Facile fabrication of rice husk based silicon dioxide nanospheres loaded with silver nanoparticles as a rice antibacterial agent

Bacterial leaf blight of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major disease of rice, leading to reduction in production by 10–50%. In order to control this disease, various chemical bactericides have been used. Wide and prolonged application of chemical bactericides resulted in the resistant strain of Xoo that was isolated from rice. To address this problem, we were searching for an environmentally friendly alternative to the commonly used chemical bactericides. In this work, we demonstrate that silicon dioxide nanospheres loaded with silver nanoparticles (SiO2-Ag) can be prepared by using rice husk as base material precursor. The results of the antibacterial tests showed that SiO2-Ag composites displayed antibacterial activity against Xoo. At cellular level, the cell wall/membrane was damaged and intercellular contents were leaked out by slow-releasing of silver ions from SiO2-Ag composites. At molecular level, this composite induced reactive oxygen species production and inhibited DNA replication. Based on the results above, we proposed the potential antibacterial mechanism of SiO2-Ag composites. Moreover, the cytotoxicity assay indicated that the composites showed mild toxicity with rice cells. Thus, this work provided a new strategy to develop biocide derived from residual biomass.

Scientific RepoRts | 6:21423 | DOI: 10.1038/srep21423 Rice husk is a commonly agricultural waste material and the world annual production is approximately 120 million tons 27 . It is reported that rice husk contains approximately 20% silicon dioxide, which makes it a potential renewable source of SiO 2 28 . And previous researches have indicated that SiO 2 nanoparticles can decrease heavy metal accumulation and increase the production of the plant 29,30 . Meanwhile, SiO 2 nanospheres have high surface and reactivity and are considered as a supporting material according to the previous study 31 . So we use rice husk as a raw material to prepare SiO 2 nanosphere, which is a suitable candidates for supporting material.
In our work, we developed a simple method to synthesize SiO 2 nanospheres using rice husk as a raw material and decorated the nanospheres with Ag NPs by the reduction of Ag ions. The results showed that the Ag NPs were successfully loaded onto the surface of the SiO 2 nanospheres (SiO 2 -Ag), and the preliminary studies showed that the composites have a superior antibacterial effect and only mild cytotoxicity to rice cells. Furthermore, the level of reactive oxygen species (ROS), the content of genomic DNA and the integrity of the cell membrane were also determined. Based on the results above, we proposed a potential antibacterial mechanism of the SiO 2 -Ag composites. In general, this study provides direct evidence that these composites have great potential to be used as antibacterial agents in agriculture and offer an environmentally friendly method to synthesize an antibacterial nanomaterial using residual biomass.

Results and Discussion
Synthesis and characterization of the SiO 2 -Ag composites. The synthesis process for the SiO 2 -Ag composites is shown in Fig. 1. In this study, we used rice husks as a raw material to synthesize SiO 2 nanomaterials by a hydrothermal method. SiO 2 was a near-perfect sphere with a smooth surface and a diameter of approximately 400 nm ( Fig. 2A,C). Then, poly-(N-vinyl-2-pyrrolidone) (PVP) was applied as the stabilizer and reductant. The Ag ions were reduced at the surface of SiO 2 nanospheres, and the typical morphology of the SiO 2 -Ag composites is shown in Fig. 2B,D,E. We propose that the bulges on the SiO 2 nanospheres are Ag NPs.
We performed high-resolution TEM (HRTEM) (Fig. 2E,F) to further analyze the nanostructures of the SiO 2 -Ag composites and found that the diameter of the Ag NPs was approximately 10 nm. This small size of the Ag NPs might have better antibacterial activity 32,33 . The interplanar spacing for the lattice fringes was approximately 0.23 nm, corresponding to the (111) lattice plane of silver 34,35 . The elemental composition of the SiO 2 -Ag composites was analyzed by energy dispersive X-ray spectroscopy (EDS), as shown in Fig. 2H; several types of peaks were clearly observed, which correspond to carbon, oxygen, copper, silicon, and silver. The as-prepared SiO 2 -Ag composites contain approximately 57.26 wt% Si and 5.47 wt% Ag.
The structural features of the SiO 2 -Ag composites have also been investigated by X-ray diffraction (XRD) analysis (Fig. 3). As shown in Fig. 3A, the typical XRD pattern of SiO 2 nanospheres was an amorphous peak with the equivalent Bragg angle at 2θ = 22°. Figure 3B indicates that the XRD pattern of the SiO 2 -Ag composites has two sharp Bragg peaks at 38.2°, 44.4° in the 2θ region, which could be assigned to the (111) and (200) planes of silver, indicating that Ag NPs were successfully loaded onto the surface of the SiO 2 nanospheres.
