Bacterial Silver Nanoparticles: Method, Mechanism of Synthesis and Application in Mosquito Control

Abstract

Silver nanoparticles (AgNPs) received tremendous attention due to their fascinated applications. Extensive research reports are available on the physical, chemical, and biological synthesis of colloidal Ag NPs. Research on biological systems mediated silver nanoparticle synthesis is essential to explore more applications. Microbial synthesis has been recognized as an eco-friendly and influential source among biological sources. Therefore, the bacteria are often considered an exciting reducer for silver and gold nanoparticles fabrication. Further, the synthesized nanoparticles incorporated different biological agents from what we need as bio reducing agents. The cell membrane of microorganisms plays a crucial role in the endogenous synthesis of nanoparticles. The cell membrane interacts electronically with the charged metal ions because it is charged. Enzymes inside the cell membrane biodegrade metal ions into nanoparticles, which eventually propagate through the cell membrane in small volumes. The fabricated silver nanoparticles were characterized by different spectroscopy techniques, to reveal the structural and functional properties. The synthesized nanoparticle reacts against many pathogens and insects and is used in medical fields. One of the pesticide industry’s significant applications is mosquito larvicidal application. This chapter dealt with the microbial-mediated synthesis of silver nanoparticles, characterization, and mosquito larvicidal applications.

1. Introduction

Nanotechnology can regulate and manipulate objects at the individual level of atoms and particles. Physicist Richard Feynman previously envisioned the hypothetical utilization of nanotechnology in 1959, and indeed, the term “Nanotechnology” was coined by Norino Taniguchi. When a parameter is stated as a measure of 10⁻⁹ meters of SI units, it is called “nano” [1].

Dimensionality, shape, composition, homogeneity, and aggregation are used to classify nanoparticles. The shape and syllable structure of nanoparticles plays a significant role in their function and harmful impacts on the environment and people. Nanoparticles can be classified into one, two, and three-dimensional nanoparticles. One-dimensional nanoparticle includes thin flicks used in electronics and sensor devices. Two-dimensional nanoparticles are high in carbon nanotubes absorption capacity and constancy. Three-dimensional nanoparticles are dendrimer quantum points. On top of that, morphology may be the basis of nanoparticles flat, spherical, and crystal in structure. They may be in a single form or the arrangement of compounds. Nanoparticles can be supplementary classified oxide nanoparticles, sulfide nanoparticles, and magnet nanoparticles [2, 3].

Various physical, chemical, and biological strategies are generally utilized to synthesize nanoparticles. Integrated nanoparticles are considered unfavorable due to high capital cost, energy requirements, anaerobic conditions, utilization of toxicity generation of furnaces, and harmful waste. Nanoparticles are less biodegradable, and utilizing toxic chemicals for synthesis, and the absence of sustainability have restricted their utilization in medical applications. Therefore, the development of ecologically safe, economical, and biological compatibility events for the assembly of nanoparticles is preferred. The fabrication of nanoparticles by natural resources is economical and alternate for physical and chemical methods. The latest advances were made in developing nanotechnology collection of Nano-sized particles. These nanoparticles are considered building blocks for developing optoelectronic electronics and numerous biochemical and chemical sensors [2, 4].

Development of ecologically safe, economical, and biological compatibility procedures for the assembly of nanoparticles is preferred. The synthesis of nanoparticles by biological means is low-priced. For the fabrication of nanoparticles, biological synthesis, including microbes, has been exploited worldwide. Bacteria, fungi, and yeasts are chosen for synthesis due to their rapid growth rate, easy cultivation, and ability to grow ambient temperature, pH, and pressure environments. Because of their adaptation to a toxic metal environment, eco-friendly microorganisms have the inherent ability to integrate nanoparticles by following the reduction mechanism through internal and external-external routes. Microbes trap metal ions from the location and convert them into the basic form using their enzymes [2].

