Bacillus licheniformis (MN900686) Mediated Synthesis, Characterization and Antimicrobial Potential of Silver Nanoparticles

: The use of bacteria in the synthesis of silver nanoparticles (AgNPs) emerges as an ecofriendly and exciting approach. In the present study, we reported the biosynthesis of AgNPs by using culture supernatant of the bacteria Bacillus licheniformis (MN900686). The biogenically synthesized AgNPs were confirmed by the change in the color of the culture filtrate from yellow to brown after the addition of AgNO 3 . Further characterization performed by means of UV vis-spectroscopy showed absorption peak at 414 nm which confirmed the formation of AgNPs. Fourier Transfer infrared (FTIR) confirmed the involvement of biological molecules in the formation of nanoparticles (NPs). The SEM revealed that the NPs have approximately 38 nm size. The agar well diffusion assay was used to determine antibacterial activity while tube dilution method was used to determine minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The human pathogenic bacterial strains i.e., P. aeruginosa (MN900691) and B. subtilis (MN900684), were used as test strains. The anti-bacterial assay against test strains revealed that these NPs showed concentration dependent increased zone of inhibition (ZOI). The maximum ZOI at 25 µL of AgNPs was 20 mm against B. subtilis after 24 hours of incubation. One-way ANOVA test showed significant ZOI ( p ≤ 0.05) against B. subtilis . The MIC was ranged from 4.3-6.6 μg/mL while MBC ranged from 8.3 to 6.6 μg/mL. Overall, this study suggested that the biogenically synthesized NPs are an effective alternative source of antimicrobials against pathogenic bacteria.

become able to synthesize nanoparticles NPs in the labs 3 . There are many types of NPs synthesized using metals such as Au, Ag, Ce, Pt, Pd and Zn 4 , but among them, most important are silver nanoparticles AgNPs . AgNPs have potential applications in medical nano-engineering and pharmaceutical fields for the development of therapeutic agents, chronic diseases diagnostics and biosensors 5 . The AgNPs have extensively been studied for their activities particularly antimicrobial activities 6,7 and emerging as promising nano antibiotics now a days 8 . AgNPs have broad spectrum based antibacterial abilities so can be utilized in biomedical and medical devices and textile fields 9 . By Abstract: The use of bacteria in the synthesis of silver nanoparticles (AgNPs) emerges as an ecofriendly and exciting approach. In the present study, we reported the biosynthesis of AgNPs by using culture supernatant of the bacteria Bacillus licheniformis (MN900686). The biogenically synthesized AgNPs were confirmed by the change in the color of the culture filtrate from yellow to brown after the addition of AgNO 3 . Further characterization performed by means of UV vis-spectroscopy showed absorption peak at 414 nm which confirmed the formation of AgNPs. Fourier Transfer infrared (FTIR) confirmed the involvement of biological molecules in the formation of nanoparticles (NPs). The SEM revealed that the NPs have approximately 38 nm size. The agar well diffusion assay was used to determine antibacterial activity while tube dilution method was used to determine minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The human pathogenic bacterial strains i.e., P. aeruginosa (MN900691) and B. subtilis (MN900684), were used as test strains. The anti-bacterial assay against test strains revealed that these NPs showed concentration dependent increased zone of inhibition (ZOI). The maximum ZOI at 25 µL of AgNPs was 20 mm against B. subtilis after 24 hours of incubation. One-way ANOVA test showed significant ZOI (p ≤ 0.05) against B. subtilis. The MIC was ranged from 4.3-6.6 μg/mL while MBC ranged from 8.3 to 6.6 μg/mL. Overall, this study suggested that the biogenically synthesized NPs are an effective alternative source of antimicrobials against pathogenic bacteria.
Key words: Bacillus licheniformis, silver nanoparticles, characterization, antibacterial activity, MIC and MBC determination using an ecofriendly approach, AgNPs applications have been increased in preparation of large number of products such as pests, electronic devices and in controlling microbe s growth and infections 10,11 .
The use of silver as suspension and in nano-particulate form has a dramatic revival in nanotechnology. It has great antibacterial potency against human pathogens. The main task in NPs synthesis is the control of their physical properties like uniform particle size, similar shape, chemical composition, morphology and crystal structure. AgNPs have great effectiveness against most of the microbial pathogens particularly against the multi-drug resistance MDR bacteria. Microorganisms have been probed as potential bio-factories for metallic NPs synthesis such as silver, copper, zinc and gold 12 .
The antibacterial activity of biogenic NPs in combination with antibiotics enhances their importance in controlling the MDR pathogenic bacteria in planktonic and biofilm mode 13 . Biogenically synthesized NPs are easy to produce biocompatible, economic, environmental friendly and offer different catalytic abilities compared to chemically synthesized ones. They have anticancer and antioxidant properties. Furthermore, they have more stability, as the natural organic material citrate, sodium dodecyl sulfate of bacteria work as natural capping layers surrounding the biogenic NPs, make these AgNPs active, stable and reusable 13 15 . AgNPs act as promising antimicrobial agent due to their long term stability and biocompatibility 16 .
It has been suggested that biogenic AgNPs produce reactive oxygen species and free radicals which cause cell death through apoptosis and prevent bacterial replication. Since AgNPs are smaller than the microorganisms, they diffuse into cell and rupture the cell wall. It has also been observed that smaller NPs are more effective than the bigger ones because of their quick penetration in the bacterial cell 17 .
In this present work, microbial production of AgNPs was investigated by using culture supernatant of the bacterial strain Bacillus licheniformis as a reducing agent. The biogenically synthesized AgNPs were characterized by UV-vis spectroscopy, fourier transform infrared spectroscopy FTIR , X-ray diffraction XRD and scanning electron microscopy SEM . The synthesized AgNPs were further evaluated for the antibacterial efficacy using agar well diffusion method against two pathogenic bacteria both Gram negative and Gram positive such as P. aeruginosa MN900691 and B. subtilis MN900684 . The minimum inhibitory concentration MIC and minimum bactericidal concentration MBC was also determined.

