Applied Microbiology and Biotechnology

, Volume 97, Issue 2, pp 859–869 | Cite as

A biomimetic approach towards synthesis of zinc oxide nanoparticles

  • Navin Jain
  • Arpit Bhargava
  • Jagadish C. Tarafdar
  • Sunil K. Singh
  • Jitendra Panwar
Environmental biotechnology

Abstract

Using natural processes as inspiration, the present study demonstrates a positive correlation between zinc metal tolerance ability of a soil fungus and its potential for the synthesis of zinc oxide (ZnO) nanoparticles. A total of 19 fungal cultures were isolated from the rhizospheric soils of plants naturally growing at a zinc mine area in India and identified on the genus, respectively the species level. Aspergillus aeneus isolate NJP12 has been shown to have a high zinc metal tolerance ability and a potential for extracellular synthesis of ZnO nanoparticles under ambient conditions. UV–visible spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction analysis, transmission electron microscopy, and energy dispersive spectroscopy studies further confirmed the crystallinity, morphology, and composition of synthesized ZnO nanoparticles. The results revealed the synthesis of spherical nanoparticles coated with protein molecules which served as stabilizing agents. Investigations on the role of fungal extracellular proteins in the synthesis of nanoparticles indicated that the process is nonenzymatic but involves amino acids present in the protein chains.

Keywords

Biomimetics ZnO nanoparticles Rhizosphere Soil fungi Metal tolerance Aspergillus 

Introduction

Nature is considered as a school for material science and its associated disciplines such as chemistry, biology, physics, or engineering (Bensaude-Vincent et al. 2002). An assortment of biological entities serves as the fundamental base for solving a variety of challenges in the field of architecture, aerodynamics, and mechanical engineering, as well as in material science (Fratzl 2007). Biology is considered as the master of so-called bottom-up fabrication which includes building up nanostructures starting from basic atoms or molecules (Naik and Stone 2005). Biological systems serve as prominent source of inspiration due to their remarkable variety of complex structures and functions which confer a huge impact on material science since several decades. Some examples of natural amalgams include crustacean’s carapaces, mollusk shells, bone, and tooth tissues in vertebrates (Sanchez et al. 2005). A number of single-celled organisms also produce inorganic materials of nanometer range intra- and/or extracellulary. Common examples include magnetotactic bacteria which synthesize magnetite (Lovley et al. 1987); diatoms which synthesize siliceous materials (Kröger et al. 1999), S-layer forming bacteria (Pum and Sleytr 1999), etc. These structures are highly controlled, range from macroscopic to nanometer scale, and result in intricate architectures that provide multifunctional properties.

Taking inspiration from these natural biological systems, recently, biologists were able to develop an alternative strategy for nanoparticle synthesis using microorganisms. A landmark study by Klaus et al. (1999) established an interface between material science and biological systems. They reported the synthesis of crystalline silver nanoparticles of well-defined composition and shapes using Pseudomonas stutzeri isolated from a silver mine. The nanoparticles were observed on the surface of bacterium with sizes ranging from a few to 200 nm. A similar approach used by Nangia et al. (2009) demonstrated the intracellular synthesis of gold nanoparticles by Stenotrophomonas maltophilia isolated from soil samples of the Singhbhum gold mines, India. The study also proposed a mechanism of production which suggests the involvement of a NADPH-dependent enzyme that reduces Au3+ to Au0 through an electron shuttle pathway for the synthesis of gold nanoparticles. In contrast, Prasad and Jha (2009) hypothesized the role of pH-dependent membrane-localized oxidoreductases for synthesis of ZnO nanoparticles in their study using Lactobacillus sporogenes. Labrenz et al. (2000) had shown the synthesis of spherical aggregates of sphalerite particles (2–5 nm) within the natural biofilms dominated by sulfate-reducing bacteria of the family Desulfobacteraceae. These observations lead to the emergence of a new branch of science called “Biomimetics,” which is defined as application of biological principles for material synthesis (Sarikaya et al. 2003). Biomimetics is sometimes also coined as material bionics or bio-inspired material research (Fratzl 2007). The subject is not just a consequence of an observation of naturally occurring structures but also involves complex biochemical and physiological processes which are still not properly understood and might play an important role in the formation of structures in nanometer range.