Nanomedicine utilizes nanoscale structures to diagnose, treat, and prevent diseases in improving human health. Nanomaterials are comparable to cellular elements, including nano quantity proteins; thus, they can target chosen sites without contacting other cellular machinery. Now scientists aim to integrate the modernity of nanomedicine with conventional molecular tools and biotechnology to develop advanced therapies for the treatment of disease and tissues repair, novel drug delivery systems, rapid and ultrasensitive analytic tools such as biosensors, biopharmaceuticals, surgical aids compatible biomaterials [2, 5].

Arthropods are extremely dangerous vectors of pathogens and parasites, which may hit epidemics or pandemics in the increasing world population of humans and animals [6]. Among them, mosquitoes (Diptera: Culicidae) represent a huge threat for millions of people worldwide, vectoring important diseases, including malaria, dengue, yellow fever, filariasis, Japanese encephalitis and Zika virus [7]. Furthermore, Culicidae transmits key pathogens and parasites that dogs and horses are very susceptible to, including dog heartworm, West Nile virus, and Eastern equine encephalitis [8, 9]. Unfortunately, no treatment is available for most of the arboviruses vectored by mosquitoes, with special reference to dengue. A promising interface between nanotechnology and arthropod control recently opened new routes to manage vector and pest populations [10].

Now the researcher is strongly promoting the development of nano-based insecticides. Nanobiotechnology is a new discipline in nanosciences that emerged from the interface between nanotechnology and biotechnology. This crossbreeding performance is green and environmentally friendly, biocompatible, inexpensive, and a great alternative to traditional chemical approaches in the pest control industry [11]. Among the bio-based insecticides, microbial insecticides are essential for improving the toxicity of mosquito larvae, causing less harm to non-target species, and reducing environmental risks. Therefore, microbially mediated nano metallic synthesis has proven to be an environmentally friendly and efficient source, and bacteria have been reported to be effective reducing agents in synthesizing silver and gold nanoparticles (NPs). In this synthesis method, several metabolites of microorganisms can be used, which simultaneously promote the reduction and stabilization of nanoparticles and the adhesion and formation of a layer of biomolecules on their surface, the corona, which increases their biocompatibility. In this context, the current work was aimed to study the method, mechanism of synthesize of silver nanoparticles using bacteria, characterize the silver nanoparticles and assess its larvicidal effectiveness against mosquito vectors.

2. Synthesis of silver nanoparticle

The specific mechanism for synthesizing nanoparticles using biological agents has not yet been developed, as diverse biological agents react inversely with metal ions. The composition of nanoparticles contains different biological molecules. In addition, the mechanism for intracellular and extracellular synthesis of nanoparticles differs in other biological agents (Figure 1). The cell wall of microorganisms plays a vital role in the endogenous synthesis of nanoparticles. The cell wall interacts electronically with the positively charged metal ions as it is negatively charged. Enzymes inside the cell wall biodegrade metal ions into nanoparticles, which eventually propagate through the cell wall in small volumes [12].

Figure 1.

Schematic flow diagram for intracellular and extracellular synthesis of nanomaterials.

2.1 Biosynthesis of metal nanoparticles using bacteria

Many reports proved that bacteria is an excellent organic apparatus for the fabrication of metal nanoparticles—for instance, Streptomyces sp. M25 was isolated from a soil sample of Western Ghats and is used to synthesize silver nanoparticles [13]. The bacterial sample was inoculated into 100 ml of YEM broth and incubated in the rotary shaker for 5 days. 10 g bacterial mass was mixed with the 100 ml 1 Mm AgNO3. The reaction mixture was kept in a rotary shaker for 24 h. The color change is the primary confirmation of the synthesis of Ag NPs. The same solution was centrifuged, and the Ag NP was collected from the supernatant. The reaction mixer was further subjected to spectrometric UV-VIS spectrophotometer, Transmission electron microscopy, X-ray diffraction (XRD), and further characterization. The result showed that the size of the Ag NPs is 10-35 nm [13].

