Biological Synthesis and Characterization of Silver Nanoparticles by Bacillus subtilis

Mahammad Rafi Shaik¹*, Dr. A. Pavani², Dr. V. Uma Maheswar Rao³, Dr. V.R.M. Gupta¹

¹Department of Pharmaceutics, Pulla Reddy Institute of Pharmacy, Domadugu, Sangareddy, Hyderabad.

²Department of Pharmaceutics, Sri Venkateswara College of Pharmacy, Madhapur, Hyderabad.

³Department of Pharmacognosy, TRR College of Pharmacy, Meerpet, Hyderabad.

*Corresponding Author Email: rafeeq.prip333@gmail.com

ABSTRACT:

Over the past few decades interest in metallic nanoparticles and their synthesis has greatly increased. This has resulted in the development of numerous ways of producing metallic nanoparticles using chemical and physical methods. However, drawbacks such as the involvement of toxic chemicals and the high-energy requirements of production make it difficult for them to be widely implemented. An alternative way of synthesizing metallic nanoparticles is by using living organisms such as bacteria, fungi and plants. The attractive possibility of green synthesis nanotechnology is to use micro organisms in the synthesis of nanoparticles. Recently, the utilization of Biological materials especially fungi and bacteria has emerged as a novel method for the development of nanoparticles. Nanoparticles are the starting points for preparing many nanostructured devices and materials. In the research we report he extracellular biosynthesis of silver nanoparticles (AgNPs) by bacteria named bacillus subtilis. In this process the toxic Ag+ ions are reduced to the non-toxic metallic AgNPs through the catalytic effect of the extracellular enzyme and metabolites of the Bacillus subtilis. Absorption UV-Visible spectroscopy is used to follow up with the reaction process. Fourier transform Infrared spectroscopy is used for quantitative analysis of the reaction products. Scanning electron microscopy study indicated that the formation of silver nanoparticles along with circular shape. Particle size analysis shows the average particle size of nanoparticles. Zeta potential indicates the surface charge of the particles and zeta potential is a powerful parameter for predicting the stability of particles. The present research is an excellent candidate for industrial scale production of silver nanoparticles.

KEYWORDS: Extracellular biosynthesis, Nanoparticles, Silver Nitrate, Bacillus subtilis, Zeta Potential.

INTRODUCTION:

Nanoparticles are particles between 1 and 100 nanometers in size. Nanoparticles can be defined as objects ranging in size from 1-100 nm that due to their size may differ from the bulk materials¹. Ultrafine particles are the same as nanoparticles and between 1 and 100 nanometers in size, fine particles are sized between 100 and 2,500 nanometers, and coarse particles cover a range between 2,500 and 10,000 nanometers^2,3.

The term “nano” comes from the Greek word “nanos” meaning dwarf and denotes a measurement on the scale of one-billionth (109) of a meter in size.^{4, 5} A strand of DNA is 2.5 nm in diameter⁶ a typical virus is around 100 nm wide⁷ and a typical bacterium is around 1-3 µm wide⁸. Nanoparticles are defined as particulate dispersions of solid particles with at least one dimension at a size range of 10-1000 nm⁹. The most important feature of nanoparticles is their surface area to volume aspect ratio, allowing them to interact with other particles easier. In order to survive in environments containing high levels of metals, organisms have adapted by evolving mechanisms to cope with them. These mechanisms may involve altering the chemical nature of the toxic metal so that it no longer causes toxicity, resulting in the formation of nanoparticles of the metal concerned. Thus nanoparticle formation is the “by-product” of a resistance mechanism against a specific metal, and this can be used as an alternative way of producing them. Nanoparticles have unique thermal, optical, physical, chemical, magnetic and electrical properties compared to their bulk material counterparts¹⁰. These features can be exploited for next generation biosensors, electronics, catalysts and antimicrobials. Metallic nanoparticles are one important and widely studied group of materials, showing great diversity and many different uses. This research will focus on how material science and biology can work together to create a “green” way of synthesing metal nanoparticles for a wide range of uses. There are important links between the way nanoparticles are synthesized and their potential uses. Silver nanoparticles (AgNPs) have been shown in numerous studies to display antibacterial properties.^11-14

Types of Nanoparticles:

Fig.1: Types of Nanoparticles

Common methodologies for synthesis of metal nanoparticles using microorganisms:

Extracellular Mechanism:

· The test strain (culture) is grown in suitable media and incubated on orbital shaker at 150 rpm at 37°C.

