Biofabrication of isotropic silver nanoparticles using cell-free metabolic extract of Bacillus kochii, and evaluation of their antimicrobial, antioxidant and catalytic potentials


 Abstract

Microbial metabolites have been reported as potent candidates in the biofabrication of nanomaterials. For the first time, the present study reported the biofabrication of ecofriendly isotropic silver nanoparticles (BkCE-AgNPs) showed potential antimicrobial, antioxidant and biocatalysts characteristics using the metabolite of Bacillus kochii. Further, B. kochii crude extract (BkCE) mediated synthesis of BkCE-AgNPs was confirmed by surface plasmon resonance (SPR) peak at 420 nm using a UV-vis spectrophotometer. Various major physico-chemical parameters such as temperature, pH, crude extract concentration, AgNO3 concentration were utilized to characterize BkCE-AgNPs. In addition, FTIR analysis revealed the functional groups of active compounds present in the BkCE that mediated the fabrication of BkCE-AgNPs. Whereas, HRTEM confirmed the synthesis of BkCE-AgNPs with a size range between 4 to 44 nm and the crystalline nature of the biofabricated BkCE-AgNPs was confirmed by EDAX. Furthermore, the BkCE-AgNPs exhibited a broad-spectrum antimicrobial activity against A. baumannii, S. typhi, E. coli, K. pneumoniae, S. aureus and P. aeruginosa. The BkCE-AgNPs also showed excellent catalytic dye degradation potential and antioxidant activities confirmed by DPPH assay, total reducing sugar test and nitric oxide scavenging assays. In conclusion, the present study could help understand the development of biofabrication of nanomaterials using potent microbial metabolites in the agricultural and medicinal industries.


