Introduction

Nanoparticle biosynthesis has recently gained attention because of the growing demand for eco-friendly solutions that involve material synthesis. There have been numerous attempts, particularly in the synthesis of silver nanoparticles (AgNPs) that utilize microorganisms as mediators. In recent years, the importance of microorganism-mediated synthesis of nanoparticles has grown owing to their ease of production and low on the environmental impact [1]. Nanotechnology is primarily concerned with producing nanoparticles that can vary in their dimensions, forms, and structures, and investigating the potential uses of these particles in human life. Many interesting investigations have been made in nanoparticles-related research due to their diverse applications and the improvements they have made in the fields of of nanoscale material-based catalysis, electronic devices and materials science. It is commonly believed that nanoparticles have better physicochemical and biological properties than their bulk material counterparts. This belief is founded on the fact that nanoparticles possess unique features, including surface area, shape, size of nanoparticles, and charge distribution. Researchers are focusing on developing nanomaterials that can be used as affordable antimicrobial agents in the fight against infectious diseases [2]. The use of biologically produced nanoparticles is becoming more and more common owing to their features, pathogenicity, and cost-effectiveness of the nanoparticles themselves. These nanomaterials, which are safe for the environment are incredibly useful in various biomedical applications, including sensing, diagnostics, and others. The most significant advantage of using these nanoparticles is that biosynthesized nanoparticles do not require any physical factors, such as increased levels of energy, pressure and temperature, during the process of biogenic synthesis [3].

In recent years, the biological applications of nanomaterials have attracted research interest as an alternative novel therapeutic agent for several human health defects. Additionally, nano-sized particles are beneficial in optical and catalytic applications [4]. The synthesis of nanoparticles involves a wide range of physical and chemical techniques, which have added to the advancement of an innovative and unexplored research territory in the synthesis of nanoparticles. Thun, the need for clean, non-toxic, and eco-friendly advances has prompted us to spotlight to the biosynthesis of nanoparticles [5]. Microorganisms such as actinomycetes can synthesize nanoparticles with precisely controlled form, size, and content. Silver nanoparticles attracted researchers because of their flexible physiochemical parameters, broader applications [6], and display of numerous microbial inhibition modes. Silver nanoparticles are in high demand because of their stable dispersions and budding applications such as effective antimicrobial, anti-inflammatory, and anti-cancerous agents [5], anti-diabetic activity [7], microelectronics, and nonlinear optics [8]. Biosynthesized AgNPs are notable for their enhanced effect on clinical pathogens and ability to prevent harmful infections [9]. In mammals, the nanoparticles can interfere with the antioxidant defence mechanism which produces ROS [10]. Different groups of microorganisms, including actinobacteria, bacteria, algae, fungi, yeasts and cyanobacteria have been reported to achieve the biosynthesis of various metal nanoparticles [8, 11]. The actinobacteria are considered an important metabolite producer [12]. They are present in different environments, including extreme habitats. Actinobacteria found in severe environments contain novel compounds [9], and have been used as proficient candidates for eco-friendly nanofactories [12]. Actinobacterial nanoparticles have excellent stability, polydispersity, and better size control [13]. In the biomedical aspects, the vector-borne diseases caused by mosquitoes are considered to be the more dangerous; they are the primary vectors for the transmission of several potentially fatal diseases, including dengue, yellow fever, malaria, lymphatic filariasis, and chikungunya. There are 390 million people infected with dengue and chikungunya worldwide, and more are estimated to have a clinical manifestation of the disease. These epidemics occur in tropical and subtropical regions [14]. Medical experts and government health officials around the world have shown serious concerns about dengue fever, which has recently emerged as a significant threat to the health of the general population. Although a vaccine has been developed, there is no effective and safe treatment for dengue fever. Tropical regions usually use methods including indoor residual spraying, insect growth regulators, insecticide-treated bed nets, and organophosphates to target mosquito larvae and reduce the risk of disease transmission. The extensive use of the abovementioned chemicals during outbreaks can be harmful to both humans and the environment. Vector control is allegedly a very difficult problem in developing nations such as India, where there is a dearth of both a high socioeconomic level and a comprehensive understanding. It is therefore crucial to develop an effective insecticide that is inexpensive and eco-friendly [15]. In the face of the greater potential of drugs as well as uses of biosynthesized nanoparticles in the service of human well-being, they are still inadequate and further research is immediately needed especially in the biomedical and human health related studies [4]. This study is aimed at examining if and how actinobacteria can synthesize silver nanoparticles. The potential of the biogenic AgNPs was evaluated by their antibacterial and mosquito larvicidal properties. Furthermore, the potential silver nanoparticle production isolate was characterized using morphological and molecular analysis.