· After incubation the broth is centrifuged and the supernatant is used for synthesis of nanoparticles.

· The supernatant is added to separate reaction vessels containing the metal ions in suitable concentrations and incubated for a period of 72 hrs.

· The color change of the reaction mixture suggests the presence of nanoparticles in the solution, and bio reduction of silver ions in the solution is monitored by sampling the aqueous solution and measuring the absorption spectrum using a UV-Visible spectrophotometer.

· The morphology and uniformity of silver nanoparticles are investigated by X-ray diffraction (XRD) and Scanning electron microscopy (SEM). While the interaction between protein and silver nanoparticles (AgNPs) are analyzed using Fourier transform infrared spectroscopy (FTIR).¹⁵

Intracellular Mechanism:

· The culture is grown in suitable liquid media incubated on shaker at optimal temperature.

· After incubation the flask is kept at static condition to allow the biomass to settle following which the supernatant is discarded and sterile distilled water is added for washing the cells.

· The flask is kept steady for 30 minutes to settle the biomass post which the supernatant is again discarded. This step is repeated for three times.

· The biomass is then separated from the sterile distilled water by centrifugation for 10 minutes.

· The wet biomass is exposed to 50 ml of sterilized aqueous solution of metals at various dilutions and incubated on shaker at suitable temperature till visual color change is observed.

· The change in color from pale yellow to brownish color indicates the formation of silver nanoparticles, pale yellow to pinkish color indicates the formation of gold nanoparticles and the formation of whitish yellow to yellow color indicates the formation of manganese and zinc nanoparticles.¹⁶

A general representation of the synthesis and applications of biogenically synthesized silver nanoparticles using plant extract. Bacterial and fungal synthesis of nanoparticles is practical because bacteria and fungi are easy to handle and can be modified genetically with ease. This provides a means to develop biomolecules that can synthesize AgNPs of varying shapes and sizes in high yield, which is at the forefront of current challenges in nanoparticle synthesis. Fungal strains such as Verticillium and bacterial strains such as K. pneumoniae can be used in the synthesis of silver nanoparticles. When the fungus/bacteria are added to solution, protein biomass is released into the solution.¹⁷ Electron donating residues such as tryptophan and tyrosine reduce silver ions in solution contributed by silver nitrate. These methods have been found to effectively create stable monodisperse nanoparticles without the use of harmful reducing agents. For example, environmentally friendly microorganisms could minimize the toxicity in the process of metallic nanoparticle production by reduction of the metal ions or by formation of insoluble complexes with metal ions in the form of colloidal particles.¹⁸

Intracellular or extracellular synthesis, growth temperature, synthesis time, ease of extraction and percentage synthesized versus percentage removed from sample ratio, all play an important role in biological nanoparticle production. Finding the right biological method can depend upon a number of variables. Most importantly, the type of metal nanoparticle under investigation is of vital consideration, as in general organisms have developed resistance against a small number of metals, potentially limiting the choice of organism. However synthetic biology. A nascent field of science is starting to address these issues in order to create more generalized chassis, able to synthesize more than one type of metallic nanoparticle using the same organism.¹⁹

“Natural” biogenic metallic nanoparticle synthesis can be split into two categories. The first is bioreduction, in which metal ions are chemically reduced into more stable forms biologically. Many organisms have the ability to utilize dissimilatory metal reduction, in which the reduction of a metal ion is coupled with the oxidation of an enzyme.²⁰

This results in stable and inert metallic nanoparticles that can then be safely removed from a contaminated sample. The second category is bioabsorption. This involves the binding of metal ions from an aqueous or soil sample onto the organism itself, such as on the cell wall, and does not require the input of energy. Certain bacteria, fungi and plants express peptides or have a modified cell wall which binds to metal ions, and these are able to form stable complexes in the form of nanoparticles.²¹

BACILLUS SUBTILIS:

Bacillus subtilis is a Gram-positive bacterium, rod-shaped and catalase-positive. It was originally named Vibrio subtilis by Christian Gottfried Ehrenberg²² and renamed Bacillus subtilis by Ferdinand Cohn in 1872.²³ (subtilis being the Latin for 'fine'). B. subtilis cells are typically rod-shaped, and are about 4-10 micrometers (μm) long and 0.25–1.0 μm in diameter, with a cell volume of about 4.6 fL at stationary phase.²⁴ As with other members of the genus Bacillus, it can form an endospore, to survive extreme environmental conditions of temperature and desiccation.²⁵ B. subtilis is a facultative anaerobe²⁶ and had been considered as an obligate aerobe until 1998.