Release property. An inductively multitype coupled plasma emission spectrometer (ICP-AES) was used to compare the release rate of Ag ions from AgNO 3 , Ag NPs and the SiO 2 -Ag composites. As shown in Fig. 4A, the Ag ions were completely released into the ultrapure water from AgNO 3 and the Ag NPs in less than 3-10 d. However, the SiO 2 -Ag composites could stably release Ag ions over 30 d. The Ag ion release rate of the composites was slower than that of AgNO 3 and the Ag NPs. After 30 d, the SiO 2 -Ag composites had released 68.1% of the Ag ions, which was significantly lower than AgNO 3 (96.4%) and the Ag NPs (78.8%). The results showed that the SiO 2 -Ag composites have a lower release speed than AgNO 3 and the Ag NPs, which had more long-term antibacterial effects than that of AgNO 3 and Ag NPs 36,37 . Antibacterial evaluation. To confirm the antibacterial effect of the SiO 2 -Ag composites, the growth inhibition of the tested bacteria Xoo was investigated by the disk diffusion method. As shown in Fig. 4B, Ag NPs and SiO 2 -Ag composites have an average diameter of the inhibition zone of 18 ± 2 mm and 23 ± 1 mm, respectively. However, the disks with the control and SiO 2 nanospheres have no inhibition zone, indicating that the Ag NPs are the effective antibacterial component of the SiO 2 -Ag composites. Minimum inhibitory concentration (MIC) testing against Xoo was carried out to further evaluate the antibacterial activity of the SiO 2 -Ag composites. As shown in Fig. 5, the density of bacterial growth was decreased in a dose-dependent manner. Xoo growth was completely   inhibited when the concentration of the SiO 2 -Ag composites was 3.2 μ g/mL (Fig. 5J), whereas the Ag NP solution exhibited the same effect at a concentration of 12.5 μ g/mL (Fig. 5A). The tests of the antibacterial properties confirmed that the antibacterial activity of the SiO 2 -Ag composites was approximately four times higher than that of the Ag NPs against Xoo.
The ability of SiO 2 -Ag to prevent viable bacteria growth is also demonstrated by fluorescence staining. Ethidium bromide (EB) and acridine orange (AO) were used as live/dead coloring agents. EB could enter through the damaged cell membrane and selectively stain dead cells, whereas AO could penetrate live and dead cells 25,38 . As shown in Fig. 6, nearly all of bacteria were viable when cultured on the control and SiO 2 -Ag composites. In contrast, the Xoo treated with the SiO 2 -Ag composites exhibited strong red fluorescence, indicating that most of the bacteria were killed (Fig. 6C). These results further support the antibacterial studies that the SiO 2 -Ag composites were clearly more effective than the Ag NPs.
Cell wall/membrane integrity assay. A TEM study was performed to observe the morphological changes of bacteria cells after treatment with the SiO 2 -Ag composites. As shown in Fig. 7A,B, the bacteria were adsorbed by the SiO 2 -Ag composites, and the morphology of bacteria cells changed from cylindrical to spherical after treatment with the SiO 2 -Ag composites for 2 h. Figure 7C,D illustrate that released Ag ions disrupted the cell wall/ membrane integrity. As a result, more Ag NPs were internalized into the bacteria cell wall/membrane, and the contents of the cell leaked out, leading to protein denaturation and cell death.
The antibacterial results demonstrate that the SiO 2 -Ag composites have better antibacterial properties compared to those of the Ag NPs. According to the literature, the antibacterial activity of Ag NPs would be reduced due to aggregation and oxidation [17][18][19] . In our work, we prepared the composites such that the Ag NPs were loaded on the surface of the SiO 2 nanospheres. These composites could effectively enhance the antibacterial activity by preventing the aggregation and oxidation of Ag NPs and by continuously releasing Ag ions. This result was consistent with previous studies 25,39 . The SiO 2 -Ag composites have a large surface area and high adsorption properties; thus, the bacteria could be easily adsorbed by the composites. Intracellular oxidative stress. It has been suggested that the production of ROS is the common pathway by which antibacterial agents induce oxidative damage in bacteria cells 40 . Many nanomaterials have been reported to exert their toxic effects through ROS [41][42][43] . Therefore, we compared the level of ROS after treatment with SiO 2 nanospheres, Ag NPs and the SiO 2 -Ag composites by fluorescence intensity. As shown in Fig. 8A, the DCF fluorescence intensity in samples treated with SiO 2 nanospheres was similar to that in the untreated cells. However, in the presence of the Ag NPs, the DCF fluorescence intensity was increased two-fold compared with exposure to the SiO 2 nanospheres and to untreated cells. In addition, the DCF fluorescence intensity of the samples treated with the SiO 2 -Ag composites was nearly 1.4 times higher than that of the samples treated with Ag NPs. These results revealed that the SiO 2 -Ag composites could significantly increase ROS production and lead to cell damage. Moreover, the SiO 2 -Ag composites would have a long-term antibacterial effect by continually releasing Ag ions.
