Nanotechnology offers the capability to engineer the properties of materials by controlling their size, and this has determined research toward a mass of possible uses for nanomaterials. Metallic nanoparticles exhibition of uncommon optical, thermal, chemical, and physical properties. The reduction of materials’ dimension has noticeable effects on the physical properties that may be suggestively different from the consistent bulk material. Some of the physical properties exhibited by nanomaterials are due to (i) large surface atom, (ii) large surface energy, (iii) spatial confinement, and (iv) reduced imperfections. One main aspect of nanotechnology concerns the development of rapid and reliable experimental protocols for the synthesis of nanomaterials over a range of chemical compositions, sizes, high monodispersity and large scale manufacture. A variation of techniques have been advanced to synthesize metal nanoparticles, including chemical reduction using a number of chemical reductants including NaBH4, N2H4, NH2OH, ethanol, ethylene glycol and N,N-dimethyformamide (DMF), aerosol technique, electrochemical or sonochemical deposition, photochemical reduction, and laser irradiation technique. A lot of interest has been created by the term “green nanotechnology”. In a wide-ranging sense, this term includes a wide range of possible applications, from nanotechnology-enabled, environmentally friendly manufacturing processes that reduce waste products (ultimately leading to atomically precise molecular manufacturing with zero waste); the use of nanomaterials as catalysts for greater efficiency in current manufacturing processes by minimizing or eliminating the use of toxic materials (green chemistry principles); the use of nanomaterials and nanodevices to reduce pollution (e.g. water and air filters); and the use of nanomaterials for more efficient alternative energy production (e.g. solar and fuel cells). With the flourishing demand of “green” nanoparticle synthesis processes, the field of nanoparticle synthesis has recently developed new routes. These include employing microorganisms, such as: Pseudomonas stutzeri, Verticilium sp, Fusarium oxysporum, Thermomonospora sp and, also, alfalfa and geranium plants. Biosynthetic methods employing either biological microorganisms or plant extracts have emerged as a simple and viable alternative to chemical synthetic procedures and physical methods. Although it is known that microorganisms such as bacteria, yeast and now fungi play an important role in remediation of toxic metals through reduction of the metal ions, this was considered interesting as nanofactories very recently. The presence of hydrogenase in the F. oxysporium broth was demonstrated. Some bacteria reduce Fe3+ oxides by producing and secreting small, diffusible redox compounds that can serve as electron shuttle between the microbe and the insoluble iron substrate.This extracellular enzyme shows an excellent redox properties and it can act as an electron shuttle in the metal reduction. It was evident that electron shuttles or other reducing agents (e.g. hydroquinones) released by microorganisms are capable of reducing ions to nanoparticles. However, biological nanoparticles synthesis generates nanoparticles at a much slower rate. In the studies synthesis of silver nanoparticles using the cell mass of bacteria and fungi or their leached cell components, the time required completing the reaction ranged from 24 to 120 h; this lengthy reaction is the one major drawback of the biological synthesis. Biological synthesis would have greater commercial viability if the nanoparticles could be synthesis more rapidly and in the large production scale. Despite stability and a green method of production, particles are not monodispersible and the rate at which they form is not comparable to chemical synthesis methods. Thermal factors have been demonstrated to affect the size and uniformity of nanoparticles. Controlling particle growth rate is possible through knowledge of a material’s properties and controlling of the reaction by changes to the time and temperature. The ability of any material to engage microwave energy is expressed by its dielectric loss factor combined with the dielectric constant. The microwave radiation heats up a material through its dielectric loss, which converts the radiation energy into thermal energy. The main challenges frequently encountered by the researchers of nanoparticle synthesis are (i) controlling the particle size/shape and (ii) achieving the monodispersity. The synthesis of nanoparticles with controlled monodispersity is a recent demand by the material developers for the advancement of nanotechnology. (Saifuddin et al., 2009)
