Bacteriogenic synthesis of morphologically diverse silver nanoparticles and their assessment for methyl orange dye removal and antimicrobial activity

Materials

Klebsiella pneumoniae (PME2), K. pneumoniae (MBC34), Micrococcus luteus (MBC23), Micrococcus luteus (MBC57), Enterobacter aerogenes (MBX6), Bacillus subtilis (MBC3), Bacillus cereus (MBC4), Bacillus megaterium (MBP11), Enterococcus faecalis (MBP13), and Serratia marcescens (MBC27) were obtained as gift samples from the Gujarat Biotech Research Centre (Gandhinagar, Gujarat, India). The materials used in the study included silver nitrate (SRL, Gujarat, India), nutrient agar media, nutrient broth and antibiotic assay media (Himedia, Mumbai, India); ethanol (SLC Chemicals, Delhi, India), Whatman filter paper no. 42. (Axiva, Mumbai, India); and methyl orange (MO) (Loba, Chemie, Gujarat, India) and double distilled water (ddw). All the chemicals were analytical grade except silver nitrate and methyl orange dye (LR grade).

Methods

UV-Visible spectroscopy

The UV-Vis measurement of all three types of AgNPs was done by adding 1 mg of AgNPs in 5 mL of ddw in three different test tubes, followed by sonication for 10 in an ultrasonicator. An aliquot (2 mL) was transferred to the quartz cuvette from the well-dispersed AgNPs samples, and the UV-Vis measurement was done in the range of 200–800 nm at a resolution of 1 nm by using a UV-Vis spectrophotometer (UV 1800; Shimadzu spectrophotometer, Shimadzu, Japan). The UV-vis measurement of the control sample was also done in the above range by using a double-beam LMSP-UV100S (Labman, Chennai, India), instrument.

FTIR

The FTIR measurement was done to identify the various functional groups present in the bacterially synthesized AgNPs. The FTIR measurement was done using the solid KBr pellet method, where the pellets were prepared by mixing 2 mg AgNPs and 198 mg KBr for all three types of AgNPs. The measurement was done in the mid-IR region 599–4,000 cm⁻¹ at a resolution of 2 cm⁻¹ by using a spectrum S6500 instrument (Perkin-Elmer, Waltham, MA, USA).

XRD

The XRD patterns for all three types of AgNPs samples were recorded using a Miniflex 800 (Rigaku, Houten, Netherlands) instrument equipped with an X’celerometer to reveal the crystallinity. XRD patterns were recorded in the 2-theta range of 20–70 by using a filter K-beta (x1) with a step size of 0.02 and a time of 5 s per step, scan speed/duration time: 10.0 degree/min., step width: 0.0200 degrees at 30 kV voltage, and a current of 2 mA.

FESEM-EDS

The morphological analysis of all three types of AgNPs was investigated using a FEI Nova NanoSEM 450 (USA). The dry AgNPs were loaded on the carbon tape with the help of a fine brush, which in turn was kept on the Al stub holder. All the samples were exposed to gold sputtering. The elemental analysis of AgNPs was carried out using an Oxford-made energy-dispersive X-ray spectroscopy (EDS) analyzer fixed to the FESEM at variable magnifications at 20 kV.

Mechanism of formation of AgNPs by bacteria

The bacterial strains, i.e., K. pneumoniae (MBC34), M. luteus (MBC23), and E. aerogenes, have numerous microbial proteins and enzymes that help in the reduction of Ag⁺ ions into Ag⁰ (Ballén et al., 2021). The actual mechanism of the biosynthesis of AgNPs by bacteria is well described in the literature. It is a simple mechanism where the oxidized silver ions get two electrons from microbial proteins and enzymes and are reduced to the stabilized Ag⁰. When the AgNO₃ aqueous solutions are mixed with the bacterial culture/supernatant, the bacterial enzymes present in the supernatant reduce the Ag⁺ ions into Ag⁰. So, during this step, the previously milky color of the aqueous silver solutions gets converted to red, indicating the development of AgNPs in the medium. Moreover, these biomolecules may also act as stabilizing and capping agents for the synthesized AgNPs (Giri et al., 2022; Terzioğlu et al., 2022). Figure 1 shows the mechanism involved in the formation of AgNPs from the bacteria. Here, the color of the medium changed from milky white to dark brown within 2 to 3 days. Earlier, a similar color change (yellow to brown) was observed during the synthesis of AgNPs by K. pneumoniae isolated from humans and sheep (Sayyid & Zghair, 2021). Saleh & Khoman Alwan (2020) used K. pneumoniae culture supernatant for the biosynthesis of AgNPs. Earlier, Javaid et al. (2018) also suggested a similar pathway for the formation of AgNPs from the bacteria via a NADH-dependent nitrate reductase enzyme. Table 1 shows the major microbial proteins and enzymes present in K. pneumoniae, M. luteus, and E. aerogenes.

