Bio-synthesis of silver nanoparticles from bacteria Klebsiella pneumonia: Their characterization and antibacterial studies

Epidemics of infectious acute diseases are caused by bacteria that cause various diseases and the increase of antibiotic resistance, which has encouraged drug companies and researchers to search for new antibacterial agents.. the aim of this study, involved the creation, characterization and antibacterial studies of silver nano-particles by using bacterial culture supernatant of bacteria Klebsiella pneumonia the production. We used an eco-friendly extracellular bio-synthetic method for the production of the silver nanoparticles. The biosynthesis SNPs solution were initially categorized by several techniques, the UV-vi. spectrophotometric record absorbance a powerful peak at 432 nm, analysis (FTIR) Fourier Transformer Infrared and (SEM) Scanning Electron Microscope. The SNPs solution showed anti-microbial activity against different types of pathogenic bacteria that used in the present study: Gram negative (Pseudomonas aeruginosa, Escherichia coli), Gram positive: (Staphylococcus aureus, B. cerius).


INTRODUCTION
Nano-technology is high-speed science that related to other sciences like Physics, Chemistry, Biology, Engineering and other sciences (Islam and Miyazaki, 2009). Its deals with Nano-particles that sized 0.1 to 100 nm; also these compounds displayed different properties like electrical conductance, magnetism, chemical reactivity, physical strength , thermal conductivity, chemical stability , and optical effects, from huge compounds due to their small size (Ju-Nam , 2008).The tiny sizes, make it a technology of highly importance in multiple uses (electronic engineering, communications), moreover, this technology has been accommodation in the field of cosmetics,

Fourier Transform Infrared Spectroscopy (FTIR)
Fourier transformed infrared (FTIR) spectrum of the sample was recorded by Fourier transform infrared (Nicolet 6700 FT-IR, Thermo Scientific) spectrophotometer. The FTIR

Scanning electron microscope (SEM)
SEM was used for characterization the morphological and size of nanoparticles in electron microscope unit, Preparation of slides by adding a small drop of suspension of biosynthesis nanoparticles on slides, and left to dry and then analyzed by (SEM), The microscope operated at an accelerated voltage at 5-10 KV and different magnification, low vacuum, a spot size 4 and working distances 5-10mm ( Li et al.,2010).

Effect of some physic-chemical condition
Different parameters were taken, such as supernatant size, silver nitrate concentration, temperature, pH. the experiment was conducted in three replicates and the results have been monitored using the UV-Visible spectrophotometer

Effect of supernatant size
Examined different volumes of bacteria culture supernatant (2 ml, 4 ml, 8 ml) in the mixture solution to obtain the optimum supernatant size of the substrate for SNPs production. the optimum supernatant size was detected on the basis of the characterized by UV Visible spectrophotometer (Hulkoti and Taranath., 2015).

Effect of concentration of silver nitrate
Designed the pilot experiment by examining three different concentrations of silver nitrate (1mM, 2 mM, 4mM) in the reaction solutions to Discovery of the optimal concentration which is the most important factors that make the interaction more economical and effective were the substrate that can be converted into the final product. the optimal concentration change in color and was detected on the basis of the change in color and characterized by UV Visible spectrophotometer ( Vikas et al ., 2015).

Effect of pH
Experiment was designed to investigate the optimal pH for the synthesis of nanoparticle particles, PH (4, 6,7,8.10) was set in plastic screw cap tubes that containing 4 ml. of supernatant that prepared in (3.2.5.3) and(1mM.) of silver nitrate all the tubes were incubated at 25° C for 24 hrs. and 100 rpm. in a shaking incubator, Acetic acid was used to adjust the pH ˂ 7, While NH3 to adjust the pH <7 , the optimum pH was detected based on the color change and characterized by UV Visible spectrophotometer. (Patra and Baek ,2014

Effect of temperature
Temperature is an essential factor affecting on AgNPs production so the pilot experiment was designed to detect the optimum temperature for AgNPs bio-synthesis, tubes were prepared that contained 4 mL of the supernatant at the optimum concentration (1mM) of the substrate at gradient temperatures (30,60, 90°C) in heating mantle until color change until color change the optimal temperature was detected based on the color change and characterized by UV Visible spectrophotometer(Husam and kredy., 2018).