In recent years the subject of nanoparticles has established certain concern in a widespread range of fields. The term “nano” comes from the Greek word “nanos” meaning dwarf and signifies a measurement on the scale of one-billionth (109) of a metre in size. A strand of DNA is 2.5 nm in diameter, a typical virus is around 100 nm wide and a typical bacterium is around 1-3 µm wide. Nanoparticles can be well-defined as particulate dispersions of solid particles with at least one dimension at a size range of 10-1000 nm. The most significant characteristic of nanoparticles is their surface area to volume aspect ratio, allowing them to interact with other particles easier. In direction to endure in environments comprising high levels of metals, organisms have altered by developing mechanisms to cope with them. These mechanisms may include altering the chemical nature of the toxic metal so that it no longer causes toxicity, causing in the development of nanoparticles of the metal concerned. Thus nanoparticle development is the “by-product” of a resistance mechanism against a specific metal, and this can be used as another way of generating them. Nanoparticles have exclusive thermal, optical, physical, chemical, magnetic and electrical properties compared to their bulk material counterparts. These features can be exploited for next generation biosensors, electronics, catalysts and antimicrobials. Metallic nanoparticles are one significant and extensively studied group of materials, presenting unlimited diversity and many different uses. This analysis will focus on how material science and biology can work together to create a “green” way of synthesizing metal nanoparticles for a varied range of uses. (Pantidos and Horsfall, 2014)
Silver nanoparticles (Ag-NPs or nanosilver) have attracted increasing interest due to their unique physical, chemical and biological properties compared to their macro-scaled counterparts. Ag-NPs have distinctive physico-chemical properties, including a high electrical and thermal conductivity, surface-enhanced Raman scattering, chemical stability, catalytic activity and nonlinear optical behavior. These properties make them of potential value in inks, microelectronics, and medical imaging. Besides, Ag-NPs exhibit broad spectrum bactericidal and fungicidal activity that has made them extremely popular in a diverse range of consumer products, including plastics, soaps, pastes, food and textiles, increasing their market value. To date, nanosilver technologies have appeared in a variety of manufacturing processes and end products. Nanosilver can be used in a liquid form, such as a colloid (coating and spray) or contained within a shampoo (liquid) and can also appear embedded in a solid such as a polymer master batch or be suspended in a bar of soap (solid). Nanosilver can also be utilized either in the textile industry by incorporating it into the fiber (spun) or employed in filtration membranes of water purification systems. In many of these applications, the technological idea is to store silver ions and incorporate a time-release mechanism. This usually involves some form of moisture layer that the silver ions are transported through to create a long-term protective barrier against bacterial/fungal pathogens.
There are many consumer products and applications utilizing nanosilver in consumer products; nanosilver-related applications currently have the highest degree of commercialization. A wide range of nanosilver applications has emerged in consumer products ranging from disinfecting medical devices and home appliances to water treatments. According to the Project on Emerging Nanotechnologies over 1300 manufacturer-identified, nanotechnology-enabled products have entered the commercial market place around the world. Among them, there are 313 products utilizing nanosilver (24% of products listed), this has made nanosilver the largest and fastest growing class of NPs in consumer products applications. According to the report of silver nanotechnology commercial inventory published in 2008, the health and fitness markets were found to be the biggest emergence of products utilizing nanosilver (131 records) compared to other categories such as appliances, medical applications, and electronics and computers. The worldwide market incorporating nanotechnology continues to grow on a rapid and consistent basis. With the world annual rate of increase ∼25%, the commercial nanotechnology industry value is predicted to increase significantly from $91 billion by 2009 to $1 trillion by 2015 and $3 trillion by 2020. Despite the historic use of nanosilver in health-related fields, however, the future prospect of silver nanotechnologies applications for other product fields, i.e., for environmental disinfections remains to be unexploited. Because of their widespread applications, the scientific community and industry has paid special attention to the research topic of Ag-NPs. (Tran et al., 2012)
In this study microbes are utilized for their unique capability to precipitate silver nanoparticles in the growth medium. It is thought the certain microbes may possess certain enzymes or any metabolic components which may cause separation and precipitation of silver nanoparticles from a silver compound AgNO3. We have reported the efficiency of bacterial strains to synthesize silver nanoparticles. Two different concentrations, 10mM and 15mM, of AgNO3 is used for the study.