MATERIAL AND METHODS:

Chemicals:

Bacillus subtilis pure culture strain was collected from Dept. of Biotechnology, Malla Reddy College of Pharmacy, Hyderabad. Deionized water was procured from AMS Enterprises, Hyd. Silver Nitrate extra pure, Peptone Bacteriological powder, Sodium chloride and disodium hydrogen ortho phosphate were procured from SDFCL, Mumbai, manganous chloride tetrahyderate obtained from Finar limited, Ahmedabad, Ferric chloride and potassium dihyderogen ortho phosphate were procured from Accord Labs, Secunderabad and Beef extract powder (Type-I) was obtained from Titan Biotech limited, Rajasthan. All other chemicals and reagents used were analytical grade.

Instruments:

Incubator (Kshitij Innovation), Centrifuge (Remi RM-12C), Hot air oven (Lab Care-Mumbai), Analytical Precision Balance (Citizon Scales Pvt Ltd-Mumbai), Ultra sonicator Bath (YJ5200DT-Citizon Scales Pvt Ltd-Mumbai).

Production of Nutrient Broth Medium:

For 100 ml of Nutrient Broth medium: 0.5 ml Metal mix, 0.05 ml Fe-solution and 5ml of phosphate buffer were mixed and made up to 100ml and autoclaved.

In a typical biosynthesis production scheme of silver nanoparticles, firstly Nutrient Broth medium (NB Medium) was prepared, Sterilized and inoculated with a freshly grown inoculum of the strains of Bacillus subtilis. The culture flasks were incubated for 24hr at 35⁰c. After incubation, the cultures were centrifuged at 6000 rpm and the Supernatant was collected.

Identification test for pure culture of Bacillus subtilis:

Certain identification tests were performed too confirm that the supernatants containing bacteria or not. For this identification Gram staining and zone of inhibition were performed.

Gram’s staining:

Gram staining is one of the most fundamental and widely used techniques for the differentiation and identification of bacteria. This differential staining technique was discovered by Dr Christian gram in 1884. It not only reveals the size and shape of bacteria but is also used to differentiate bacteria in to gram- +ve and gram -ve cells. Hence, it is known as differential staining.

In this staining, the cells are stained with basic stain (crystal violet) and treated with an iodine- kI mixture to fix the stain. Then, the stain is washed with alcohol or acetone (decolorizer) and counter stained with a polar dye of a different color (eg: saffarin). All bacteria take up the initial violet stain but only gram +ve cells retain it during the subsequent steps (Bacillus subtilis).²⁷

Zone of inhibition:

Different concentrations of antibiotic solution from stock solution of known concentration of antibiotic (ampicillin) was prepared and then NB medium was sterilized using autoclave pour in to Petri plates and allow for solidifying then 0.1 ml of inoculum is added and spread uniformly.²⁸

Biosynthesis of Silver Nanoparticles:

Aq.AgNO3 (10^-3M) was separately added to vessels containing different concentrations of supernatants and resulting mixture was allowed to stand for 48 hrs at room temperature for color change.²⁹In this process silver nanoparticles were produced through reduction of silver ions to metallic silver. After 48 hrs centrifuge to 6000 rpm for 10 mins to get silver nanopaticles. Pellet was collected and dried in hot air oven at 37⁰C for 10 mins.

CHARACTERIZATION OF NANOPARTICLES:

Reduction of silver ions and formation of was justified by measuring the UV Visible spectra, Silver nitrate and SNP05 characteristic peak of nanoparticle in the visible range of 380-500 nm was not shown by Pure Silver nitrate solution whereas SNP05 will show a characteristic peak at 440nm indicating formation of AgNPs. Similarly a strong absorption peak was obtained in the range of 420-430nm indicating the formation of AgNPs.