Microorganisms present in metal-rich regions exhibit high metal resistance which is mostly due to absorption or adsorption of metals and their chelation by extra- or intracellular proteins (Pócsi 2011). It is well demonstrated that high metal stress may lead to affect the various microbial activities (Giller et al. 2009). Well-adapted microbes isolated from native metal-rich soil conditions can be a better source for bio-inspired synthesis of metal nanoparticles as an indigenous microbial ecotype results from the long-term adaptation to soil with extreme properties.

The sophistication and success of natural bottom-up fabrication processes inspired the present attempt of creating a biomimetic approach using a zinc metal-tolerant fungal isolate for the synthesis of zinc oxide (ZnO) nanoparticles. ZnO is a unique material that exhibits semiconducting, piezoelectric, and pyroelectric properties and has versatile applications in transparent electronics, ultraviolet (UV) light emitters, piezoelectric devices, chemical sensors, spin electronics, personal care products, and coating and paints (Wang 2004; Wahab et al. 2010; Akhtar et al. 2011). In biological systems, various forms of zinc have significant roles in a wide variety of metabolic processes such as carbohydrate, lipid, nucleic acid, and protein synthesis as well as their degradation. In addition, zinc is an integral component of many enzyme structures and is the only metal to be represented in all six enzyme classes viz. oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases (Auld 2001).

With the objective of developing an eco-friendly and low-cost protocol for synthesis of ZnO nanoparticles, the present study investigates the relationship between the metal tolerance ability of soil fungi and their potential towards synthesis of ZnO nanoparticles. The study was carried out in two phases. The first was to investigate the metal tolerance profile of fungi isolated from a zinc-rich region and the second was to examine the potential of a fungal isolate exhibiting maximum metal tolerance towards synthesizing ZnO nanoparticles. Attempts were also made to investigate the role of fungal extracellular proteins in the nanoparticle synthesis process. The present study shows that the fungus Aspergillus aeneus isolate NJP12 exhibited maximum metal tolerance as well as that it has the potential of ZnO nanoparticle synthesis. We substantiate that our eclectic approach promises to yield revolutionary advances in the development of a low-cost, clean, and environmentally benign protocol for fabrication of metal nanoparticles. To the best of our knowledge, the present study is the first report on synthesis of ZnO nanoparticles using a fungus as a model organism.

Materials and methods

Sampling site and isolation of fungi

Soil samples were collected in February and July 2009 from the Zawar mines area (24°21′ N, 73°44′ E) which is located on the bank of the River Tiri, about 38 km south of Udaipur town in the Aravalli hill regions of Rajasthan, India. The mine is historically well known for zinc deposits and is owned by Hindustan Zinc Limited, Udaipur, India, the world’s second largest producer of zinc. The deposits have estimated ore reserves of 28.7 million tons (Mt) containing 4.76% zinc with an annual ore production capacity of 1.20 Mt as in the year 2010 (http://www.hzlindia.com/zawar.aspx). The region has a seasonally tropical climate and minimum and maximum temperatures of 5.0 and 38.4 °C, respectively, with a total annual rainfall of 637 mm as recorded by the Agro-Meteorological Department, Udaipur, India.

A total of five sampling sites have been studied. Soil samples were collected from naturally grown plants (Calotropis procera and Tephrosia purpurea) from three spatially separated points at each site, with a minimum of 5-m distance between each sampling point. The upper layers of soil were scrapped off to remove foreign particles and litter before taking samples. Soil firmly adhering to the root, designated as rhizosphere soil, was collected by brushing the root part of the plants. The soil samples were stored in self-sealing polyethylene bags, placed in an insulated carrier for transport, and then immediately refrigerated at 4 °C. Before processing (in most cases within 2 days), soil samples were passed through a sieve (2-mm mesh size) to remove stones and coarse roots. A subsample of each soil was air dried and used for estimation of various physicochemical properties. In order to rule out the possibility of seasonal variation, an additional set of soil samples was also collected after a 6-month interval from the subsets of sites and processed separately.

