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Biological Synthesis of Zn, Ag and Au
Nanoparticles
Using Bacteria and Fungi
1
About Nanoparticles
Word ‘Nano’ comes from Greek word ‘nanos’ meaning dwarf. These are tiny
particles with dimensions on the nanometer scale (1-100 nanometers). They
exhibit unique properties due to their small size and high surface area-to-
volume ratio, making them valuable in various applications such as
medicine, electronics, and materials science.
2
Approaches for the synthesis of nanomaterials
• The “top-down” approach, which involves the breaking
down of large pieces of material to generate the
required nanostructures from them.
• Bottom-up approach in nanotechnology is making larger
nanostructures from smaller building blocks such as
atoms and molecules. Self assembly in which desired
nano structures are self assembled without any external
manipulation.
3
Biological Synthesis (Green synthesis)
It is a rapid, ecofriendly, and easily scaled-up technology.
Why Green Synthesis?
• Easy.
• Efficient.
• Eco- Friendly.
• Eliminates use of toxic chemicals.
• Consume less energy.
• Produce safer products and by products.
• Does not impart any hazardous effect on environment.
4
Biological Synthesis of Nanoparticles by Microorganisms
(Bacteria)
• Bacteria has been most extensively researched for synthesis of nanoparticles because of
their fast growth and relative ease of genetic manipulation.
• Intracellular – Inside the cell, in cytoplasm.
• Extracellular – Outside the cell on the surface or between the cells inside a colony.
Methods of synthesis
5
• Mechanism
Uptake of Metal Ions
The cell takes up metal ions,
often from the surrounding
environment, through
transporters or diffusion.
Bio-Reduction
Intracellular reduction processes
occur due to the presence of
enzymes, co-factors, and other
biomolecules. These facilitate the
conversion of metal ions into
nanoparticles.
Nucleation and Growth
Nucleation of nanoparticle
seeds occurs, followed by the
controlled growth of
nanoparticles within the
cellular confines.
6
Microbial production of silver nanoparticles
It was investigated using the bacterial strain Escherichia coli.
Method
The bacterial strain E.coli were cultured, for broth medium and LB
medium. The culture flasks were incubated on an orbital shaker at
27°C and agitated at 220 rpm. The biomass was harvested after 24
hours of growth and centrifuged at 12000 rpm for 10 minutes.
Synthesis of Silver nanoparticles
The sample added separately to the reaction vessel containing
silver nitrate (AgNO3) at specific concentration and control was
also run along with the experimental condition. The primary
conformation of synthesis of nanoparticles in the medium was
characterized by the changes in color from yellowish white to
brown.
Production of biomass
7
Microbial production of silver nanoparticles
Characterization of silver nanoparticles
The bioreduction of the Ag+ ions in the solution was
monitored and sample of 2ml was withdrawn at
different time intervals and the absorbance was
measured using UV–visible spectrophotometer with
samples in quartz cuvette.
Particle sizing measurements
Particle size analyzing experiments were carried out
by means of laser diffractometry. Measurements
were taken in the range between 0.04 up to 500 μm.
8
Microbial production of Zn nanoparticles
• ZnO nanoparticles are produced intracellular by bacteria such as Lactobacillus.
Methods
Zn Solution preparation
1 M stock solution for Zn2+ is prepared. Then
solution Zn2+ was filtered into the bacterial culture
medium and added.
Isolation of lactic acid bacteria
Using (MRS) medium, lactic acid bacteria was isolated from Cow
milk and incubated. The streak plate technique was used to
obtain single colonies with various morphologies.
9
Microbial production of Zn nanoparticles
Tolerance determination
Determine the NaCl tolerance and bile
salt tolerance of isolated lactobacillus.
Biosynthesis of ZnO nanoparticles
The cell biomass then suspended in sterilized deionized water containing
Zn2+ at concentrations and incubated. The cells were collected at 5000
rpm for 10 minutes by centrifugation and washed with saline before being
hung in a buffer. Cells were interrupted by alternating ultrasound cycles in
100 W for 5 minutes to get the ZnO nanoparticles.
10
Microbial production of Zn nanoparticles
11
Extracellular synthesis
• It includes bio-mineralization, bio sorption or precipitation.
• Here cell wall reductive enzymes or soluble secreted enzymes are involved in the
reductive process of metal ions.
• With the change in pH of the solution, various shapes and sizes were formed.
The culture supernatants of Enterobacteria like Klebsiella pneumonia, E.coli, Enterobacter
cloaceae rapidly synthesize silver nanoparticles by reducing Ag+ to Ag.