Materials and Methodologies

Source of Marine Actinobacteria

Actinobacteria were isolated from materials gathered from maritime environments, including ECR Lake, ECR brine pit (Uppalam) (Lat. 10.37 N, Long. 79.85 E) Muttukadu estuary (Lat. 12.79 N, Long. 80.20 E) and Kovalam beach (Lat. 12.78 N, Long. 80.25 E). Using a sterile spatula, soil samples were collected and placed in sterile polythene bags before being transported to the laboratory. The samples were named as ES-ECR lake, EU-ECR brine pit (uppalam), ME-Muttukadu estuary and KL-Kovalam beach.

The actinobacteria Isolation Process

One gram of pre-treated soil and sediment samples were mixed with 10 mL of distilled water, vigorously shaken, and diluted up to 10−5. The serially diluted samples were used to inoculate onto starch casein agar (SCA) medium [16] amended with antifungal agent fluconazole (100 mg/mL). After that, 7–14 days of incubation occurred at room temperature with the plates, the figures of the mother inoculum obtained from soil sample and the pure colonies obtained from the mother inoculum of the actinobacteria provided in the supplementary file Fig. 2.2A, B, respectively.

Biological Silver Nanoparticles Synthesis

Following inoculation with the pure actinobacteria culture, three Erlenmeyer flasks containing starch casein broth were shaken in an orbital shaker set to 120 rpm and 30 °C for six days. After the incubation period was complete, the biomass was harvested from the broth as a mat using Whatman No.1 filter paper, centrifuged at 7000 rpm for 15 min. and washed with double distilled water [17]. Subsequently, 10 g of biomass was ground thoroughly using a mortar and pestle and suspended with 100 mL of distilled water. The biomass was mixed with 50 mL of 1 mM silver nitrate for the silver nanoparticle synthesis. Concurrently, a control without silver nitrate was also prepared with the experimental flasks. The reaction mixture was kept in a shaking incubator at 100 rpm in the dark for overnight. After incubation, the color change in both the reaction mixtures was observed, the detailed scheme visible confirmation of silver nanoparticle formation from the light yellow to dark brown coloration during the constant time interval is provided in the supplementary file Fig. 2.3A initial period, Fig. 2.3B 24 h, and Fig. 2.3C 72 h. Further, the biosynthesized NPs were separated by ultracentrifugation and the double distilled water was used to remove all contaminants [18].

Characterization of the Silver Nanoparticles

The nanoparticles were characterize using various analytical techniques; this is considered an important role in evaluating the functional aspects of synthesized particles. UV–Vis spectrophotometer was used to keep an eye on the levels of synthesized AgNP (Labram HR Evolution) in the range of 400 to 800 nm. To determine the composition and structure of the material, X-ray diffraction (XRD) was used (BRUKER USA D8 ADVANCE) with Cu-Kα1 radiation in the 2θ range from 10° to 80°. The AgNPs were analyzed using FT-IR and the KBr pellet method and scanned over a wave number range of 500–4000 cm1 for the determination of various functional groups. The surface appearance and nanoscopic structure of the synthesized AgNPs were studied using scanning electron microscopy (SEM) with energy dispersive spectroscopy (EDS) (Bruker nano inc).

