1 Introduction

Nanotechnology is one of the fast growing fields especially in the area of biotechnology and medicine [1]. Nanoparticles are nanoscale particles (1-100 nm) which have unique chemical and physical properties. Due to their smaller size and extraordinary properties, they are commonly used in various fields of science and technology. Various methods are being used now-a-days for the synthesis of nanoparticles like physical, chemical and biological methods. Physical methods are highly exothermic reactions with lesser yields. Chemical methods exert toxic effects due to the production of large amount of hazardous by-products. On the other hand, biological nanoparticle synthesis is comparatively economical, ecofriendly and appreciable. In biological process of metal nanoparticles synthesis, principles of green chemistry are applied. Microorganisms (bacteria, fungi and actinomycetes) and plants are used as a reducing agent for conversion of silver metal to stabilized silver nanoparticles of various sizes [2]. Microbial infections have now become a global health concern due to the antibiotic resistance developed by pathogenic microbes and hence, confronting proper medical treatments. This increasing antibiotic resistance is responsible for elevated risk of health disorders, rate of mortality, expenditure and low life probability. We need an alternative method as a permanent solution for this emerging problem [3]. Silver has excellent potential to be used as an antibacterial and antiseptic agent since remote past. Silver nanoparticles are important metal particles which are commonly used in photocatalysis, photonics, micro-electronics and antimicrobial activities. Silver nanoparticles are unique inorganic nanoparticles because of their unique size and properties like catalysis, surface plasmon resonance (SPR), biomedicine, nanoelectronics and sensing. The antimicrobial activity of bacterial AgNPs can offer a unique option to reduce the use of antibiotics thereby minimizing the development of antibiotic resistance [4]. The current study deals with the bacterial AgNPs having antimicrobial potential and characterization of these biosynthesized AgNPs through spectroscopic and microscopic techniques.

2 Materials and methods

3 Results

3.2 Bacterial silver nanoparticles synthesis

3.2.1 Extracellular synthesis of bacterial silver nanoparticles

Change in color of reaction mixture manifested the synthesis of AgNPs. Maximum absorbance was recoded at 400 nm. Optimum AgNPs production potential was exhibited by Serratia marcescens (S4c1) (Figure 1).

Figure 1

Biosynthesis of bacterial AgNPs.

3.2.2 Intracellular synthesis of bacterial silver nanoparticles

Among the four bacterial strains used, the bacterial strain Serratia marcescens (S4c1) has shown maximum production of AgNPs by intracellular method (Figure 1).

3.3 Factor affecting bacterial silver nanoparticles synthesis

3.3.1 Cell supernatant and residue

Cell supernatant was observed to be more effective in the synthesis of AgNPs by some bacterial strains while on the other hand some bacterial strains utilized intracellular method in which cell residue was responsible for the reduction of AgNO₃ (Figure 1).

3.3.2 AgNO₃ concentration

Increase in AgNO₃ concentration caused improvement in the amount of AgNPs synthesized (Figure 2). Bacterial strains Serratia marcescens (S4c1), Brevundimonas diminuta (S5a) and Bacillus subtilis (Mt3b) have shown optimum production of AgNPs upto a certain threshold concentration of AgNO₃ which was recorded to be upto 2 mM and further increase resulted in gradual reduction in AgNPs synthesis. On the other hand, bacterial strain Bacillus cereus (So3II) has shown quite different behavior and AgNPs production was quite low at 1 and 2 mM concentration of AgNO₃ which gradually increased at 3 mM AgNO₃.

Figure 2

Effect of AgNO₃ concentration on AgNPs synthesis.

3.3.3 Reaction time

After 72 h of incubation time, the production of AgNPs was increased hundred times with the bacterial strains Serratia marcescens (S4c1), Brevundimonas diminuta (S5a) and Bacillus subtilis (Mt3b) compared to 4 h incubation while Bacillus cereus (So3II) has shown quite different behavior and reduction in AgNPs production was recorded after 72 h of reaction time as compared to 4 h of reaction time (Figure 3).

Figure 3

Effect of reaction time on AgNPs synthesis.