12
Microbial production of Au nanoparticles
Materials and Reagents:
• Bacterial Culture Preparation: Inoculate a culture flask with the selected bacterial strain
and incubate it overnight at the optimal temperature and pH for growth.
• Growth Medium Adjustment: Prepare a fresh growth medium with the gold precursor
(e.g., HAuCl4). Adjust the pH of the medium to promote the reduction of gold ions.
• Incubation with Gold Precursor: Add the gold-containing medium to the bacterial
culture flask.
Incubate the flask with constant shaking to ensure uniform mixing of the medium and
bacterial culture.
13
Microbial production of Au nanoparticles
Monitoring and Optimization: Periodically sample the solution and measure the changes
in color. The development of a ruby red or purple color is indicative of AuNP formation.
Biomass Separation: Centrifuge the culture to separate the bacterial biomass from the
AuNPs and supernatant.
Characterization: Characterize the synthesized AuNPs using Transmission Electron
Microscopy (TEM), UV-Vis spectroscopy, X-ray Diffraction (XRD), and Dynamic Light
Scattering (DLS) to analyze size, shape, and other properties.
14
Intracellular and Extracellular Synthesis of nanoparticles
Aspect Intracellular Synthesis Extracellular Synthesis
Location of Synthesis Inside the microbial cell Outside the microbial cell
Microbial Synthesis Bacteria, fungi, algae Bacteria and fungi
Key Process Reduction of metal ions within the
cell.
Reduction of metal ions
outside the cell.
Control over size and
shape
Good control, can be precise. Limited control, more
challenging.
Biocompatibility High biocompatibility due to
nanoparticles formed inside cells.
Depends on presence of any
toxic byproducts.
Purification and
collection
Require additional steps for cell lysis
and purification
Easier to separate
nanoparticles from the
extracellular medium.
Applications Drug delivery, catalysis and sensing. Drug delivery, catalysis, sensing
and nanomedicine.
15
Synthesis of nanoparticles by fungi
• Fungi and Yeast are very effective secretors of extracellular enzymes.
16
Potential fungal isolates used for biosynthesis of nanoparticles.
Biogenic synthesis of nanoparticles using
fungi
• Intracellular and Extracellular.
In intracellular synthesis, the metal precursor is
added to the mycelial culture and is internalized
in the biomass. Extraction of the nanoparticles is
required after the synthesis, employing chemical
treatment, centrifugation, and filtration to disrupt
the biomass and release the nanoparticles
In extracellular synthesis, the metal precursor
is added to the aqueous filtrate containing only
the fungal biomolecules, resulting in the
formation of free nanoparticles in the
dispersion.
17
Biosynthesis of Zinc Oxide Nanoparticles using Fungus
a. Fungi like Aspergillus,
Penicillium etc. transferring
onto a fresh agar plate and
allowing it to grow.
b. Once the culture is
established, transfer it to
Erlenmeyer flask. Incubate the
flask with agitation (if using a
shaker) until the fungus
reaches the desired growth
phase.
a. When the fungal culture
has grown, add a known
volume or concentration of
the zinc precursor solution to
the fungal culture medium.
b. Incubate the culture
mixture under appropriate
conditions (temperature, pH,
and agitation if necessary) for
a specified period.
Dissolve the selected
zinc salt (e.g.,
Zn(NO3)2 or ZnSO4) in
deionized water to
prepare a stock
solution of known
concentration.
The formation of Zn nanoparticles is often indicated by a color change from colorless or pale to brown
or reddish-brown.
Use techniques like UV-Visible spectroscopy to measure the absorbance spectrum, which can provide
information about the formation of nanoparticles.
18
19
Biosynthesis of Silver Nanoparticles using Fungus
Isolation and Culturing of
Fungi:
a. Isolate and identify a
suitable
fungal strain. Common strains
used include Aspergillus,
Fusarium, and Trichoderma.
b. Cultivate the selected fungal
strain on a suitable growth
medium under controlled
conditions .Allow the fungus to
grow until it reaches the
desired biomass.
Preparation of Silver
Nitrate Solution:
Prepare a stock
solution of silver nitrate
(AgNO3) by dissolving a
known amount in Milli-
Q water. This solution
will be used as a source
of silver ions (Ag+).
Inoculation and Incubation:
a. Add the fungal culture to a
flask containing a growth
medium.
b. To this flask, add silver nitrate
solution.
c. Adjust the pH of the mixture
(typically pH 7-8).
d. Incubate the mixture in an
incubator at a controlled
temperature and allow the
reaction to proceed. Ag+ ions
will be reduced by the fungal
biomass to form silver
nanoparticles
20
Biosynthesis of Gold Nanoparticles using fungi
Isolate and Culture the Fungus: Isolate a pure culture of the selected fungus.