Microorganisms source
Bacillus licheniformis MN900686 was obtained from microbiology lab, Government College University, Lahore to be used for the synthesis of AgNPs, cultured on nutrient agar slant and incubated at 37 for 24 hrs. The antimicrobial activity was checked against two human pathogenic bacteria both Gram negative and Gram positive such as P. aeruginosa MN900691 and B. subtilis MN900684 , obtained also from microbiology lab, Government College University, Lahore.

Supernatant collection
Sterilized nutrient broth was inoculated with fresh culture of the bacterial strain B. licheniformis. The culture flasks were incubated for 24 hours in rotatory shaker incubator at 37 . After the incubation period, the bacterial culture was centrifuged at 12000 rpm for 15 minutes. The supernatant was saved and the pallet was discarded.

Extracellular synthesis of AgNPs
10 mM silver nitrate AgNO 3 solution was added in the supernatant for AgNPs synthesis in 2:1 2 ratio was supernatant and 1 ratio was AgNO 3 solution while another reaction mixture without AgNO 3 was used as control. The solutions were incubated for 72 hours on rotatory shaker at 150 rpm at 37 and kept in dark to prevent any photochemical reaction during the experiment. After 72 hrs, the solutions color was turned from yellow to brown having mixture of AgNO 3 and bacterial suspension while no color change was observed in control supernatant without AgNO 3 . The AgNPs were purified by centrifugation at 12000 rpm for 15 minutes twice and collected for further characterization.

Characterization of AgNPs
The appearance of brown color indicated that the AgNPs formation occurs in the reaction mixture due to reduction of Ag ions 1,18 . The mixture was further characterized, by UV-Visible spectroscopy, fourier transform-infrared FTIR spectroscopy, scanning electron microscopy SEM , and X-ray diffraction XRD in order to authenticate the formation and check the specificity, size etc. of AgNPs.

UV-visible spectroscopy
The supernatant was tested qualitatively by UV-visible spectroscopy using AE-S70-1U UV-visible spectrophotometer and silver nitrate solution was used as control. UV-vis spectrophotometer from 370 to 970 nm operated at a resolution of 1 nm was used as a function of wavelength for spectral analysis of AgNPs. The peak of AgNPs varies from 400 to 470 nm. Occurrence of peak between 400-470 nm showed formation of more AgNPs and reduction of silver nitrate.

Fourier transform-infrared FTIR spectroscopy
The FTIR spectral analysis establishes the bio-molecules which are responsible for stabilization and capping of AgNPs as well as to check the functional groups of the AgNPs. The completely dried samples of AgNPs were used in order to perform FTIR. The spectrum was recorded on FTIR: IR Prestige-21 P/N 206-72010. SHIMADZU Fourier transform infrared in the transmission range of 500 -4000 cm 1 .

Scanning electron microscopy SEM
The size and morphology of the AgNPs were examined by scanning electron microscope EM6200 .

X-ray diffraction XRD
The crystalline nature of the AgNPs was characterized by XRD technique using an X-ray diffractometer Phillips PW 1729/40 operated at 40 kv, 40 mA, step size of 0.2 over the 2θ range of 20-80 . Glass slides coated with AgNPs were tested.

