Research Article

Biobased approach for the synthesis, characterization, optimization and application of silica nanoparticles by fungus Fusarium oxysporum

M. Kannan1*, K. Uma Sangareswari1, P. Suganya1, R. Ganesan2, K. Rajarathinam3

1Research Department of Microbiology, V.H.N.S.N. College (Autonomous), Virudhunagar – 626 001. Tamil Nadu, India 2School of Energy, Environment and Natural resources, Madurai Kamaraj University, Madurai - 625 021. Tamil Nadu, India 3Research Department of Botany, V.H.N.S.N. College (Autonomous), Virudhunagar - 626 001. Tamil Nadu, India

*For correspondence

Dr. M. Kannan,

Research Department of Microbiology, V.H.N.S.N. College (Autonomous), Virudhunagar – 626 001. Tamil Nadu, India.

Email:microkannan@gmail.com

Received: 18 November 2015

Accepted: 11 December 2015

ABSTRACT

Objective: Nanoparticles, a building block of nanotechnology offers a broad application in several areas of science. Among various methods for nanoparticle synthesis, the utilization of microbes is one of the solutions for bioanalysis of biocomponents. Microbes which are non-pathogenic to humans are employed to reduce the metal ions. The objective of the study was to synthesize of inorganic meterials on nano and micro length scale.

Methods: In this work, Fusarium oxysporum from wilted tomato plant was selected and silica nanoparticles were synthesized. The silica nanoparticles synthesis characterization was done using UV-spec., FTIR and SEM analysis. The appropriate conditions for silica nanoparticles synthesis optimization and the separation of nanoparticle biosynthetic protein were carried out. Then the efficiency of nanoparticle in DNA protection from enzymatic cleavages and dye decolourization also determined.

Results: The studies showed the confirmation of isolated F. oxysporum and the synthesized silica nanoparticles surface plasmon band occurs at 280 nm. In FTIR analysis, the presence of a resonance at ca.1113 cm-1 and absorption bands at ca.1623.62 indicated the amide band in the particle.

Conclusions: Thus, fungus mediated silica nanoparticles synthesis could be used in many biological researches.

Keywords: Fusarium oxysporum, Silica nanoparticles, Dye decolourization, DNA protection from enzymatic cleavages

Introduction

There has been an explosive growth of nanoscience and technology in the last few years, primarily because of the availability of new strategies for the synthesis of nanomaterials as well as new tools for the characterization and manipulation. Nanotechnology initiative is the understanding and control of matter at dimensions of roughly 1-100 nanometre. Nanotechnology offers new solutions for the transformation of bio systems and provides a broad technological platform for applications in several areas, bioprocessing industry, molecular medicine.1 Nanoparticles are viewed by many as fundamental building blocks of nanotechnology. Nanoparticles play an important role in a wide variety of fields including advanced materials, pharmaceuticals and environmental detection and monitoring. A nanoparticles or nano powder is microscopic particles whose size is measured in nanometers (nm) 1 nm=10-9 m. It is well known that many unicellular organisms such as bacteria and algae are capable of synthesizing inorganic materials, both intra and extracellularly.

Silica occurs in minerals consisting of pure silica dioxide in different crystalline forms, sand, amethyst, agate, quartz, rock crystal, flint, Jasper, and opal are some of the forms in which silicon dioxide appear. Silica also occurs as silicates. The silica based nanoparticles find its use in bioanalysis if these are conjugated to recognize biocomponents. Silica nanoparticles doped with fluorescent dyes have also used as labelling reagent for biological applications. The metallic nanoparticles are known to exhibit different characteristic colors. Hence, UV-Vis. can be utilized to study the unique optical properties of nanoparticles.2,3 Fourier Transforms Infrared (FTIR) Spectroscopy deals with the vibration of chemical bonds in a molecule at various frequencies depending on the elements and types of bonds. Scanning Electron Microscopy (SEM) is one of the most widely used techniques for characterization of nanomaterials and nanostructures.

The utilization of microorganisms in the synthesis of nanoparticles is a relatively recent phenomenon. The microorganisms minimize the toxicity by reduction of the metal ions or by formation of insoluble complexes with metal ion in the form of colloidal particles. A curiosity, environmental compulsion and understanding that nature has evolved the processes for synthesis of inorganic materials on nano and micro length scale have created the great interest for done this research work.