Bacillus safensis LAU 13 is the gram-positive spore-forming bacteria isolated from the waste dumpsite, and it was used for the biosynthesis of silver nanoparticles using the supernatant of B. safensis LAU 13. 1 mM of AgNO₃ was diluted into the 40 ml of distilled water and add 1 ml of the supernatant of B. safensis LAU 13. The synthesized silver nanoparticle is spherical shaped, having a size of 5–95 nm, and it is confirmed by UV-VIS spectrophotometer, Fourier transform infrared spectroscopy, and Transmission electron microscope. Bio synthesized silver nanoparticles were used for bioassay against first instar Anopheles larvae in 10–100 μg/ml [14].

2.2 Mechanism of bacterial silver nanoparticle synthesis

All the bacteria cannot reduce the metal ions. The capability of the reduction mechanism depends upon the bacterial defense mechanism. If the bacteria are exposed to the metal environment, metal ions like Ag + enter the cell and bind to the bacterial DNA. Silver particles have a positive charge, and DNA is contrarily charged. It changed the nature of DNA, resulting in a loss of structure and replication ability. Ag + binds with protein, especially thiol-containing proteins, and inhibits the function of proteins. The reductase enzyme of bacteria reacts metal (active Ag + − inactive Ag0) into inactive and is not lead to cell death. Most of the reductase enzymes are NADPH dependent, and bacteria can have or secrete this cofactor NADH dependent enzyme. The quantity of reductase enzymes varies between microorganisms. Extracellular and intracellular reductions do the defense mechanism. Extracellular means the bacteria can release the reductase enzyme to the external environment and reduce the metal ions. Intracellular implies the removal of metal ions by reductase that takes place inside the cell. If the bacteria do not have a reductase mechanism, they will die [12].

2.3 Method for intracellular synthesis of silver nanoparticles

Prepare the pure bacterial seed culture in 100 ml nutrient broth and incubate it on a rotary shaker (150 rpm) for 24 hours.

1. After incubation, centrifuge the cell suspension and separate the bacterial pellet.

2. Suspend the pellet into the 100 ml distilled water. Then add 1 mM silver nitrate into the pellet suspension.

3. Incubate pellet suspension overnight at 60°C, pH 10.

4. The silver nanoparticle will deposit on the bacterial cell. Whitethorn changes the color of suspension (white to dark brown) (Figure 2).

Figure 2.

Mechanism of intracellular synthesis of silver nanoparticles - bacteria can oxidize metal by an action of reductase enzyme (direct mechanism of gaining electrons from reduced minerals). The redacted Nano metal ions are attached to the surface of the bacterial cell wall.

2.4 Separation of nanoparticles

The synthesized nanoparticles are separated from reaction mixture by adapting the following method [12, 15]:

Solution 1: 0.9% NaCl.

Solution 2: NaCl 0.5 M + Sucrose 0.5 M (equal volume of each solution).

Solution 3: NaCl [17.5 g/L] + KCL [0.74 g/L] + MgSO₄. 7H₂O [12.3 g/L] + Tris HCL [0.15 g/L].

pH = 7.5[Salt solution]

1. Centrifuge the bacterial suspension holding nanoparticle at 4000–9000 rpm for 10 minutes.

2. Suspend the pellet using solution 1 (equal volume of the pellet) and centrifuge at 4000–9000 rpm for 10 minutes. Repeat the step three times in order to remove the unwanted materials.

3. Homogenize the bacterial pellet by mortar with 20 mg Egg White Lysozyme and solution 2 (equal volume of the pellet) and centrifuged at 12000 rpm for 10 minutes.

4. Transfer the pellet to an enormous container, add solution three and incubate this mixture at room temperature for 18 hours.

2.5 Nanoparticle purification

1. Centrifuge the lysate at 12000 rpm and discard the supernatant (salt solution).

2. Suspend the pellet into the distilled water and wash twice at 5000–8000 rpm. Separated nanoparticles suspend into milli-Q water.