UV-Vis analysis for preliminary confirmation of the formation of AgNPs

Figure 2 shows a typical absorbance peak in the range of 425–505 nm for all three AgNPs synthesized by bacteria by UV-Vis instrument, indicating the formation of AgNPs (Saleh & Khoman Alwan, 2020). Earlier, Saleh & Khoman Alwan (2020) obtained a peak at 432 nm for the AgNPs synthesized by K. pneumoniae (Saleh & Khoman Alwan, 2020), 420 and 440 nm by M. luteus (Vimalanathan et al., 2013), 450 nm by cyanobacterium Oscillatoria limnetica (Hamouda et al., 2019), 450 nm by endophytic bacteria Enterobacter roggenkampii BLS02 (Kumar & Dubey, 2022), and 405–407 nm for Klebsiella pneumoniae by Kalpana & Lee (2013). The AgNPs formed only when the AgNO₃ to bacterial supernatant ratio was about 4:6, during which there was higher absorption intensity. Moreover, the amount of bacterial culture filtrate and reducing agents in the supernatant governs the yield of the AgNPs (Kalpana & Lee, 2013).

Identification of functional groups of silver NPs by FTIR

A typical FTIR spectra of all the AgNPs (AgNPs-K, AgNPs-M, and AgNPs-E) synthesized by bacteria is shown in Fig. 3, which was used for the identification of various functional groups present in AgNPs. All the samples have a common band at 599 cm⁻¹ and 963, 1,299, 1,349, 1,693, 2,299, 2,891, and 3,780 cm⁻¹. The band at 599 cm⁻¹ is attributed to the metallic Ag. The band at 963 cm⁻¹ is attributed to the amide V band arising due to out-of-plane NH bending of peptide linkages (Kalpana & Lee, 2013). A small intensity band at 1,051 cm⁻¹ is attributed to the primary amine C–N stretch. Another small intensity band at 1,349 cm⁻¹ in all the samples is attributed to the C-C bond. A small intensity band in all the samples at 1,699 cm⁻¹ is attributed to the OH group in the samples. Moreover, this band is also attributed to the C=O stretching of amide I bands of peptide linkage. The band at 1,229 cm⁻¹ is attributed to the CN stretching of peptide linkage. The band at 1,349 cm⁻¹ is attributed to the (C–C) stretching vibration of aliphatic amines, as previously documented by Ibrahim et al. (2019). In this study, AgNPs were developed from endophytic bacteria, which showed a band at 1,359 cm⁻¹ (Ibrahim et al., 2019). All the samples exhibit an atmospheric carbon band at 2,891 cm⁻¹ is attributed to the methylene C–H asymmetric or symmetric stretch. All the samples showing a small band from 3,400 to 3,800 cm⁻¹ centered at 3,780 cm⁻¹ are attributed to the -OH molecule.

Earlier, Saleh & Khoman Alwan (2020) obtained four distinct peaks for the AgNPs synthesized by K. pneumoniae at 3,332.78 cm⁻¹, 2,115.35, 1,635.60, and 1,096.92 cm⁻¹ and suggested that the band at 3,332.78 cm⁻¹ is due to the stretching vibration of the OH bond of alcohol and phenols. The band at 2,115.35 cm⁻¹ is attributed to the C-H stretching of the methylene groups of protein and the N-H stretching of amine salt. The band at 1,635.60 cm⁻¹ was attributed to the carbonyl groups (C=O) of the amino acid residues. In contrast, the band at 1,096.92 cm⁻¹ was attributed to the (C-O) stretching of alcohols and esters, carboxylic acids, and the C–N stretching of aliphatic amines (Saleh & Khoman Alwan, 2020). Based on the above information, it was further concluded that the presence of protein in the supernatant acts as a stabilizing and capping agent for stabilization, which binds to the synthesized AgNPs through free cysteine or amine groups in proteins (Saleh & Khoman Alwan, 2020). The results were in close agreement with the investigation carried out by Kalpana & Lee (2013) where major intensity bands were obtained at 2,964.55, 1,262.22, 1,095.89, 1,021.96, 800.73 cm⁻¹, and small intensity bands at 2,960.64, 1,650.01, 865.33, 701 and 477.07 cm⁻¹ for the AgNPs synthesized by K. pneumoniae.