Isolation of fungi was carried out for each sampling site by plating the inoculum on Martin Rose Bengal Agar medium (HiMedia, Mumbai, India, pH 7.2) after serial dilutions of pooled soil samples (homogenized soils of three samples taken per sampling site) during both first and second collection of soils. Bacterial contamination was inhibited by supplementing the medium with chloramphenicol (Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 10 μg mL−1 after autoclaving. Petri plates were incubated at 28 °C for 4 days under dark conditions. Individual fungal colonies were selected and further purified by repeated subculturing on Potato Dextrose Agar (PDA) medium (HiMedia, Mumbai, India, pH 5.6). Preliminary identification of fungal isolates was performed on the basis of morphological characteristics.

Physicochemical characteristics of rhizosphere soil

Rhizospheric soil samples were analyzed for pH and electrical conductivity (EC) on an 1:2.5 soil/water suspension using digital pH and EC meter, respectively. Organic carbon was estimated by the method of Walkley and Black (1934) using 1.0 N potassium dichromate for titration and 0.5 N ferrous ammonium sulfate for back titration. Available phosphorus (Olsen P) in soil samples was determined by chlorostannus-reduced molybdophosphoric blue color method (Olsen et al. 1954) after extraction with 0.5 M sodium bicarbonate for 30 min. Available N, K, and micronutrients (Cu, Fe, Pb, and Zn) in soil samples were estimated as described by Jackson (1967).

Molecular characterization of fungal isolates

Liquid cultures of fungal isolates were prepared using 25 mL of mineralic Czapek’s Dox Broth medium (HiMedia, Mumbai, India, pH 7.3) in 100-mL Erlenmeyer flasks to obtain fresh mycelia for DNA extraction. Separation of mycelia was carried out by centrifugation at 8,000 rpm at 4 °C for 10 min. The obtained mycelia were mechanically crushed in liquid nitrogen. The genomic DNA was extracted from 100 mg of fungal mycelia using HiPurA Plant Genomic DNA Miniprep Purification Kit (HiMedia, Mumbai, India) according to the manufacturer’s instructions.

Polymerase chain reaction (PCR) primers namely ITS-1 (5′ TCC GTA GGT GAA CCT GCG G 3′) and ITS-4 (5′ TCC TCC GCT TAT TGA TAT GC 3′) developed by White et al. (1990) were used to amplify the internal transcribed space (ITS) region of ribosomal DNA, which encompasses the 5.8S gene and the ITS1 and ITS2 regions. PCR amplification was performed in a total volume of 50 μL containing: 1 U Taq DNA polymerase (Promega, Mannheim, Germany), 2.5 mM MgCl2, 160 μM dNTP mix (MBI Fermentas, St. Leon-Rot, Germany), 50 pmol of each of the ITS-1 and ITS-4 primers (Sigma-Aldrich, St. Louis, MO, USA), and 50 ng genomic DNA in dH2O. The reactions were performed in a gradient thermal cycler with the following conditions: 1 min denaturation at 95 °C, 30 s annealing at 50 °C, 90 s elongation at 72 °C, for 34 cycles with a final elongation step of 72 °C for 10 min.

Amplified ITS regions were sequenced with an ABI Prism DNA sequencer (Applied Biosystems, Carlsbad, CA, USA) using either the ITS-1 and/or the ITS-4 primer for DNA labeling by the BigDye terminator method (Applied Biosystems, Foster City, CA, USA). The sequenced data obtained from the ITS-4 primer were inversed using Gene Doc software (Nicholas et al. 1997) and clubbed with the sequence data obtained with the ITS-1 primer, to obtain the complete sequence of the ITS region. Comparison of nucleotide sequences was performed using the Basic Local Alignment Search Tool (BLAST) network services of the National Centre for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov). Molecular characterization of fungal isolates was done on the basis of similarity with the best aligned sequence of BLAST search.

Accession numbers

The ITS1-5.8S-ITS2 gene complex sequences of obtained fungal isolates were submitted to the GenBank database of NCBI with the following accession numbers: HM222932–34, HQ710532–46, and JF298825 (Table 2). A. aeneus isolate NJP12 has been deposited in the Microbial Type Culture Collection and Gene Bank at the Institute of Microbial Technology, Chandigarh, India with the MTCC number 10830 and available at public domain.