Introduction
Extracellular synthesis of nanoparticles using bacteria is being carried out using bacterial biomass, bacterial supernatant and cell-free extract. Bacterial biomass-mediated synthesis involves reducing metal ions that contact the cell surface of bacteria, proteins and enzymes in the local environment (Parikh et al. 2011). Cell pellet of Bacillus licheniformis (Kalimuthu et al. 2008) and Lactobacillus(Ganesh Babu and Gunasekaran 2013) strains have been reported in the extracellular synthesis of nanoparticles. The biomolecules present in supernatants played a crucial role in reducing the silver ions and stabilizing them. Previous studies reported that culture supernatants of Bacillus indicus, B. cecembensis, Pseudomonas antarctica, P. proteolytica, P. meridiana and Arthrobacter kerguelensis readily reduced the silver ions when compared to the microbial biomass-cell surface (Shivaji et al. 2011). However, the organic matrix of media components negatively affected their colloidal dispersion, characterization and recovery. Hence, cell-free extract of bacteria-mediated synthesis of nanoparticles deserves merit (Wei et al. 2012;Boopathi et al. 2012) Besides exopolysaccharides (Saravanan et al. 2018), pigments (El-Sayyad et al. 2018) as components for cell-free extract mediated synthesis, bacterial metabolite mediated synthesis also be utilized.
Bioactive metabolite from bacteria was used as chemotherapeutic agents to treat human and animal diseases (Schwartsmann et al. 2001). There is a huge demand for novel bioactive metabolites to treat drug-resistant human and animal pathogens (Nathan 2004) and biocontrol agents against plant insert and pathogens (Islam et al. 2011). Bioactive metabolite from marine bacteria was least explored. In the aquatic bacterial community, Bacillus sp. belong to phenogenetically and phylogenetically heterogeneous groups. Bacillus sp. are ubiquitous in the maritime world and can withstand adverse conditions (Rampelotto 2010) Previous studies reported that several marine Bacillus isolates were able to produce secondary metabolites, including lipopeptides, polypeptides, macrolactones, fatty acids, polyketides, lipoamides and isocoumarins (Hamdache et al. 2011;Mondol et al. 2013a). In addition, Bacillus kochii has showed antagonistic activity against human pathogens (Chinnachamy;Mondol et al. 2013b;Matobole et al. 2017).
Nanoparticles such as gold, silver, zinc, titanium, selenium, copper and magnesium were synthesized and studied in all application prospectus. Silver was used as an antiseptic for treating warfare injures and burns. Silver nanoparticles (AgNPs) were exploited in various elds like health care, biomedicals, wastewater treatment, pharmaceuticals, agriculture and others (Zhang et al. 2016). AgNPs possessed strong bactericidal and broad-spectrum sterilization effects for many pathogens and multidrug-resistant (MDR) bacteria, and AgNPs were reported to kill over 600 kinds of bacteria in a short time (Nagasundaram et al. 2014). The latest nding showed that AgNPs had higher inhibition against pathogens and aided in creating protective coatings upon dental resins, bone adhesive, ion exchange bers and medical tools. In addition to biomedical applicability, AgNPs found increasing effectivity in catalysis of azo dyes, solid, liquid wastewater treatment, plant growth promotor and crop production (Ibrahim et al. 2021). In recent years, the overuse of antibiotics led to a vast increase of MDR pathogens.
These MDR pathogens are the perils to society. To challenge these pathogens, searching for suitable candidates is one of the most signi cant research tasks, where AgNPs play a crucial role. AgNPs possessed antioxidant solid properties, leading to developing new areas with enhanced targeted actions (Akintola et al. 2020). In addition, the modern world depends on industrial-, textile-, plastic-and cosmeticbased products that release many hazardous dyes in the environment. Degradation of these toxic dyes is an essential task in the environmental and pollution control eld. Several physicochemical and biological methods have been used to degrade azo dye (Sarkar et al. 2017). With the advantage of the size to volume ratio, nanoparticles emerged as suitable candidates in degrading the dyes (Salem and Fouda 2021). Nanoparticles degraded the dyes in a two-step reaction that involved the accumulation of electrons of sodium borohydride on the surface of nanoparticles followed by the distribution of azo dyes through its molecules on their surface; reduction was carried out by super cial electrons. The surface of the nanocatalyst depended on the properties of the capping molecules, which was directly proportional to the kinetics of the degradation reaction (Zhang et al. 2011).
In the present investigation, we reported the biofabrication of isotropic nanoparticles (BkCE-AgNPs) using the crude secondary metabolite of B. kochii (BkCE). In addition, we characterized BkCE-AgNPs with various physicochemical and analytical methods and con rmed their antibacterial, antioxidant and biocatalytic potentials with various assays.

Chemicals
Polymerase chain reaction (PCR) reagents and 16S rRNA universal primers fd1 and rp2 were purchased from Sigma-Aldrich. Inc., India. PCR product puri cation kit and silver nitrate (  India (Prof. N. Sakthivel, puns2005@gmail.com;(https://www.pondiuni.edu.in). HPLC grade solvents were purchase from Merck chemicals, India and all other chemicals, reagents and solvents were purchased from standard commercial sources and of the analytical grade.
Isolation and Molecular identi cation of the bacterial strain A sea-sediment water sample was collected in a sterile bottle from the old harbor seashore, Puducherry, India (geospatial coordinates: 11°55′21.5″N, 79°50′06.6″E). The seawater was serially diluted in sterile saline water and then plated onto Zobell marine agar (ZMA). To obtain pure strain, that capacity to resist the silver ion and produce nanoparticles, the isolated colonies were further sub-cultured on ZMA supplemented with a one mM lter-sterilized AgNO 3 . Single colonies were selected after 48 hours of incubation and designated as strain SW6.
Unsheared total genomic DNA was extracted from the strain SW6 using the phenol: chloroform method (Pathma and Sakthivel 2013). The universal bacterial primer pair fd1 (5'-GAGTTTGATCCTGGCTCA-3') and rp2 (5'-ACGGCTACCTTGTTACGACTT-3') was used to amplify the 16S rRNA gene and it was con rmed by 1% agarose gel electrophoresis. The product was puri ed using a Himedia PCR puri cation kit and sequenced with Illumina -DNA automated sequencer with 16S rRNA universal fd1 and rp2 primers using the facility at Macrogen Inc. (Seoul, Korea). The 16S rRNA sequence was deposited in GenBank. The phylogenetic analysis of strain SW6 was constructed using the neighborjoining (NJ) method and MEGA v10.0 (Ravindra Naik et al. 2008).