In the current investigation, authors have also obtained bands for the K. pneumoniae synthesized AgNPs at 599, 963, 1,229, 1,693, 2,891, and 3,780 cm⁻¹, which correspond to the bands obtained by Kalpana & Lee (2013). Similarly, in another study also, four distinct FTIR bands were observed at 1,643, 1,586, 1,397, and 1,042 cm⁻¹, and it was concluded that the AgNPs synthesized by bacteria exhibited improved stability because the coating of AgNPs by bacterial and media components (Peiris et al., 2018). AgNPs-M and AgNPs-E displayed similar bands as those of AgNPs-K with slight variation in their intensity.

Phase identification of silver nanoparticles by XRD

The XRD investigation was carried out to identify the crystalline phase of the AgNPs. A typical XRD pattern of all the bacterially synthesized AgNPs is shown in Fig. 4, where all the AgNPs exhibit three characteristic peaks of silver NPs at 27.6°, 32.1°, and 46.2°, and three small intensity peaks at 54.7°, 57.4°, and 76.7°. The major intensity peaks in all three types of bacterially synthesized AgNPs are at 32.1°, followed by 46.2° and 27.6°. The XRD planes in all three types of AgNPs were 101, 111, 200, 220, and 311, as matched with the Joint Committee on Power Diffraction Standards (JCPDS) 03-0921. The diffraction peaks obtained in the current investigation at 27.6°, 32.1°, and 46.2° and small diffraction peaks at 54.7°, 57.4°, and 76.7° for the AgNPs-K. AgNPs-M also has the same peaks as AgNPs-K, i.e., at 27.6°, 32.1, 46.2, 54.7, and 57.4°. All the XRD peaks for AgNPs-M were of almost the same intensity except for 27.6°, which was comparatively stronger than AgNPs-K. The XRD peaks for AgNPs-E were also at the same places as those of AgNPs-M and AgNPs-K, but the peak at 27.6° was of stronger intensity than AgNPs-K and weaker than AgNPs-M. Moreover, the peak at 46.2° in both AgNPs-M and AgNPs-E was stronger than the AgNPs-K. Further, the peaks at 54.7° (311) and 57.4° (222) were like a small, broad hump in both AgNPs-K and AgNPs-M, but these two peaks were sharper in AgNPs-E.

The results were in close agreement with the previous results obtained by Kalpana & Lee (2013) and Ibrahim et al. (2019), where Kalpana & Lee (2013) obtained diffraction peaks at 37.76°, 45.87°, 64.08°, and 77.11°, which were indicated by the (111), (200), (220), and (311) reflections of metallic Ag. The data obtained here was matched with the database of JCPDS file no. 03-0921 (Kalpana & Lee, 2013). Ibrahim et al. (2019) obtained diffraction peaks for the AgNPs synthesized by an endophytic bacteria, Bacillus siamensis strain C1, at 27.81, 32.34, 46.29, 57.47, and 77.69°, corresponding to (101), (111), (200), (220), and (311) crystal planes, respectively, for the AgNPs. Earlier, the study by Vimalanathan et al. (2013) also obtained similar results, where the major peaks were at 28, 32, and 47°, two small intensity peaks at 55° and 57°, and a small diffraction peak at 76°.

The crystalline size of all three AgNPs synthesized by bacteria was determined by using the Scherrer formula as given in Eq. (2),

(2) $$D={\displaystyle \frac{k\lambda }{\beta Cos\theta }}$$where,

D = crystalline size,

k = constant (0.9), and

β = the FWHM values of the diffracted peaks.

The highest intensity peak was used to find all the parameters in the Scherrer equation. The Gaussian peak fits were used to find the FWHM values and exact theta values. The calculated crystalline size was found to be around 16.88, 18.00, and 16.44 nm for AgNPs-K, AgNPs-M, and AgNPs-E. Therefore, it is well examined that the synthesized AgNPs showed a crystalline nature and 16.88, 18.00, and 16.44 nm crystallite sizes.