Antibacterial Assay of the AgNPs

The biosynthesized AgNPs were tested for antibacterial efficacy against clinical isolates viz. using the agar well diffusion method, Escherichia coli, Bacillus subtilis, and Klebsiella pneumoniae. The overnight cultures of the test pathogens were prepared by adjusting them the turbidness to match the 0.5 McFarland sample standards. Subsequently, the dried AgNPs (1 mg/mL) were dissolved in sterile distilled water [19]. The AgNPs (10 µL) were added to 10 µL of crude actinobacterial metabolites (1:1) and antibiotics (cefotaxime 25 µg/mL) as the control sample. A total of 20µL of test mixture was transferred in each of the wells and incubated overnight at 37 °C [20]. The inhibition zone around the well was recorded. Microplate reader readings at 600 nm were used to determine the MIC of the AgNPs against the test organisms. In 96-well dilution plates, a twofold increased dilution series of the AgNPs at 10, 20, 30, 40, and 50 g/mL were prepared for the MIC. The log phase of each individual strain at a concentration of 105 CFU/mL was used.

Larval Toxicity Test

The standard method (WHO, 1996) was employed for the larvicidal activity on mosquito larvae and pupae. DMSO broth (1 mL) was added to 125 mL of dechlorinated tap water in wax-coated paper cups. The larvae (n = 25) at the third in-star stage (Cx. quinquefasciatus Ae. aegypti, and An. stephensi) were introduced [21]. Larvicidal activity analysis was performed against all three different larval species using five different concentrations of synthesized silver nanoparticles [22].

Molecular Profiling of Actinobacteria

Genomic DNA was isolated and purified from the most potential isolate, Actinobacterium KL-3, using the QIAGEN DNA isolation kit (Qiagen). The 16S rRNA gene sequences were amplified using 27f: (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1522r: (5′-AAGGAGGTGATCCANCCRCA-3′) primers. Thereafter, the annealed gene product was amplified using Taq polymerase and sequenced using an ABI PRISM 3730 genetic analyzer. Finally, MEGA 5.0 was used to examine the percentage of sequence similarity index and create the phylogenetic tree.

Result and Discussions

The maritime environments of ECR Lake includes 110 solitary colonies, including ECR brine pit, Muttukadu estuary, and Kovalam beach on and around the Chennai coast, Tamil Nadu. Amongst them, 8 colonies were morphologically different. They were produced by white, ash, green, yellow, and pink-colored aerial mycelium on an SCA medium. Among the 8 isolates, four isolates produced diffusible pigments on the isolation medium. The ability to synthesize silver nanoparticles was tested in a wide variety of morphologically distinct actinobacteria. The screening findings showed that there were two possible isolates, that could potentially synthesize the AgNPs (SRM-EU5 and SRM-KL3). Based on previous studies [6, 9], a sizable fraction of actinobacterial strains can synthesize silver nanoparticles, making them useful in a wide range of contexts. The actinobacteria inoculated in the silver nitrate solution was pale yellow and turned brown after a while. Even after being incubated for 96 h, the color of control did not change. The isolate KL3 was found to have a faster-reducing capacity; this was confirmed by the color change observed after 6 h. The Ag+ reduction may be due to the enzyme system of the actinobacteria. Thus, the isolate KL3 was selected for AgNP synthesis and characterization. As an apparent sign of AgNPs production, the color of the reaction mixer changed from yellow to yellowish-brown. After 10 min of reaction, a strong surface plasmon resonance (SPR) band at 420 nm was apparent, and the absorbance steadily increased at periodic intervals, as shown in the UV–Vis spectra of the mixture of isoamyl acetate and the AgNO3 solution recorded against the time of reaction. Velayutham and Ramani Bai reported that the increase in intensity may be due to a surge in nanoparticle production as a result of the decrease in silver ions in the aqueous solution [2, 23, 24]. A absence of a color change indicated the absence of silver nitrate, whereas a color change indicated the removal of Ag+ and surface plasmon resonance was enhancement [11, 25]. Comparatively, actinobacteria have many advantages in the synthesis of nanoparticles because they are naturally available and produce versatile novel metabolites that may help in reduction [26] as well as being safe to handle [26, 27]. Electron microscopy SEM and TEM, spectroscopy UV–Vis, FT-IR, and energy dispersive X-ray (EDX) analyses are common and valuable techniques used to characterize nanoparticles [28,29,30]. In addition, a UV–Vis spectrophotometer validated the biosynthesized AgNPs by showing a distinct peak with maximum absorbance at 428 nm. In general, the silver nanoparticles exhibit their characteristic peaks between 300 and 500 nm. NPs with characteristic peaks of about 400 nm will increase in size and vanish once the particle size drops out of nano-dimensions [29, 31]. The crystallinity of the synthesized AgNPs was evaluated using an XRD spectrum. Strong peaks at 2θ values of 27°, 32°, and 46° were noticed in the XRD spectra of the AgNPs. The peak revealed that the AgNPs have a face-centered cubic (fcc) structure [1]. In this study, peak ranges from the lattice planes were seen at 38.17, 44.27, and 64.77 which are attributable to the facets of silver, correspondingly confirming the presence of a crystalline form that was prepared via biosynthesis using Andrographis serpyllifolia. These results were partially corroborated with our present study which was conducted by the actinobacteria-mediated silver synthesis as shown in supplementary file Fig. 1.