3.3.4 Reaction temperature

The ideal temperature for the production of AgNPs was found to be 60°C. All the bacterial strains i.e., Serratia marcescens (S4c1), Brevundimonas diminuta (S5a), Bacillus cereus (So3II) and Bacillus subtilis (Mt3b) exhibited optimum potential for the production of bacterial AgNPs at 60°C.

3.4 Characterization of bacterial silver nanoparticles

3.4.1 Fourier transform infrared spectroscopic (FTIR) analysis

IR spectrum of bacterial AgNPs was examined for the possible presence of compounds responsible for biological reduction of silver ions and stability of silver nanoparticles. The IR spectrum of bacterial AgNPs was scanned in the range of 450-4000 cm⁻¹ which showed intense bands at 3466.77 cm⁻¹ representing the stretching vibrations of amines while band at 1637.88 cm⁻¹ represented phenyl ring substitution and its banding. Also band at 1637 cm⁻¹ has shown carbonyl stretch in the amide linkages of protein. NO₂ group exhibited vibrations at 1390 cm⁻¹ (Figure 4). For the reduction of Ag⁺ to AgNPs, bacterial proteins might have reacted and bound with metals particles and caused its reduction to silver nanoparticles. Bacterial proteins developed a coating on AgNPs resulting in its stability (Figure 4).

Figure 4

IR spectra of bacterial AgNPs.

3.4.2 Transmission electron microscopic (TEM) analysis

Transmission electron microscopic analysis showed that bacterial AgNPs are poly dispersed, spherical and ranges from 1-100 μm in size (Figure 5).

Figure 5

Transmission electron microscopic (TEM) analysis of bacterial AgNPs.

3.4.3 UV-vis spectroscopic analysis

Serratia marcescens (S4c1) has shown maximum absorbance at 400 nm for both extracellular and intracellular nanoparticles i.e., 1.344 and 1.4293, respectively (Figure 1).

3.5 Antimicrobial activity of bacterial silver nanoparticles

Bacterial silver nanoparticles have shown excellent antimicrobial activity against all bacterial strains used. Strong antibacterial activity was recorded with AgNPs synthesized using Bacillus subtilis (Mt3b) against Butyricum exhibiting an inhibition zone of 20 mm. AgNPs obtained from Brevundimonas diminuta (S5a) proved effective against Pasturella with an inhibition zone of 10 mm. Nanoparticles obtained from Bacillus cereus (So3II) have shown strong antimicrobial potential against Aeromonas with an inhibition zone of 10 mm. AgNPs from Serratia marcescens (S4c1) have also shown antibacterial potential against Aeromonas with an inhibition zone of 8 mm (Figure 6).

Figure 6

Antimicrobial potential of bacterial AgNPs.

3.6 Antifungal activity of bacterial silver nanoparticles

Bacterial silver nanoparticles showed the potential to inhibit the growth of Phytophthora palmivora, a fungus causing several plant diseases. Extracellular nanoparticles obtained from Bacillus subtilis (Mt3b) have shown strong antifungal potential against Phytophthora palmivora. Extracellularly synthesized AgNPs obtained from Bacillus subtilis (Mt3b) exhibited a percentage radial inhibition of 81.81%. The AgNPs obtained from Serratia marcescens (S4c1), Brevundimonas diminuta (S5a) and Bacillus cereus (So3II) have shown percentage radial inhibition up to 77.27, 63.63 and 45.45%, respectively. Intracellular nanoparticles were not effective as an antifungal agent against Phytophthora palmivora (Figure 7).

Figure 7

Antifungal potential of bacterial AgNPs.