• Grow the fungus on a suitable culture medium in a sterile environment, following
standard microbiological techniques.
Prepare Fungal Biomass: Allow the fungus to grow until it reaches the logarithmic growth
phase.
• Harvest the fungal biomass by centrifugation and wash it thoroughly to remove any
culture medium components.
Gold Salt Reduction: Prepare a gold salt solution (e.g., HAuCl4) in deionized water.
• Add the fungal biomass to the gold salt solution and incubate the mixture at a controlled
temperature, typically around 25-30°C.
• Over time, the fungal biomass will reduce the gold ions to form AuNPs. The color of the
solution will change from pale yellow to a characteristic red or purple, indicating the
formation of AuNPs
21
Characterization: Measure the absorbance spectrum of the reaction mixture using a
spectrophotometer. AuNPs typically exhibit a strong absorbance peak in the visible range,
around 520-550 nm.
• Analyze the size and shape of the synthesized AuNPs using techniques such as
transmission electron microscopy (TEM) or dynamic light scattering (DLS).
Purification: Centrifuge the reaction mixture to separate the fungal biomass and any
unreacted gold ions from the AuNPs.
22
References
• EL-GHWAS, D. E. (2022). Characterization and biological synthesis of zinc oxide nanoparticles by new strain
of Bacillus foraminis. Biodiversitas Journal of Biological Diversity, 23(1).
• Suba, S., Vijayakumar, S., Vidhya, E., Punitha, V. N., & Nilavukkarasi, M. (2021). Microbial mediated
synthesis of ZnO nanoparticles derived from Lactobacillus spp: Characterizations, antimicrobial and
biocompatibility efficiencies. Sensors International, 2, 100104.
• Moormann, G. C., & Bachand, G. D. (2021). Biosynthesis of Zinc Oxide Nanoparticles using Fungal Filtrates
(No. SAND2021-9437R). Sandia National Laboratories (SNL-NM), Albuquerque, NM (United States). Center
for Integrated Nanotechnologies (CINT).
• Guilger-Casagrande, M., & Lima, R. D. (2019). Synthesis of silver nanoparticles mediated by fungi: a review.
Frontiers in bioengineering and biotechnology, 7, 287.
23
24

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Microbial bio Synthesis of nanoparticles.pptx

  • 1. Biological Synthesis of Zn, Ag and Au Nanoparticles Using Bacteria and Fungi 1
  • 2. About Nanoparticles Word ‘Nano’ comes from Greek word ‘nanos’ meaning dwarf. These are tiny particles with dimensions on the nanometer scale (1-100 nanometers). They exhibit unique properties due to their small size and high surface area-to- volume ratio, making them valuable in various applications such as medicine, electronics, and materials science. 2
  • 3. Approaches for the synthesis of nanomaterials • The “top-down” approach, which involves the breaking down of large pieces of material to generate the required nanostructures from them. • Bottom-up approach in nanotechnology is making larger nanostructures from smaller building blocks such as atoms and molecules. Self assembly in which desired nano structures are self assembled without any external manipulation. 3
  • 4. Biological Synthesis (Green synthesis) It is a rapid, ecofriendly, and easily scaled-up technology. Why Green Synthesis? • Easy. • Efficient. • Eco- Friendly. • Eliminates use of toxic chemicals. • Consume less energy. • Produce safer products and by products. • Does not impart any hazardous effect on environment. 4
  • 5. Biological Synthesis of Nanoparticles by Microorganisms (Bacteria) • Bacteria has been most extensively researched for synthesis of nanoparticles because of their fast growth and relative ease of genetic manipulation. • Intracellular – Inside the cell, in cytoplasm. • Extracellular – Outside the cell on the surface or between the cells inside a colony. Methods of synthesis 5
  • 6. • Mechanism Uptake of Metal Ions The cell takes up metal ions, often from the surrounding environment, through transporters or diffusion. Bio-Reduction Intracellular reduction processes occur due to the presence of enzymes, co-factors, and other biomolecules. These facilitate the conversion of metal ions into nanoparticles. Nucleation and Growth Nucleation of nanoparticle seeds occurs, followed by the controlled growth of nanoparticles within the cellular confines. 6
  • 7. Microbial production of silver nanoparticles It was investigated using the bacterial strain Escherichia coli. Method The bacterial strain E.coli were cultured, for broth medium and LB medium. The culture flasks were incubated on an orbital shaker at 27°C and agitated at 220 rpm. The biomass was harvested after 24 hours of growth and centrifuged at 12000 rpm for 10 minutes. Synthesis of Silver nanoparticles The sample added separately to the reaction vessel containing silver nitrate (AgNO3) at specific concentration and control was also run along with the experimental condition. The primary conformation of synthesis of nanoparticles in the medium was characterized by the changes in color from yellowish white to brown. Production of biomass 7