Crude extract from strain SW6
A total of 10% of an overnight culture of strain SW6, B. kochii was added to 2 L (v/v) Luria-Bertani (LB) broth. The fermentation condition was optimized at 130 rpm, 24 ºC for four days. The fermented crude secondary metabolite was extracted from cell-free supernatant using an equal volume of ethyl acetate.
The ethyl acetate extract was collected and concentrated in a rotary evaporator (Buchi, Switzerland). The dried crude extract was weighed, dissolved in sterile MilliQ water and ltered for further experiments.
Optimization of physicochemical factors for nanoparticle synthesis Synthesis of AgNPs using crude extract of strain SW6 was optimized by a one-factor trial method. Effect of different temperatures (60-90 ºC with 10 ºC interval, and 95 ºC), pH (6-10 with one pH interval), AgNO 3 concentrations (0.5, 1, 1.5 and 2 mM) BkCE concentrations (1, 3, 5 and 7 mg/ml) and time interval (5 min to 50 min) were studied. The samples were ten times diluted and recorded using a UV-Vis spectrophotometer (Systronic, Gujarat, India) with a scan range of 350-600 nm and color change was also observed. The size of BkCE-AGNPs at different pH was analyzed using DLS (Malvern Nano ZS, WR14 1XZ United Kingdom). The optimized conditions were used for the large-scale synthesis of BkCE-AGNPs.

Puri cation of BkCE-AgNPs
The solution containing biofabricated AgNPs was centrifuged at 9000 rpm for 30 min. Settled nanoparticles were further re-dispersed twice with MilliQ water and centrifuged to remove less bounded compounds over the nanoparticles. The nanoparticles were oven-dried at 40 °C for 24 h and stored at 4 ºC for further studies .

UV-visible (UV-vis) spectroscopy and Dynamic light scattering (DLS) analyses
Change of colour from white to brown was visualized in a reaction mixture of 1 mM AgNO 3 solution with 10% of crude extract of strain SW6 (5 mg/ml) at pH 9 and kept in a water bath set at 90 ºC. The reaction mixture was diluted ten times for monitoring AgNPs periodically in a UV-Vis spectrophotometer (Systronic, Gujarat, India) with the wavelength 400 -750 nm spectral range. For DLS analysis, 1 ml of colloidal nanoparticle solution was sonicated for 20 min in a water bath sonicator and analyzed using the ZETA Seizer Nano series (Malvern Nano ZS, WR14 1XZ United Kingdom) instrument .

Fourier transform infrared spectroscopy (FTIR) analysis
FTIR was used to determine the functional groups of the molecules present in the crude extract and capping and stabilizing the nanoparticles. The nanoparticles were puri ed by centrifugation and washed using sterile water to remove the least bound molecules. The dried pellets of crude extract and puri ed AgNPs were ground with KBr in the ratio of 1:100 and subjected to FTIR measurements on a Thermo Nicolet model 6700 spectrometer (Thermo Fisher Scienti c, Tempe, AZ, USA) with IR source ranging from 500-4000 cm -1 where KBr alone was used as a reference.
X-ray diffraction (XRD) analysis X-ray diffraction (XRD) was used to analyze both molecular and crystal structures. Thin lms of the solution were drop-coated onto a glass slide in the area of 1 x 1.5 cm at 50 ºC and analyzed using X'pert Pro X-ray diffractometer (Netherlands). The pattern was then scanned in the range of 2θ from 30 to 80° using nickel monochromatic Cu-Kα 1 radiation with λ of 1.5406 Å at voltage 40 kV and current of 30 mA.