Metal tolerance profiles of fungal isolates

A maximum tolerable concentration (MTC) assay was performed to determine the zinc metal tolerance ability of fungal isolates. The experimental plates were prepared by supplementing PDA medium with varying amounts of zinc sulfate to obtain final concentrations of Zn+2 ions in the ranges of 200, 400, 800, 1,600, and 3,200 μg mL−1. Plates without Zn+2 ions were used as control. Each plate was subdivided into four equal sectors and an inoculum of test fungi (106 cfu mL−1) was spotted on the media surface. After inoculation, the plates were incubated at 28 °C for 4 days under dark conditions to examine the fungal growth. The experiment was done in triplicate. The maximum concentration of Zn+2 ions in the medium which allowed the growth of a fungus was taken as MTC.

Extracellular biosynthesis of zinc oxide nanoparticles

The fungal isolate showing the highest MTC value was selected for extracellular synthesis of ZnO nanoparticles. For this, the fungal isolate was maintained at 28 °C by regular subculturing on fresh PDA medium slants. The stock culture (4 days old) was inoculated in 100 mL of MGYP medium (0.3% malt extract, 1.0% glucose, 0.3% yeast extract, 0.5% peptone; pH 7.0) in 250-mL Erlenmeyer flasks. Inoculated flasks were incubated at 28 °C for 72 h on a rotary shaker (150 rpm) under dark conditions. Fungal mycelia were separated from the culture medium by centrifugation (8,000 rpm, 10 min, and 4 °C) and washed thrice with sterile water in order to remove all traces of media. Typically, 10 g of biomass (fresh weight) was resuspended in 100 mL of sterile deionized Milli-Q water and further incubated for 72 h under the same conditions as described above. After incubation, biomass was separated by filtration using Whatman filter paper no. 1 (Whatman Inc., Florham Park, NJ, USA), and the fungal cell-free filtrate containing extracellular secretions was collected. For synthesis of nanoparticles, aqueous zinc acetate solution (Sigma-Aldrich, St. Louis, MO, USA) at a final concentration of 1.0 mM was added to flasks containing 100 mL of fungal cell-free filtrate and incubated for 72 h under the same conditions as described above. Controls containing fungal cell-free filtrate (without zinc acetate; positive control) and pure zinc acetate solution (without fungal cell-free filtrate; negative control) were also run simultaneously along with experimental flasks in three replicates. Viability of the fungal cells after incubation in Milli-Q water for 72 h was also checked. For this, the fungal mycelia were inoculated on fresh PDA plates in triplicate and incubated for 4 days at 28 °C in dark conditions.

Characterization of zinc oxide nanoparticles

Synthesis of nanoparticles was monitored using UV–visible spectroscopy by sampling of aliquots (1 mL) at different time intervals. Absorption spectra were measured using a Jasco V-630 UV–visible spectrophotometer (Jasco Corporation, Tokyo, Japan) operated within the range of 200–900 nm at a resolution of 1 nm.

For Fourier transform infrared (FTIR) spectroscopy measurements, biotransformed products present in the fungal cell-free filtrate were freeze-dried and diluted with potassium bromide in a ratio of 1:100. FTIR spectra of samples were recorded on a Shimazdu IR Prestige-21 FTIR spectrometer (Shimadzu, Nakagyo-ku, Japan) with a diffuse reflectance mode (DRS-8000) attachment (Shimadzu Corporation, Nakagyo-ku, Japan). All measurements were carried out in the range of wavenumbers 400–4,000 cm−1 at a resolution of 4 cm−1.

Samples for transmission electron microscopy (TEM) were prepared by drop coating the as-synthesized nanoparticle solution onto carbon-coated copper grids. After about a minute, extra solution was removed using a blotting paper, and the grids were kept in a vacuum desiccator, prior to measurement. TEM micrographs were taken by analyzing the prepared grids on a Hitachi H-7650 TEM instrument (Hitachi High-Technologies Corporation, Tokyo, Japan) at an acceleration voltage of 100 kV. Energy dispersive spectroscopy (EDS) of drop coated grids of samples was carried out using Bruker attachment (Bruker AXS Ltd., Coventry, UK) with TEM instrument.

X-ray diffraction (XRD) measurements of the freeze-dried samples were carried out using a Rigaku MiniFlex II Benchtop XRD System (Rigaku Company, Texas, USA) operated at a voltage of 20 kV and current of 15 mA with CuKα radiation. Phase analysis was carried out by comparing the calculated values of interplanar spacing and corresponding intensities of diffraction peaks with theoretical values from the Powder Diffraction File database (PCPDF-WIN; JCPDS-ICDD 2008).