Purification of biosynthesized silver nanoparticles
Silver colloids were centrifuged at (6000 rpm, 25 minutes) after optimization of synthesis conditions , the supernatant was discarded, replaced with de ionized distilled water, and washed three times with millipore filter to remove the residue of supernatant, while the pellet that found in the bottom of the tube is dried at 40°C (18-24 h) and collected dried powder gently and stored for other tests (Gurunathan et al., 2009).

Determination of antimicrobial activity of SNPs :
The SNPs synthesized from Klebsiella pneumonia were tested for antimicrobial activity by welldiffusion method against pathogenic organisms such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Bacillic Ceruis Wells of 6-mm diameter were made on Müller-Hinton agar plates using gel puncture. Each strain was swabbed uniformly onto the individual plates using sterile cotton swabs . 100μl from 150 μg/ml concentration of AgNPs was distributed into one of the wells , and bacteria culture supernatant is placed in the other well as a control and the third end of the petri dish in which placed a loaded nano silver particle was done by depositing every 10 tablets of filter paper in 5 ml of 150 µg/ml SNPs for two-days and incubated at the same degree and measured different levels of inhibition zones.( Sanchooli et al .,2018)

Visual Examination
K. pneumoniae was inoculated in nutrient broth, 4mM of AgNO3 added to the bacteria culture supernatant (BCS), then incubation 24hr in dark condition, the reduction will causes the color changes in mixture of bacterial supernatant from light yellow to brown color ‫ﻭ‬then the color becomes darker by increasing the incubation period‫ﻭ‬ this refers to the ability of SNPs biosynthesis through the bio-reduction of Ag ions and the formation of , while observed in the control sample no change in color that containing bacteria supernatant without AgNO 3 when incubated in the same conditions. Figure (1

UV-Visible spectrometry
UV-Visual spectrometry is the technique which is used in visualization of nano-silver particles changes in the medium, because the ability of SNPs to absorb light in the visible region due to the surface plasmone resonance (SPR) phenomenon, when analyzed at room temperature by UV-Vis spectrophotometer and the absorbance of the sample was read at the wave lengths of 200-800 nm., Results after 24 hours showed the measurement of the visible absorption spectrum of ultraviolet rays different absorption peaks at limited wave lengths and the absorption intensity gradually increased to the highest absorption indicates a continuous decrease in silver nitrate lead to an increase in the concentration of silver nano-particles indicates their formation in the reaction mixture.    Fig (4) : FTIR analysis of the biosynthesized SNPs by K. pneumoniae .

Physic-Chemical conditions specific to the biosynthesized SNPs supernatant Volume
In the present study; three different volumes were used:( 2 ‫4ﻭ‬ and 8 ml) the effect of the bacterial culture supernatant volume on the synthesis of SNP at 4mM of silver nitrate was depended. However‫4ﻭ‬ml of bacterial culture supernatant exhibited maximum SNPs synthesis, this volume ( 4 ml. ) is depended in the first time in this study as a minimum volume that can obtain SNPs from 4mM silver nitrate. This concentration was favorable in this study; because it gave the smallest nanoparticle size that have the optimum ability to penetrate the bacterial envelop and destroy it due to their size, because the smallest particles easily penetrate microorganisms and have greater toxicity effects; however, the larger ones cause less toxicity (Azam et al., 2012). UV spectra results of present study showed a positive correlation between the increasing intensity of surface plasmon absorbance and the volume of bacterial culture supernatant This result agreed with Divya et al ., (2016) . Who noticed that the absorbance increased from 307 to 309nm. when the size of the culture supernatant increased from ( 20-30 ml )for bacteria Escherichia coli.