Influence of SiO 2 -Ag composites on genomic DNA. It has been reported that Ag NPs interact with DNA and inhibit DNA replication, resulting in rapid antibacterial activity 44,45 . In this study, agarose gel electrophoresis analysis was used to investigate the possible antibacterial mechanism of the SiO 2 -Ag composites. As shown in Fig. 8B, the intensity of the genomic DNA band was decreased in a dose-dependent manner. The intensity of the genomic DNA band was lowest when the cells were treated with 12.5 μ g/mL of SiO 2 -Ag composites. In contrast, the intensity of the genomic DNA band of the untreated cells formed a clear band. The results of agarose gel electrophoresis analysis were consistent with the MIC values. According to the above results, the potential antibacterial mechanism of the SiO 2 -Ag composites was proposed as follows ( Fig. 9): the bacterial cells absorb on the surface of the SiO 2 -Ag composites by electrostatic forces, and Ag ions were released from the Ag NPs and transported to the cytoplasm. The Ag ions directly interact with intracellular mitochondria, resulting in the generation of ROS and the inhibition of DNA replication. Subsequently, the integrity of the cell wall/membrane was disrupted, and the intracellular contents leaked out.
Cytotoxicity assay. To test the toxicity of the SiO 2 -Ag composites, we selected rice cell viability to elucidate the cellular response to a toxin. The rice cell suspension was exposed to different concentrations of SiO 2 nanospheres, Ag NPs or the SiO 2 -Ag composites for 24 or 48 h (Fig. 10). The results showed that the cell viability with Ag NPs was lower than that of the SiO 2 nanospheres and SiO 2 -Ag composites after a 24 h culture. However, after culturing for 48 h, the viability of cells treated with Ag NPs was significantly lower than that of the SiO 2 nanospheres and SiO 2 -Ag composites. Increasing the incubation time and concentration of the Ag NPs resulted in a significant decrease in cell viability. In our study, the different results between the Xoo and rice cells might have occurred because the structure of bacteria and plant cells is different and the content of silver in the SiO 2 -Ag composites was low and had no impact on the rice cells. More detailed reasons require further study. Therefore, we anticipate that the SiO 2 -Ag composites are promising antibacterial agents that would control rice diseases effectively and provide rice plants with nutrients.
In summary, we have developed a simple and environmentally friendly method using rice husks as a raw material to synthesize SiO 2 nanospheres. These materials were then decorated with Ag NPs by the reduction of Ag ions in the presence of PVP as a stabilizer and reductant. TEM, SEM and XRD indicated that Ag NPs with small sizes were well dispersed onto the surface of SiO 2 nanospheres. This structure could prevent the aggregation and oxidation of Ag NPs. We also confirmed that the SiO 2 -Ag composites displayed antibacterial activity against Xoo that was approximately four times higher than that of the Ag NPs. Meanwhile, the antibacterial mechanism of the SiO 2 -Ag composites was explored. The Ag ions released from the SiO 2 -Ag composites could induce the production of ROS, leading to the inhibition of DNA replication and disruption of the cell wall/membrane. More importantly, the cytotoxicity assay indicated that the SiO 2 -Ag composites showed only mild toxicity towards rice cells. Thus, the SiO 2 -Ag composites have a great potential application in rice disease management as antibacterial    Methods Synthesis of SiO 2 nanospheres. SiO 2 nanospheres were synthesized using rice husks. The rice husks were washed with distilled water and milled into powder. Next, 2.0 M NaOH was mixed with the rice husk powder at a ratio of 1:7 (w/v) in a 200 mL three-neck round-bottom flask equipped with a thermometer and heated to 100 °C for 4 h. The extract was separated from the solids by vacuum-assisted filtration and diluted with different volume ratios of distilled water and ethanol at 25 °C. Then, sulfuric acid (1.0 M) was added drop-wise into the system until the pH of the system was approximately 9.0. Using an ultrasonicator, polyethylene glycol was completely dissolved into the solution, followed by the drop-wise addition of a solution of 1 M sulfuric acid to lower the pH to 3. The mixture was left standing for 10 min at 25 °C and then centrifuged for 5 min. The products were washed several times with distilled water and dried at 60 °C for 5 h. To obtain the SiO 2 nanospheres, the samples were calcined at 550 °C for 1 h in a muffle furnace to remove residual organics in the SiO 2 nanosphere sample.