Analysis of fungal cell-free filtrate

UV–visible spectra of fungal cell-free filtrates were recorded to confirm the presence of proteins. The total protein concentration in the fungal cell-free filtrates was determined by the method of Lowry et al. (1951) using bovine serum albumin as standard. To investigate the involvement of proteins in nanoparticle synthesis, proteins were precipitated from the fungal cell-free filtrates using the standard trichloroacetic acid method (Simpson 2004). One hundred milliliters of supernatant fraction without proteins was used for synthesis of ZnO nanoparticles under the same conditions as mentioned above. In order to understand the nature of proteins present in the fungal cell-free filtrate, a separate experiment was performed. Briefly, 100 mL of fungal cell-free filtrate was boiled in a water bath for 20 min, cooled, and used for synthesis of ZnO nanoparticles under the same conditions as described above. A flask containing 100 mL of fungal cell-free filtrate without heat treatment was used as control. Systematic monitoring of pH of the reaction medium was also observed at different time intervals to understand the chemical nature of system.

Results

Physicochemical characteristics of rhizosphere soil

Physicochemical characteristics of rhizospheric soils of naturally grown plants collected from the Zawar mines, Udaipur, India are shown in Table 1. In general, the soil was slightly alkaline in nature. Nitrogen, phosphorus, and potassium contents of the soil samples were calculated as 63.4, 30.0, and 150.0 mg kg−1, respectively. Determination of micronutrient content revealed the abundance of Zn in the rhizosphere soil with a mean value of 121.0 mg kg−1 along with moderate concentrations of Cu, Fe, and Pb.
Table 1

Physicochemical characteristics of rhizospheric soil samples collected from the Zawar mines, Udaipur, India

Parameter

Value

pH

7.72 ± 0.02

EC

0.48 ± 0.01 dS m−1

Available N

63.39 ± 4.5 mg kg−1

Available P

30.0 ± 1.2 mg kg−1

Available K

150.0 ± 10.9 mg kg−1

Zn

121.0 ± 14.3 mg kg−1

Cu

0.02 ± 0.001 mg kg−1

Fe

10.0 ± 0.61 mg kg−1

Pb

3.4 ± 0.08 mg kg−1

n = 30; 15 samples from the first and 15 samples from the second collection of soils

Identification of fungal isolates

Preliminary identification of fungi was performed on the basis of morphological parameters such as color, spore shape, arrangement, and hyphal branching pattern after staining with cotton blue. Molecular characterization of fungi was carried out using the universal primers for the amplification of the internal transcribed spacer (ITS) regions of the fungal rRNA operon as described by White et al. (1990).

A total of 19 fungal isolates were observed on the basis of distinct morphological parameters. However, molecular characterization has revealed the presence of fungal isolates of 13 different species from five different genera (Table 2). All detected fungal isolates belong to the phylum Ascomycota except Mortierella sp. isolate NJP14 which belongs to the phylum Zygomycota. An abundance of the genus Aspergillus (in total 11 isolates from 7 different species representing 58% of all isolated species) was observed.
Table 2

Sequence analysis of fungal isolates with their reference organisms

Fungus

Isolate

Accession number

Sequence length

BLAST results

ITS1

5.8S

ITS2

Maximum score

Query coverage (%)

Maximum identity (%)

Closest match

Reference

Aspergillus aeneus

NJP12

HM222934

152

157

164

891

100

99

EF652474T

Peterson (2008)

Aspergillus flavus

NJP08

HM222933

180

158

168

1,037

100

99

AF027863T

Haugland et al. (2004)

Aspergillus niger

NJP09

HQ710538

185

157

170

1,018

94

99

EF661191T

Peterson (2008)

Aspergillus ochraceus

NJP04

HQ710534

169

157

100a

837

98

100

AY373856T

Haugland et al. (2004)

Aspergillus ochraceus

NJP13

HQ710541

142a

157

176

957

100

100

EF661419T

Peterson (2008)

Aspergillus oryzae

NJP01

HQ710532

181

157

170

1,061

98

99

AY373857T

Haugland et al. (2004)

Aspergillus oryzae

NJP06

HQ710536

181

157

170

1,053

98

99

AY373857T

Haugland et al. (2004)

Aspergillus oryzae

NJP10

HQ710539

183

157

169

1,051

99

99

AY373857T

Haugland et al. (2004)

Aspergillus oryzae

NJP15

HQ710543

181

157

168

1,064

100

99

AY373857T

Haugland et al. (2004)

Aspergillus oryzae

NJP18

HQ710546

181

157

170

1,077

99

99

AY373857T

Haugland et al. (2004)

Aspergillus sp.