  • 8. Microbial production of silver nanoparticles Characterization of silver nanoparticles The bioreduction of the Ag+ ions in the solution was monitored and sample of 2ml was withdrawn at different time intervals and the absorbance was measured using UV–visible spectrophotometer with samples in quartz cuvette. Particle sizing measurements Particle size analyzing experiments were carried out by means of laser diffractometry. Measurements were taken in the range between 0.04 up to 500 μm. 8
  • 9. Microbial production of Zn nanoparticles • ZnO nanoparticles are produced intracellular by bacteria such as Lactobacillus. Methods Zn Solution preparation 1 M stock solution for Zn2+ is prepared. Then solution Zn2+ was filtered into the bacterial culture medium and added. Isolation of lactic acid bacteria Using (MRS) medium, lactic acid bacteria was isolated from Cow milk and incubated. The streak plate technique was used to obtain single colonies with various morphologies. 9
  • 10. Microbial production of Zn nanoparticles Tolerance determination Determine the NaCl tolerance and bile salt tolerance of isolated lactobacillus. Biosynthesis of ZnO nanoparticles The cell biomass then suspended in sterilized deionized water containing Zn2+ at concentrations and incubated. The cells were collected at 5000 rpm for 10 minutes by centrifugation and washed with saline before being hung in a buffer. Cells were interrupted by alternating ultrasound cycles in 100 W for 5 minutes to get the ZnO nanoparticles. 10
  • 11. Microbial production of Zn nanoparticles 11
  • 12. Extracellular synthesis • It includes bio-mineralization, bio sorption or precipitation. • Here cell wall reductive enzymes or soluble secreted enzymes are involved in the reductive process of metal ions. • With the change in pH of the solution, various shapes and sizes were formed. The culture supernatants of Enterobacteria like Klebsiella pneumonia, E.coli, Enterobacter cloaceae rapidly synthesize silver nanoparticles by reducing Ag+ to Ag. 12
  • 13. Microbial production of Au nanoparticles Materials and Reagents: • Bacterial Culture Preparation: Inoculate a culture flask with the selected bacterial strain and incubate it overnight at the optimal temperature and pH for growth. • Growth Medium Adjustment: Prepare a fresh growth medium with the gold precursor (e.g., HAuCl4). Adjust the pH of the medium to promote the reduction of gold ions. • Incubation with Gold Precursor: Add the gold-containing medium to the bacterial culture flask. Incubate the flask with constant shaking to ensure uniform mixing of the medium and bacterial culture. 13
  • 14. Microbial production of Au nanoparticles Monitoring and Optimization: Periodically sample the solution and measure the changes in color. The development of a ruby red or purple color is indicative of AuNP formation. Biomass Separation: Centrifuge the culture to separate the bacterial biomass from the AuNPs and supernatant. Characterization: Characterize the synthesized AuNPs using Transmission Electron Microscopy (TEM), UV-Vis spectroscopy, X-ray Diffraction (XRD), and Dynamic Light Scattering (DLS) to analyze size, shape, and other properties. 14
  • 15. Intracellular and Extracellular Synthesis of nanoparticles Aspect Intracellular Synthesis Extracellular Synthesis Location of Synthesis Inside the microbial cell Outside the microbial cell Microbial Synthesis Bacteria, fungi, algae Bacteria and fungi Key Process Reduction of metal ions within the cell. Reduction of metal ions outside the cell. Control over size and shape Good control, can be precise. Limited control, more challenging. Biocompatibility High biocompatibility due to nanoparticles formed inside cells. Depends on presence of any toxic byproducts. Purification and collection Require additional steps for cell lysis and purification Easier to separate nanoparticles from the extracellular medium. Applications Drug delivery, catalysis and sensing. Drug delivery, catalysis, sensing and nanomedicine. 15
  • 16. Synthesis of nanoparticles by fungi • Fungi and Yeast are very effective secretors of extracellular enzymes. 16
