Home Physical Sciences Characterization of silver sulfide nanoparticles from actinobacterial strain (M10A62) and its toxicity against lepidopteran and dipterans insect species
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Characterization of silver sulfide nanoparticles from actinobacterial strain (M10A62) and its toxicity against lepidopteran and dipterans insect species

  • Fayez M. Saleh , Hattan Gattan , Bassam M. Al-Ahmadi , Abdullah F. Shater , Zuhair M. Mohammedsaleh , Awatif M. E. Omran , Rasha Jame , Noha Omer , Yahya S. Al-Awthan , Elham M. Alhathli , Hamza S. Al-Shehri , Syed Khalid Mustafa , Adel D. Althaqafy , Hatem A. Al-Aoh and Chellasamy Panneerselvam EMAIL logo
Published/Copyright: December 31, 2023
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Abstract

In this research, cell-free extracts from magnesite mine-isolated actinobacterial strain (M10A62) were used to produce silver sulfide nanoparticles (Ag2SNPs). Streptomyces minutiscleroticus JX905302, actinobacteria capable of producing Ag2SNPs, was used to synthesize Ag2NPs. The UV–vis range was used to confirm the biosynthesized Ag2NPs; Fourier transform infrared spectroscopy (FT-IR), atomic force microscopy (AFM), transmission electron microscopy (TEM), X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDAX), and dynamic light scattering analysis were employed to characterize them further. Surface resonance plasma (SRP) for Ag2SNPs was obtained at 355 nm using UV–visible spectroscopy; FT-IR detected bimolecular and eventually microbial-reduced Ag2SNPs from S. minutiscleroticus culture extract. Furthermore, AFM and TEM analysis confirms that the synthesized Ag2SNPs were spherical in shape. Dynamic light scattering revealed a negatively charged Ag2NPs surface with a diameter of 10 nm. The XRD spectrum showed the crystalline nature of the obtained particles. EDAX revealed a pure crystalline nature, and a significant silver particle signal confirms the presence of metallic silver and sulfide nanoparticles together with the signals of Cu and C atoms. After 40 and 48 h of treatment at 150–200 µg·ml−1, Ag2SNPs produced the highest mortality in Spodoptera litura, H. armigera, Aedes aegypti, and Culex quinquefasciatus larvae. Hence, the biosynthesized Ag2SNPs may be useful for potential pest control in integrated pest management and vector control program as a safer, cost-effective, selective, and environmentally friendly approaches.

1 Introduction

The focus of nanoparticles is on the development of nanotechnology in several fields, such as material science, medical, agricultural, and environmental remediation [1,2]. Nanoparticles (NPs) have increased catalytic, mechanical, optical, and magnetic properties due to their high surface area-to-volume ratio [3]. Among various nanomaterials (gold, silver, copper, iron, aluminum, cobalt, titanium, and zinc), silver NPs have offered novel designs of NPs (1–100 nm) that were used in various pest management programs [4,5]. Recently, silver sulfide NPs (Ag2SNPs) synthesized from green plants and microbial origin have gained an important place in nanofabrication with a wide range of applications, including anticancer, anti-inflammatory, anthelmintic and wastewater treatment, and antimicrobial properties [6,7].

The phylum Actinomycetota is primarily composed of gram-positive bacteria with a high nitrogenous base content. By acting as a reducing agent, the metabolites secreted by these bacteria can promote the fusion of NPs [8]. For instance, AgNPs synthesized by Nocardiopsis sp. MBRC-1 (marine antimicrobial strain) showed potent antimicrobial and cytotoxic properties [9]. Similar results were obtained when Streptomyces xinghaiensis OF1-derived AgNPs were tested against pathogenic bacteria and yeast [10]. Due to their metal nature, AgNPs must be suited for a biological system to reduce their cytotoxic effect and the ways in which they interact and connect with biological cells [11].