Morphological analysis of silver nanoparticles by FESEM and elemental analysis by EDS

Figures 5A–5F shows FESEM micrographs of AgNPs synthesized by K. pneumoniae (AgNPs-K). Figures 5A and 5B show a porous flakes-like structure that is embedded with bright-colored AgNPs. Figures 5C and 5D clearly shows rhombohedral-shaped AgNPs, whose size varies from 22–66 nm. These two images clearly show that the AgNPs are embedded in porous, sheet-like structures. Figures 5E and 5F shows aggregated spherical-shaped structures. Previously, several investigators have demonstrated similar morphology for the bacterially synthesized AgNPs.

Figure 5G shows the EDS spot of the AgNPs-K, while Fig. 5H exhibits the EDS spectra and elemental table of the AgNPs-K. The EDS spectra in Fig. 5H show peaks for Ag, Cl, Na, P, S, C, O, and N. Among these, the elements contributing the most to the sample were Ag (37.8% At wt.), Cl (29.8%), and P and S 0.4% each. The P and S were not present in trace amounts in the AgNPs-K. The major impurities in the synthesized AgNPs-K are NaCl, which alone comprises 50% due to the improper washing of the samples. Moreover, these two may also come from the nutrient broth used for growing the bacteria. At the same time, the presence of C, S, and P indicates the association of biomolecules with the synthesized AgNPs-K. Earlier, Sayyid & Zghair (2021) reported cube-shaped to irregular heterogeneous forms of AgNPs synthesized by K. pneumoniae, whose average size was 40.47 nm (Sayyid & Zghair, 2021). Moreover, investigators further observed that the morphology by TEM was a pseudo-spherical shape of size 40–80 nm.

Saleh & Khoman Alwan (2020) obtained spherical-shaped particles of size 26.84 to 44.42 nm, which were highly aggregated. Further, the investigators concluded that the conglomeration of the AgNPs occurs during the drying process.

Rasheed et al. (2023) obtained nano-rod-like AgNPs of size 100–200 nm from the Conocarpus erectus plant, while oval-shaped AgNPs of size 110–150 nm were synthesized from Pseudomonas spp., and by applying the chemical reduction method, flower-like AgNPs of size 100–200 nm were obtained. So, the smallest size was reported from Pseudomonas spp., which was oval-shaped.

Figures 5I–5L shows FESEM micrographs of AgNPs synthesized by the M. luteus (AgNPs-M). Figures 5I and 5J shows a high aggregation of the synthesized AgNPs-M. Figures 5K and 5L shows spherical-shaped AgNPs-M, whose size varies from 21–45 nm. Figure 5M shows the EDS spot of the AgNPs-M, while Fig. 5N exhibits the EDS spectra and elemental table of the AgNPs-M. The EDS spectra of AgNPs-M in Fig. 5N show peaks for Ag, Cl, Na, P, S, C, O, and N. Out of all these, the significant elements were mainly Ag (61% At wt.), Cl (21%), C (8.4%), N (3.7%), and O (2.6%). Other detected elements, such as Na, P, and S, were present in trace amounts, and the major impurity in the final sample was Cl due to improper sample washing. Moreover, it also came from the bacterial media, i.e., nutrient broth. At the same time, the presence of C, N, S, and P indicates the presence of biomolecules with AgNPs-M.

Figures 5O–5R shows FESEM micrographs of AgNPs synthesized by E. aerogenes (AgNPs-E). Figures 5O and 5P shows a porous flakes-like structure embedded with the bright color of AgNPs. Figures 5Q and 5R clearly shows rhombohedral-shaped AgNPs-E, whose size varies from 24–60. The images clearly show that the AgNPs-E are embedded in the porous flakes-like structures. The particles are showing high aggregation, as evident from the SEM micrographs. Figure 5S shows the EDS spot of the AgNPs-E, while Fig. 5T exhibits the EDS spectra and elemental table of the AgNPs-E. Figure 5T shows the spectra of Ag, Cl, Na, P, S, C, O, and N. Out of all these, the significant elements were mainly Ag (52.1% At wt.), Cl (21.7%), C (9.8%), O (8.2%), Na (4.0%), and N (3.1%). In addition to this, P and S were present in trace amounts, whose total composition was near 1.1%. The major impurities in the synthesized AgNPs-E are NaCl, which alone comprises 25.7%, which indicates the improper washing of the sample. Moreover, these two may also come from the nutrient broth used for growing the bacteria. The presence of C, S, N, and P indicates the association of enzymes and proteins from the E. aerogenes with the synthesized AgNPs-E. Table 2 shows the major elements present in all three types of AgNPs synthesized by bacteria.