FT-IR analysis was performed in this study to determine the presence of various functional groups produced by the silver nanoparticles. In this study, there were peaks around 730, 797, and 1270 cm−1 the peaks from 730 cm−1 and 797 cm−1 confirmed strong carbon and hydrogen bending, and the peaks between 1210 and 1163 cm−1 exhibited carbon and oxygen stretching, confirming the presence of ester groups in the sample [32]. There were characteristic peaks between 3,417 cm−1 (corresponding to OH stretching due to the alcoholic group) and 1578 cm−1 (corresponding to the C=C ring stretching, which was partially consistent with the present study, as displayed in supplementary file Fig. 2).

The size and shape of the nanoparticle were determined using SEM. The SEM image revealed that the actinobacteria has a high potential to synthesize AgNPs and that S. parvisporogenes produced globular particles with sizes ranging from 23 to 27 ± 0.02 nm. This was considered an excellent size range and surface properties, similar to previous studies [1] and [32]. The morphology and content of the AgNPs were visualized using SEM analysis in conjunction with EDAX. According to both SEM and TEM analyses, the AgNPs were mostly observed to be poly dispersed spheres, and the size of the nanoparticles was found to be around 24 and 5 nm respectively. These findings can be considered as supportive evidence to the notion that S. parvisporogenes can serve as an effective biological source for the synthesis of nanomaterials. The results of this study are shown in the supplementary file Fig. 3. In the TEM images, the smaller particles are depicted as spheres, while the bigger particles often take the appearance of disks. The absence of particle aggregation was another sign that the reaction mixture was intrinsically stable. The EDS spectrum showed a high percentage of elemental Ag along with organic components such as O and C atoms, and the EDAX spectra make it abundantly clear that the weight% of the AgNPs that were depleted by S. parvisporogenes was identical to that of silver. The presence of silver in the sample was indicated by a representative peak at 3 keV. This peak demonstrated that the reduction of AgNPs was achieved with a binding energy of between 97 and 98% as shown Fig. 1. The above mentioned results were similar to the previous findings of [1] and [32].

Fig. 1
figure 1

EDX analysis of synthesized AgNPs by Streptomyces parvisporogenes

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Similarly, previous studies reported that the size of the actinobacterial nanoparticles ranged from 20 to 100 nm with different shapes [9, 33, 34]. In the antibacterial assay, 10µL of the biosynthesized nanoparticles along with 10 µl of actinobacterial metabolites and antibiotic (cefotaxime) as control were examined. Isolate KL3 AgNPs exhibited strong antimicrobial activity in vitro against Escherichia coli, Bacillus subtilis, and Klebsiella pneumoniae. The AgNPs along with metabolites showed maximum inhibition zone against the test organisms. The actinobacterial metabolite alone (without AgNPs) also showed observable activity against the tested pathogens. The inhibitory data showed that the Escherichia coli and Klebsiella pneumoniae were more sensitive to the synthesized AgNPs than Bacillus subtilis. The AgNPs synthesized by actinobacteria have potential antibacterial activity against some pathogenic bacteria [28, 35]. This inhibitory effect may be due to variations in the cell wall composition structure [36]. This experimental result indicates that the biosynthesized AgNPs exhibited good bacterial effects and could help develop drug to combat pathogenic bacteria [7]. The AgNPs were found to possess broad-spectrum activity against various clinical pathogenic bacteria [37].

