Biochemical Engineering Journal 43 (2009) 303–306 Contents lists available at ScienceDirect Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej A green low-cost biosynthesis of Sb2 O3 nanoparticles Anal K. Jha a , Kamlesh Prasad b , K. Prasad c,∗ a University Department of Chemistry, T.M. Bhagalpur University, Bhagalpur 812007, India Department of Food Engineering and Technology, Sant Longowal Institute of Engineering and Technology, Longowal, Sangrur 148106, India c University Department of Physics, T.M. Bhagalpur University, Lower Nathnagar Road, University Campus, Bhagalpur 812007, India b a r t i c l e i n f o Article history: Received 5 August 2008 Received in revised form 19 October 2008 Accepted 21 October 2008 Keywords: Nanoantimony trioxide Nanoparticle Bio-nanotechnology Green approach a b s t r a c t A green low-cost and reproducible yeast (Saccharomyces cerevisiae) mediated biosynthesis of Sb2 O3 nanoparticles is reported. The synthesis is performed akin to room temperature in the laboratory ambience. X-ray and transmission electron microscopy analyses are performed to ascertain the formation of Sb2 O3 nanoparticles. Rietveld analysis indicated that Sb2 O3 nanoparticles have face centered cubic (FCC) unit cell structure. Individual nanoparticles as well as a few number of aggregate almost spherical in shape having a size of 2–10 nm are found. Possible involved mechanism for the synthesis of nano-Sb2 O3 has also been proposed. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Nanomaterials are at the leading edge of the rapidly developing field of nanotechnology. Their unique size-dependent properties make these materials superior and indispensable in many areas of human activity [1]. Ranging from medicine, biology, food technology and electronics to aerospace engineering, nanomaterials hold prodigious promises. The synthesis of ultrafine oxide nanoparticles have been investigated using various methods such as sol–gel, hydrothermal, solvothermal, flame combustion, emulsion precipitation, fungus-mediated biosynthesis, etc. [2]. Antimony trioxide (Sb2 O3 ) is a semiconducting material and is excellent catalyst for the production of PET plastic used in the packaging of mineral water and soft drinks. Its safe use in the production of PET bottles has been confirmed by the World Health Organisation (2003) and the European Food Safety Authority (2004). Sb2 O3 greatly increases flame retardant effectiveness when used as a synergist in combination with halogenated flame retardants in plastics, paints, adhesives, sealants, rubber and textile back coatings [3,4]. It is known that Sb2 O3 /Sb2 O5 is the potential chemical for the synthesis of antimony gluconate which is considered to be the effective medicine against Kala azar (Visceral Leishmaniasis). Common salts of antimony are irritant, and thus an oral administration produces nausea, vomiting and diarrhoea; therefore they are administered parenterally. Also, it is a cumulative drug. Antimony compound are avoided in ∗ Corresponding author. Tel.: +91 641 2501699; fax: +91 641 2501699. E-mail address: k.prasad65@gmail.com (K. Prasad). 1369-703X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2008.10.016 case of pulmonary tuberculosis, jaundice, nephrites and dysentery [5]. Nanoscale antimony compounds could prove to be less toxic to the body because they can cross the renal barrier. Sb2 O3 is also used for several purposes such as fining agent or as degasser (to remove bubbles) in glass manufacturing, an opacifier in porcelain and enameling services, a white pigment in paints, etc. Recently, nanoparticles of Sb2 O3 have been synthesized in a controlled condition using a structure-directing surfactant cetyltrimethylammonium bromide [4]. Also, Ye et al. [6] have reported the successful synthesis of Sb2 O3 fibrils and tubules with length of up to several millimeters and diameters of nanometers to submicrometers using a vapor–solid synthetic route. However, the nanorods of Sb2 O3 have been prepared by using a microemulsion method for the system of sodium bis-2-ethylhexylsulfosuccinate–water–toluene [7]. The structure of a one-dimensional crystal of Sb2 O3 encapsulated within a single-walled carbon nanotube and conformation of the latter has been solved simultaneously by high-resolution transmission electron microscopy by Friedrichs et al. [8]. European Union has recently been issued the legislation on waste electrical/electronic