibiotics with silver nanoparticles hasbetter antimicrobial effects and provided helpful insightinto the development of new antimicrobial agents. Duránand coworkers showed that extracellularly produced silvernanoparticles using Fusarium oxysporum can be incorpo-rated into textile fabrics to prevent or minimize infection

14 Rev. Adv. Sci. Eng., 3, 1–18, 2014


with pathogenic bacteria such as Staphylococcus aureus.105

Also in the textile application of nanotechnology, lemonsilver nano-dyed cotton and silk fabric pieces were put onFusarium and Alternaria culture.73 Substantial inhibitionof both the fungal species was obtained in terms of growthrestriction.Satyavani et al. reported biomedical potential of silver

nanoparticles synthesized from calli extract of Citrul-lus colocynthis on human epidermoid larynx carcinoma(HEp-2) cell line.78 Biosynthesized silver nanoparticlespossess considerable anticancer effect compared with com-mercial nanosilver by decreasing progressive developmentof tumor cells. Silver nanoparticles caused cell deaththrough apoptosis. The use of silver nanoparticles shouldemerge as one of the novel approaches in cancer therapyand, when the molecular mechanism of targeting is betterunderstood, the applications are likely to expand further.In addition, Bacillus licheniformis produces silver

nanoparticles and these nanoparticles have the potentialof anti-angiogenic.106 Bovine retinal endothelial cells weretreated with different concentrations of silver nanoparticlesfor 24 h in the presence and absence of vascular endothe-lial growth factor, and 500 nM (IC50) silver nanoparticlesolution was able to block the proliferation and migrationof bovine retinal endothelial cells. The cells showed a clearenhancement in caspase-3 activity and formation of DNAladders, evidence of induction of apoptosis. The resultsshowed that silver nanoparticles inhibit cell survival. It isanticipated that nanoparticle-mediated targeted delivery ofdrugs might significantly reduce the dosage of anticancerdrugs with better specificity, enhanced efficacy, and lowtoxicities.

6.2. SensorsNanoparticles possess interesting electronic and opticalproperties and can be used in biosensor applications.The sensitivity and performance of biosensors have beenimproved by using nanomaterials. Use of nanomaterialsin biosensors allows the use of many new signal trans-duction technologies in their manufacture. Because oftheir submicron size, nanosensors, nanoprobes and othernanosystems are revolutionizing the fields of chemicaland biological analysis, to enable rapid analysis of mul-tiple substances in vivo. Many new signal transductiontechnologies have been introduced in biosensors, bio-probes and other biosystems using nanomaterials producedthrough living organisms.107

The gold nanotriangles with unique and highlyanisotropic planer shapes might find application in photon-ics, optoelectronics, and optical sensing. Furthermore, theeffect of different organic solvent vapors like methanol,benzene and acetone on the conductivity of tamarind leafextract reduced gold nanotriangles suggests the applicationof gold nanotriangles to future chemical sensors.108

Zheng et al. reported that Au–Ag alloy nanoparticlesbiosynthesized by yeast cells were applied to fabricate a

sensitive electrochemical vanillin sensor.109 Electrochemi-cal investigations revealed that the vanillin sensor based onAu–Ag alloy nanoparticles-modified glassy carbon elec-trode was able to enhance the electrochemical responseof vanillin for at least five times. Under optimal work-ing conditions, the oxidation peak current of vanillin atthe sensor linearly increased with its concentration in therange of 0.2–50 �M with a low detection limit of 40 nM.This vanillin sensor was successfully applied to the deter-mination of vanillin from vanilla bean and vanilla tea sam-ple, suggesting that it may have practical applications invanillin monitoring systems.In other study, AuNP-based glucose oxidase (GOx)

biosensors were developed based on observations that goldnanoparticles can increase the enzyme activity of GOx.110

The linear response range of the glucose biosensor is20 �M to 0.80 mM glucose with a detection limit of17 �M (S/N = 3). This type of biosensor was successfullyapplied to determine the glucose content in commercialglucose injections.

