Index

● Title: Isolation and Characterization of Selenium nanoparticle producing bacteria

● ABSTRACT
● INTRODUCTION
● REVIEW OF LITERATURE
● METHODOLOGY
○ 1. Enrichment of Bacteria:
○ 2. Cultivation of Bacteria:
○ 3. Production of Selenium nano-particles by Bacteria:
○ 4. Isolation and Identification of Bacteria producing Se nanoparticles:
● OBJECTIVES
● REFERENCES

Name : Priyanka Zala

Enrollment No. 92100220053

Title: Isolation and Characterization of Selenium nanoparticle producing

bacteria

ABSTRACT
Nanotechnology is a powerful platform for manipulating matter at the level of atoms and
molecules in order to alert their properties. It is the control of matter at atomic molecular levels
in a size range of about 1 to 100 nm. Metallic nanoparticles have fascinated scientists for over a
century and are now heavily utilized in biomedical science for the synthesis of metallic
nanoparticles. Biological methods would help to remove harsh methods and harsh processing
conditions, enabling the synthesis at physiological pH, temperature, and pressure, and at the
same time at a lower cost. Large numbers of microorganisms have been found capable of
synthesizing a nanoparticle. When microorganisms are confronted with high concentrations of
metal ions, they reduce them to their element state. Metal like Selenium is an essential trace
element required by many organisms. Many bacteria, fungi, and plants are reported to synthesize
Selenium nanoparticles. Selenium nanoparticles have many applications. Selenium nanoparticles
are effective agents for the treatment of Alzheimer’s disease and also act as antioxidants,
antidiabetic, and antimicrobial activity. With this, several soil samples were collected and
screened for selenium nanoparticle production. The obtained isolate was enriched, cultivated,
and utilized for its potential to produce selenium nanoparticles. The produced selenium
nanoparticles were analyzed for their various physicochemical properties.

Key words: Nanoparticles, Metallic ion, Selenium Potential for production

INTRODUCTION
Nanotechnology is the manipulation of matter on an atomic, molecular and
supramolecular scale. In the past, the description of nanotechnology referred to the particular
technological goal of precisely manipulating atoms and molecules for the fabrication of
macro-scale products. Nowadays, it is referred to as molecular nanotechnology. The National
Nanotechnology Initiative, the US, defines nanotechnology as the manipulation of matter with at
least one dimension sized from 1 to 100 nanometers. This definition reflects the fact that
quantum mechanical effects are significant at this quantum-realm scale. So the purpose shifted
from a particular technological goal to research and technologies that deal with the unique
properties of matter which occur below the given size threshold. It is, therefore, common to see
the plural form “nanotechnologies” as well as “nanoscale technologies” to refer to the broad
range of research and applications whose common trait is size.

Nanotechnology, as defined by size, is naturally very broad, including fields of science as

diverse as surface science, organic chemistry, molecular biology, semiconductor physics, energy
storage, microfabrication, molecular engineering, etc. Nanotechnology can create many new
materials and devices with a vast range of applications, such as nanomedicine, nanoelectronics,
and biomaterials energy production and consumer products.

In nanotechnology, a particle is defined as a small object that behaves as a whole unit

with respect to its transport and properties. Nanoparticles are particles between 1 to 100
nanometers in size with a surrounding interfacial layer. The interfacial layer is an integral part of
nanoscale matter, fundamentally affecting all of its properties. The nanoparticles are responsible
for their different Chemical, Optical, and Mechanically magnetic properties as compared to the
extensive bulk material. Nanoparticles have unique properties because of their colossal face area,
high surface energy, and quantum confinement.