1. MATERIALS AND METHODOLOGY
1.1 Materials used : bacterial isolates
1.1.1 Isolation of bacterial: Samples of different types of bacteria were taken from the soil and isolated using basic sub culturing technique.
1.1.2 Broth preparation
Nutrient Broth
• Peptone 0.5gm
• Yeast/Beef 0.5gm
• NaCl 0.5gm
• Distilled water 100ml
3.1.3 Silver Nitrate Stock
• Silver Nitrate 1mM
• Distilled Water 10ml
3.1.4 Gram staining
• Crystal violet
• Iodine
• Alcohol
• Saffranin
• Slide
• Water
• Sprit lamp
• Inoculation loop
• Microscope
3.2 Methodology
3.2.1 Sample collection
• Samples of different types of bacteria were taken from the soil and isolated using basic sub culturing technique for their comparison of characteristics and morphology.
3.2.2 Broth preparation
• The Nutrient Broth is used widely for the cultivation of bacteria. Nutrient Broth can be used as a pre-enrichment medium and may also be used in accordance with many standard methods procedures for testing food, milk and dairy products.
Nutrient Broth is a liquid medium that contains peptone and beef extract. Nutrients necessary for the replication and growth of a large number of nonfastidious microorganisms are provided by this simple formulation. Water soluble substances including carbohydrates, vitamins, organic nitrogen compounds and salts are present in beef extract.
Protocol
• A clean flask was taken and required amount of peptone, yeast extract and NaCl were added.
• Added distilled water such that the total volume of suspension was 200ml.
• Then sterilized the medium by autoclaving at 15 pounds per square inch (100kPa) for 15 minutes.
• After the media was sterilized, it was divided into 6 autoclaved flasks of volume 25ml.
3.2.3 Silver Nitrate Stock
• Silver nitrate stock solution of a particular concentration is used for the production of silver nanoparticles in the prepared bacterial medium.
Protocol
• Autoclaved the distilled water and tarson tube to be used.
• Silver nitrate was measured to make a stock solution of 1M.
• Syringed filtered distilled water was added to make 10ml solution.
• The tube was covered using aluminium foil and kept at low temperature.
3.2.4 Analysis of Silver precipitation
Principle
Bacteria have the capability to precipitate silver nanoparticles in the provided growth medium. Therefore, two different concentrations of silver nitrate were used.
Protocol
• The prepared broth medium were inoculated with 3 different bacterial isolates and kept in the incubator at 35°C for the growth to occur.
• After approximately 48 hours, the bacterial medium were added with silver nitrate stock solution with different concentrations.
• For 10mM concentration, 0.25ml silver nitrate solution was added in the medium.
• For 15mM concentration, 0.375ml silver nitrate solution was added in the medium.
• All the flasks containing respective concentrations of silver nitrate and bacterial strains were kept in the incubator at 35°C until precipitation was observed.
Colour change-
• The change in colour of the silver nitrate stock treated bacterial medium was observed.
• Photographs were taken at regular interval of time.
• After the characterization on the basis of colour change, the solution was again covered and kept in incubator for further observation.
• When compared with the control, dark grey coloured precipitate was observed.
3.2.5 Characterization of Nanoparticles
Method for Spectra analysis-
• The following method was used in our experiment: Visible and UV-Visible Spectroscopy.
• In this, spectrophotometer was turned on and adjusted to the wavelength of 560nm.
• The sample around 2ml was placed in cuvette which was cleaned and washed properly.
• After 2 minutes, the absorbance was read.
• After the completion, the lamps were switched off, the sample was disposed and the area was cleaned properly.