Materials and Methods

Sample collection

For the biosynthesis of silica nanoparticles, Fusarium oxysporum was selected because, the local environment suits for this fungi. Hosts of F. oxysporum include: potato, sugarcane, garden bean, cowpea, prickly pear, cultivated zinnia, pansy.4 So the present study was planned to isolate the F. oxysporum from wilted tomato plant leaf. For fungus isolation, aseptically collected the wilted tomato plants and the infected plants were certified by Research Department of Botany, V.H.N.S.N. College (Autonomous), Virudhunagar.

Surface sterilization of infected plant tissue and culturing

The infected part of the diseased leaves, stems and roots were cut with the help of sterilized scalpel, and kept in the first beaker containing saturated borax solution 0.1%, for 10 to 15 minutes. Thereafter it is taken out from the first beaker with the help of glass rod, and thoroughly washed in the second beaker containing mercuric chloride solutions 0.1% only for 15 seconds. Once again the infected materials is thoroughly washed in the third beaker containing distilled water. Now, these sterilized inoculums are being transformed into a sterilized petriplate, and then inoculated in a Potato Dextrose Agar (PDA) plates. The plates were incubated at 220C for 5-7 days.5

Fungal staining and colony morphology

The fungal isolates were cleaned up by sub-culturing successively and were identified by their resting structure morphology, by means of the slide culture technique.6,7 Colony morphology of the organisms were studied by growing them on Czapekdox agar, Malt extract agar, Oat meal agar, Potato dextrose agar, Rose bengal agar and Sabouraud dextrose agar. Cultures were incubated at 220C for 5-7 days. The fungus colony diameter, morphology were recorded.

Biosynthesis of silica nanoparticles by isolated F. oxysporum

For the synthesis of nanoparticles, the fungus F. oxysporum was grown in 500 ml erylenmeyer flasks each containing MGYP media (100 ml), composed of malt extract (0.3%), glucose (1.0%), yeast extract (0.3%) and peptone (0.5%) at 25–28°C under shaking at 200 rpm for 96 h. The mycelial mass were then separated from the culture broth by centrifugation (5000 rpm) at 10°C for 20 min. and the settled mycelia were washed thrice with sterile distilled water. Some of the harvested mycelial mass (20 g) was then used for the synthesis of silica nanoparticles. This methodology was followed with small modification.8

The harvested mycelia mass (20g wet weight) was then resuspended in 100 ml aqueous solutions of 10-3 M potassium silicofluoride (pH-3.1) and kept on a shaker (200 rpm) at 270C. After incubation the reaction solution was observed. Nanoparticles containing fungal mycelia were filtered under laminar flow through Wattman filter paper. The reaction solution was removed and the absorption was measured by UV-vis spec. Then allowed to calcinations at 1800C for 5 hrs. is required for crystallization of silica nanoparticles. After, the products were analysed by FTIR and SEM.

In control experiments, the fungal biomass was resuspended in double distilled water in the absence of aqueous solution of potassium silicofluoride and the filtrate obtained thereafter was characterized by UV-vis spec. This reaction did not result in the formation of silica nanoparticles. In another control experiment, the hydrolysis of aqueous solution of potassium silicofluoride in double distilled water in the absence of fungal biomass was studied by UV-vis spectrophotometer and FTIR.

Characterization of biosynthesized nanoparticles

The biosynthesized nanoparticles were characterized by the following techniques:

UV-Vis spectroscopy: The biosynthesized nanoparticles were subjected to UV-Vis spectra, quartz cuvette (1cm) with Shimadzu UV-160 (Japan, Kyoto) spectrophotometer was used. The process was monitored at every 12 hrs upto 48 hrs for F. oxysporum. Based on the UV-Vis spec. readings, the silica nanoparticles biosynthesized by F. oxysporum was subjected to FTIR and SEM analysis.

Fourier transform infrared spectroscopy: The FTIR analysis of the biosynthesized products present in the filtrate were dried, pulverized and dried samples were recorded using a Perkin-Elmer infrared spectrophotometer with a resolution of 4 cm-1, in the range of 400–4000 cm-1.

