TiO2 nanoparticle biosynthesis and its physiological effect on mung bean (Vigna radiata L.).

TiO2 nanoparticle (NPs) biosynthesis is a low cost, ecofriendly approach developed using the fungi Aspergillus flavus TFR 7. To determine whether TiO2 NPs is suitable for nutrient, we conducted a two part study; biosynthesis of TiO2 NP and evaluates their influence on mung bean. The characterized TiO2 NPs were foliar sprayed at 10 mgL-1 concentration on the leaves of 14 days old mung bean plants. A significant improvement was observed in shoot length (17.02%), root length (49.6%), root area (43%), root nodule (67.5%), chlorophyll content (46.4%) and total soluble leaf protein (94%) as a result of TiO2 NPs application. In the rhizosphere microbial population increased by 21.4-48.1% and activity of acid phosphatase (67.3%), alkaline phosphatase (72%), phytase (64%) and dehydrogenase (108.7%) enzyme was observed over control in six weeks old plants owing to application of TiO2 NPs. A possible mechanism has also been hypothesized for TiO2 NPs biosynthesis.

Introduction

Titanium is a strong, lustrous, corrosion resistant metal. Its common compound, titanium di-oxide, is a popular photo-catalyst, and is used in the manufacture of pigments [1]. The Ti+4 ionic state dominate titanium chemistry, owing to its high oxidation state, showing a high degree of covalent bonding. In plants, titanium has been reported to stimulate production of more carbohydrates, encouraging growth and photosynthesis rate [2], [3], [4].

TiO2 is a non-toxic white pigment for use in manufacture of paints, plastics, paper, ink, rubber, textile, cosmetics, leather, and ceramics [5]. Photo catalytic degradation of pesticides with TiO2 and other catalyst has shown promise as a potential water remediation method [6]. It has also been noted that titanium dioxide breaks down the ethylene gas produced in storage rooms into carbon-dioxide and water, thus it is also used to treat the air in fruit, vegetable, and cut flower storage areas to prevent spoilage and increase the product’s shelf life [7].

In the rhizosphere, root exudation is a key process for n class="Chemical">carbon tranpan>sfer inpan>to the soil, inpan>fluenpan>cinpan>g the role of soil microbial communpan>ities inpan> the dpan> class="Chemical">ecomposition of organic matter and in native nutrient cycling [8]. Root exudates are the substances released by roots and may affect growth and activity of soil microorganisms in the rhizosphere [9]. Root exudates act as a chemo-attractants to attract microbes toward roots and have been shown to increase the mass and activity of soil microbes [10].

n class="Chemical">Nanpan>otpan> class="Chemical">echnology is one of the most important tools in modern science yet only a few attempts have been made to apply these advances for increasing crop productivity [4], [11]. It is possible to develop microorganisms as bionanofactories for synthesis of agriculturally important particles. TiO2 NPs are promising as efficient nutrient source for plants to increase biomass production due to enhanced metabolic activities, and utilization of native nutrients by promoting microbial activities. Fungi are relatively recent addition to the list of microorganism used in the synthesis of nanoparticles. The use of fungi is potentially exciting since they secrete large amounts of enzymes and are simpler to manage in the laboratory. In the biosynthesis of metal nanoparticles by a fungus, extracellular secreting enzymes are produced which reduce the metal salt of macro or micro scale into nano-scale diameter through catalytic effect. Negative electro kinetic potential of microorganisms enables to attract the cations and act as a trigger for biosynthesis of metal and metal oxide nanoparticles [12], [13]. This study attempts to synthesize TiO2 NPs using Aspergillus flavus TFR 7 as an ecofriendly biological approach and evaluate their effect on mung bean (Vigna radiata L).

Experimental details

Characteristic of experimental soil

An experimental soil (20 cm depth) was collected from Jodhpur, India (26°18′N 73°01′E), then air dried and sieved through 2 mm mesh. The soil was classified as loamy sand. Organic carbon was estimated by following the method of Walkley and Black [14]. Nitrogen, phosphorous and potassium were analyzed by Jackson [15]. In addition, pH and electrical conductivity were also measured.