3.2.6 Identification of Bacterial isolates
3.2.6.1 On the basis of morphology
Principle
Gram’s staining technique which is used to differentiate bacteria of the basis of their cell wall composition .The bacterial which appeared purple in colour are called as Gram’s positive while the bacteria which appear pink in colour are Gram’s negative in nature .Gram-positive cells have a thick peptidoglycan cell wall that is able to retain the crystal violet- iodine complex that occurs during staining, while Gram –negative cells have only a thin layer of peptidoglycan. Thus Gram –positive cells do decolorize with ethanol, Gram –negative cells do decolorize, this allows the Gram- negative cells to accept the counter stain safranin. Gram- positive cells will appear blue to purple, while gram- negative cells will appear pink to red.
Procedure
• Prepared the smear and heat-fix smear.
• Stained the slide by flooding it with crystal violet for 1 min.
• Poured off excess dye and washed gently in tap water.
• Added Gram’s iodine for one min. for 60 second by washing with iodine then added and left it on the smear until the min. is over.
• Washed with tap water and drained.
• Washed with 95% alcohol drop by drop until no more colour flows from the smear.
• Washed the slides with distilled water and drained.
• Counter stained with safranine for 1 min.
• Washed, drained, blotted and examined under immersion oil at 100x objective.
3.2.7 Instruments Used
3.2.7.1 Electronic Balance
Fig 1. Electronic balance
An electronic balance (model no. LCB4A) was used to weigh sweet potato flour all the ingredients required for preparation of biscuits. The maximum limit of the balance was 300g.
3.2.7.2 Incubator
Fig 2. Incubator
An incubator is a device used to grow and maintain microbiological cultures or cell cultures. The incubator maintains optimal temperature humidity and other conditions such as the carbon dioxide (CO2) and oxygen content of the atmosphere inside.
3.2.7.3 Autoclave
Fig 3. Autoclave
An autoclave is a pressure chamber used to sterilize equipment and supplies by subjecting them to high pressure saturated steam at 121 °C (249°F) for around 15–20 minutes depending on the size of the load and the contents.
3.2.7.4 Laminar air flow
Fig 4. Laminar air flow
A laminar flow/ closet or tissue culture hood is a carefully enclosed bench designed to prevent contamination of semiconductor wafers, biological samples, or any particle sensitive materials. Air is drawn through a HEPA filter and blown in a very smooth, laminar flow towards the user.
3.2.7.5 Spectrophotometer
Fig 5. Spectrophotometer
A spectrophotometer is commonly used for the measurement of transmittance or reflectance of solutions, transparent or opaque solids, such as polished glass, or gases. However they can also be designed to measure the diffusivity on any of the listed light ranges that usually cover around 200nm – 2500nm using different controls and calibrations. Particle surface and the surrounding fluid.
2. RESULTS AND DISCUSSION
4.1 Sample collection
• Samples of different types of bacteria were taken from the soil and isolated using basic sub culturing technique for their comparison of characteristics and morphology.
4.2 Broth preparation
• The Nutrient Broth is used widely for the cultivation of bacteria. Nutrient Broth can be used as a pre-enrichment medium and may also be used in accordance with many standard methods procedures for testing food, milk and dairy products.
Nutrient Broth is a liquid medium that contains peptone and beef extract. Nutrients necessary for the replication and growth of a large number of nonfastidious microorganisms are provided by this simple formulation. Water soluble substances including carbohydrates, vitamins, organic nitrogen compounds and salts are present in beef extract.
4.3 Silver Nitrate Stock
• Silver nitrate stock solution of a particular concentration is used for the production of silver nanoparticles in the prepared bacterial medium.
4.4 Analysis of Silver precipitation
• Bacteria have the capability to precipitate silver nanoparticles in the provided growth medium. Therefore, two different concentrations of silver nitrate were used and the changes were observed
Colour change-
• The change in colour of the silver nitrate stock treated bacterial medium was observed.
• For 10mM concentration of Silver nitrate inoculation, the color change was observed from pale yellow to grey.