Scanning electron microscopy: The biosynthesized nanoparticles powder was subjected to scanning electron microscopy.

Separation of nanoparticles biosynthetic protein

For the separation of protein, 20 g of the fungal biomass was suspended in 100 ml of sterile distilled water and aqueous solution of potassium silicofluoride for a period of 24 h at 270C under shaking conditions. The reaction solution was salted out overnight using ammonium sulphate precipitation followed by centrifugation. The proteins obtained thereafter were dissolved in the minimal volume of deionized water and dialyzed (using a 3 KDa cut off dialysis membrane). The purified protein fraction was obtained by ion exchange chromatography. Then the purified protein was analysed by 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) carried out at pH 8.2 and the gel were stained with comassive blue. The results were compared with standard protein molecular weight marker.

Determination of electron shuttling compounds using TLC

In order to determine the water soluble quinines that might function as an electron shuttle, the culture was filtered and the filtrate was adjusted to pH 3 with 1M HCl. The acidified solution was then passed through a column with ion exchange resin (Amberlite) for absorption of the pigments. Compounds were removed from the column by elution with acetone, the acetone removed using evaporation and the aqueous phase extracted 3 times with ethyl acetate. All ethyl acetate extraction were combined and reduced by evaporation. After that 2 ml samples were repeatedly spotted on a silica gel until a spot was visible under UV light at 254 nm. Samples were resolved using a chloroform-methanol-acetic acid (195:5:1) or benzene-nitromethane-acetic acid (75:25:2) system designed to mobilize polar pigments. Plates were air dried, and spots visualized under UV light.9

Optimization of biosynthesized nanoparticles

The biosynthesis of nanoparticles were investigated for the optimum pH, temperature (0C), metal concentration (mM) and time of incubation (h) for the synthesis of silica nanoparticles using F. oxysporum. The OD value recorded on the UV-Vis spec.

Decolorization of dye

To apply the biosynthesized silica nanoparticles against the removal of an azo group dyes to reduce some environmental pollutions. A typical direct azo dye of biphenyl amine, methyl orange is commonly used in textile industries. Therefore, we chose it as a model pollutant in this study.

Dye solutions were prepared in double distilled water at 50 mgL-1 of methyl orange and then 0.25%, 0.5%, 1% (w/v) of biosynthesized silica nanoparticle was added. The flask was stirred at 150 rpm by shaker upto 300 min. The experiment was performed in triplicate and conducted at room temperature. Samples were collected at 60,120,180,240 and 300 minutes. The samples were centrifuged at 6,000 rpm for 5 minutes. The residual concentrations of dye were quantified by UV-Vis spectrophotometer at 461 nm for methyl orange. Methyl orange before and after treatment by silica nanoparticles were also qualified by observing the change of UV-vis spectrum. To investigate the effect of pH on decolorization of methyl orange by biosynthesized nanoparticles, the dye solution were adjusted pH at 3,5,7,9 using 1M HCl and 1M NaOH before treatment by nanoparticles.

Stability of silica nanoparticle-bound DNA

The stability of DNA was analysed with the Plasmid DNA pUC 18 (Geni 105840). Plasmid DNA and biosynthesized silica nanoparticles (NP) complex samples (the molar ratio of NP : DNA = 400:1) were incubated for 30 min in DNase I reaction buffer (40 mM Tris–HCl, pH 8.0, 10 mM MgSO4, 1 mM CaCl2) at room temperature, followed by addition of 2mL of DNase I solution. The samples were then incubated at 37°C for 45 min, followed by addition of 5 mL 20 mM EGTA [ethylene glycol bis(2-aminoethyl ether)-N,N,N'N'-tetraacetic acid] to stop the reaction of DNA alone and 5 mL 5% SDS to stop the reaction and release the NP-bound DNA. The nanoparticles bounded biomolecules were separated by 0.6% agarose gel electrophoresis.