From the EDS data of all three types of AgNPs, it was found that Ag was present in the highest percentage in AgNPs-M and at least 37.8% in AgNPs-K. Among all the three types of AgNPs, Cl was present most in AgNPs-K and least in AgNPs-M (21.0%). The oxygen was present in the highest amount in AgNPs-E and the least in AgNPs-K, i.e., 8.2% and 2.8%, respectively. The carbon was highest in AgNPs-E (9.8%) and least 7.0% in AgNPs-K. Out of all the three types of AgNPs, Na was highest in AgNPs-K (21.8%) and least in AgNPs-M (0.4%). Among all the three samples of AgNPs, N was present highest in AgNPs-M (3.7%) and 3.1% in AgNPs-E, and it was not detected in AgNPs-K. The P and S were present almost identically in all the samples but least in AgNPs-K, i.e., 0.4%.

In the current investigation, the authors found a broad peak of silver ions at 3 keV in all three types of AgNPs, which confirmed the reduction of Ag⁺ to Ag⁰. Moreover, here, the authors have mainly considered peaks in EDS for Ag, Cl, and carbon. The peaks for Ag, Cl, and S were consistent with the results obtained for the AgNPs synthesized by endophytic bacteria by Ibrahim and his team. Ibrahim et al. (2019) and his research group also concluded that the broad peak of silver ions was formed at 3 keV, which indicated the reduction of Ag⁺ to Ag⁰. Table 3 shows the comparative analysis of all the previously reported bacterially synthesized AgNPs with the current investigation.

From all the previous investigations, it was revealed that the largest size of AgNPs was 40.47 ± 89 nm synthesized by using K. pneumoniae, whose shape was cube to spherical and, AgNPs synthesized by B. cereus, was smallest in size, i.e., 2–16 nm with spherical in shape. In the current investigation, the size of the synthesized AgNPs varied from 21 to 66 nm. Previously, three separate research studies reported on synthesized AgNPs from different species of K. pneumoniae, which were mainly spherical and cube-shaped. In most cases, the synthesized AgNPs had a spherical shape, with only a few exceptions, and in two instances, cube-shaped AgNPs were obtained. Regarding elemental composition and purity, AgNPs synthesized by B. siamensis strain C1 were purest, where Ag was 91.8%. At the same time, the remaining was impurity mainly by Cl and S, whereas in our case, the Ag (%) varied from 37.8% to 61.6%, i.e., less than reported previously (Ibrahim et al., 2021). The lower purity could be due to improper washing of the AgNPs during centrifugation. From the UV-Vis study, it was found that the absorbance peak of AgNPs synthesized from different bacteria could be varied from 400 to 510 nm based on the size and shape of the synthesized AgNPs. From XRD and FTIR, it was found that the majority of the peaks and bands remain the same in all the syntheses with slight variations, respectively.

Batch adsorption study of MO dye by AgNPs

MO dye shows the highest absorbance at 464 nm when examined using a UV-Vis spectrophotometer. The AgNPs-M, AgNPs-K, and AgNPs-E treated MO were measured for their color intensity in an aqueous dye solution for up to 120 min at a regular interval of 30 min. With time, the concentration of MO dye in the sample decreased gradually, evident from the UV-visible spectra (Figs. 6A–6C). So, the maximum removal of MO dye was found after 120 min using AgNPs-M. The analysis at an interval of every 30 min shows a gradual decrease in the concentration and absorbance of the dye; hence, the reduction in the graph can be observed easily as it progresses from 0 to 120 min. Figures 6A–6C show the MO dye absorbance by UV-Vis spectroscopy at different time intervals. Figure 6A (AgNPs-K), Fig. 6B (AgNPs-M), Fig. 6C (AgNPs-E), and Fig. 6D (control). The control flask had 50 ppm dye and no AgNPs. The UV-Vis spectra show no change in absorbance peaks from 0 to 120 min.

