POSSIBILITIES OF PHYSICAL METHODS IN DEVELOPMENT
OF MICROBIAL NANOTECHNOLOGY
K CHENNA REDDY, ASSISTANT PROFESSOR, chenna_atp@gmail.com
C SREENIVASULU, ASSISTANT PROFESSOR, chitrasreenu44@gmail.com
C VINOD, ASSISTANT PROFESSOR, vinodatp78@gmail.com
Department of Chemistry, Sri Venkateswara Institute of Technology,
N.H 44, Hampapuram, Rapthadu, Anantapuramu, Andhra Pradesh 515722
Abstract: We provide the findings of practical research in nanobiotechnology that several groups of
Georgian scientists have conducted using various physical and chemical methodologies. To create the silver
and gold nanoparticles, several novel terrestrial actinomycete strains were used, together with the blue-
green algae Spirulina platensis, which had been previously discovered in Georgian soils and rocks. To
characterise the synthesised nanoparticles and find the best conditions for synthesis, a range of spectral and
analytical approaches were used. There is a detailed description of the research methodologies employed
and the outcomes that were produced using them. The methods are compared based on their benefits and
their ability to describe the nanoparticle manufacturing process.
Keywords: microbial synthesis, nanoparticle, gold, silver, nanotechnology, biotechnology
Introduction
One innovative biotechnological strategy that has evolved in recent years is the manufacture of nanosized
materials using the physical characteristics and metabolic activities of microbial cells.1 The production of
highly structured metallic nanoparticles has been made possible by a wide variety of microorganisms,
including yeasts, fungi, and bacteria. To create nanoparticles, some cellular processes in microbes allow
surface functional groups (peptides, proteins, nucleic acids) to interact with metal ions in water-based
solutions.2- 4. Electronics, IT, catalysis, medicine, pharmacology, sensing, and photonics are just few of the
many uses for gold and silver nanoparticles. Oncology, cardiology, immunology, neurology, and
endocrinology are among of the medical fields that have shown therapeutic promise with these.5- 7
It is critical to find novel, simple, and environmentally safe ways to hunt for efficient microbial strains that
can produce nanoparticles of gold and silver. Actinomycetes and the blue green algae Spirulina platensis are
two examples of the microorganisms that have attracted a lot of attention because of the medicinal
possibilities they provide. This article describes and discusses the potential outcomes of the collaborative
research that Georgian scientists have conducted over many years in an effort to create biotechnological
approaches.
Materials and methods
Materials
A great deal of microbiological and plant biodiversity may be found in some regions of Georgia. Microbes
are of particular interest because of the enormous biotechnological potential they provide for use in
medicine. Several groups of Georgian scientists worked together to examine several microorganisms that
are typical of the Georgian environment. Researchers in Georgia looked at the distribution of terrestrial
actinomycetes strains in a variety of rock types, soil types, and the rhizosphere. In order to create strategies
for nanoparticle production, researchers looked at several groups of newly identified microbes from
Georgia. Gold and silver nanoparticles for pharmaceutical and medicinal applications were also produced
from the blue-green algae Spirulina platensis. Below, in Table 1, you can see the microorganisms that were
investigated.
You may find a detailed description of the procedures used to cultivate the examined bacterial culture and
produce biomass using gold and silver nanoparticles elsewhere [14–18].
Methods
A variety of spectral and analytical methods was used to characterize the synthesized gold and silver nanoparticles.
Table 1. Studied microorganisms
Names of bacteria Species of bacteria Site of bacteria isolation
Arthrobacter genera Arthrobacter globiformis 151B Isolated from the Kazreti region in Georgia
Arthrobacter oxydans 61B
Streptomyces genera Streptomyces glaucus 71MD Isolated from the rhizosphere of soybeans in Georgia
Streptomyces sp. 211A Isolated from the Cinnamonic calcareous soil of Sagarejo
region in Georgia
Extremophile bacteria Streptosporangium spp. 94A Isolated from the Black soil of Shiraki Valley in Georgia
Thermophilic Thermoactinomycete spp. 44Th Isolated from the red soil of Adjara Region
actinomycetes Thermomonospora spp. 67Th Isolated from the cinnamonic calcareous soil of Tetritskaro
region in Georgia
Blue-green alga Spirulina platensis Strain IPPAS B-256
A spectrophotometer "Cintra 10" (GBC Scientific Equipment Pty Ltd, Australia) with a wavelength range
of 190 - 1100 nm was used to record the UV-vis spectra of the samples. We used a Dron-2.0 diffractometer
to measure the X-ray diffraction (XRD). The radiation was produced by the BCV-23 X-ray tube that had a
Cu anode (CuKα: λ = 1.54178 Å).A JEOL SX-100 (Japan) TEM system running at 100 kV was used for the
transmission electron microscopy (TEM). A drop of solution containing gold or silver nanoparticles was
placed on carbon-coated transmission electron microscopy grids to prepare the samples.