4 Discussion

Bacterial production of silver nanoparticles is a defensive mechanism against metal toxicity. This defensive mechanism of bacteria is employed in the synthesis of silver nanoparticles and is advantageous over conventional chemical method for being ecofriendly. Bacterial cell wall play a pivotal role in the synthesis of silver nanoparticles because bacterial cell wall is negatively charged and it interacts electrochemically with positively charged metal ion (Ag⁺) and hence, caused bioreduction of metal ions to metal nanoparticles AgNPs [8]. All the four bacterial isolates [Serratia marcescens (S4c1), Brevundimonas diminuta (S5a), Bacillus cereus (So3II) and Bacillus subtilis (Mt3b)] used in the current study showed nitrate reductase potential (Table 1). Nicotinamide adenine dinucleotide (NADH) dependent nitrate reductase enzyme is a powerful tool for converting silver ions to silver nanoparticles extracellularly in some bacteria and fungi [9]. All the bacterial strains utilized in the current study have the potential to produce AgNPs by utilizing both extracellular and intracellular method of AgNPs production. The bacterial strain Serratia marcescens (S4c1) was most effective in production of bacterial AgNPs (Figure 1). Different factors such as temperature, incubation time and concentration of silver nitrate influence production of silver nanoparticles. Bacterial cell pellet was more effective as compared to cell supernatant for the synthesis of silver nanoparticles in some bacterial strains (Figure 1). The amount of silver nitrate is also an important factor for the synthesis of AgNPs. The production of AgNPs was recorded to be increased exponentially with increase in concentration of AgNO₃ but it is not the case for every strain and varies from species to species (Figure 2). Extended reaction time was responsible for the greater production of silver nanoparticles (Figure 3). FTIR analysis confirmed the stabilization of nanoparticles with amines interaction (Figure 4). Surface bound proteins of bacteria play an important role in the production and stabilization of silver nanoparticles. The proteins were observed to cap the nanoparticles using amino acids and amines, hence, caused reduction of Ag⁺ to AgNPs [8]. Silver ions (Ag⁺) bind with silver nanoparticles which prevent disruption of secondary structure of proteins [9]. Thamilselvi and Radha [6] also reported extended incubation method for the synthesis of silver nanoparticles. Bacterial silver nanoparticles act as an effective biocide agent to inhibit the growth of both gram positive and gram negative bacteria as recorded using disc diffusion method. Six different gram-positive and gram-negative bacterial strains were used in the current study to evaluate the antibacterial potential of bacterial silver nanoparticles. Silver nanoparticles synthesized by Bacillus subtilis (Mt3b) were applied to inhibit the growth of bacterial strain Butyricum (Figure 6). Bacterial nanoparticles inhibit the growth of pathogenic bacteria which might be due to the production of free radicles from silver which cause lipid degradation of the membrane and hence, change membrane structure and function. Free radicles are also responsible for changing the structure of bacterial cell wall [10]. The mechanism involved in the antibacterial activity of AgNPs is that pathogenic bacterial strains uptake silver ions easily from the surroundings which results in blocking the ATP and DNA synthesis pathways and produce reactive oxygen species (ROS) that results in direct damage of bacterial cell membrane resulting in cell lysis [11]. Phtyophthora palmivora is an Oomycetic fungus infecting multiple hosts worldwide like coconut, papaya, mango and many other plants of economic importance. This pathogen is a causative agent of many diseases like kole roga, fruit rot and bud rot. In the current study extracellular AgNPs of Bacillus subtilis (Mt3b) was most effective bio-control agent of this fungus (Figure 7). Silver nanoparticles inhibited production of protective substances like spores in fungi. Silver nanoparticles are responsible for the inactivation of sulfhydral group in the fungal cell membrane and produce various toxic compounds hence, disrupt and change the structure of membrane attached proteins and lipids ultimately resulting in cell death of fungus thus exhibiting antifungal potential.

5 Conclusion

In conclusion, the current study has emphasized on the optimization and impact of various factors affecting the synthesis of bacterial AgNPs. In the present wok, bacterial AgNPs synthesized by the bacterial strains exhibited prominent antimicrobial and antifungal potential. In the current era of increasing antibiotic resistance, the antimicrobial activity of bacterial AgNPs can be effectively exploited for human benefit. In vivo analysis is among the future prospects of our current work, which if positive, will allow us to exploit these bacterial AgNPs with nanobiotic potential to help minimize the antibiotic resistance which is causing great harm not only to mankind but to the whole ecosystem.