High-resolution transmission electron microscopy (HRTEM) -SAED analysis
The nanoparticles were loaded on carbon-coated copper grids and left undisturbed for the solvents to dry to make a uniform lm. The size and morphology of the nanoparticles were analyzed on the HRTEM FEI Tecnai F30 model (FELMI-ZFE, Graz, Austria).

High-performance liquid chromatography (HPLC) analysis
The supernatant, after pelleting the synthesized AgNPs, was collected and ltered. The SW6 crude exact and the supernatant was analyzed using HPLC (Shimadzu-LC-20A, Shimadzu, Kyoto, Japan) equipped with an SPD 20A UV-Vis photodiode detector (192 nm-700 nm). The Phenomenex-Gemini C-18 column (250 x 4.6 mm; 5µm 110 Å) packed with C18 silica particles was used. The mobile phase HPLC water and methanol in the ratio 7:3 at the ow rate of 1 ml/min was used and observed at 254 nm.

Antimicrobial testing
The antimicrobial potential of the nanoparticles was determined using the standard Kirby-Bauer disc diffusion method. Using swabbing technique, 18-24 h old (approximately 10 6 colony-forming units/mL) of A. baumannii, S. typhi, E. coli, K. pneumoniae, S. aureus and P. aeruginosa on MHA to form a bacterial lawn. The sterile paper discs having 6 mm diameter were loaded with 20 μg nanoparticles (20 µl of 1mg/ml stock), 20 µl of 1mM AgNO 3 and 20 µl of crude extract (5 µg/ml) suspended in deionized water and 20 µg of streptomycin disc as control. The discs were placed over the bacterial lawn and kept for incubation for 24 h at 37 °C. and observed for inhibition of test organism and repeated thrice (Pugazhenthiran et al. 2009;Wang et al. 2015).

Minimum inhibitory concentration (MIC), Minimum bactericidal concentration (MBC) and evaluation of growth kinetics of pathogenic organisms
The MIC and MBC of nanoparticles were done using the method described in the guidelines of (Clinical and Laboratory Standards Institute 2018). The MIC test was performed in 96-well round-bottom microtiter plate using the standard MHB microdilution method, to which 100 µl of bacterial inoculums concentration of 10 6 CFU/mL was added. Then 100 μL of the nanoparticles stock solution (200 μg/mL) was added and diluted two-fold with the bacterial inoculums from column 12 to column 4. Column 4 of the microtiter plate contained the lowest concentration of AgNPs, while column 12 contained the highest concentration. Column 1 and 2 served as a negative (only medium) and positive control (MHB and bacterial inoculums), respectively. The microwell plate (Bio-Rad, USA) reading was measured at 595 nm every three hours. After 24 h, 10 µl of each pathogen subjected to different concentrations of nanoparticles were plated on an MHA plate and incubated at 37 °C for 24 h (Wang et al. 2015).
In vitro antioxidant assays DPPH free radical scavenging assay The free radical scavenging activity was determined using a modi ed 2,2'-diphenyl-1-picrylhydrazyl (DPPH) assay (Brand-Williams et al. 1995;Choi et al. 2002). Brie y, an aliquot of 0.9 ml of DPPH (100 µM) was added to 0.1 ml of crude extract and AgNPs containing concentrations ranging from 50 -250 µg/ml. The reaction mixture was kept in the dark for 30 min and absorbance was measured at 517 nm using a UV-Vis spectrophotometer (Systronic, Gujarat, India). Butylated hydroxytoluene (BHT) was used as standard. The percentage inhibition for DPPH was calculated according to the following equation (Brand-Williams et al. 1995) : Free radical scavenging effect (%) = (Control absorbance -Sample absorbance)/ (Control absorbance) x 100 (1) Reducing power assay The reducing power activity was performed as described previously (Czinner et al. 2000). Brie y, 0.5 ml of crude extract and nanoparticles at different concentrations ranging from 50-250 µg/ml were mixed with 0.5 ml of phosphate buffer (200 mM) followed by the addition of 0.5 ml of potassium ferricyanide (1%). The mixture was incubated at 50 ºC for 20 min, and then 0.5 ml of trichloroacetic acid (10%) was added. After that, 0.5 ml of the above mixture was added to 0.5 ml of double-distilled water and 0.1 ml of ferric chloride solution (0.1%). After 5 min, the absorbance was measured at 700 nm using a UV-Vis spectrophotometer (Systronic, Gujarat, India). Butylated hydroxyaniline (BHA) was used as standard.
Nitric oxide free radical scavenging assay Nitric oxide (NO) free radical scavenging assay was estimated as described previously (Milanezi et al. 2019) with some modi cation. In this test, 0.3 ml of sodium nitroprusside (10 mM) was prepared in phosphate buffer saline followed by the addition of 0.1 ml of various concentrations (200-600 µg/ml) of crude extract and nanoparticles, and incubated at 10 min under UV hand lamp (λ max 365 nm, UVITE CLF-206.LS, 6W, intensity at 15 cm is 580 μW / cm 2 ). Then, an equal volume of Griess reagent (1% sulphanilamide in 2.5% phosphoric acid and 0.1% naphthylethylene diamine dihydrochloride in 2.5% phosphoric acid) was added to the reaction mixture. The absorbance was measured at 546 nm using a UV-Vis spectrophotometer (Systronic, Gujarat, India) and ascorbic acid (ASA) was used as standard. NO free radical scavenging effect (%) was calculated using the equation mentioned above in equation (1).