The common cutworm and cotton bollworm (Spodoptera litura and H. armigera) are well-known lepidopteran agricultural pests that almost occupy entire agricultural crops due to their polyphagous nature and are responsible for major crop damage [12]. Besides crop pests, mosquitoes, Aedes aegypti and Culex quinquefasciatus, served as a vector for spreading many deadly diseases worldwide [13]. Since their outbreak as a pest, the persistent use of chemical insecticides to combat these insect pests may lead to the development of resistance against them through various resistance mechanisms [14]. Apart from resistance, chemical insecticide residues may harm the environment, humans, and other non-target organisms [15]. Therefore, alternatives to chemical pesticides for insect pest management must be investigated. Nanotechnology has provided a different strategy for the pest control program to overcome pesticide resistance [16].

Numerous lines of research have demonstrated the antimicrobial characteristics of AgNPs; however, the insecticidal properties of Ag2SNPs produced through biological synthesis have not been studied properly [17,18]. Comparably, various biomedical applications and mosquito larvicidal activities of selenium NPs synthesized from the M10A62 strain [19,20]. Hence, the present study investigated the biosynthesis of Ag2SNPs and its possible application in pest control using the Streptomyces minutiscleroticus actinobacterial strain (M10A62) isolated from a magnesite mine soil sample.

2 Materials and methods

2.1 Actinobacterial strain

S. minutiscleroticus M10A62 (GenBank Accession Number: JX905302) actinobacterial strain was isolated from a magnesite mine soil sample from Salem district, Tamil Nadu, India. This strain was isolated on casein starch agar (CSA) medium with the supplement of ampicillin (15 μg·ml−1) and fluconazole (20 μg·ml−1) to inhibit undesirable microorganism development and purified on ISP-2 (International Streptomyces Project 2) medium [21]. The phenotypic characterization was done by TEM analysis to confirm the presence of aerial and substrate mycelium; further, 16s rDNA sequencing was used to confirm the strain of Streptomyces sp. [19].

2.2 Biosynthesis of Ag2SNPs

The Ag2SNPs were synthesized by using a shaking incubator. For 5 days at 250 rpm, the S. minutiscleroticus M10A62 strain was transferred to a 250 ml conical flask filled with 100 ml of yeast and malt extract broth and placed in a shaking incubator. Further, the biomass and cell fluids were separated by centrifugation, which ran for 30 min at 4˚C and 6,000 rpm. 5 g of fresh, wet biomass was added to a 100 ml aqueous solution containing 1 mM AgNO3 (silver nitrate) and Na2S⸱9H2O (sodium sulfide nonahydrate), rinsed three times with sterile distilled water, and mixed (HiMedia). After 48 h of consistent shaking at 250 rpm, at room temperature, the entire mixture was centrifuged for 30 min at 10,000 rpm to obtain a cell extract that was used in subsequent research.

2.3 Characterization of Ag2SNPs

2.3.1 UV–visible spectral analysis

The visual color shift of the media from white to black served as a preliminary confirmation of the biosynthesis of Ag2SNPs [28]. Afterwards, the mixture was centrifuged at 5,000 rpm for 15 min to separate the nanoparticles from the liquid. The reduction of nanoparticles was observed in a UV–vis spectrophotometer (Cyber Lab dual-beam spectrophotometer) at the wavelength of 200–700 nm by using 2 ml of aqueous solution at 1 nm resolution [22].

2.3.2 Fourier transform infrared spectroscopy (FT-IR) analysis

FT-IR was used to examine the nanoparticles containing allied functional groups (free amines, amides, or cysteine residues of protein) using the FT-IR model EXI. The lowest amount of dried powder sample was ground with 100 mg of potassium bromide of FT-IR quality before being formed into a pellet [23]. The compressed sample was held in the sample holder, and infrared spectra with a resolution of 4 cm−1 in the wavelength range of 400–4,000 cm−1 were collected. By contrasting functional peaks with already-existing peaks, the resulting nanoparticle spectrum was identified.

2.3.3 Atomic force microscopic (AFM) analysis

AFM analysis was carried out to monitor surface images of Ag2SNPs by using Nanosurf-AFM. The samples were prepared by mixing nanoparticles with methanol, and a drop of the mixture was coated in a silicon slide and further evaporated to form a thin film. Finally, a thin film (1 cm × 1 cm) containing the sample was observed using AFM [24].