Temperature
The temperature could play important role in particle formation, shape and size especially for silver nanoparticles‫ﻭ‬therefore‫ﻭ‬in this study, Various temperatures degree were used (30ºC, 60° C ,90 ° C) to reach the optimum forms of nanoparticles. During color change and measurements of absorption by UV radiation with peak at 423 nm the results showed that the optimum temperature for the biological reduction of Ag was 90 ° C. Figure(4-13) , this temperature ( 90° C ) allowed the particles to be created at a faster rate. This result clarify the effect of temperature on the biosynthesis of nanoparticles‫ﻭ‬the reaction solution containing the bacterial supernatant and the silver nitrate was change the color from pale yellow to brown within 24 h. the In present study the synthesis of silver nano-particles is faster under alkaline conditions as compared to acidic ‫ﻭ‬the synthesis of silver nano-particles increased as the pH towards the alkaline area where it reaches the maximum at pH 10‫ﻭ‬the appearance of the level absorption ranges at pH 4 and 6 indicates that there was no synthesis of SNPs due to they damaged by the acidity‫ﻭ‬ Also‫ﻭ‬ in the acidic milieu the biosynthesis take a longer time to change from yellow to dark brown. Moreover‫ﻭ‬ high PH leads to make the nanoparticles to be tend to fusion resulting the aggregation of these particles (Gurunathan et al., 2009).
The highest peak was 425 at pH 10‫ﻭ‬alkaline pH condition facilitated the reduction and stabilizing capacity of nitrate reductase enzyme,catalyzing the synthesis which probably activated and become more alkaline‫ﻭ‬ and this may be the reason for increase synthesis of AgNPs and elevation of absorbance that observed at higher pH values (Prasad et al., 2010).

AgNO3 concentration
In this study, the biological factors and the concentration of silver nitrates have a significant and visible effect on the biological synthesis of SNPs and this is illustrated by visual examination of color alteration from pale yellow to dark brown in the solution and surface plasmon absorbance peaks of SNPs The mixture prepared with 4mM AgNO3 solution showed surface plasmon absorbance peaks at 421nm ,the peak density was increased by increasing the concentration of AgNO3  10 (1,2,4) mM, which indicates a faster rate of bioreduction with increased concentration of AgNO 3 , the biosynthesis of silver nano-particles depends also on the dealings of the other factors (Lee and El.sayed ., 2006). The results of our study show that the optimal concentration for silver bio-reduction was 4mM as as shown, due to the communication between the ability of proteins in the reaction medium and enzymes reductase in addition to the Ag+ concentration to to obtain the equilibrium suitable very important for the biosynthesis silver nano-particles The use of nano-particles is increasing in the current century because they possess specific chemical and physical properties • Mineral nanoparticles are very important because they show good antibacterial properties that appear as the current interest in researchers because of the increased resistance to many microbial strains to anti-microbial agents. (Hiramatsu et al., 2014 ). Silver NPs have been tested in various fields of biological sciences, medicine and the world of drug delivery its conceder very important because they are nontoxic to the human and animal body when used at low and specific concentrations it's have a wide and effective effect as antimicrobials agent causes inhibition of both gram (+) and gram (-) (Willing et al.,2018 ). Three concentrations ‫ﻭ05(‬ 100 150) µg/ml of SNPs were tested against the bacteria in the present study by using well diffusion method; to identify the concentration that reveal broader zone of inhibition, as shown in figure (6). The concentration 150 µg/ml gave broader zone of inhibition‫ﻭ‬ while the concentrations ‫ﻭ05(‬ 100) µg/ml of SNPs are less inhibition zone‫ﻭ‬ this result may regarded to the fact that increase the concentration of SNPs leading to increase the antibacterial efficacy. Also‫ﻭ‬saturated filter paper in silver nanoparticles at 150 µg/ml concentration was used (Yamamoto et al., 2004 ). This study revealed that there is significant increase in the inhibition efficacy (P<0.05), when we used the concentration 150 of SNPs to all bacteria that tested in the present study.