3. Separated nanoparticles purified by gel electrophoresis and column chromatography [2, 12, 15].

2.6 Extracellular synthesis of silver nanoparticles

1. Prepare 100 ml of seed culture in nutrient broth and incubate at 37°C for 72 hrs.

2. After incubation, centrifuge the broth culture at 5000-8000 rpm for 15 minutes.

3. Discard the pellet and transfer the supernatant into a new flask. Add the 1 mM silver nitrate and set pH 10.

4. Incubate this mixture overnight at 60°C. Centrifuge the culture supernatant at 7000 rpm and discard the supernatant.

5. Suspend the pellet into distilled water and wash two times at 5000 rpm.

6. Purified nanoparticles are again suspended into milli-Q water for further use [14] (Figure 3).

Figure 3.

Mechanism of extracellular synthesis of silver nanoparticles - the bacteria can release the metabolite or type of reductase enzyme to the environment. It oxidizes the metal to an inactive form.

2.7 The optimum condition for the synthesis

One of the most critical factors in bacteria-mediated Ag NP synthesis is a high pH. In the presence of silver ions, high pH catalyzes the opening of monosaccharide rings to open chain aldehyde forms, which then undergo oxidation to the appropriate carboxylic acid while simultaneously reducing silver ions to Ag NPs. Reductases of oxidoreductase enzymes are also activated by high pH [16]. Some bacterial proteins are involved in the synthesis of silver nanoparticles. It binds the thiol region at alkaline conditions (there is no need for agitation). In addition, alkaline ions are very much required for the reduction of metal ions. Under the alkaline state, it enhances the enzyme activity to do a reduction mechanism. In acidic conditions, it will take up to 4 days for silver nanoparticle synthesis. In alkaline conditions, the nanoparticle will be synthesized within 4 hours [12].

The high temperature will increase the dynamics of ions and the formation of more nucleation regions due to the obtainability of OH ions and the conversion of silver metal to the silver nanoparticle. At 60°C, nanoparticles are redacted up to 2–15 nm; in acidic conditions (50 nm), size is not reduced [12].

3. Characterization of silver nanoparticle

Once the synthesis procedure is completed, it is necessary to characterize the nanoparticles to know their structure, size, purity, and efficacy by studying their physiochemical properties, size, shape, surface area, and homogeneity. UV- visible spectroscopy, FTIR, TEM, SEM-EDAX, X-ray diffraction (XRD), and atomic force microscopy (AFM) are the tools used to characterize the synthesized nanoparticles [17].

3.1 UV: visible spectroscopy

UV-visible spectroscopy is a primary tool to analyze the availability of nanoparticles in the reaction mixture at 200–500 nm. It is based on the transition of electrons from one molecular orbital to another due to the absorption of electromagnetic radiation of UV and visible regions. It is the type of absorption spectroscopy when electromagnetic radiation interacts with matter, and the incident light can be reflected off, absorbed by, or transmitted through a sample. Electromagnetic radiation is absorbed by atoms or molecules, transitioning from lower energy to an excited state. Then the energy matched the difference in energy between two energy samples [18].

3.2 Fourier transform infrared spectroscopy

FTIR is used to analyze the surface chemistry of silver nanoparticles. The range that covers the electromagnetic spectrum is 1 micrometer to 100 micrometers. This spectroscopy is a type of vibrational spectroscopy. At the temperature above absolute zero, the bonds within molecules will vibrate. There are two main types of bond vibrations - stretching and bending. A stretching vibration occurs along the line of the chemical bond, whereas a bending vibration is any vibration that does not occur along the line of the chemical bond. It provides that all the functional groups are present in silver nanoparticles [18].

3.3 Transmission electron microscope

In the TEM, a condenser lens focuses the electron beam onto the specimen, transmitting electrons through the specimen. The portion of the beam absorbed by the specimen is minimal; to be absorbed, an electron must lose all its energy to the specimen. Some electrons scattered through the specimen focus on forming an image, like how an image in a light microscope is formed. A phosphorescent screen, a photographic plate, or a high-resolution camera can be used to view the image [18].

3.4 Scanning electron microscope

The scanning electron microscope views the surface nature of specimens. The sample is fixed, dried, and coated with a thin layer of heavy metal, such as gold or silver, scanned with a very narrow beam of electrons. Molecules in the specimen are excited, and they release secondary electrons. Molecules in the specimen are excited, and they release secondary electrons captured by a detector, generating an image of the specimen’s surface. The resolving power of scanning electron microscopes, limited by the thickness of the metal coating, is only about 10 nm; they produce three-dimensional in SEM [18].