2.3.4 X-ray diffraction (XRD) analysis

An XRD (SHIMADZU XRD 6000) was used to analyze the purity and phase formation in Ag2SNPs by using a 40 kV voltage and 30 mA current. The nanoparticle samples were centrifuged at 5,000 rpm for 20 min to ensure purity and re-dispersed in 10 ml of sterile deionized water. Then, the samples were freeze-dried, powdered, and used for structural characterization. The lucid nature of the nanoparticles was identified by comparing the XRD peaks with the Joint Committee on Powder Diffraction Standards (JCPDS) pattern [25].

2.3.5 Transmission electron microscopic (TEM) analysis

The morphological structure of the Ag2SNPs was determined by the TEM model (HITACHI H-600). The silver sulfide sample was prepared by mixing nanoparticles with methanol and ground well. Further, the samples were dehydrated with acetone and infiltrated. Then, the drop of the sample solution was coated in a silicon slide and evaporated to form a thin film, and 1% osmium tetraoxide was poured over the slide, which acts as a post-fixative agent. By using TEM analysis at 80 kV, the size and form of the nanoparticles in the carbon-coated copper grid were determined [26].

2.3.6 AFM analysis

AFM was used to analyze the size of the synthesized Ag2SNPs with selected area electron diffraction [27]. The samples were diluted in distilled water in a 1:9 ratio for AFM analysis. Then, two drops of the dilution were placed in a sample holder and allowed to air dry.

2.3.7 Dynamic light scattering (DLS) analysis

To determine the nanoparticle size dispersion, DLS was used. By injecting 2 ml of deionized water into the interior flow cell, the remaining particles that were present in the flow cell were removed. In order to determine a baseline scattering intensity, the light scattering measurements were logged for 2 min. A 1 ml syringe with 0.8 ml of nanoparticle solution was streaming into the flow cell at the end of the baseline intensity setup. To investigate the scattering intensity within the detector limit, only 0.7 ml of the 0.8 ml solution was pumped into the flow cell. The flow rate was halted once the 0.7 ml of solution had entered the flow cell, and the light scattering measurement was taken for 25 min. Subsequently, sterilized deionized water was used to flush out the nanoparticle’s solution [28].

2.3.8 EDAX analysis

The percentage of the elemental composition of the nanoparticles was determined by EDAX studies by using Bruker AXS Inc., USA, Quantax-200 micro-analysis system coupled with TEM [29].

2.4 Insecticidal activity of Ag2SNPs on lepidopteran insects

The toxicity of biosynthesized Ag2SNPs was investigated on two important lepidopteran insects using the leaf dip method [30]. The H. armigera and S. litura (Accession no. NBAII-MP-NOC-01) egg mass was purchased from the National Bureau of Agricultural Insect Resources live insect repository (NBAIR), Bangalore, Karnataka, India. The first instar larvae hatched from each culture were maintained in the laboratory on castor leaves (25 ± 1°C, 70 ± 5% RH, and 12:12 h light: dark) without exposure to any insecticide. Fresh, clean, and young castor leaves were soaked in different concentrations of Ag2SNPs (10, 50, 100, 150, and 200 µg·ml−1) for 20 s and left for a few minutes until getting dry; the control received water only. Approximately 25 early third instar H. armigera and S. litura larvae were released in Ag2SNPs dipped and control leaves. Three replications were maintained for each concentration. After 14, 24, 36, 40, and 48 h of treatment, the larval mortality was determined.

2.5 Insecticidal activity of Ag2SNPs on dipterans insects

The insecticidal activity of Ag2SNPs was evaluated on two dipterans insects as per the method of Muthusamy and Shivakumar [31]. The Ae. aegypti and Cx. quinquefasciatus were collected in the form of egg mass and egg raft from the Institute of Vector Control and Zoonoses (IVCZ, Hosur, Tamil Nadu, India). The eggs were brought into the laboratory and cultured under controlled conditions (25 ± 1°C, 70 ± 5% RH, and 12:12 h light: dark) on fresh tap water containing trays covered with mosquito net cloth. During the culture period, dog biscuits were provided as larval food. The newly hatched early third instar mosquito larvae were used for the insecticidal efficacy of Ag2SNPs. Serial concentrations of Ag2SNPs were prepared (as mentioned above) in a 250 ml paper cup containing distilled water. There were three replicates for each dose, and 25 larval (uniform-sized) were released in each paper cups; the control received only water. The larval mortality was assessed at 7, 14, 24, 36, 40, and 48 h of post treatment.