Results and Discussion

Isolation and characterization of fungi F. oxysporum

After the incubation period the plant pathogenic fungi F. oxysporum was observed under the microscopy for morphological studies. The hyphae were septate and hyaline. Conidiophores were simple. Macroconidia was moderately curved, stout, thick-walled, have 3-5 septate, the aerial mycelium were white to orange in colour on potato dextrose agar (Figure 1d).

The fungal morphology and mycelia growth of F. oxysporum observed on five different cultivation media (Figure 1) and the results were tabulated (Table 1). Among these six media, mycelial growth of the F. oxysporum on oat meal agar were greater than other media.

(a) Oat meal agar; (b) Czapekdox agar; (c) Sabouraud dextrose agar

(d) Potato dextrose agar; (e) Malt extract agar; (f) Rose Bengal agar

Figure 1: Colony morphology of F. oxysporum on different selective media.

Figure 1(g): Microscopic view (40X).

Biosynthesis of nanoparticles by isolated F. oxysporum

In silica nanoparticle biosynthesis, the flask with isolated culture biomass showed in pale yellow but the flask containing potassium silicofluoride and biomass showed the colourless on completion of the reaction (Figure 2). Biological synthesis or biosynthesis refers to the phenomena which takes place by means of biological processes or enzymatic reactions. These eco-friendly processes, referred to as green technology, can be used to obtain better metal nanoparticles from microbial cells.10 The fungus F. oxysporum, when exposed to metal ions releases enzymes that reduce the metal ions to yield highly stable nanoparticles in solution. Recently, it was found that aqueous chloroaurate ions may be reduced extracellularly using the fungus F. oxysporum to generate extremely stable gold or silver nanoparticles in water. F. oxysporum behaved considerably differently, the reduction of the metal ions occurring extracellularly resulting in the rapid formation of highly stable gold and silver.8,11

Table 1: Colony morphology of F. oxysporum on selected cultivation media.

Cultivation media

Growth

Colony morphology

Sabouraud Dextrose Agar

Potato Dextrose Agar

Oat Meal Agar

Czapek-Dox Agar

Malt Extract Agar

Rose Bengal agar

++

++

+++

+++

++

+

White to pink

White to orange

White cottony

White cottony

White sparse

White cottony

C1- Metal control; C2- Culture control; T- Biosynthesized nanoparticle; A- Biosynthesized nanoparticle powder

Figure 2: Biosynthesis of silica nanoparticles by F. oxysporum.

Silica nanoparticles are more hydrophilic and biocompatible, they are not subject to microbial attack, and no swelling or porosity change occurs with changes in pH.12 Silica nanoparticles are favourable because they are inexpensive, easy to produce, easy to separate via centrifugation during particle separation, and have surface hydroxide groups that make them easy to functionalize. The use of silicon (Si) nanoparticles as an imaging agent provides an alternative to conventional proton imaging that allows sensitive targeted imaging. They are hyperpolarizable, non-toxic and using silicon minimizes background noise problems because it is found in insignificant amounts in the body.13

Characterization and optimization of nanoparticles biosynthesis

UV-vis spec analysis

The UV-Vis spectra recorded for the aqueous potassium silicofluoride for Fusarium oxysporum reaction medium as a function of the time of reaction. It was observed that the silica surface plasmon band occurs at ca. 280 nm. Complete oxidation of the potassium silicofluoride ions by F. oxysporum occurs after nearly 48 hours of reaction (Figure 3).

(a) Metal control. (b) Culture control.

(c) UV-Vis spectra for silica nanoparticles obtained by F. oxysporum as a function of reaction time.

Figure 3: Biosynthesis of silica nanoparticles by F. oxysporum.

FTIR analysis

Fourier Transform Infrared (FTIR) analysis of silica nanoparticles from the fungus F. oxysporum-potassium silico fluoride reaction medium after 48 hrs of reaction showed the presence of a resonance at ca. 1113 cm-1 (Fig 4a).