Catalytic reduction of hazardous dyes
Three different azo dyes, such as 4-nitrophenol, phenol red and methylene blue, were used to test the biocatalytic property of the nanoparticles. Control experiments were carried out in the absence of nanoparticles and the presence of the dyes and NaBH 4. The freshly prepared 0.3 ml of 150 mM NaBH 4 solution was separately added to 0.3 ml of 10 mM 4-nitrophenol, phenol red and methylene blue and the reaction volume was made up to 3 ml by the addition of deionized water, and then 0.1 ml of BkCE-AgNPs (100 µg/ml) were added to each reaction mixture (Nakkala et al. 2018;Kumar et al. 2019) The absorbance of each reaction mixture was monitored at regular intervals for both control and test solutions.

Results And Discussion
Isolation and identi cation of the bacterium Strain SW6 isolated from seawater near Pondicherry old harbor, Puducherry, India, was tested positive for its potential for the biosynthesis of AgNPs. Genomic DNA of SW6 was extracted, puri ed and used as a template for the ampli cation of 16S rRNA coding gene-speci c primers. The 16S rRNA gene sequence that resulted in a continuous stretch of 1438 bp was submitted to NCBI (GenBank accession number: MW979601). Analyzing in EZtaxon e-server, the strain SW6 showed the highest sequence similarity of 98.07 % with B. kochii. Also. the NJ molecular phylogenetic tree analysis con rmed that the strain SW6 belonged to the species B. kochii with a 100% substantial bootstrap value, as shown in Fig.1.