NJP02

HM222932

154

156

170

983

99

99

EF652481

Peterson (2008)

Cladosporium sp.

NJP19

JF298825

154

157

152

952

97

99

FJ936159

Schubert et al. (2009)

Cladosporium sp.

NJP05

HQ710535

157

157

148a

845

99

95

EU167574

Simon et al. (2009)

Eupenicillium javanicum

NJP17

HQ710545

175

157

169

931

94

97

GU981613T

Houbraken et al. (2011)

Mortierella sp.

NJP14

HQ710542

173

158

258

1,000

100

96

HQ630345

Nagy et al. (2011)

Penicillium commune

NJP11

HQ710540

175

156

168

1,051

100

99

AY373905T

Haugland et al. (2004)

Penicillium commune

NJP07

HQ710537

175

156

168

1,029

100

99

AY373905T

Haugland et al. (2004)

Penicillium commune

NJP03

HQ710533

175

156

169

1,044

100

99

AY373905T

Haugland et al. (2004)

Penicillium crustosum

NJP16

HQ710544

175

156

168

1,035

99

99

AY373907T

Haugland et al. (2004)

aIncomplete ITS sequence

TType strain

Metal tolerance profile of fungal isolates

All the 19 fungal isolates were subjected to a screening for metal tolerance towards zinc, and the results were expressed in terms of MTC. A higher proportion (90%) of fungal isolates showed significant tolerance with a varying degree of magnitude. The genus Aspergillus exhibited a more prominent level of zinc metal tolerance as compared to other fungal isolates. It is evident from the results that A. aeneus isolate NJP12 and Aspergillus sp. isolate NJP02 have tremendous zinc metal tolerances with a MTC value of 2,800 and 2,400 μg mL−1 respectively (Fig. 1). Due to its maximum MTC value, A. aeneus isolate NJP12 was selected for further studies on extracellular synthesis of ZnO nanoparticles.
Fig. 1

Zinc metal tolerance profile of fungal isolates

Extracellular biosynthesis of zinc oxide nanoparticles

The extracellular synthesis of ZnO nanoparticles was carried out by exposure of a precursor salt solution (Zn+2 ions; 0.65 mM) to fungal cell-free filtrate obtained by incubating the fungus NJP12 in an aqueous solution. The viability experiment results showed that fungal cells were viable till the end of reaction (72 h).

Characterization of zinc oxide nanoparticles

UV–visible spectroscopy was used to monitor the synthesis of ZnO nanoparticles. Figure 2a showed a gradual increase in absorbance at ca. 375 nm with respect to time of reaction, representing the synthesis of ZnO nanoparticles. At initial time intervals (0, 6, 12, and 24 h), synthesis was relatively slow, but at later time intervals (36, 48, and 72 h), significant changes in the magnitude of absorbance were observed. pH values of 7.4 and 7.8 were observed at the start (0 h) and end (72 h) of the reaction, respectively. This minimal change in pH makes the present protocol “eco-friendly” which is highly advantageous as compared to chemical synthesis protocols which show high pH variations (Sharma et al. 2009). The stability of the nanoparticle solution stored at room temperature in dark conditions for more than 3 months after completion of reaction was determined by UV–visible spectroscopy measurements.
Fig. 2

a UV–visible spectrum representing gradual synthesis of ZnO nanoparticles with respect to time. The absorbance is expressed in terms of arbitrary unit (a.u.) b FTIR spectrum of freeze-dried samples of ZnO nanoparticles. The absorbance is expressed in terms of arbitrary unit (a.u.)