3 Results and discussion

The Actinobacteria are a significant bacterial group that is present in both terrestrial and aquatic settings. It has economic importance as a source of many antibiotics and the decomposing ability of many organic matters [32]. The present study revealed that five bacterial isolates with vegetative mycelium development on selective media were isolated. However, based on the quantitative analysis of metal-producing ability, actinomycete isolate S. minutiscleroticus M10A62 strain was used in this research to produce Ag2SNPs in the fight against target insect pests.

Figure 1 illustrates the morphological characteristics of the M10A62 strain, and the texture and color of white aerial mycelium are depicted in Figure 1a. The SEM image (Figure 1b) shows the morphology of spores with recti flexible (RF) arrangement of smooth surface. After being identified as a potential source of AgNPs, physicochemical characteristics such as temperature, pH, and various reaction combinations were optimized [33]. The conversion of AgNO3 and Na2S⸱9H2O into Ag2SNPs was validated during M10A62 strain incubation by color changes to a brown, yellow, indicating the production of Ag2SNPs (Figure 2a and b), is well in accord with the reports of Ramya et al. [20].

Figure 1 
               Morphological characteristics of S. minutiscleroticus M10A62 (a); (b) SEM analysis of strain M10A62.
Figure 1

Morphological characteristics of S. minutiscleroticus M10A62 (a); (b) SEM analysis of strain M10A62.

Figure 2 
               Production of Ag2SNPs by M10A62 strain (a and b).
Figure 2

Production of Ag2SNPs by M10A62 strain (a and b).

The UV–vis spectrum data revealed the confirmation of Ag2SNP production, and the surface plasma resonance (SPR) phenomenon was responsible for the color shift [34]. The absorption maxima of synthesized Ag2SNPs were obtained at 355 nm (Figure 3). The FT-IR measurement was used primarily to identify the possible biomolecules that are acting as reducing agents in Ag2SNP synthesis by actinobacterial culture filtrate (Figure 4). The FT-IR spectra of Ag2SNPs showed various bands at different wave numbers with respective functional groups (Table 1). The observation for Ag2SNPs indicates that the protein binds to nanoparticles by means of free amines, amides or cysteine residues of protein bind to the negatively charged groups of enzymes present in the cell wall of actinobacterial mycelium through the electrostatic interaction [35,20]. Further, AFM analysis shows that the synthesized Ag2SNPs were mostly spherical in shape with an average size ranging from 0.5 to 5 µm, respectively (Figure 5).

Figure 3 
               UV–visible spectra of S. minutiscleroticus derived Ag2SNPs.
Figure 3

UV–visible spectra of S. minutiscleroticus derived Ag2SNPs.

Figure 4 
               FT-IR spectrum of S. minutiscleroticus derived Ag2SNPs.
Figure 4

FT-IR spectrum of S. minutiscleroticus derived Ag2SNPs.

Table 1

FT-IR peaks and their functional groups for Ag2SNPs

S. No Peaks (cm−1) Functional groups
1 3,777 Strong sharp O–H stretching
2 3,471 Amide I
3 2,926 C–H stretching
4 2,139 Alkyene
5 1,776 C═O stretching
6 1,650 Amide II
7 1,597 Amide conjugated
8 1,356 Amide III
9 1,128 C–N stretching
10 1,019 C–O stretching
11 647 Residues of NO 3
Figure 5 
               AFM images of S. minutiscleroticus derived Ag2SNPs.
Figure 5

AFM images of S. minutiscleroticus derived Ag2SNPs.

The TEM pictures revealed that the actinobacterial strain M10A62 produced well-disseminated nanoparticles that were attached to capping protein molecules. The morphology and size of the Ag2SNPs were 50–85 nm in size with spherical in shape (Figure 6). TEM analysis results were well matched with Ramya et al. [19], who studied the biological properties of actinobacterial-synthesized selenium nanoparticles. Figure 7 shows the XRD pattern of the Ag2SNPs exhibited strong reflections at 2θ values of 46.25°, 44.12°, 32.23°, and 37.10° corresponding to the planes −111, 111, 112, and 120 (JCPDS card No. 14-0072). Comparably, similar results from pomegranate peel extract and Nicotiana tabacum leaf extract derived XRD pattern were documented [36,37]. The dynamic light dispersion experiment revealed that Ag2SNP particle size was about 10 nm (Figure 8). Figure 9 illustrates that the EDAX analysis of sulfide nanoparticles exhibited a strong signal at 3 keV due to SPR [10,38].