Optimization of physicochemical factors for nanoparticle synthesis
BkCE reduced the AgNO 3 solution to BkCE-AgNPs at a higher rate at 90°C, pH 9 with one mM AgNO 3 and 5 mg/ml BkCE concentration after investigating with a one-factor trial method as shown in Fig. 2. BkCE-AgNPs intensity increased with an increase in temperature. At 90ºC, maximum peak intensity was observed, while 95 ºC showed little reduction in peak absorbance. The high temperature, 90 °C, facilitated the molecules in BkCE to colloid and reacted with AgNO 3 for reducing the silver ions to metallic silver. decreased BkCE-AgNPs synthesis. BkCE concentration was optimized at 5 mg/ml compared to 1, 3 and 7 mg/ml. In the case of pH, the intensity of BkCE-AgNPs was directly proportional to increasing pH. At pH 10, the peak was wider and shifted to 440 nm, indicating the aggregation of nanoparticles. To con rm this, the size of the nanoparticles was examined using DLS and it was found that at pH 9, smaller nanoparticle size was observed than all other pHs. Hence, pH 9 is used as an optimum pH for the synthesis of BkCE-AgNPs. With all optimum parameters, synthesis of BkCE-AgNPs at different time intervals was analyzed from 5 to 50 min where at 40 min, the intensity was recorded the highest. AgNPs represented the CC stretching group, whereas the peaks at 1045 and 1035 cm −1 of BkCE and BkCE-AgNPs showed cyclohexane ring vibration (Nandiyanto et al. 2019). The FTIR spectrum of BkCE and BkCE-AgNPs thus, con rmed that the molecules of BkCE capped and stabilized the BkCE-AgNPs, as evident in Fig.3.

XRD analysis
BkCE-AgNPs showed distinct 2θ peaks (angle at 38°, 55°, 64° and 72.2°) that attributed to the planes 111, 200, 220 and 222 as shown in Fig. 4. These peaks con rmed the formation of the face-centered cubic lattice of AgNPs when compared with standard pure crystalline silver peak pattern published by the Joint Committee on Powder Diffraction Standards (JCPDS) le no. 04-0783, and 31-1238 (Judith Vijaya et al. 2017).

HRTEM-SAED analysis
HRTEM images of BkCE-AgNPs revealed that the nanoparticles were spherical with a size range of 4 to 44 nm in diameter. Fig. 5 showed the histogram of various sizes of BkCE-AgNPs. The average diameter of BkCE-AgNPs was 19.96 ± 8.27 nm. The images also revealed that the nanoparticles were in close contact with each other (Khan, Saeed, and Khan 2019) and the dendrogram analysis showed maximum particles were around 12 to 22 nm in diameter. In a similar report, AgNPs synthesized using B. licheniformis were also reported as spherical and 40 nm in diameter (Shanthi et al. 2016). The selected area diffraction pattern showed that the BkCE-AgNPs were a mixture of monocrystalline and polycrystalline nature. The concentric ring corresponding to the planes (111), (200), (220) and (300) con rmed the crystalline facecentered cubic lattice AgNPs (Alahmad et al. 2021).

HPLC analysis
The chromatogram of BkCE showed four different peaks at retention-time (RT) 3.35, 4.38, 6.39 and 8.64, while the supernatant after synthesis showed three peaks at RT 3.34, 4.38 and 8.64 min similar to BkCE peaks at the same RT, but its intensity was reduced. Two new peaks appeared at RT 5.69 and 6.90 min in the supernatant in contrast to RT 6.39 of BkCE extract. This result inferred that the compound corresponding to the peak at RT 6.39 min in the BkCE was oxidized to reduce the silver ions to AgNPs, and hence, the new peak was identi ed in the supernatant as shown in Fig.6. The reduced intensity of other peaks was noted as they acted as capping agents.