equipment (WEEE) and restriction of hazardous substances (RoHS). To meet the requirements and growing technological demand, there is a need to develop a green approach for nanomaterials synthesis that should not use toxic chemicals in the synthesis protocol. It is observed that the interaction between inorganic nanoparticles and biological structures are one of the most promising areas of research in modern nanoscience and technology [9,10]. Yeast being a member of the class Ascomycetes (also called sac fungi) in kingdom fungi has been taken into regular use as media supplement in different culture 304 A.K. Jha et al. / Biochemical Engineering Journal 43 (2009) 303–306 procedures and this organism itself has been a very good source of different enzymes and vitamins. It seems from the literature survey that only one report [4] is available on the synthesis of Sb2 O3 nanoparticles. Therefore, keeping in view the above, in the present effort, the baker’s yeast (Saccharomyces cerevisiae) has been taken in order to assess its potential as putative candidate fungal genera for the transformation of Sb2 O3 nanoparticles (abbreviated hereafter n-Sb2 O3 ). Tolerance of the organism towards Sb2 O3 has also been assessed. Furthermore, we have tried to explore a cost-effective, green and reproducible approach for the purpose of scaling up and subsequent downstream processing. Also, an effort has been made to understand the nanotransformation mechanism of biosynthesis. 2. Materials and methods Nanoparticles of Sb2 O3 were prepared by using baker’s yeast. Yeast cells were allowed to grow as suspension culture in the presence of suitable carbon and nitrogen source for 36 h. This was treated as source culture. A small portion of it (25 mL) was filtered and diluted four times by adding 30% Et-OH containing nutrients. This diluted culture was again allowed to grow for another 24 h until it attains a light straw colour. Now, 20 mL of 0.025 M SbCl3 solution was added to the culture solution and it was heated on steam bath up to 60 ◦ C for 10–20 min until white deposition starts to appear at the bottom of the tube, indicating the initiation of transformation. The culture solution was then cooled and allowed to incubate at room temperature in the laboratory ambience. After 3–4 days, the culture solution was observed to have distinctly markable coalescent white clusters deposited at the bottom of the tube (Fig. 1). A remarkable change in pH was observed at this stage. The formation of Sb2 O3 nanoparticles was checked by X-ray diffraction (XRD) technique using a X-ray diffractometer (XPERT-PRO) with Cu K␣ radiation  = 1.5406 Å over a wide range of Bragg angles (20◦ ≤ 2 ≤ 90◦ ). Rietveld analyses [11] were carried on the samples to estimate the unit cell parameters, their crystal structure, profile matching, etc. The refinements were carried out using the program FULLPROF 2000 [12] under Windows XP together with WinPLOTR. TEM micrograph of nano-Sb2 O3 were obtained using Hitachi H-7500 transmission electron microscope at 20 K and 100 nm magnification. The specimen was suspended in distilled water, dispersed ultrasonically to separate individual particles, and two drops of the suspension was deposited onto holey-carbon-coated copper grids. 3. Results Fig. 2 depicts the observed, calculated and difference X-ray diffraction profiles for Sb2 O3 -nanoparticles after final cycle of refinement. Rietveld refinements were done on n-Sb2 O3 system, selecting the space group Fd3m (227). The Bragg peaks were modeled with pseudo-Voigt function and the background was estimated by linear interpolation between selected background points. It can be seen that the profiles for observed and calculated one are perfectly matching. XRD analyses indicated that n-Sb2 O3 has a face centered cubic (FCC) unit cell having the lattice parameter: a = 11.138 Å which is in the very good agreement with the literature report (PCPDF Nos. #72-1334 and #75-1565). The value of 2 comes out to be of the order of 3, which is considered to be very good for estimations. The profile fitting procedure adopted was minimizing the 2 function [11]. Fig. 3 shows the TEM micrograph at 100 nm of the Sb2 O3 nanoparticles being formed using Saccharomyces strain. The Fig. 1. Photograph showing deposited n-Sb2 O3 at bottom of the tube. Inset shows the yeast culture solution. micrograph clearly illustrates individual nanoparticles as well as a number of aggregates. The