6.3. CatalystsNanoparticles have been widely used to improve variousreaction rates as reductants and/or catalysts due to theirlarge surface areas and specific characteristics.Yong and coworkers studied the catalytic properties of

chemically reduced Pd(0) and bioPd(0) by measuring theamount of hydrogen released during a test reaction withhypophosphite.36 This procedure was also used in case ofthe Pd nanoparticles formed in the bacterial surface ofC. necator.41 Cell-free controls exhibited a significantlyhigher rate of hydrogen production. In consequence, it wassuggested that increasing the biomass/ Pd(II) ratio of C.necator during Pd(II) reduction leads to a higher frac-tion of periplasmic Pd(0) compared to extracellular Pd(0)and resulted bioPd(0) with progressively lower catalyticcapacity.Growth of Sesbania seedlings in chloroaurate solution

resulted in the accumulation of gold with the formationof stable gold nanoparticles in plant tissues.66 The cat-alytic function of the nanoparticle-rich biomass was sub-stantiated by the reduction of aqueous 4-nitrophenol. Thisis the first report of gold nanoparticle-bearing biomatrixdirectly reducing a toxic pollutant. This will help to retainthe nanoparticles for continuous usage in bioreactors. Alsocatalytic performance of the biosynthesized Pd nanoparti-cles using Gardenia jasminoides Ellis’ water crude extractwas investigated by hydrogenation of p-nitrotoluene.72 Thecatalysts showed a conversion of 100% under conditionsof 5 MPa, 150 �C for 2 h. The selectivity of p-methyl-cyclohexylamine achieved 26.3%. The catalyst was recy-cled five times with no agglomeration and maintainedactivity, which was attributed to the appropriate protectionof the antioxidants. On the basis of the study, it appearsto be a new promising biosynthetic nanocatalyst for thedevelopment of an industrial process.

Rev. Adv. Sci. Eng., 3, 1–18, 2014 15


6.4. BioremediationIn the previous sections, it was described the incorpora-tion of metal nanoparticles to the life, for example, asantibacterial agent in cotton fabrics. However, also impor-tant is the treatment of the effluents derived from theseprocesses, by recovering the silver nanoparticles using abiotechnological approach. The bacteria involving Chro-mobacterium violaceum, which is able to metabolize orstore metal ions, was involved in the bioremediation ofeffluents containing silver in order to avoid any environ-ment damage. C. violaceum produces around 1–4 mM freecyanide and it can metabolize several metals as cyanidecomplex. Among these metals are gold, nickel, and silver.This treatment was based on biosorption and showed to bevery efficient.105

Recently, synthesis of Pt-, Ru-, and Pd nanoparti-cles, their structure and catalytic properties were underconsideration. In order to solve environmental problems,nanocatalysts were investigated in processes of oxida-tive degradation of phenol and reductive denitrification ofnitrates for purification of sewage and natural water.111

7. CONCLUDING REMARKS ANDRESEARCH CHALLENGESFOR BIOSYNTHESIS

The emerging biosynthesis using living or dead biomassand derived products is undoubtedly an important fieldwhich faces considerable research challenges if the aimis to achieve benefit from nanotechnology minimizing theimpact on human health and the environment. It has beenproposed as a cheap and effective alternative biotechnol-ogy. Despite the progress, there are still some researchquestions to solve.112

• Most of these methods are still in a development stageand the problems experienced are stability and aggregationof nanoparticles, control of crystal growth, morphology,size and size distribution.• It is necessary to predict biological impacts, ecologicalimpact, and degradation at end-of-life of the nanoparti-cles. To achieve this aim, these methods should producewell-defined structures and purity profiles. The surfacechemistry of biogenic nanoparticles should be properlyrecognized.• An important challenge is scaling up for production levelprocessing. The large-scale is interesting because of thelack of surfactants, templates, or other auxiliary substancesto stabilize and control nanoparticle shape which could betoxic and persist as residual contaminants in the product.The use of continuous-flow microreactors should be fur-ther examinated because they have shown advantages fornanoparticles production.• The understanding of nanoparticle formation mecha-nisms and reaction stoichometries has to be improved.Nowadays, little is known about the mechanistic details ofthese transformations but this knowledge is necessary for

an economic and rational development of new syntheticmethods. For example, genetic engineering techniques canpotentially be used to improve the particle properties andto control their composition.

The shift from bacteria to fungi and plants as a means ofdeveloping natural “nanofactories” has the added advan-tage that downstream processing and handling of thebiomass would be much simpler. At present, the varia-tion of composition using biosynthesis of nanomaterialsis extremely limited and confined to metal, some metalsulfide, and very few oxides. An extension of the proce-dures to enable reliable synthesis of nanocrystals of otheroxides (TiO2, ZrO2, etc.), nitrides, carbides, etc., couldmake biosynthesis a commercially feasible option. Greennanosynthesis is an early stage but further research wouldallow develop the approach and implement the applicationsin the future.

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