Percentage removal of MR dye by all the AgNPs

AgNPs-M removed MO dye 2.34% at 30 min, 4.37% at 60 min, 14.83% at 90 min, and 19.24% at 120 min. AgNPs-K removed MO dye at 1.5% at 30 min, 4.06% at 60 min, 9.56% at 90 min, and 15.03% at 120 min. AgNPs-E removes MO dye 1.52% at 30 min, 2.36% at 60 min, 3.86% at 90 min, and 4.15% at 120 min. By comparing all the AgNPs mentioned above, it was observed that AgNPs-M has the highest dye removal efficiency and AgNPs-E has the lowest dye removal efficiency. Figures 7A–7C shows the percentage removal of MO dye by all three types of AgNPs.

AgNPs, synthesized by Aspergillus sp., photocatalytically degraded the reactive yellow dye in an aqueous solution where the initial dye concentration was about 20–100 mg/L, and about 1 g/L of retentate biomass of the fungus removed 82–100% dye. As the initial dye concentration increased, the decolorization efficiency of the biomass retentate decreased from 9.2% to 32.3% (Gola et al., 2021). In one of the investigations, Pseudomonas sp. synthesized AgNPs achieved 100% removal of MB dye within 30 min (Rasheed et al., 2023), whose more detailed outcomes are provided in Table 4. AgNPs synthesized from Salvinia molesta demonstrated the maximum adsorption capacity (121.04 mg/g) of methylene blue dye on the surface of AgNPs by Langmuir isotherm (Batool, Daoush & Hussain, 2022). About 83% of MO dye was removed by using chemically synthesized AgNPs in the presence of NaBH₄ under optimized conditions (Bhankhar et al., 2014). Table 4 depicts the summarized form of all the investigations and current investigations where AgNPs were used for the removal of MO from wastewater.

Based on the investigations mentioned in Table 4, it was found that earlier, only a single attempt had been made to remove MO dyes from contaminated water by using AgNPs synthesized by chemical route, resulting in a removal percentage of 83% within a span of 2 min (Bhankhar et al., 2014). There was only one attempt where dyes (MB, 4-NP, RB5, and RR120) were removed by using AgNPs synthesized by Pseudomonas sp., where the removal efficiency varied from 40–100% under optimized conditions (Rasheed et al., 2023). Moreover, in the current investigation, AgNPs-M has the smallest average size (21–45 nm), which exhibited the highest MO dye removal, i.e., 19.24% in 2 h. In addition, the removal percentage of MO was just 4–19.24%, almost 5 to 15 folds less than the previous attempt by using AgNPs synthesized by the chemical method. The lower MO dye removal percentage in the current investigation might be attributed to the usage of a very low dose of AgNPs (1 mg/100 mL), in comparison to the previous study, whereas earlier, about 20 to 100 mg/50 mL of AgNPs synthesized from Pseudomonas sp. for the removal of varies organic dyes (20 mg AgNPs) (Rasheed et al., 2023). Zaman et al. (2023) and his research team successfully achieved a 94% degradation of MB in an aqueous solution using photocatalysis under solar irradiation.

To achieve a higher removal efficiency of dye, a higher dose of AgNPs is suggested. Moreover, further improvement in the dye removal by AgNPs could be done by using different types of chemical reducing agents (NaBH₄) along with the AgNPs (Rasheed et al., 2023). From the investigation, it was also concluded that the morphology of the AgNPs also affects the removal efficiency of the dyes, i.e., smaller particles have a high removal efficiency (Zaman et al., 2023), as evident from the current investigation and study done by Rasheed et al. (2023). Moreover, the removal of dyes under irradiation (solar, UV, etc.) may significantly enhance the dye degradation or removal, which could also be governed by pH and temperature.

Evaluation of the antimicrobial activity of the synthesized AgNPs

AgNPs-K exhibits an impressive ZOI measuring 11 mm when tested against B. megaterium and E. faecalis. B. subtilis exhibits a moderate ZOI measuring 10 mm, while B. cereus demonstrates a 9 mm ZOI due to the impact of AgNPs. The AgNPs formulation (40–50 μg/mL) synthesized from K. pneumoniae exhibited higher antibacterial activity against E. coli in comparison to S. aureus, which might be due to variations in the thickness and composition of their cell walls, like peptidoglycan (Sayyid & Zghair, 2021). Saleh & Khoman Alwan (2020) used (50, 10, and 150 µg/mL) concentrations of AgNPs synthesized from K. pneumoniae and evaluated them against E. coli, P. aeruginosa, S. aureus, and B. cereus and found the highest concentration, i.e., 150 µg/mL, was found to be most effective against these pathogens compared to 50 and 100 µg/mL. It can be inferred that a higher concentration of AgNPs leads to a greater level of antibacterial activity (Saleh & Khoman Alwan, 2020; Liu et al., 2024).