• For 15mM concentration of Silver nitrate inoculation, the color change was observed from pale yellow to dark grey.
Figure 6. 10mM AgNO3 inoculate in bacterial isolates medium
Figure7. 15mM AgNO3 inoculate in bacterial isolates medium
4.5 Characterization of Nanoparticles
Method for Spectra analysis-
• The effect of silver nitrate on the bacterial medium was observed by taking the absorbance of the medium at 560nm at the time interval of 5- 6 days for both the concentrations.
• On 5th day, for 10mM concentration-
Table 1: Absorbance for 10mM conc. Of AgNO3 on 5th day of inoculation
S.NO. SAMPLE NAME ABSORBANCE (560nm)
1. CD13 3.125
2. CD3 3.529
3. C6 3.310
Graph 1: Absorbance for 10mM conc. Of AgNO3 on 5th day of inoculation
• On 6th day, for 10mM concentration-
Table 2: Absorbance for 10mM conc. Of AgNO3 on 6th day of inoculation
S.NO. SAMPLE NAME ABSORBANCE (560nm)
1. CD13 4.000
2. CD3 4.000
3. C6 3.321
Graph 2: Absorbance for 10mM conc. Of AgNO3 on 6th day of inoculation
• On 5th day, for 15mM concentration-
Table 3: Absorbance for 15mM conc. Of AgNO3 on 5th day of inoculation
S.NO. SAMPLE NAME ABSORBANCE (560nm)
1. CD13 3.862
2. CD3 4.000
3. C6 4.000
Graph 3: Absorbance for 15mM conc. Of AgNO3 on 5th day of inoculation
• On 6th day, for 15mM concentration-
Table 4: Absorbance for 15mM conc. Of AgNO3 on 6th day of inoculation
S.NO. SAMPLE NAME ABSORBANCE (560nm)
1. CD13 4.000
2. CD3 4.000
3. C6 4.000
Graph 4: Absorbance for 15mM conc. Of AgNO3 on 6th day of inoculation
4.6 Identification of Bacterial isolates
4.6.1 On the basis of morphology
Gram’s staining technique which is used to differentiate bacteria of the basis of their cell wall composition .The bacterial which appeared purple in colour are called as Gram’s positive while the bacteria which appear pink in colour are Gram’s negative in nature .Gram-positive cells have a thick peptidoglycan cell wall that is able to retain the crystal violet- iodine complex that occurs during staining, while Gram –negative cells have only a thin layer of peptidoglycan. Thus Gram –positive cells do decolorize with ethanol, Gram –negative cells do decolorize, this allows the Gram- negative cells to accept the counter stain safranin. Gram- positive cells will appear blue to purple, while gram- negative cells will appear pink to red.
Figure 8. Morphology of first isolate- CD13
Figure 9. Morphology of second isolate- C6
Figure 10. Morphology of third isolate- CD3
3. CONCLUSION
Today modern science is inclining toward the study of nanoparticles, their production and their application in various spheres of life. Nanoparticle technology with its immense projected scope in science is attracting a lot of research. Especially the metal nanoparticles are becoming a favorite topic of discussion among the scientists relating to various fields. Conventional methods of silver nanoparticle production include physical and chemical methods, which are of course costly and also carry some environmental and health hazards. Thus it becomes necessary to find out an alternative to the conventional methods. Biological production of silver nanoparticles is a new approach which is cheaper and least in hazards.
The study has given very positive results with regard to the nanoparticle production. This study has also shown the potential of the bacteria to carry out such job, which is a new approach as compared to the conventional methods. All the three bacteria CD3, CD13 and C6 were able to produce precipitates. Gram positive bacteria CD3 culture gave better results as compared to other two although other cultures also showed significant results. As all the three bacterial strains are non-pathogenic and showing excellent results, so they can be further used in pharmaceutical and cosmetics etc. This preliminary study have shown very optimistic results which can be further used to discover the potential of bacteria new dimensions in the production of nanoparticles.