FILTRATE.002 1976 4400.00 450.00 45.71 67.25 4.00 %T 1 1.00

Fusarium oxysporum REF 4000 64.37 2000 65.81 600 END 12 PEAK(S) FOUND

3999.30 64.36 3782.44 64.28 3448.71 45.69 2927.12 56.86 2363.36 56.70 2344.68 8.25 1623.62 53.44 1412.04 59.63 1113.05 51.68 743.91 63.57 654.20 64.49 480.14 64.74

K2SiF6.002 1976 4400.00 450.00 0.38 73.74 4.00 %T 1 1.00 Métal control

REF 4000 58.87 2000 56.94 600 END 19 PEAK(S) FOUND

3995.08 58.81 3893.46 60.92 3807.95 59.49 3787.83 58.44 3678.21 57.86 3658.42 7.44

3435.70 49.55 2926.10 53.95 2365.15 53.43 2345.27 54.13 2109.33 56.79 1605.26 4.94 1220.35 44.81 1108.70 43.74 743.22 0.38 608.31 62.08 529.98 63.62 522.08 63.18 482.65 5.14

Figure 4: FTIR analysis of silica nanoparticles by F. oxysporum & potassium silicofluoride.

The 1000-1100 cm-1 band is attributed to excitation of the antisymmetric si-o-si stretching mode of the vibration and was absent from the spectrum of pure potassium silicofluoride (Fig 4b). The presence of the absorption band at ca.743.91 cm-1 indicates the presence of some unreacted ions in the particles. The presence of protein in the silica nanoparticles, the absorption bands at ca. 1623.62 indicated by the amide bond in the particle, this bonds absent in the potassium silicofluoride.

SEM analysis

The surface morphology of the silica nanoparticles were studied by scanning electron microscopy method. The SEM micrograph Figure 5 (a) showed the control of metal oxide. Figure 5 (b) showed nanoparticles aggregates. In the micrograph observed in the size range between 20 and 50 nm. The nanoparticles were not in direct contact even within the aggregates, indicating stabilization of the nanoparticles by a protein capping agent.

(a) Untreated metal oxide; (b) Biosynthesized of silica nanoparticles by F. oxysporum.

Figure 5: Scanning electron microscopy characterization of nanoparticles.

UV-VIS spectrophotometer, Fourier Transform Infrared Spectroscopy (FTIR), Scanning electron Microscope (SEM), to understanding of size, morphology and protein- nanoparticles interaction.14 Typically, nanoparticle characterization includes the measurement of particle size, surface charge, surface functionality, and optical and spectral characteristics.

Biosynthetic proteins by SDS-PAGE & determination of electron shuttling compounds by TLC

The protein fraction responsible for silica nanoparticles biosynthesis was separated by SDS-PAGE and showed in Figure 6a. The molecular weight of F. oxysporum was approximately about 24 KDa.

The Thin layer chromatography analysis on silica gel plates using chloroform-methanol-acetic acid (195:5:1) showed a spot with Rf value of 0.62. TLC result (Figure 6b) showed a first spot control of a F. oxysporum culture filtrate and other spot containing reaction solution F. oxysporum - potassium silicofluoride. After the process, the plates were visualized under UV-light. F. oxysporum produce electron shuttling compound that band was pink in colour.

S M

S- Silica nanoparticle, M- Marker; C - Control (F. oxyporum), T - Biosynthesized silica nanoparticle

Figure 6: SDS – PAGE data of Protein and electron shuttling compounds by TLC.

Protein and electron shuttling compounds for stimulated the synthesis of nanoparticles. Protein assays indicate that an NADH-dependent reductase, which is a well-known enzyme, is the main responsible factor of biosynthesis processes. This reductase gains electrons from NADH and oxidizes it to NAD+. The enzyme is then oxidized by the simultaneous reduction of metal ions.15 The extracellular enzymes, several naphthoquinones with excellent redox properties, were reported in F. oxysporum that could be act as electron shuttle in metal reduction.16

In F. oxysporum, this enzyme is conjugated with an electron donor (quinine), reduces the metal ion, and changes it to elemental form.17 In the case of rapid extracellular synthesis, because the reduction happens in very few minutes, complex electron shuttle materials may be involved in the biosynthesis process. This is due to the fact that nanoparticles are stabilized in solution by capping proteins, which are secreted from microorganisms. One important enzyme that may be responsible for this is Cytochrome C.18

Optimization of biosynthesized nanoparticles

The OD value obtained from UV-Vis spectra was used to determine the optimum biosynthesis of nanoparticles. Here the synthesis of silica nanoparticles by F. oxysporum yield high amount at the optimum pH 3, temperature 270C, metal concentration 10-3M and suitable incubation period 48 hrs (Figure 7). The plant pathogen F. oxysporum acting on amorphous silica in rice husks to transform it into crystalline silica nanoparticle after 24 h at room temperature.19

Figure 7: Optimization of biosynthesized silica nanoparticles under different parameters.