Fourier transform infrared spectroscopy (FTIR) gives data of proteins and other compounds present in the mixture that interact with metal ions. The identification of functional groups leads to determine the reducing agent and the capping agent responsible for synthesis and stability of nanoparticles. It was run in the diffuse reflectance mode at a resolution of 4 cm^-1.

The scanning electron microscope (SEM) images the sample surface by scanning it with a high energy beam of electrons. When the beam of electrons strikes the surface of the specimen and interacts with atoms of sample, signals in form of secondary electrons, back scattered electrons and characteristic X-rays are generated that contain information about sample’s surface topography, composition etc.

Zeta Potential analysis is a technique for determining the surface charge of nanoparticles in solution (colloids). Nanoparticles have a surface charge that attracts a thin layer of ions of opposite charge to the nanoparticle surface. This double layer of ions travels with the nanoparticle as it diffuses throughout the solution. The electric potential at the boundary of the double layer is known as the Zeta potential of the particles and has values that typically range from +100 mV to -100 mV. The magnitude of the zeta potential is predictive of the colloidal stability. Nanoparticles with Zeta Potential values greater than +25 mV or less than -25 mV typically have high degrees of stability. Dispersions with a low zeta potential value will eventually aggregate due to Van Der Waal inter-particle attractions. Zeta Potential is an important tool for understanding the state of the nanoparticle surface and predicting the long term stability of the nanoparticle.

RESULTS AND DISCUSSION:

Extra vascular synthesis of silver nanoparticles: In NB Medium Bacillus subtilis was grown by using spread plate method. In this growth can be observed by turbidity and then centrifuged to collect the supernatant.

Fig.3: Collection of Supernatant

Gram staining:

Gram staining was performed to identify the collected supernatants are contain of Bacillus subtilis, in this was performed by the normal gram staining procedure and identified the rod shape structure that confirms the supernatants contained of Bacillus subtilis and used for the synthesis of silver nanoparticles.


Fig.4: Result of gram staining test for collected supernatant	Fig.5: Zone of Inhibition

Zone of inhibition:

Place the filter disc which was dipped in antibiotic solution on surface of NB medium in the respective Petri plates for 18-24 hrs, after inoculation the zone of inhibition was observed.

FORMULATION TABLE:

Table No: 1 Formulation table for the biosynthesis of silver nanoparticles

Formulation Code	Supernatant (ml)	AgNO₃ solution (ml)
SNP01	0.25	5
SNP02	0.5	5
SNP03	0.75	5
SNP04	1	5
SNP05	2	5
SNP06	3	5

Reduction of silver ions and formation of Silver Nanoparticles was justified by measuring the UV-Visible spectra of pure Silver nitrate solution and best formulation of SNP05. Characteristic peak of nanoparticle in the visible range of 380-500nm was not shown by pure silver nitrate solution whereas SNP05 showed a characteristic peak at 440nm indicating formation of AgNPs. Similarly a strong absorption peak was obtained in the range of 420-430nm indicating the formation of AgNPs.


Extracellular filtrate of the Bacillus subtilis	*Extracellular filtrate of the B. subtilis* after exposure to AgNO3 solution for 48hrs**

Fig.6: Biosynthesis of Silver Nanoparticles-color change reaction UV-Visible Spectroscopy:

Fig.7 (a): UV-Visible graph of Pure Silver

Fig.7(b): UV-Visible graph of SNP05

Fourier Transform Infrared Spectroscopy (FTIR):

FTIR analysis can give not only qualitative (identification) analysis of materials, but, with relevant standards, can be used for quantitative (amount) analysis. FTIR can be used to analyze samples up to ~11 millimetres in diameter and either measure in bulk or the top ~1 micrometer layer. Following are the FTIR graphs of Pure Silver nitrate and best formulation SNP05.

Scanning Electron Microscope (SEM):

It was shown that relatively spherical and uniform AgNPs were formed. The SEM image of silver nanoparticles was due to interactions of hydrogen bond and electrostatic interactions between the bioorganic capping molecules bound to the AgNPs. The nanoparticles were not in direct contact even within the aggregates, indicating stabilization of the nanoparticles by a capping agent. The larger silver particles may be due to the aggregation of the smaller ones, due to the SEM measurements.