Isolation and identification of soil fungi

The fungi was isolated from rhizosphere soil by initial plating on Martin Rose n class="Chemical">Bengal Agar medium (Hi-Media, Inpan>dia, pH 7.2) followed by serial dilutionpan>s over pan> class="Species">potato dextrose agar medium supplemented with chloramphenicol (Sigma–Aldrich, St. Louis, USA) at a concentration of 10 μg mL−1. Isolated fungi was identified up to molecular level by partial sequencing of 18S and 28S rRNA and complete sequence of internal transcribed sequence 1 (ITS-1), ITS-2 and 5.8S rRNA. The sequence was compared with gene library data available on National Centre of Biotechnology Information (www.ncbi.nlm.nih.gov) using nucleotide blast algorithms, to identify isolated fungal strain using bioinformatics tool ‘blastn’.

Synthesis of TiO2 nanoparticles using soil fungus

To synthesize n class="Chemical">TiO2 nanpan>oparticles, pan> class="Species">A. flavus TFR 7 was developed in broth medium (pH 5.8) supplemented with of 0.3% malt extract, 1% sucrose, 0.3% yeast extract, and 0.5% peptone. The culture was kept on shaker at 150 rpm at 28 °C for 72 h to develop fungal ball of mycelia. These mycelia were separated out by filtration Whatman filter paper no. 1 (Whatman, UK) followed by triple washing with deionized water. Reaped mycelia (10 g fresh biomass) were re-suspended in 100 mL deionized water and incubated for 48 h at 28 °C under the same shaking condition as above. The obtained cell free filtrate containing extracellular enzymes was used for synthesis of TiO2 NPs, in which precursor salt (Bulk TiO2) was mixed at a concentration of 10−3 M and incubated for 36 h at 150 rpm and 28 °C to yield fine monodisperse TiO2 NPs,

Characterization of synthesized TiO2 nanoparticles

Synthesized nano-crystals were characterized morphologically by transmission eln class="Chemical">ectronpan> microscopy (TEM; JEOL JEM-2100F) inpan>cludinpan>g high resolutionpan> (HR)–TEM mode for crystal phase conpan>firmationpan>, anpan>d enpan>ergy dispersive X-ray sppan> class="Chemical">ectroscopy (EDS; Thermo Noran equipped with TEM) for surface elemental analyses. Since particles were dispersed in water, hydrodynamic diameter was analyzed using dynamic light scattering (DLS; Beckman DelsaNano C, USA).

Seed germination and exposure of nanoparticles

The certified seed (obtained from institutional seed house) were surface-sterilized using 10% n class="Chemical">sodium hypochlorite solutionpan> followed by triple wash with deionpan>ized pan> class="Chemical">water. After that, five seeds were sown at 3 cm depth in each pot. The pots were placed in a greenhouse with 16 h photoperiod and 30/20 °C day–night temperature, 60% relative humidity and 360 μmol m−2 s−1 photoactive radiation intensity. After 10 days of germination, seedlings were thinned to three per pot. The pots were completely randomized and re-positioned weekly to minimize uneven environmental effects.

The experiment was carried out with three treatments viz. control (without n class="Chemical">TiO2 applicationpan>), ordinpan>ary pan> class="Chemical">TiO2 (1.6 μ), nano TiO2 with each of six replicates. The TiO2 particles (10 ppm) were exposed by foliar application to avoid direct soil contact using a fine nebulizer (25 mL per pot). The concentration and amount of nanoparticle solution was optimized in a preliminary screening experiment (data not shown here).

Phenological and physiological effect of nanoTiO2

Plants were harvested after four weeks of foliar application to investigate n class="Chemical">phenology and physiological state of plant. To analyze, shoots were cut at the soil surface and roots were carefully shaken to remove excess soil, and clumps of soil trapped between roots were removed, and number of nodules, root length, area and diameter were measured using Delta T Scan Software (Delta Scan, UK). To prepare the sample, roots were dipped in a n class="Chemical">methylene blue dye for 6 h while shoot length was measured on a meter scale.

Biochemical parameter, dehydrogenase enzyme assay for microbial activity in rhizosphere was assessed according to Tabatabai [16], and n class="Chemical">phosphorous mobilizing enzymes including acid and alkaline phosphatase activity was assessed according to Tabatabai and Bremner [17]. In addition to these parameter phytase [15], n class="Chemical">chlorophyll content [18], soluble leaf protein content [14], [19] rhizospheric microbial populations were also assayed.