Decolourization of methyl orange by silica nanoparticles

The absorbance peaks of the methyl orange in the visible region 461 nm. The UV-Vis spectra of dye methyl orange solutions before and after decolorization by silica nanoparticles (Figure 8). Silica nanoparticles (SiNp) have much faster decolourization activity than those of potassium silicofluoride (K2SiF6) under the same experimental condition. The absorbance decreasing indicating that chromophore group, the basic functional group of dyes for its visible colour, was broken down. Figure 9 illustrates that the removal efficiencies of methyl orange by biosynthesized nanoparticles.

Methyl orange, which is a stable basic azo dye, is a compound that contains azo groups (−N=N−). Due to its high stability, Methyl orange is commonly used as a titration indicator and a staining agent.20 The metal oxide nanoparticles were used as a complete degradation of Rhodamine B dye.21

(a) Methyl orange before treatment.

(b) Methyl orange after treatment.

Figure 8: UV-Vis spectra of methyl orange solution (461 nm) and UV-Vis spectra of methyl orange before and after decolourization by silica nanoparticles.

C-Control, T-Time of reaction (180,240,300min.)

Figure 9: Decolourization of methyl orange dye by biosynthesized nanoparticles.

Many researchers have studied on the semiconductor nanoparticles as a photocatalyst in decolorization of dye wastewater.22 Although silica is essentially inert for many reactions, it shows noticeable activity towards certain catalytic23-25 and photocatalytic reactions.26-29

The effect of pH on dye decolouization by silica nanoparticles

Decolourization of methyl orange by silica nanoparticles at various pH are shown in (Figure 10). From the results, lowering pH from 5,6,7,8 increased the removal kinetic rates. At pH 5, the decolourization efficiencies of methyl orange using silica nanoparticles, after 300 minutes treatment. As a consequence, pH would strongly affect the degradation of methyl orange by silica nanoparticles.

Figure 10: Effect of pH on dye decolourization by silica nanoparticles.

Stability of Nanoparticle treated DNA against enzyme DNase I treatment

Silica nanoparticles are sufficient and efficient in protecting DNA from enzyme cleavage, DNase solution were added to plasmid DNA and to plasmid DNA- silica nanoparticles complex. After incubation, 20 mM EGTA was added to the DNA-alone solution to stop the reaction and 5% SDS was added to the DNA–NP solutions to denature the protein to stop the digestion. The plasmid DNA molecule alone was digested extensively by DNase I solution, while silica nanoparticles-bound DNA remained intact at this concentration. Thus, the silica nanoparticles provided substantial protection of the DNA molecules from the DNase I cleavage (Figure 11). The amine functionalized silica nanoparticles bind with DNA to inhibit DNase 1 activity.30 The similar finding was observed in the present study.

M- Plasmid DNA Marker, T- silica nanoparticle –Bound DNA, C - DNase I treated band

Figure 11: Stability of silica nanoparticle-bound DNA.

Conclusions

The biosynthesis of silica nanoparticles by a simple, easily culturable and non-pathogenic to human being was used for the productivity of silica nanoparticles. The fungus Fusarium oxyporum was capable of utilizing metal complexes that can be used in large-scale synthesis also. Because the fungus treated with silica nanoparticles have electron shuttling compounds and nanoparticle biosynthetic proteins. Due to its inhibitory activity against DNase, it could be employed in vast areas of biological studies.

Acknowledgements

The authors are grateful to the management of Virudhunagar Hindu Nadars' Senthikumara Nadar College (Autonomous), Virudhunagar, Tamil Nadu, India, for providing the facilities to carry out the work. Grateful thanks to other groups helped in this project, for their timely advices and support.

Funding: No funding sources

Conflict of interest: None declared

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