The utilization of AgNPs-M for studying antibacterial activity against various bacteria such as B. subtilis, B. cereus, B. megaterium, and E. fecalis reveals distinct variations in the size of ZOI. AgNPs-M shows a maximum ZOI of 12 mm against B. megaterium and a minimum ZOI against B. cereus of 8 mm. It offers a moderate ZOI of 9 mm against E. faecalis and 11 mm against B. subtilis. B. megaterium showed a higher susceptibility compared to E. faecalis, while B. subtilis displayed a moderate susceptibility, and B. cereus exhibited a lower susceptibility towards AgNPs-M. B. cereus gives a greater ZOI of 11 mm due to the effect of AgNPs-E. The smallest ZOI was observed while testing against B. megaterium, measuring 8 mm. Additionally, B. subtilis and E. faecalis both exhibit a ZOI of 10 mm.

Earlier Aspergillus sp. mediated synthesized AgNPs exhibited a ZOI of about 13 and 10 mm against E. coli and S. aureus, respectively, obtained for the Gola et al. (2021). Additionally, the synergistic effect of AgNPs and penicillin against E. coli and S. aureus exhibited 0.49 and 0.36-fold increases in the ZOI, respectively, compared to the AgNPs and penicillin alone (Gola et al., 2021). Paenarthrobacter nicotinovorans MAHUQ-43 mediated synthesized AgNPs (13–27 nm, crystalline, spherical-shaped) showed antibacterial activity against B. cereus ATCC 10876 and P. aeruginosa ATCC 10145, where the MIC and MBC for both pathogens were 12.5 and 25 µg/mL, respectively. The ZOI at 30 µL against P. aeruginosa was 15.5 ± 0.8 and 13.6 ± 0.5 mm against B. subtilis, whereas at 60 µL the ZOI against P. aeruginosa was 24.7 ± 0.9 mm and against B. subtilis it was 19.3 ± 1.0 mm (Huq & Akter, 2021). In one more investigation, AgNPs synthesized by B. cereus exhibited ZOI, i.e., 32.12 ± 0.55 mm (Staphylococcus epidermidis), 38.25 ± 0.05 mm (S. aureus), 33.05 ± 1.33 mm (E. coli), 30.73 ± 0.25 mm (Salmonella enterica), and 35.44 ± 1.08 mm (Porteus mirabilis) by agar-disc diffusion method at a concentration of about 200 µg/mL of AgNPs (Ibrahim et al., 2021). Figure 8 shows the antibacterial activity of Petri plates of the AgNPs against tested bacterial species. Table 5 shows a comparison of the antibacterial activity of the current investigation with previously reported studies.

The difference between the antibacterial effect against GPB and GNB bacteria over here might be due to the morphological differences in both types of bacteria (Jubeh, Breijyeh & Karaman, 2020). GPB has a thick peptidoglycan layer, while GNB has a thin peptidoglycan layer but a thick lipopolysaccharide layer (Pasquina-Lemonche et al., 2020). Several other investigators also provided a similar explanation for the antimicrobial activity of the synthesized AgNPs (Kalpana & Lee, 2013; Singh & Mijakovic, 2022). Investigators reported that spherical AgNPs have higher antibacterial/antimicrobial activity due to their high SVR (Al-Ogaidi & Rasheed, 2022). Moreover, due to their large surface area, AgNPs could bind with various ligands. AgNPs can easily infiltrate the bacterial cell membrane, resulting in enhanced antimicrobial activity (Kalwar & Shan, 2018). This effect could potentially be enhanced even further by reducing the size of AgNPs (Anees Ahmad et al., 2020; Wypij et al., 2021). AgNPs get attached to the cell membrane, slowly releasing Ag ions, leading to the formation of pores in the cell wall of pathogens and ultimately enhanced permeability and leakage of cellular components through the plasma membrane, resulting in the death of the pathogens (Tripathi et al., 2017; Chen et al., 2020; Matras et al., 2022).