FTIR analysis of freeze-dried samples showed an intense band in the vicinity of wavenumbers 400–600 cm−1 that centered around wavenumber 430 cm−1 and is attributed to ZnO vibrations (Fig. 2b; Becheri et al. 2008). The presence of bands at wavenumbers 1,625 and 1,550 cm−1 corresponds to the bending vibrations of the amide I and amide II of proteins, respectively (Ahmad et al. 2003). The band at wavenumber 3,450 cm−1 has been reported to occur due to stretching vibrations of amide I superimposed on the side of hydroxyl group band (Saeeda et al. 2009). Moreover, the bands at wavenumbers 2,995 and 2,350 cm−1 (Fig. 2b) are due to the stretching vibrations of amide II and presence of atmospheric CO2, respectively (Saeeda et al. 2009; Becheri et al. 2008).

TEM measurements of the samples were performed to visualize the size and morphology of ZnO nanoparticles. A TEM micrograph (Fig. 3a) showed the well-distributed spherical ZnO nanoparticles surrounded by a thin protein layer. The size distribution analysis showed that the particle size ranged from 100–140 nm. EDS analysis of drop coated grids of samples was performed to determine the elemental composition of nanoparticles (Fig. 3b). EDS spectra showed strong peaks at 0.5, 1.1, 8.6, and 9.5 keV which are due to O Kα, Zn Lα, Zn Kα, and Zn Kβ, respectively (Bahadur et al. 2008). The atomic percent values of Zn and O observed were 0.38 and 0.18, respectively. Other peaks observed for copper and carbon were due to the supporting carbon-coated copper grid used for sample preparation.
Fig. 3

a TEM micrograph showing the spherical shape of ZnO nanoparticles b EDS spectrum representing the elemental composition of ZnO nanoparticles

Figure 4 shows the XRD pattern of synthesized ZnO nanoparticles. Analysis of XRD spectra showed well-defined peaks at 2θ values of 32.05°, 34.69°, and 36.53° which correspond to (100), (002), and (101) planes of ZnO, respectively. The observed lattice values were in agreement with the hexagonal phase of ZnO (PCPDF-WIN; JCPDS-ICDD 2008).
Fig. 4

XRD spectrum of ZnO nanoparticles with Bragg’s diffraction values shown in parentheses. The absorbance is expressed in terms of arbitrary unit (a.u.)

Analysis of fungal cell-free filtrate

An absorption peak at ca. 280 nm observed in the UV–visible spectrum of fungal cell-free filtrate indicated the presence of proteins in the fungal cell-free filtrate (not shown). Concentration of protein in the fungal cell-free filtrates was determined as 458.2 ± 2.8 μg mL−1. Incubation of precursor zinc ions with a supernatant fraction without proteins (obtained by protein precipitation) showed no evidence of ZnO nanoparticle synthesis which indicates that proteins play an important role in the synthesis of ZnO nanoparticles. An experiment with incubation of denaturated (heat treated) and native (untreated) proteins with precursor zinc ions resulted in the synthesis of ZnO nanoparticles in both cases. Interestingly, comparison of absorbance spectra indicates a higher rate of reaction in case of denatured proteins as compared to native proteins (Fig. 5).
Fig. 5

Effect of denaturation of proteins on synthesis of ZnO nanoparticles. The absorbance is expressed in terms of arbitrary unit (a.u.)

Discussion

The present study approaches to establish the relationship between metal tolerance ability of a soil fungus and its potential for synthesis of ZnO nanoparticles. The Zawar mines, Udaipur, India were opted as sample collection site which is a natural zinc metal-enriched region. The rhizospheric soil samples of naturally growing plants were used as a source for isolation of fungi. It is well known that rhizosphere regions exhibit high microbial populations due to presence of more organic contents (Griffiths et al. 2001). Analysis of physicochemical properties of soil samples is a prerequisite for a better understanding of microbial population. Physicochemical analysis of collected rhizospheric soil samples showed the alkaline nature of the soil with abundant zinc concentration.