System for Microscopy and Analysis (Moscow, Russia)/Quanta 3D FEG (United States of America)
scanning electron microscopes were used for the examination. The experimental microscope's operational
characteristics were a voltage range of 1–30 kV and a magnification range of 100–200000 ×. An energy-
dispersive X-ray analysis spectrometer (EDAX, USA) was used to perform microprobe examination on
clusters of gold and silver nanoparticles.19 The experimental samples were analysed for gold and silver
using flame atomic absorption spectrometry (AAS) using "Analyst-800" and "Beckman-495" spectrometers.
The neutron activation analysis (NAA) at the reactor IBR-2 of the Frank Laboratory of Neutron Physics of
the Joint Institute for Nuclear Research (Dubna, Russia) was used to assess the elemental composition of
samples, concentrations of gold and silver, and other similar parameters. The experimental setup and sample
irradiation protocols are detailed in another section.20 Genie 2000 was used to analyse the NAA data and
determine the element concentrations.21 The biosorption process on bacterial cells was studied using
equilibrium dialysis with atomic-absorption analysis, which was also used during nanoparticle synthesis.To
enhance the processes involved in the formation of nanoparticles, the bacterial biomass was subjected to
sonication using an ultrasonic generator at a frequency of 35 kHz for a duration of 10 to 30 minutes.
Results and discussion
The biomolecules, proteins, and enzymes found in bacterial cells undergo a reduction of metal ions during
the microbial creation of metal nanoparticles in a solution of a metal compound. In an interaction between a
silver nitrate (AgNO3) aqueous solution and a bacterial suspension, for instance, silver ions are reduced
from Ag (I) to Ag (0), leading to the aggregation of silver nanoparticles. Similarly, in a chloroauric acid
(HAuCl4) water solution, the reduction of Au(III) ions to Au(0) is the process that forms the gold
nanoparticles.
Detection of nanoparticles and assessment of experimental conditions for nanoparticle formation by
bacterial cells were the primary uses of ultraviolet-visible UV-vis spectrometry.Nanoparticles' spectral
extinction peaks are sharp because of localised surface plasmon resonances in the visible and near-infrared
bands.22 When metallic nanoparticles interact strongly with incoming electromagnetic radiation, it leads to
the collective excitation of conducting electrons, which in turn causes extinction.23 Absorption spectra
measured using ultraviolet-visible surface plasmon resonances showed that gold had a peak at 530 nm and
silver at 425 nm. Nanoparticles were formed at concentrations ranging from 10-2 to 10-4 M in each instance
to determine the best concentrations of AgNO3 and HAuCl4 in aqueous solutions. Figure 1 illustrates the
results of an experiment that was conducted on Spirulina platensis biomass to produce gold nanoparticles
(a) and silver nanoparticles (b) with respect to absorbance maximums against the dosage of AgNO3. The
ideal concentration for bacterial manufacture of nanoparticles was determined to be 10-3 M in every
instance. One thing to keep in mind is that compared to the silver nanoparticles, the spectra acquired from
the gold ones are obviously more prominent.
Figure 1. UV-vis spectra of gold nanoparticles in Spirulina platensis biomass obtained for different HAuCl4 doses (a) and silver
nanoparticles absorbance maximums versus the dose of AgNO3 (b).
Figure 2.The absorption spectra of Au nanoparticles detected in the suspension of Streptomyces glaucus 71MD (a) and Arthrobacter
oxydans 61B (b) at different time reaction with HAuCl4 10-3 M water solution.
The UV-vis spectra of the actinomycetes Streptomyces glaucus 71MD (a) and Arthrobacter oxydans 61B
(b) in suspension were obtained at various reaction periods with a HAuCl4 10-3 M water solution, as shown
in Figure 2. An absorption peak at 530 nm, corresponding to Au, appears in the given spectra and its
strength grows with reaction time. Gold nanoparticles mostly have spherical forms, as seen by the shapes of
these peaks. tests using biomass Streptomyces glaucus 71MD showed an optimal reaction time of hours for
Au nanoparticle formation, but tests with Arthrobacter oxydans 61B showed a favourable reaction time of
days.