measurement of size was performed along the largest diameter of the particles. The particles are found almost spherical in shape having a size of the order of 2–10 nm. Fig. 4 shows the distribution of particle sizes which ranged from 2 to 10 nm. Majority of the n-Sb2 O3 were scattered with only a very few of them showing aggregates of varying sizes as observed under TEM. The difference in size is possibly due to the fact that the nanoparticles are being formed at different times, which may limit the nanoparticle size due to constraints related to the particles nucleating inside the organisms. Fig. 2. Rietveld refined pattern of n-Sb2 O3 in the space group Fd3m. Symbols represent the observed data points and the solid lines their Rietveld fit. A.K. Jha et al. / Biochemical Engineering Journal 43 (2009) 303–306 305 Fig. 4. Particle size distribution (%) of n-Sb2 O3 . 4. Discussion Fig. 3. TEM photograph of n-Sb2 O3 . The membrane-bound (as well as cytosolic) oxidoreductases and quinones might have played an important role in the process. The oxidoreductases are pH sensitive and work in alternative manner. At a lower value of pH, oxidase gets activated while a higher pH value activates the reductase [13,14]. Along with this, a number of simple hydroxy/methoxy derivatives of benzoquinones and toluquinones are elaborated by lower fungi (especially Penicillium and Aspergillus species) [15]. Yeast might be treasuring any other such quinones because it belongs to the same class of fungi Fig. 5. Schematic representation of a possible mechanism for the biosynthesis of n-Sb2 O3 . 306 A.K. Jha et al. / Biochemical Engineering Journal 43 (2009) 303–306 thereby facilitating the redox reactions due to its tautomerization. The transformation seems to be negotiated at two distinct levels, at the cell membrane level immediately after addition of the SbCl3 solution which triggers tautomerization of quinones and low pH-sensitive oxidases and makes molecular oxygen available for the transformation. Such a stress generated response had earlier been suggested in case of Candida glabarata cell, challenged with cadmium ion in form of elaboration of an enzyme phytochelatin synthase and a protein HMT-1 which effectively aborted the CdS nanocrystals from cytosol [16]. Once entered into the cytosol, the Sb3+ might have triggered the family of oxygenases harboured in the endoplasmic reticulum (ER), chiefly meant for cellular level detoxification through the process of oxidation/oxygenation [14]. This hypothetical mechanism of n-Sb2 O3 synthesis is illustrated in Fig. 5. Furthermore, in a few recent communications [17,18] surprisingly enough, the yeast has been placed in another class of micro-organisms and not with fungi. Authorities of mycology have placed yeast in phylum Ascomycota (sac fungi) in order Saccharomycetales and family Saccharomycetae to which our experimental organism Saccharomyces cerevisiae (or baker’s yeast) belongs. However, the term ‘yeast’ has no taxonomic standing and simply denotes a growth form much in the same way that of mangroves among the families of the vascular plants. Nevertheless, some groups of fungi are known for the presence of yeast stage in their life cycles, while others are characterized by their absence. The other members of the family include Pichia, Candida, Kluyveromyces and Dekkera (a yeast found in spontaneous fermentations of beer) [19]. Therefore, compared to MKY3 Candida and Aspergillus, the present procedure is less expensive more reproducible, emphatically least/non-toxic and a truly green approach. 5. Conclusion The present biosynthesis method is a green low cost approach, capable of producing Sb2 O3 -nanoparticles nearby room temperature. The synthesis of n-Sb2 O3 might have resulted due to tautomerisation of membrane-bound quinones or the pH-sensitive oxidoreductases. Alternatively, an activity of ER-bound family of oxygenases in the cytosol can also have a role in the process. Acknowledgements Authors are extremely grateful to the peers for critically evaluating the manuscript and for their constructive suggestions. References [1] O.V. Salata, Applications of nanoparticles in biology and medicine, J. Nanobiotechnol. 2 (2004) 1–6. [2] C.N.R. Rao, A. Müller, A.K. Cheetham, The Chemistry of Nanomaterials, Wiley–VCH Verlag GmbH & Co. KGaA, Weinheim, 2004, pp. 98–198. [3] B. Pillep, P. Behrens, U.-A. Schubert, J. 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