Zeta Potential:

Fig.10: Scanning Electron Microscope images of Silver Nanoparticle-SNP05

Nanoparticles with Zeta Potential values greater than +25 mV or less than -25 mV typically have high degrees of stability. Dispersions with a low zeta potential value will eventually aggregate due to Van Der Waal inter-particle attractions.

Zeta Potential is an important tool for understanding the state of the nanoparticle surface and predicting the long term stability of the nanoparticle. Following results of Zeta potential reveals that Silver Nanoparticle with formulation code SNP05 shows the Zeta Potential value of -39 mV which means the prepared Nanoparticles having good number of Stability.

Fig.11: Zeta Potential of silver nanoparticle-SNP05

Fig.12: Particle size analysis of silver nanoparticle-SNP05

Particle Size Analysis:

The particle size and size distribution of nanoparticles can be determined using numerous commercially available instruments. Instruments can be used for the analysis of dry powders and powders dispersed in suspension. In general, there are two basic methods of defining particle size. The first method is to inspect the particles and make actual measurements of their dimensions. Malvern Particle size analyzer is used for analysis of Particle size and shows Z-average is 174.4 d. nm which confirms that prepared Nanoparticles having acceptable particle size.

CONCLUSION:

In this research, we have shown the use of Bacillus subtilis in the biosynthesis of extracellular Silver nanoparticles. The appearance of a light brown color in solution suggested the formation of silver nanoparticles. Thus, it was evident that the metabolites excreted by the culture exposed to silver could reduce silver ions, clearly indicating that the reduction of the ions occurs extrcellularly through reducing agents released in to the solution by Bacillus subtilis. These reactions only occurred in the light and the nanopaticles were not produced in darkness.

Culture medium was prepared sterilized and inoculated with fresh culture and incubated for 24 hrs at 37⁰C. After incubation time the culture was centrifuged at 6000 rpm and the supernatant was collected. Identification tests were done for the supernatant for the confirmation of organism present in supernatant by grams staining and Zone of inhibition. In gram staining it shows rod shape structure confirmed the presence of Bacillus subtilis. Zone of inhibition is carried out by antibiotic solution of Ampicillin and shows very good inhibition of growth by this it is confirmed that he presence of Bacillus subtilis in supernatant. This supernatant was collected and used for the biosynthesis of silver nanoparticles.

The silver nanoparticles were characterized by UV-Visible spectroscopy. This technique has proved to be a very useful technique for the analysis of nanoparticles. In figure no.7 strong but broad surface Plasmon peak located at 440 nm was observed for the silver nanoparticles prepared with Bacillus subtilis. By the FTIR spectrum revealed that the presence of silver nanoparticles synthesized from Bacillus subtilis. Figure no.10 shows the SEM micrograph recorded from the silver nanoparticle. In this micrograph silver nanopartcles in the size range of 50-100nm were observed.

Zeta Potential is an important tool for understanding the state of the nanoparticle surface and predicting the long term stability of the nanoparticle. Following results of Zeta potential reveals that Silver Nanoparticle with formulation code SNP05 shows the Zeta Potential of -39 mV this value provide full stabilization of the nanoparticles, which may be the main reason in producing particle sizes with narrow size distribution index by particle size analysis with an average size of 174.4 d.nm.

However, development of simple and eco-friendly synthetic route would help promoting further interest in the synthesis and application of metallic nanoparticles. In this respect, nature has provided exciting possibilities of utilizing biological systems for this purpose. This comes from the fact that microorganisms while interacting with metal ions have shown to reduce the ions into metallic nanoparticles. Thus, Bacillus subtilis have shown ability to reduce metal ions to form metallic nanoparticles.

ACKNOWLEDGEMENT:

Authors wish to thank management and Principal of Pulla Reddy Institute of Pharmacy, Hyderabad for providing necessary facilities for the completion of this work. We also acknowledge our gratitude to Dr. Ashwaq Hussain and Dr. Yasmin Begum for continuous support.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 28.06.2017 Modified on 21.07.2017

Research J. Pharm. and Tech. 2017; 10(7): 2367-2374.

DOI: 10.5958/0974-360X.2017.00419.X