Figure 3a shows the X-ray diffraction (XRD) spectra of gold nanoparticles in Arthrobacter oxydans 61B
biomass following a 12-day reaction with 10-3 M HAuCl4 (chloroauric acid), and Figure 3b shows the
XRD spectra of silver nanoparticles in Spirulina platensis biomass following a 1-day reaction with AgNO3
(silver nitrate). The four distinctive peaks (111), (200), (220), and (311) may be seen in Figure 3, which
corresponds to a face centred cubic (fcc) structure of gold (or silver). Crystalline gold nanoparticles were
produced by reducing Au (III) and Ag (I) ions in Spirulina platensis and Arthrobacter oxydans 61B cells,
according to the findings. The Sherrer’s formula24,25 was applied for evaluating sizes of the gold nanoparticles on
the basis one of the peaks in the diffractogram for different samples:
K
d cos
where K is the dimensionless shape factor, for cubic crystals it is 0.9 – 1; λ is the X-ray wavelength, for Cu Kα =
1.54178 Å; β is the line width at half the maximum intensity in radians, θ is the Bragg angle, and d is the sizeλ of
nanoparticles in nm.
Grain sizes below 100 nm may be modelled using the Sherrer's formula. We used the (111) interferential
maximum to get an approximation of the nanoparticle size. We have θ = 38o here. Only instrumental
broadening of β (≈0.3°) was included in the calculations, and the impact of crystal defects on the form of the
interferential maximum was not evaluated. This technique found that the gold nanoparticles in Arthrobacter
oxydans 61B biomass had a size of around 22 nanometers. Findings from other methodologies are
consistent with this one.
Figure 3. The XRD diffractogram for gold nanoparticles synthesized by Arthrobacter oxydans 61B treated with chloroauric acid for 12
days (a) and silver nanoparticles synthesized by Spirulina platensis treated with silver nitrate for 1 day (b).
Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were carried out for visualization and
approximate assessment of sizes of the formed nanoparticles.
Figure 4. TEM micrographs recorded from drop-cast films of gold nanoparticles formed by the reaction of the HAuCl 4 solution with biomass
Thermoactinomycete spp. 44Th after 6 days (a) the diffraction pattern of the selected area recorded from the gold nanoparticles (b) and Au
nanoparticle sizes distribution (c).
The transmission electron micrograph (TEM) picture captured from the drop-cast film of gold nanoparticles
synthesised after 6 days of reacting the chloroauric acid solution with 44Th biomass of
Thermoactinomycete spp. is shown in Figure 4a. Figure 4b shows that the face-centered cubic (fcc)
structure of gold nanoparticles is consistent with the diffraction pattern in the chosen region. With an
average size of 20 nm, the particle size histogram derived from this picture (Figure 4c) reveals that the gold
nanoparticles' sizes vary between 5 nm and 60 nm.Figure 5 shows scanning electron micrographs (SEMs) of
silver nanoparticles (a) and gold nanoparticles (b) produced by the actinomycete Streptosporangium spp.
94A and the actinomycete Streptomices spp. 211A, respectively. The scanning electron micrographs show
that the particles are mostly round and do not aggregate into large structures.The provided EDAX spectra
show the energy as a function of the relative counts of the detected X-rays. The existence of silver
nanoparticles in the Streptomices spp. 211A biomass (a) and gold nanoparticles in the Arthrobacter
globiformis 151B biomass (b) were confirmed by the spectra shown in Figure 6. Figure 6a shows that
Streptomyces spp. 211A has four distinct Ag peaks. Proteins and enzymes found in biomass also contribute
to the X-ray emission signals from C, O, and P. recorded. Arthrobacter globiformis 151B exhibits many Au
peaks along with signals from C, O, K, P, and Ca (Figure 6b).
Figure 5. SEM image of silver nanoparticles formed on the surface of actinomycete Streptomices spp. 211A (a) and gold nanoparticles
formed on the surface of actinomycete Streptosporangium spp. 94A (b).
Figure 6. EDAX spectra recorded from Streptomices spp. 211A biomass with silver nanoparticles (a) and from Arthrobacter globiformis
151B biomass with gold nanoparticles (b).
Using equilibrium dialysis and atomic absorption analysis, researchers investigated the biosorption process
on bacterial cells during nanoparticle synthesis. Since the sorption is dependent on the type and content of
the cell wall, the Freundlich equation, which predicts that there are heterogeneous sorption sites on bacterial
surfaces, was followed by the concentrations of the metal adsorbed by the bacteria in the solution at
equilibrium dialysis.
Freundlich adsorption isotherms describe the adsorbent's capacity as well as the equilibrium connection
between the adsorbent and adsorbate:
where Cb represents the adsorbed metal concentration, Ct the equilibrium metal ion concentration in the
solution, K the biosorption constant, and n the sorptive capacity, the empirical constants in this context.