Synthesis of SiO 2 -Ag composites.
To form a homogeneous SiO 2 -Ag composite suspension, 100 mg of the SiO 2 nanosphere powder in 100 mL of deionized water was sonicated for 30 min, and 6 g PVP was dissolved in the previous solution. Then, 20 mL of an AgNO 3 (1 mM) aqueous solution was rapidly added into the above solution. This mixture was stirred vigorously for 12 h in the dark at 80 °C. The resulting product was collected by centrifugation at 5,000 rpm for 10 min and further washed in deionized water several times to remove residual Ag ions. The dry SiO 2 -Ag composites were obtained after drying under vacuum for 3 h at 60 °C.  Fluorescence imaging. Bacteria were incubated in the LB liquid medium supplemented with SiO 2 nanospheres, Ag NPs and the SiO 2 -Ag composites. The bacterial cells were collected by centrifugation and washed three times with phosphate buffer saline (PBS) and then stained using EB and AO for 15 min. After washing with PBS, the samples were observed by fluorescence microscopy.

Cell morphological change.
To observe the morphological changes of bacterial cells after treatment with the SiO 2 -Ag composites, the Xoo cells were exposed to the SiO 2 -Ag composites (12.5 μ g/mL) in microtiter plates with silicon chips in the bottom. After the cultures grew for 2 h, the silicon chip was harvested and processed for TEM. First, the silicon chip was removed from the microtiter plates and washed three times with buffer. Then, the samples were fixed in 2.5% glutaraldehyde for 2 h. After fixation, the silicon chip was rinsed with buffer twice. The samples were washed with a cacodylate buffer and fixed in 1% osmium tetraoxide. Then, sample embedding was carried out using a standard protocol. The slices were deposited on bare #200 mesh grids and stained with uranyl acetate for 5 min. Finally, the grids were dried in a desiccator and examined using TEM. Intracellular reactive oxygen species measurement. 2,7-dichlorofluoroscein diacetate (DCFH-DA) was used to further identify the intracellular generation of ROS in the treated bacterial cells. The DCFH-DA could enter the cell and react with ROS, which formed the highly fluorescent compound dichlorofluorescein (DCF). Experimental procedures were followed as described previously 41,47 . The fluorescent signal intensity of DCF (with an emission wavelength of 525 nm) was recorded using a fluorescence spectrophotometer with an excitation wavelength of 488 nm.
Genomic DNA isolation. The genomic DNA was extracted from the Xoo cells treated with different concentrations of SiO 2 -Ag composite (12.5, 3.2, 0.8 μ g/mL) for 4 h. Then, the DNA was isolated by the phenol chloroform extraction method 48 . The isolated DNA was then analyzed on a 1% agarose gel using EB.
Cytotoxicity assay. Rice suspension cells were cultured according to the literature procedure 49 . The seeds were surface-sterilized in 75% ethanol for 1 min and 0.1% mercury chloride for 10 min and rinsed five times with sterile distilled water. The seeds were incubated in plastic petri dishes containing modified N6 medium. The sealed dishes were cultured in the dark to induce calli from mature rice seeds at 28 °C. Every 7 d, the calli were subcultured in the subculture medium. After 4 weeks, the calli were transferred to 125 mL conical flasks containing 40 mL of liquid AA medium and placed on a rotary shaker at 110 rpm at 28 °C in the dark. To supplement nutrients, the suspension cells were subcultured at 5 d intervals for 2-3 months by replacing the old nutrient solution medium every 5 d.
Then, the culture medium was replaced with 100 μ L of different concentrations of SiO 2 nanospheres, Ag NPs or the SiO 2 -Ag composites. The cells were further incubated for 24 or 48 h, and then, 25 μ L of MTT (3-(4,5-dimethyl-2-yl)-2,5-diphenyl tetrazolium bromide) (5 mg/mL) was added to each culture medium until the final concentration was 1 μ g/mL. After incubation for another 2 h, the absorbance was measured at 570 nm using a microplate reader. Cell viability was normalized to that of rice cells cultured in the cell media. Measurements were repeated three times for each concentration.