Antimicrobial testing by Disc Diffusion method
The antibacterial potential of BkCE-AgNPs was investigated against human pathogens A. baumannii, S. typhi, E. coli, K. pneumoniae, S. aureus and P. aeruginosa using the disk diffusion method. The average zone of inhibition (ZOI) result represented in Fig.7, indicated that BkCE-AgNPs had signi cant antibacterial e cacy against all the above pathogens. BkCE-AgNPs showed the highest ZOI 15.2 mm ± 0.28 and 14.7 mm ± 0.65 for A. baumannii and P. aeruginosa respectively. Against A. baumannii, BkCE-AgNPs showed higher ZOI than standard streptomycin of the same concentration and higher inhibition zone than AgNPs synthesis using Discorea bulbifera (Chopade et al. 2013). BkCE-AgNP ZOI against P. aeruginosa was equal to AgNPs synthesized using Ipomea carnea (Daniel et al. 2014)) and more than AgNPs synthesized using E. coli and S. aureus (Peiris et al. 2018). BkCE-AgNPs showed a 13.5 mm ± 0.5 zone against S. aureus, which was more signi cant than the chemically synthesized AgNPs using sodium borohydride (Vu et al. 2018). BkCE-AgNPs showed an inhibition zone of 13.2 mm ± 0.25 against E. coli which was higher than the reported AgNPs derived from fungus Guignardia mangiferae (Balakumaran et al. 2015). BkCE-AgNPs showed 12.5 mm ± 0.5 and 13.4 mm ± 0.32 ZOI against S. typhi and. K. pneumoniae was higher than previous reports of 15.62 µg/ml concentration of AgNPs derived from an extract of Ziziphus oenoplia (Soman and Ray 2016). This result evidenced that BkCE-AgNPs could be employed to protect from a broad range of bacteria. The BkCE and 1mM AgNO 3 showed an inhibition zone of 14.2 mm and 7.5 mm, respectively, only against S. aureus. Table 1 represented the broadspectrum antimicrobial activity of control and tests against human pathogenic bacteria.

Minimum inhibitory concentration (MIC), Minimum bactericidal concentration (MBC) and evaluation of growth kinetics of pathogenic organisms
Broth microdilution assay with different concentrations of BkCE-AgNPs was used to investigate the effect of BkCE-AgNPs on the growth of A. baumannii, E. coli, K. pneumoniae, P. aeruginosa, S. typhi, and S. aureus. The results showed that minimal inhibitory concentration (MIC) of BkCE-AgNP for A. baumannii, E. coli, K. pneumoniae, P. aeruginosa, S. typhi and S. aureus were 6.5, 12.5, 12.5, 6.5, 12.5 and 12.5 µg/ml, respectively. The growth kinetics graph in Fig. 9 showed that different concentrations of BkCE-AgNPs suppressed other degrees of multiplication of pathogenic strains. The result noted that the MIC of BkCE-AgNPs against A. baumannii was highly potent than the 500 µg/ml concentration of AgNP-cephalosporins conjugate as reported (Chopade et al. 2013). MIC concentration of AgNPs was double than BkCE-AgNPs (Ahmad et al. 2017). MICs of AgNPS synthesized using streptomycin were reported to be 16 and 256 µg/ml against P.aeruginosa and K. pneumoniae, respectively (Wypij et al. 2018) that were higher than our reported concentration. Similarly, the MICs of BkCE-AgNPs were reported as lowest against S. aureus and S. typhi when compared to the Lippia citriodora -AgNPs (Elemike et al. 2017).
The MBC of BkCE-AgNPs against A. baumannii, E. coli and K. pneumoniae were 25 µg/ml. MBC for S. typhi and S. aureus were noted as 50 µg/ml, and the lowest MBC was observed against P. aeruginosa at 12.5 µg/ml. The MIC and MBC values against different pathogens, as shown in Fig. 10, revealed the effective concentrations of BkCE-AgNPs required to inhibit the growth of the human pathogens that could threaten the health of hosts and the environment. Graphical representation of both MIC and MBC values of BkCE-AgNPs against human pathogens was represented in Fig. 11.
Antioxidant assay DPPH free radical scavenging assay The BkCE-AgNPs showed 54 % of DPPH radical scavenging activity at 150 µg/ml, while BkCE showed only 24% activity as represented in Fig.12A. The BkCE-AgNPs exhibited better activity than the previous report of 43.5% at 1000 mg of AgNPs obtained using Cleistanthus collinus leaf extract (VennilaRaj 2013) and 49.94% at 500 mg of AgNPs synthesized from one stem extract of Clinacanthus nutans (Chiu et al. 2021).