Gold nanoparticles in Streptomyces spp. 211A biomass were studied using Freundlich adsorption linearized
isotherms (A for homogenised and B for particulate homogenised), as shown in Figure 7.
Figure 7. Freundlich adsorption isotherms for Au nanoparticles in Streptomyces spp. 211A biomass (A – for homogenized and B – for
particulate homogenized).
Figure 8. UV-vis spectra recorded as a function of time of reaction aqueous solution of HAuCl4 with Spirulina platensis (after and without
sonication).
The blue-green algal biomass containing Au nanoparticles was sonicated for 10 minutes at 35 kHz using an
ultrasonic generator in order to investigate the potential for intensifying nanoparticle formation. The optical
microscopy analysis reveals that the Spirulina platensis biomass broke down into tiny pieces. Afterwards, a
10-3 M concentration of HAuCl4 aqueous solution was added to the suspension in order to produce Au
nanoparticles. Figure 8 shows the results of measuring the UV-vis spectra for the reaction at various time
intervals.Figure 8 shows that compared to the alga treated without sonication, the absorption peak of Au
nanoparticles is four times greater after sonication. One possible explanation is that sonication increases the
overall surface area of the tiny bacterial pieces, lending credence to the idea that nanoparticles grow on their
outsides.
Figure 9a shows a transmission electron micrograph of gold nanoparticles that were produced when the
algae was subjected to sonication. Figure 9b illustrates the size distribution of the Au nanoparticles, and the
mean size is around 15 nm, while it was 25 nm before sonication [17]. So, during sonication, the creation of
nanoparticles is enhanced and their sizes are reduced.
Figure 9.The TEM image of Au nanoparticles formed in the reaction of HAuCl4 solution with Spirulina platensis biomass subjected to
sonication (a) and Au nanoparticles size distribution histogram (b).
Figure 10.The total gold concentrations in biomass Streptomyces spp. 211A determined by NAA (a) and by AAS (b).
We determined the total Au and Ag content in the biomass of the bacteria we tested using the analytical
procedures of neutron activation analysis (NAA) and atomic absorption spectrometry (AAS). Figure 10
shows examples of analytical determinations of total concentrations of gold in the bacterial biomass of
Streptomyces spp. 211A using NAA (a) and AAS (b).Total metal buildup followed the same analogous
dynamic in all instances: concentrations of metal rose sharply in the first few hours before gradually
levelling out. The first step included the primary adsorption of metal ions onto the extracellular surface of
bacterial cells. Subsequently, metal ions were brought into cells and built up inside them.Considering the
potential medicinal use of the produced biomass with Au and Ag nanoparticles, NAA was also used to
investigate the multi-elemental composition of the bacterial samples. In Figure 11, we can see the example
for The glaucomatous streptomyces bacteria (71MD). The NAA findings indicate that the biomass was not
too polluted with harmful elements, allowing for the potential industrial, medicinal, and pharmaceutical
applications of materials synthesised using Au and Ag nanoparticles.
Figure 11. The distribution of elements in Streptomyces glaucus
71MD sample.
Conclusions
Based on the results of the experiments, the microorganisms that were studied can interact with 10-3 M
aqueous solutions of chloroauric acid (HAuCl4) and silver nitrate (AgNO3) to produce gold and silver
nanoparticles. These microorganisms include new strains of actinomycetes from the Arthrobacter genera
(Arthrobacter globiformis 151B and Arthrobacter sp.61B), extremeophiles Streptomyces spp. 211A and
Streptomyces glaucus 71MD, thermophiles Thermoactinomycete spp. 44Th and Thermomonospora spp.
67Th, and the blue-green algae Spirulina platensis.The majority of the gold and silver nanoparticles
produced by bacterial biomass are extracellular and have a crystalline structure. Bacteria typically have
spherical forms and diameters ranging from 5 to 60 nanometers, with an average size of 15 to 35
nanometers among various strains. Atomic adsorption spectroscopy (AAS), neutron activation analysis
(NAA), scanning electron microscopy (SEM) with energy dispersive X-ray diffraction (EDAX), and
ultraviolet-visible and X-ray diffraction (UV-vis) spectrometry are all potent tools for studying Au and Ag
nanoparticles in bacterial biomass and determining how they are formed. Investigating the surface
biosorption process during nanoparticle production by the microorganisms under research required a series
of experiments using equilibrium dialysis techniques coupled with AAS.According to the results of the
research, the microorganisms under study have enormous medical and industrial potential as a source of
possible new technologies for the production of gold and silver nanoparticles that are clean, simple,
nontoxic, and ecologically acceptable.
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