3.5 Atomic force microscopy (AFM)

Atomic force microscopy (AFM) offers ultra-high resolution in particle size measurement by physically scanning samples at the sub-micron level with an atomic probe tip. Based on forces between the tip and the sample surface, the instrument generates a topographical map of the sample [15]. Depending on the qualities of the sample, it is usually scanned in contact or non-contact mode. In contact mode, the topographical map is created by tapping the probe across the sample surface, while in the non-contact method, the probe hovers over the conducting surface. The ability to image non-conducting samples without special treatment is AFM’s main advantage, allow ing imaging of delicate biological and polymeric nano and microstructures. The most accurate description of size and size distribution is provided by AFM, which does not require any mathematical treatment. Furthermore, the AFM technique provides a realistic picture of particle size, which aids in understanding the impact of diverse biological circumstances [19].

3.6 X-ray diffraction

XRD is one of the most common analytical techniques for phase identification and crystalline structure determination in solid-state materials. To determine the structure of a molecule, X-rays focused on constrictive use of destructive interference caused by scattering radiation from a single crystal’s regular and repeating lattice [19].

4. Nanotechnology in mosquito control

The life of mosquitoes is intertwined with human life, and they have been a significant factor in human society for many years. Although they have been recognized as criminals for hundreds of years, their nuisance has not diminished. We have been suffering from the effects of mosquitoes since the beginning of humankind. These cause hundreds or more diseases against the human race. Millions of people have died as a result. Although, the elimination of mosquitoes is octagonal. Even though various countries have implemented many health promotion programs against mosquitoes, it is still a significant problem globally. Mosquitoes transmit dangerous diseases known to humans, such as malaria, yellow fever, dengue, encephalitis, filariasis. More than 3500 species have been recorded worldwide across five continents. Mosquito-borne diseases, such as malaria, arboviruses, and filariasis, and their vectors are present worldwide [20].

Mosquitoes are severe vectors of critical human parasites and microbes. Culex quinquefasciatus is a domestic mosquito that thrives close to human activities and habitation. West Nile virus, St. Louis encephalitis, and lymphatic filariasis are among the pathogens Cx. quinquefasciatus can transmit to humans and animals. Cx. quinquefasciatus can transmit pathogens such as avian malaria and zoonotic dirofilariasis to livestock, birds, domestic and wild animal species, resulting in loss of productivity and death. Cx. quinquefasciatus is also a nuisance because its bites can cause local dermatitis or acute systemic allergic reactions in many people [21].

There is no aquatic habitat anywhere; it does not lend itself to being a breeding ground in the world for mosquitoes. They are temporary and permanent colonial, very dirty and clean, big and small waters, even the most miniature accumulation buckets filled with water, flower vases, tires, hoof, etc., possible sources of prints and leaf axes. Flood mosquitoes in temporarily flooded areas, rivers, or lakes with water fluctuation Aedes vexans or Ochlerotatus sticticus formed in large numbers and within a range of miles. They can become a significant nuisance even in places far away from their breeding grounds.

Many methods are used to control the mosquitoes, but they have disadvantages; chemical control was one of the most widely used conventional methods for mosquito control [20]. Since chemical pesticides are relatively inexpensive, they usually produce immediate control. Generally, the chemical control is carried out by the indoor residual spraying of insecticides such as DDT, organophosphates, chlorpyrifos-methyl, fenthion, fenitrothion, malathion, pirimiphos-methyl, temephos, carbamates bendiocarb, propoxur, pyrethroids alpha-cypermethrin, bifenthrin, cyfluthrin, cypermethrin, deltamethrin, etofenprox, lambda-cyhalothrin, permethrin, hexachlorocyclohexane, benzene hexachloride, malathion, and synthetic pyrethroid. However, the development of resistance against these chemicals in various mosquito populations has been reported [20]. Therefore, biological control can provide a practical and environmentally friendly approach that can be used as an alternative to reduce the number of mosquitoes. Microbially mediated AgNPs are powerful tools for combating mosquitoes and agricultural insects [20].