Results and discussion

Physicochemical characteristics of rhizosphere soil

The characteristic of the experimental soils studied are presented in Table 1. The soil was alkaline in nature (pH 7.8) with low electrical conductivity (0.34 dS m−1), organic carbon (0.29%) and NPK contents.

Table 1

Characteristics of the experimental soil.

Parameter	Quantity
Sand (%)	83.2 ± 4.2
Slit (%)	6.4 ± 0.8
Clay (%)	6.3 ± 0.5
pH	7.8 ± 0.1
EC (dSm−1)	0.4 ± 0.04
Organic carbon (%)	0.29 ± 0.01
Total N (mg Kg−1)	456 ± 13.5
Total P (mg Kg−1)	719 ± 19.1
Total K (mg Kg−1)	681 ± 16.8

Isolation and identification of fungi

Isolated fungal strain was identified as n class="Species">A. flavus designpan>ated with laboratory strainpan> TFR7 onpan> the basis of 5.8S rDpan> class="Chemical">NA gene (Complex of -18S-ITS1-5.8S-ITS2-28S) sequence similarity. The gene sequence was submitted to NCBI GenBank and got accession no. of strain, JQ675308 which is available on NCBI the database (http://www.ncbi.nlm.nih.gov/nuccore/383929211).

Biosynthesis and characterization of TiO2 NPs

The biosynthesis of n class="Chemical">TiO2 NPs was carried out by exposure of a precursor salt as bulk TiO2 solution of 10−3 M concentration to extracellular enzyme obtained by A. flavus TFR 7 in an aqueous solution. The reaction was carried out for 36 h. Synthesized nanoparticles were characterized for morphological analyses. Particle size distribution was analyzed by DLS. Histogram shows average particle size (based on intensity distribution) ranges from 18 nm (Fig. 1). The polydispersity index (PDI) was 0.302 reflects monodisperse nature of the particle. Since DLS measure hydrodynamic diameter, so it was further confirmed with TEM analysis.

Fig. 1

Size distribution of biologically synthesized TiO2 nanoparticles.

Size distribution of biologically synthesized n class="Chemical">TiO2 nanpan>oparticles.

TEM measurements showed well distribution of n class="Chemical">TiO2 NPs with the average size of 12–15 nm (Fig. 2). Difference in size measurement of TEM and DLS is due to hydrodynamic core that surrounds the particle when dispersed in solvent. The crystal and lattice structure of biosynthesized TiO2 NPs can be observed in HR-TEM micrograph (Fig. 3). The EDS spectrum (full scan mode) of drop coated TiO2 NPs shown in Fig. 4, confirms the purity of titanium metal. The spectrum shows strong peak intensity of titanium metal at 4.6 keV (94 atom%) corresponds to purity of biosynthesized TiO2 NPs.

Fig. 2

TEM micrograph of biologically synthesized TiO2 nanoparticles.

Fig. 3

High Resolution TEM micrograph of biologically synthesized single TiO2nanoparticles.

Fig. 4

EDX spectrum of biologically synthesized TiO2 nanoparticles.

TEM micrograph of biologically synthesized n class="Chemical">TiO2 nanoparticles.

High Resolution TEM micrograph of biologically synthesized single n class="Chemical">TiO2nanoparticles.

Physiological effect of TiO2 nanoparticles in mung bean

The results demonstrated a significantly higher plant growth in those plants, which were treated by n class="Chemical">TiO2 NPs. With respect to control, plants exposed with TiO2 NPs showed significant improvements in shoot length (17%), root length (49.6%), root area (43%) and root nodule (67.5%) due to foliar application of TiO2 NPs was noticed (Table 2). Clear morphological differences in the phenology of mung bean plant can also be observed in Fig. 5.

Table 2

Phenological parameter of mung bean plants at six weeks of crop age.

Treatment	Shoot length (cm)	Root length (mm)	Root area (mm2)	Root nodule
Control	47.0	32.4	16.5	11.7
Ordinary TiO2	49.6	39.1	19.7	13.3
Nano TiO2	55.0	48.5	23.6	19.6
LSD (p = 0.05)	0.23	0.09	0.07	1.39

Fig. 5

Phenology of mung bean plant under varying treatment; Control; OTiO2: ordinary titanium di oxide; n TiO2: nano titanium di oxide.