Reducing power assay
BkCE and BkCE-AgNPs showed reducing power activity directly proportional to increasing the concentrations, as seen in Fig. 12B. At 250 µg/ml, BkCE-AgNPs showed 0.585 nm absorbance compared to 0.619 of BHA, a well-known antioxidant. The BkCE-AgNPs showed reducing power activity higher than the reported AgNPs synthesized using Lippia nodi ora extract (Sudha et al. 2017) and Iresine herbstii (Dipankar and Murugan 2012).
NO free radical scavenging assay Even though reactive nitrogen species are essential in the human system, acting as bio supervisory molecules, the excess radicals implicate many disorders and pathogenic conditions such as sepsis. In the present study, we reported that BkCE-AgNPs could be employed in scavenging excess NO. BkCE-AgNPs showed 53 % scavenging activity at 600 µg/ml, while ascorbic acid (ASA) exhibited 70 % scavenging activity at the same concentration. The results implied that BkCE-AgNPs showed more signi cant scavenging activity than BkCE, as evident in Fig.12C. Methylene blue is a cationic thiazine dye used in many industries, especially in textile industries. It is used as a dye for cotton, silk and fabric materials. Hence, the industrial e uents contain lots of dyes which, when discarded in the environment, cause an adverse impact on the aquatic system and human health, causing diarrhea, nausea and other health issues (Vutskits et al. 2008). In our study, the control methylene blue showed the UV-Vis absorption peaks at 670 and 610 nm. This peak was reduced gradually within 40 min upon the addition of BkCE-AgNPs and NaBH 4 . In general, sodium borohydride reacts with methylene blue to produce leucomethylene blue, a reversible product. The percentage of methylene blue degradation by BkCE-AgNPs along with NaBH 4 was found to be 73% at 40 min and the rate of reaction was 0.0324 min -1 , as shown in Fig. 13A.
The 4-nitrophenol dye is used to manufacture drugs such as paracetamol, fungicides, and insecticides and is widely used in leather industries. The US Environmental Protection Agency (EPA) categorized it as a potent pollutant due to its high solubility and stability in water. In humans, this dye causes eyes, skin and respiratory tract irritation, and it also forms methaemoglobin that causes cyanosis, confusion and unconsciousness. In our study, 4-nitrophenol showed a strong peak at 400 nm with NaBH 4 due to the formation of 4-nitrophenolate ions in alkaline conditions (Kumar et al. 2019). With the addition of BkCE-AgNPs, the peak intensity of 4-nitrophenol at 400 nm exponentially decreased. The complete reduction of 4-nitrophenol was found to be at 4 min, after which there was no further decrease in peak intensity. The degrading percentage of 4-nitrophenol and rate of reaction were found to be 95.56% at 8 min and 0.0612 min -1 , respectively, as shown in Fig. 13B.
Phenol red is a textile dye categorized as triphenylmethane dyes that are highly soluble in water. It is also used as a reagent for measuring the basic pH of water. It was reported that phenol red inhibited the growth of renal epithelial cells and caused irritation to the eyes, respiratory system and skin (Mittal et al. 2009). In our study, at 560 nm, the maximum absorbance of the dye was observed. The maximum decrease in absorption intensity of phenol red was achieved at 6 min in the presence of BkCE-AgNPs along with NaBH 4 solution. The percentage inhibition and rate of reaction were found to be 89 % at 5 min and 0.1453 min -1 , respectively, as shown in Fig. 13C.

Conclusion
In this work, the fermented crude extract of B. kochi, BkCE was used successfully to synthesize BkCE-AgNPs for the rst time after optimizing the physicochemical parameters. BkCE-AgNPs were characterized using various spectroscopic and microscopic analyses. It was observed BkCE-AgNPs showed SPR band at 420 nm while HRTEM images con rmed the spherical nanoparticles ranged around  UV-vis absorption spectra and the rate of reaction during the catalytic degradation of (A) methylene blue, (B) 4-nitrophenol and (C) phenol red the presence of BkCE-AgNP and NaBH4 at time course intervals