Figure 6 
               TEM image of S. minutiscleroticus derived Ag2SNPs.
Figure 6

TEM image of S. minutiscleroticus derived Ag2SNPs.

Figure 7 
               XRD pattern of S. minutiscleroticus derived Ag2SNPs.
Figure 7

XRD pattern of S. minutiscleroticus derived Ag2SNPs.

Figure 8 
               DLS of S. minutiscleroticus derived Ag2SNPs.
Figure 8

DLS of S. minutiscleroticus derived Ag2SNPs.

Figure 9 
               EDAX spectrum of S. minutiscleroticus derived Ag2SNPs.
Figure 9

EDAX spectrum of S. minutiscleroticus derived Ag2SNPs.

Numerous researchers have studied the toxic effect of nanoparticles on pathogenic bacteria and other pathogens of animals [39,40,41]. There have been relatively few investigations on the biocidal effect of biosynthesized nanoparticles [42,43]. However, no reports were evidenced in Ag2SNPs against insects as insecticidal compounds. The biosynthesized Ag2SNPs from the M10A62 strain were shown to have a high mortality rate on S. litura and H. armigera at 150 and 200 g·ml−1 after 40 and 48 h post-treatment (Figure 10a and b). Followed by lepidopteran, Ag2SNPs showed 100% mortality at 150 and 200 µg·ml−1 on Ae. aegypti after 40 and 48 h post-treatment (Figure 10c), whereas in Cx. quinquefasciatus Ag2SNPs produced 100% mortality at 100, 150, and 200 µg·ml−1 after 36, 40, and 48 h treatment (Figure 10d). Similarly, in support of our research, Jafir et al. [16] found that the silver nanoparticles from Ocimum basilicum had effective insecticidal activity against S. litura at a dose of 1,500 mg·l−1. Further, the Annona glabra-derived NPs displayed potent larvicidal activity against the dengue vector Ae. aegypti and Aedes albopictus [44]. The essential oil-wrapped AgNPs showed good toxicity against the larvae and pupae of Ae. albopictus [45]. Cassia hirsute-derived AgNPs showed LC50 4.43 ppm against Cx. quinquefasciatus [46]. Consequently, the results of this work bring up a new application for Ag2SNPs from actinobacterial strain M10A62 and can be used as an alternative technique for pest management.

Figure 10 
               Insecticidal activity of Ag2SNPs at different time intervals. (a) Insecticidal activity against S. litura. (b) H. armigera. (c) Ae. aegypti. (d) Cx. quinquefasciatus; Mortality (%) represents mean of three replicates, bar indicates standard deviation (SD) of the mean.
Figure 10

Insecticidal activity of Ag2SNPs at different time intervals. (a) Insecticidal activity against S. litura. (b) H. armigera. (c) Ae. aegypti. (d) Cx. quinquefasciatus; Mortality (%) represents mean of three replicates, bar indicates standard deviation (SD) of the mean.

4 Conclusion

In this research, an eco-friendly and target-specific stable Ag2SNPs were fabricated from actinobacterial strain M10A62. The synthesized nanoparticles were confirmed by various bio-physical techniques, and the obtained nanoparticles were mostly spherical in shape with an average size ranging from 50 to 85 nm. Based on the results of this research, the Ag2SNPs had significant toxicity on both crop and human pests. Overall, the actinobacterial-derived nanoparticles could be used as alternative insecticides in pest management programs since they are more affordable and safer. The process of biogenic synthesis may be optimized in the future, and field applications may be developed.

  1. Funding information: Authors state no funding involved.

  2. Conflict of interest: Authors state no conflict of interest.

  3. Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Received: 2023-08-07
Accepted: 2023-11-15
Published Online: 2023-12-31

© 2023 the author(s), published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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