Marimuthu et al. [22] reported that synthesized metal nanoparticles derived from B. thuringiensis showed remarkable larvicidal activity against A. subpictus and A. aegypti. Similarly, AgNPs obtained from B. thuringiensis (Bt) and F. oxysporumculture filtrate showed strong larvicidal activity against A. aegypti, A. stephensi and C. quinquefasciatus mosquitoes [23]. In addition, several studies have reported that the bacterial strains of L. monocytogenes, B. subtilius, and S. anulatus-mediated AgNPs showed larvicidal activity [24]. The Pseudomonas aeruginosa synthesized Ag NPs were treated against Cx.quinquefasciatus and showed a higher susceptibility to the synthesized AgNPs and showed that 100% mortality was observed after 1 hour of incubation [25].

4.1 Procedure for studying Larvicidal activity of silver nanoparticle

4.1.1 Stock solution

1. 20 ml (solvent) + 200 mg (nanoparticle) = 1000 ppm.

2. 2 ml of stock solution is serially diluted in 18 ml of solvent (working solution) = 100 ppm.

4.1.2 Procedure

1. The fourth instar of mosquito larvae is taken in a 250 ml container. Each container has 200 ml distilled water and 10 mosquito larvae. It makes up of three replicates.

2. Add the silver nanoparticle to 1 ppm–100 ppm concentration. Furthermore, monitor the larval behavior in 24 hrs – 48 hrs. Twenty-four hours of fasting mosquito larvae were used in the test, and the control organism was fed two times per day.

3. The action of silver nanoparticle mosquito larval behavior will be changed activity like slow swimming, stand in a single place, larval color was changed, damage the body parts and last mortality.

4. The mortality will be observed and tabulated [26].

4.2 Mechanism of actions of nanoparticles on mosquitoes

Surprisingly, despite a mass of information on their toxicity against specific pests and vectors, exact information on nanoparticles’ potential mode of action against insects is limited. This information is crucial for predicting the toxicological effects of using nanoparticles as insecticides in the actual world. Silver nanoparticle cytotoxicity and genotoxicity mechanisms have been extensively studied, as their toxicity in biological models is strongly influenced by their size, shape, and charge [27].

According to accepted theory, the toxicity of several nanoparticles is considered to be achieved by causing oxidative stress in arthropod tissues [28]. Furthermore, nanoparticle penetration through the exoskeleton may increase their toxicity. The nanoscale material then binds to sulfur from proteins or phosphorus from DNA in the intracellular space, causing organelles and enzymes to denature rapidly. As a result of the decreased membrane permeability and disturbance in the proton motive force, cellular function and cell death may be lost. The green-capped nano-Ag significantly reduced total protein levels. It also reduced the activities of acetylcholinesterase and - α and ß-carboxylesterase [28].

5. Conclusion

The efficacy of green-fabricated nanoparticles is promising, and this excited much research groups worldwide, opening new ways to manage arthropod pests and vectors. However, while some researchers have tried to clarify how silica, alumina, silver, gold, titania, and graphene nanoparticles act as toxins against arthropods, our knowledge in this research field is still scarce.

Green fabrication processes rely on selected compounds to rule out the insecticidal impacts of botanicals and microbial products used as reducing and capping agents. This allows us to avoid hard-to-reproduce results due to tested green reducing agents [29].

Finally, more effort is necessary to validate the proposed nano pesticides in field conditions while simultaneously monitoring their stability, environmental fate, and sublethal effects on non-target organisms, focusing on genotoxicity and acceptable physiological and behavioral modifications. It is essential to understand the various mechanisms leading to chronic toxicity of nanoparticles invertebrates, focusing on humans.

Acknowledgments

The authors are grateful to the Management Ayya Nadar Janaki Ammal College and the Principal for providing well-equipped research facilities during this work.

Conflict of interest

“The authors declare no conflict of interest.”