Determination of Minimal Inhibitory Concentration

The antibacterial activity of the AgNPs was evaluated at a concentration of 105 CFU/mL against Klebsiella pneumonia, Bacillus subtilis and Escherichia coli; the details of the in-vitro antibacterial activity are shown in the supplementary file Fig. 3.1A–C. The AgNPs were synthesized in concentrations of 10, 20, 30, 40, and 50 µg/mL. Figure 2 shows the results of an OD 600 nm screening of the final bacterial concentration. The bacterial concentrations decreased when the concentration of the AgNPs increased [25, 38]. For MIC, Klebsiella pneumoniae was utterly inhibited in growth at 40 µg/mL of AgNPs, while Escherichia coli and Bacillus subtilis were inhibited entirely in growth at 50 µg/mL of AgNPs.

Fig. 2
figure 2

Growth inhibition of test organisms in the presence of different concentration of AgNPs: a Escherichia coli, b Bacillus subtilis and c Klebsiella pneumoniae

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The data presented here demonstrated that the silver nanoparticles (AgNPs) biosynthesized in this study had effective larvicidal activity against An. stephensi (LC50 17.511 and LC90 50.572), A. aegypti (LC50 22.481 and LC90 61.920) and Cx. quinquefasciatus (LC50 29.548 and LC90 73.123 (Table 1).

Table 1 Acute toxicity of synthesized AgNPs by S. parvisporogenes KL3 against mosquito larvae
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The mean and standard deviation of the mortality rates were determined over five replicates; there were no fatalities reported in the control group. The means that are not statistically distinct from one another (P < 0.05) are those that are separated from one another inside a row. LC50 is the deadly concentration at which 50% of exposed organisms die, and LC90 is the lethal concentration at which 90% of exposed organisms die. Similarly, the LC50 for S. alboflavus extract was reported to be 1.48 ± 0.09, LC90 3.33 ± 0.22 against Ae. aegypti, and 1.30 ± 0.09, LC90 3.13 ± 0.21 against An. Stephensi [39]. Further, Amelia-Yap et al. [40] also studied the larvicidal activity of Streptomyces cacaoi sub sp. cacaoi-M20 and found that it had a positive effect on Cx. quinquefasciatus.

Since relying on just one technological solution could be prohibitively expensive and inefficient [41], it is necessary to create and employ hybrid technologies to overcome the drawbacks of each technology and make the method inexpensive [42]. Thus, there is a slight possibility of using combined technologies (microbe-nanoparticle) to achieve-sustainable alternatives to overcome this problem. Morphological and molecular characterization methods were used to pinpoint the possible isolate KL3. The micromorphology of the isolate KL3 showed ash-colored aerial spores and dark green-colored substrate mycelia on starch casein agar. The isolate KL3 produced a unique smooth spore surface that was spirally twisted with 4–5 bends, and the SEM image showed a spherical sporophore [43]. The micro-morphological features of the isolate allowed us to place it in the genus Streptomyces. Streptomyces is morphologically characterized by its dark green substrate mycelia [39]. For definitive confirmation, the 16S rRNA gene of Streptomyces species was performed and KL3 was sequenced. An entry number of MH986192 was assigned to the gene sequence in GenBank (NCBI). The resulting gene sequence was compared to other sequences and used to construct a phylogenetic tree. After comparison with the 16S rRNA sequences of actinobacteria available in GenBank, 99% homology with the taxa S. parvisporogenes was confirmed (Fig. 3).

Fig. 3
figure 3

Phylogenetic analysis of Streptomyces parvisporogenes KL3 using 16S rRNA sequence

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Conclusion

This study described the first use of Streptomyces parvisporogenes KL3 in an environmentally friendly and cost-effective synthesis of AgNPs which have unique optical properties. Biogenic nanoparticle synthesis is widely advocated owing to its low toxicity and other significant advantages. Advances in nano-engineering have opened novel avenues for the development of cost-effective and eco-friendly treatment options. Therefore, actinobacterial AgNPs could be helpful in the development of new drugs that combat various bacterial and vector-borne diseases. The silver ions are reduced extra-cellularly by the actinobacteria to silver ions in the nanometer range. Owing to the synthesis of smaller-sized nanoparticles, mosquito vectors and bacterial infections can be easily combated by Streptomyces parvisporogenes KL3. Moreover, the use of AgNPs together with microbial systems will greatly increase their stabilization potential and public health relevance.