Antibacterial activity of AgNPs
The antibacterial activity of B. licheniformis synthesized AgNPs was analyzed through agar well diffusion method against the test pathogens i.e., P. aeruginosa and B. subtilis 13, 19 21 . Muller Hinton agar plates were prepared and well of 6 mm diameter was made by using sterile cork borer. The test pathogens culture was adjusted to 0.5 Mc-Farland turbidity standard and was spread on the media plate uniformly. The AgNPs at different concentrations 10 μg/mL, 15 μg/mL, 20 μg/mL, and 25 μg/mL were used. A well loaded with DMSO without AgNPs was maintained as negative control Fig. 1 . The plates were incubated in an inverted position for 24 hrs at 37 . The susceptibility of the test organisms was determined by measuring the diameter of the zone of inhibition around each well and measured in mm to test the antibacterial activity.

Minimum inhibitory concentration MIC and minimum
bactericidal concentration MBC MIC and MBC of synthesized AgNPs was measured using tube dilution method and following 22 with little changes. 3 mL nutrient broth was added into the test tubes. Almost 10 μL of bacterial fresh culture which was already adjusted to 0.5 McFarland turbidity standard was added to the tubes having broth. After that, different concentrations of AgNPs were added in these test tubes. The test tubes were incubated at 37 for 24 hours. Following incubation, MIC was recorded as the lowest most concentration without any visible growth. For MBC determination, 10 μL of lower most MIC was spread on nutrient agar plates. The concentration at which the 99 of the growth was inhibited was recorded as MBC.

Statistical analysis
Experiments were run in triplicates. Microsoft Excel 2019 was used to draw graphs while SPSS version 10 was used to calculate Means, Standard error and ANOVA test. The figure of FTIR data was made on origin 2019A. The pictures of SEM and XRD were from one replicate.

Biogenic synthesis of AgNPs
Synthesis of AgNPs by using B. licheniformis was confirmed by the change of color from yellow to brown after 72 hours incubation of the reaction mixture. The formation of AgNPs indicated that certain reducing agent were present in the supernatant released by the tested bacteria are actually involved in the reduction of Ag ions to AgNPs. In control group, the reduction of Ag ions did not occur due to the absence of reducing agent produced by bacteria. It is assumed that the Ag ions required NADPHdependent nitrate reductase enzymes for their reduction, which were released by bacteria in their extracellular environment 23 .

UV-visible spectroscopy
The AgNPs were analyzed using UV visible spectrophotometer. The absorption spectrum for AgNPs was measured from 370 nm -970 nm. The absorption peak was observed around 414 nm Fig. 1 . The AgNPs showed broad peaks due to different sizes of the NPs.

Fourier transform-infrared FTIR spectroscopy
The FTIR showed number of bands in the region 500-4000 cm 1

Scanning Electron Microscopy SEM
The size and morphology of AgNPs were examined by SEM analysis. The synthesized NPs were mostly spherical in shape with size ranging from 30-46 nm in a scale bar of 0.5 μm Fig. 3 . There were certain NPs aggregation suggesting that the protein molecules play important role as capping agents for biosynthetic NPs by preventing agglomeration and providing stability to the synthesized NPs.

X-ray diffraction XRD
The XRD pattern of the AgNPs showed unique diffraction peaks at 2θ of about 37, 44.8, 64.9, and 77 which indicated the presence of 111, 200, 220 and 311 orientations, respectively Fig. 4 . The sharp peaks of AgNPs appeared as a result of capping agents, which stabilize the NPs. The

Antibacterial activity of AgNPs
Bacillus licheniformis synthesized AgNPs showed significant antibacterial activity against human pathogenic strains such as P. aeruginosa and B. subtilis. DMSO was used as negative control during antibacterial test. AgNPs showed highest zone of inhibition against B. subtilis. The maximum ZOI 20 mm was observed against B. subtilis. The negative control DMSO was unable to inhibit the growth of any test strain Fig. 5 . One-way ANOVA showed that all the AgNPs activity was significant as compared to control.

MIC and MBC Determination
The Maximum MIC value 6.6 μg/mL was noted against P. aeruginosa strain while lowest value was 4.3 μg/mL against B. subtilis Fig. 6 . The MBC values were calculated following spreading. The maximum MBC value was 8.3 μg/mL against P. aeruginosa while minimum value was 6.6 μg/mL against B. subtilis Fig. 6 .