  • 17. Potential fungal isolates used for biosynthesis of nanoparticles. Biogenic synthesis of nanoparticles using fungi • Intracellular and Extracellular. In intracellular synthesis, the metal precursor is added to the mycelial culture and is internalized in the biomass. Extraction of the nanoparticles is required after the synthesis, employing chemical treatment, centrifugation, and filtration to disrupt the biomass and release the nanoparticles In extracellular synthesis, the metal precursor is added to the aqueous filtrate containing only the fungal biomolecules, resulting in the formation of free nanoparticles in the dispersion. 17
  • 18. Biosynthesis of Zinc Oxide Nanoparticles using Fungus a. Fungi like Aspergillus, Penicillium etc. transferring onto a fresh agar plate and allowing it to grow. b. Once the culture is established, transfer it to Erlenmeyer flask. Incubate the flask with agitation (if using a shaker) until the fungus reaches the desired growth phase. a. When the fungal culture has grown, add a known volume or concentration of the zinc precursor solution to the fungal culture medium. b. Incubate the culture mixture under appropriate conditions (temperature, pH, and agitation if necessary) for a specified period. Dissolve the selected zinc salt (e.g., Zn(NO3)2 or ZnSO4) in deionized water to prepare a stock solution of known concentration. The formation of Zn nanoparticles is often indicated by a color change from colorless or pale to brown or reddish-brown. Use techniques like UV-Visible spectroscopy to measure the absorbance spectrum, which can provide information about the formation of nanoparticles. 18
  • 19. 19
  • 20. Biosynthesis of Silver Nanoparticles using Fungus Isolation and Culturing of Fungi: a. Isolate and identify a suitable fungal strain. Common strains used include Aspergillus, Fusarium, and Trichoderma. b. Cultivate the selected fungal strain on a suitable growth medium under controlled conditions .Allow the fungus to grow until it reaches the desired biomass. Preparation of Silver Nitrate Solution: Prepare a stock solution of silver nitrate (AgNO3) by dissolving a known amount in Milli- Q water. This solution will be used as a source of silver ions (Ag+). Inoculation and Incubation: a. Add the fungal culture to a flask containing a growth medium. b. To this flask, add silver nitrate solution. c. Adjust the pH of the mixture (typically pH 7-8). d. Incubate the mixture in an incubator at a controlled temperature and allow the reaction to proceed. Ag+ ions will be reduced by the fungal biomass to form silver nanoparticles 20
  • 21. Biosynthesis of Gold Nanoparticles using fungi Isolate and Culture the Fungus: Isolate a pure culture of the selected fungus. • Grow the fungus on a suitable culture medium in a sterile environment, following standard microbiological techniques. Prepare Fungal Biomass: Allow the fungus to grow until it reaches the logarithmic growth phase. • Harvest the fungal biomass by centrifugation and wash it thoroughly to remove any culture medium components. Gold Salt Reduction: Prepare a gold salt solution (e.g., HAuCl4) in deionized water. • Add the fungal biomass to the gold salt solution and incubate the mixture at a controlled temperature, typically around 25-30°C. • Over time, the fungal biomass will reduce the gold ions to form AuNPs. The color of the solution will change from pale yellow to a characteristic red or purple, indicating the formation of AuNPs 21
  • 22. Characterization: Measure the absorbance spectrum of the reaction mixture using a spectrophotometer. AuNPs typically exhibit a strong absorbance peak in the visible range, around 520-550 nm. • Analyze the size and shape of the synthesized AuNPs using techniques such as transmission electron microscopy (TEM) or dynamic light scattering (DLS). Purification: Centrifuge the reaction mixture to separate the fungal biomass and any unreacted gold ions from the AuNPs. 22
  • 23. References • EL-GHWAS, D. E. (2022). Characterization and biological synthesis of zinc oxide nanoparticles by new strain of Bacillus foraminis. Biodiversitas Journal of Biological Diversity, 23(1). • Suba, S., Vijayakumar, S., Vidhya, E., Punitha, V. N., & Nilavukkarasi, M. (2021). Microbial mediated synthesis of ZnO nanoparticles derived from Lactobacillus spp: Characterizations, antimicrobial and biocompatibility efficiencies. Sensors International, 2, 100104. • Moormann, G. C., & Bachand, G. D. (2021). Biosynthesis of Zinc Oxide Nanoparticles using Fungal Filtrates (No. SAND2021-9437R). Sandia National Laboratories (SNL-NM), Albuquerque, NM (United States). Center for Integrated Nanotechnologies (CINT). • Guilger-Casagrande, M., & Lima, R. D. (2019). Synthesis of silver nanoparticles mediated by fungi: a review. Frontiers in bioengineering and biotechnology, 7, 287. 23
  • 24. 24