Photosynthetic pigment, n class="Chemical">chlorophyll anpan>d total soluble leaf proteinpan> conpan>tenpan>t was inpan>creased by 46.4% anpan>d 94%, resppan> class="Chemical">ectively (Table 3) due to TiO2 NPs at 10 mg L−1concentration. Results of phenology and physiology, clearly indicates that biosynthesized TiO2 NPs is promising for plant nutrition. Results presented in Table 4, exhibited that population of rhizospheric microbes (fungi, bacteria and actinomyceteae) was also increased between 21.4% and 48.1% by application at critical growth stage (six weeks) of mung bean crop. Indirectly, TiO2 NPs also enhance activity of dehydrogenase (108.7%), phytase (64%), acid phosphatase (67.3%) and alkaline phosphatase (72%) in the rhizosphere (Table 5) that may be due to increased microbial population over the control. Increased activity of phytase and phosphatase enzyme activity may help in native phosphorous nutrient mobilization in rhizosphere [20].

Table 3

Total soluble protein and chlorophyll content mung bean plants at six weeks of crop age.

Treatment	Total soluble protein (mg Kg−1)	Chlorophyll content (mg Kg−1)
Control	8.4	52.3
Ordinary TiO2	11.3	60.7
Nano TiO2	16.3	76.5
LSD (p = 0.05)	0.18	0.5

Table 4

Microbial population in rhizosphere of six weeks old mung bean plants.

Treatment	Fungi (CFU × 10−4)	Bactria (CFU × 10−6)	Actinomycetes (CFU × 10−5)
Control	21.0	38.7	13.7
Ordinary TiO2	24.0	45.7	15.0
Nano TiO2	26.7	47.0	20.3
LSD (p = 0.05)	0.04	0.08	0.05

Table 5

Enzymes activity in rhizosphere of six weeks old mung bean plants.

Treatment	Acid phosphatase (EU × 10−4)	Alkaline phosphatase (EU × 10−4)	Phytase (EU × 10−2)	Dehydrogenase (pkat g−1)
Control	5.2	4.3	2.5	5.7
Ordinary TiO2	7.4	6.7	3.7	9.5
Nano TiO2	8.7	7.4	4.1	11.9
LSD (p = 0.05)	0.09	0.04	0.04	0.14

Microbial population in rhizosphere of six weeks old n class="Species">mung bean plants.

Enzymes activity in rhizosphere of six weeks old n class="Species">mung bean plants.

Extracellular secretionpan> of enpan>zymes offers the advanpan>tage to obtainpan> pure, monpan>odisperse nanpan>oparticles, which are free from cellular componpan>enpan>ts, associated with organpan>isms anpan>d easy downpan>-stream processinpan>g. Results inpan>dicated that A. TFR 7 is capable to synpan>thesize finpan>e pan> class="Chemical">TiO2 NPs. To understand the mechanism behind biosynthesis of TiO2 NPs, a simple mechanism is drawn (Fig. 6), showing TiO2 NPs nanoparticle synthesis using fungus extracellular enzyme secrets. Capping protein, secreted by fungus itself, encapsulates the TiO2 nanoparticle and increases its stability whereas associated proteins may help in mineralization of precursor salt [21], [22]. Detail studies for identification of these proteins and biochemistry investigations are still underway. Such biologically synthesized, functional TiO2 NPs are economically cheap to synthesize, easy downstream processing and environmentally safe. These promising TiO2 NPs may act as nanonutrient fertilizer to enhance crop production by stimulating plant metabolic activities. As a nanonutrient, best response of TiO2 NPs can be perceived by foliar application 10 mg L−1 on 14 days old plant. In plant leaves nanoparticles may adsorb to plant surface and taken up through natural nano or micrometer scale openings. Several pathways exists which are predicted for nanoparticle association and uptake in plants [23], [24]. Present invention may open new door for plant nutrition research and fertilizer industries.

Fig. 6

Mechanism for biosynthesis of TiO2 nanoparticles.

Conclusion

In the summary, we synthesized an n class="Chemical">eco-frienpan>dly anpan>d conpan>venpan>ienpan>t approach for the synpan>thesis of pan> class="Chemical">TiO2 NPs using A. flavus TFR 7 in which no harmful chemical reagent or surfactant template was required, consequently enables the bioprocess with the advantage of being environmental friendly. Synthesized TiO2 NPs may be used as plant nutrient fertilizer to enhance crop production.