Selection of well-adapted fungi was performed by investigating the metal tolerance profile of obtained fungal isolates. Due to its high zinc metal tolerance ability, A. aeneus isolate NJP12 was selected for further studies on synthesis of ZnO nanoparticles. The protocol employed for nanoparticle synthesis was similar to our earlier study for synthesis of silver nanoparticles which involves exposure of precursor ions to proteins present in the fungal cell-free filtrate (Jain et al. 2011). The absorption maxima at 375 nm observed in UV–visible spectra of forming ZnO nanoparticles (Fig. 2a) can be attributed to a band-to-band emission of ZnO which represents a direct band gap of 3.3 eV (Prasad et al. 2006; Sridevi and Rajendran 2009). The presence of a broad absorption peak in FTIR spectra (Fig. 2b) indicates the synthesis of ZnO nanoparticles of varying sizes. TEM analysis of drop coated grids further confirmed the presence of ZnO nanoparticles of various sizes with proteins as capping molecule on individual ZnO nanoparticles (Fig. 3a). Previous studies on biosynthesis of silver nanoparticles using fungal cell-free filtrate also showed the presence of proteins on the surface of individual nanoparticles which conferred their stability (Balaji et al. 2009; Jain et al. 2011). EDS and XRD analysis in this study further confirmed the composition and crystallinity of the obtained ZnO nanoparticles, respectively (Fig. 3b and 4).

The understanding of a plausible mechanism involved in biosynthesis of nanoparticles is a key step to scale-up the process for mass level production. Few studies have shown the role of proteins present in fungal cell-free filtrates for nanoparticle synthesis. In a previous study, we demonstrated the presence of two proteins (32 and 35 kDa) in a fungal cell-free filtrate and their involvement in silver nanoparticle synthesis (Jain et al. 2011). Ahmad et al. (2003) proposed the involvement of NADH-dependent reductases present in a cell-free filtrate of Fusarium oxysporum for the synthesis of silver nanoparticles. In the present study, efforts were made to investigate the role of fungal extracellular proteins towards synthesis of ZnO nanoparticles. No evidence of nanoparticle synthesis was observed in reaction medium lacking proteins which further confirm the involvement of proteins in the synthesis of ZnO nanoparticles.

Denaturation of proteins present in the fungal cell-free filtrate was performed to understand the nature of proteins. Synthesis of ZnO nanoparticles by both denaturated (heat treated) and native (untreated) proteins revealed that the native form of proteins is not mandatory for nanoparticle synthesis. It can also be inferred that the nanoparticle synthesis process is nonenzymatic as the activity of enzymes depends on their structure which changes during denaturation. Hence, with the current experimental evidences, it can be concluded that the synthesis process depends on interaction with amino acids present in the proteins. The high reaction rate for ZnO nanoparticles synthesis as observed by absorption spectra (Fig. 5) in case of denatured (heat treated) proteins further validates that the rate of synthesis depends on the interaction of amino acids with metal (Zn+2) ions. Heating of protein molecules results in the breakdown of hydrogen bonds which allows dismantling the hydrophobic core which in turn enhances the interactions between amino acids and zinc ions. Our results substantiate the previous reports which demonstrate that interactions between amino acids and metal ions are responsible for synthesis of metal nanoparticles (Xie et al. 2007). However, the present findings are in disagreement with earlier reports which hypothesize the involvement of enzymes in the synthesis of nanoparticles (Ahmad et al. 2003; Nangia et al. 2009). To the best of our knowledge, this is the first study which proposes the involvement of simply amino acids in the fungal-mediated synthesis of metal nanoparticles. Further investigations including isolation, purification, and characterization of proteins along with their possible interactions with zinc ions are in progress in our laboratory.

Notes

Acknowledgments

This research was supported by the National Agricultural Innovation Project (NAIP), Indian Council of Agricultural Research (ICAR) through its sub-project entitled “Nano-technology for Enhanced Utilization of Native Phosphorus by Plants and Higher Moisture Retention in Arid Soils” Code number “NAIP/C4/C-2032.” Facilities provided by Electron Microscopy & Nanoscience Laboratory, Punjab Agricultural University, Ludhiana are gratefully acknowledged. Navin Jain thanks the Council of Scientific and Industrial Research, Government of India for providing a research fellowship. The authors are thankful to Dr. Ursula Kües for constructive and meaningful suggestions, which helped us to improve the manuscript up to the desired level.

Conflicts of interest

The authors declare that they have no conflicts of interest.

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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Navin Jain
    • 1
  • Arpit Bhargava
    • 1
  • Jagadish C. Tarafdar
    • 2
  • Sunil K. Singh
    • 2
  • Jitendra Panwar
    • 1
  1. 1.Centre for Biotechnology, Department of Biological SciencesBirla Institute of Technology and SciencePilaniIndia
  2. 2.Central Arid Zone Research InstituteJodhpurIndia

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