Discussion
Biogenically synthesized AgNPs have been proved as effective and valuable compounds against pathogenic bacteria. They also have excellent antimicrobial and antiviral activity 24,25 . Although, a lot of methods are available which can be used for the synthesis of AgNPs. Much of these techniques are of chemical nature compared to biogenic or green synthesis of AgNPs, a more eco-friendly approach 25,26 and non toxic 3 . The bacterial synthesis of AgNPs seems more budget friendly as compared to chemical synthesis.
In this research work, the bacterial strain B. licheniformis MN900686 was used for the biogenic synthesis of AgNPs. The color of solution was turned brown from yellow, which confirmed the formation of AgNPs. Zhang et al. 26 described that the change of color indicates the ability of supernatant to form AgNPs. Previously, Iravani et al. 27 also reported that the formation of AgNPs changes the color to brown. The more saturated brown color indicates the more AgNPs in the solution 28 . The change in color was due to the excitation of the surface plasmon vibrations in metal NPs 29 .
Following visual observation, the solution was further analyzed by UV visible spectrophotometer. UV-visible graph showed a peak at 414 nm which confirmed the formation of spherical-shaped AgNPs. The peak at aforementioned wavelength also confirmed the size ranging less than 100 nm 30 . The FTIR of the sample was performed to check the involvement of biological molecules. The bands present at 3550, 3062, 2945, 2358, 1337 and 1391 cm 1 were corresponded to the stretching vibrations of alcohol O-H , primary amines N-H , alkane C-H , amine C-N and carbonyl C O groups, respectively 31 . The SEM revealed that the AgNPs are of spherical shape and were around 38 nm in size. It is established fact that the things with smaller size have more surface area, hence making them more effective for in-depth potential. The formation of various sharp peeks using XRD, indicated the presence of different molecules which are involved in the stabilization of the AgNPs. Similar results were presented by Kalyanasundaram et al. 32 .
The agar well diffusion method was used to check the antibacterial efficacy of AgNPs against P. aeruginosa and B. subtilis. AgNPs tested against P. aeruginosa at a different concentration of 10, 15, 20, and 25 μg/mL revealed the mean zone of inhibition as 0, 8, 9, and 10 mm in diameters, respectively. Similarly, mean zone of inhibition against B. subtilis were recorded as 14, 16, 18, and 20 mm at 10, 15, 20, and 25 μg/mL concentrations, respectively. The antimicrobial activity was found to be maximum for B. subtilis and moderate for P. aeruginosa. The mean potential as depicted by zones of inhibition of synthesized AgNPs in the screening test determined a less significant effect against growth of Gram-negative bacteria compared to Gram-positive bacteria. This might be due to the structural differenti-  ation of the cell wall compositions of the two groups of bacteria 33,34 . Different studies have established that the metal NPs penetrate through the bacterial cell membrane and disturb its functions 35,36 . Thus, AgNPs possess significant antibactericidal activity against MDR isolates such as P. aeruginosa and B. subtilis. Recently 37 , reported that AgNPs release Ag ions from the surface and these ions are responsible for the bactericidal efficacy of AgNPs. The more Ag ions release will kill more bacteria which results in bigger ZOI. Further, tube dilution method was used for the determination of MIC and MBC. The Maximum MIC value 6.6 μgmL 1 was noted against P. aeruginosa strain while lowest value was 4.3 μg/mL against B. subtilis. The maximum MBC value was 8.3 μg/mL against P. aeruginosa while minimum value was 6.6 μgmL 1 against B. subtilis. Similar, results were reported by Hamouda et al. 38 . Findings of current study showing AgNPs having lowest minimum inhibitory concentration against pathogenic bacteria suggests the broad spectrum nature of their antimicrobial activity 39,40 .

Conclusion
Bacillus licheniformis can be utilize to synthesize bioactive AgNPs efficiently using inexpensive substances in an ecofriendly and nontoxic manner. UV.vis spectrophotometer confirmed the reduction of AgNO 3 to AgNPs through reductase enzyme released by B. licheniformis in supernatant solution. The FTIR data also confirmed the presence of stabilizing agents, which provide stabilization to the AgNPs. The zone of inhibition in antimicrobial screening test indicated that the AgNPs synthesized via this technique have excellent antimicrobial efficacy against above mentioned pathogenic bacteria. It also suggested that the AgNPs synthesized by this process can be used in various medical fields following toxicity evaluation.

Conflict of Interests
On behalf of all authors, the corresponding author states that there is no conflict of interest.

Author Contributions
IL designed and supervised the study. ST performed all the experimental work and drafted the manuscript. SA, MU, AS, AS helped in data analysis. RI and FA revised the manuscript. All authors approved the final version. CC BY 4.0 Attribution 4.0 International . This license allows users to share and adapt an article, even commercially, as long as appropriate credit is given. That is, this license lets others copy, distribute, remix, and build upon the Article, even commercially, provided the original source and Authors are credited.