Metal nanoparticles of Ag, Au, Ce, Fe, Se, Si, Ti, and Zn have a special place in the area
of nanotechnology as they offer a unique opportunity not only as theranostic agents, proteins,
siRNA, etc. Among all nanoparticles, Selenium nanoparticles are one of the most studied.
Selenium is an essential trace element required by many organisms. Many bacteria, fungi, and
plants are reported to synthesize selenium nanoparticles. Many bacteria synthesize nanoparticles,
and mainly Bacillus species produce nanoparticles. The Bacillus species produce a reddish color
reaction mixture indicating Selenium nanoparticles. Selenium nanoparticles have many
applications, such as anticancer, antioxidant, antiviral, and antimicrobial activity.
REVIEW OF LITERATURE
Selenium is a chemical element with the symbol Se and atomic number 34. Selenium has
a standard atomic weight of 78.971 [1]. Selenium comes from the Greek word Selene meaning
"Moon," discovered by Jons Jacob Berzelius and Johan Gottlieb Gahn [2].
Se was found immobilized in sedimentary rocks and soils, exhibited high persistence, and
is meticulously influenced by soil's oxidation-reduction potential, pH, and solubility [3].
Selenium occurs naturally in several inorganic forms, including selenide, selenate, and
selenite. Selenium is most commonly found as an impurity, replacing a small part of the sulfur in
sulfide ores of many metals. Certain soils are selenium-rich, and Selenium most occurs in soluble
forms such as selenate[4].
In living systems, Selenium is found in the amino acids selenomethionine,
selenocysteine, and methyl selenocysteine. In these compounds, Selenium plays a role similar to
that of sulfur. Another naturally occurring organoselenium compound is dimethyl selenide [5].
Selenium is a constituent of the unusual amino acids selenocysteine and
selenomethionine. In humans, Selenium acts as a trace element nutrient. It is utilized as a
cofactor for the reduction of antioxidant enzymes such as glutathione peroxidases [6]. The
enzyme plays important roles in anti-oxidation, reproduction, and muscle function. Selenium is
used in the electronic industries for manufacturing industries as a decolorizer, in point industry as
pigment, industry metallurgy as lubricants, and various aerospace devices Se used in the
agricultural industry for plant breeding [7].
Selenium has gained importance in medicine for the effective management of the disease.
Various inorganic and organic forms of Se, such as sodium selenite, Selenium chloride,
selenomethionine, and selenocysteine, are also used as a nutritional supplement along with
various medicine in minor doses>200/day for adults [8].
Selenium may inhibit Hashimoto's disease, in which the body's own thyroid cells are attacked as
foreign. A reduction of 21% in TPO antibodies is reported with the dietary intake of 0.2 mg of
Selenium [9].
Nanotechnology is the science of developing and utilizing materials, systems, or devices
at roughly 1-100 nm scale. Nanomaterials, due to the size effect phenomenon, have unique and
new functions. Nanoparticles are made of several atoms or molecules with different Sizes and
morphology [10].
Among nanoparticles, metallic and metalloid nanoparticles have been widely used
because of their catalytic, absorbent, optical, electrical, and magnetic properties. Top-down and
bottom-up these two methods are widely used for the synthesis of nanoparticles [11].
Selenium is a metalloid element that is focused on different oxidation statuses and
properties in nature. Selenium element has photolytic and conductor properties. It is useful in
pesticides, the glass industry, and as a food additive in animals and poultries meals [12].
Liu et al. (2004) have synthesized selenium nanoparticles by a reverse microemulsion system
using sodium selenosulfate as a selenium source. They establish that hydrochloric acid
concentration and reaction temperature have a great influence on the morphology of selenium
nanoparticles [13].
Abdelouas et al. (2000) produced selenium nanoparticles through the application of
cytochrome C3 obtained from Desulfovibrio Vulgaris, a sulfate-reducing bacterium. This
cytochrome was able to reduce selenate to Selenium and so the production of selenium
nanoparticles [14].
Zhang et al. (2004) reported that the reduction of Selenium was performed by ascorbic
acid in the presence of different polysaccharides such as chitosan, acacia gum, and
carboxymethyl cellulose. In the results, it is revealed that very stable spherical selenium
nanoparticles were produced that are stable in solution for six months in the presence of
polysaccharides [15].
Zhang et al. (2005) have studied the toxicity of bulk selenium and selenium nanoparticles
in rat liver. As a result, it was discovered that selenite had increased the serum level of alanine
aminotransferase and aspartate aminotransferase and inhibited the activity of catalase and
superoxide dismutase while having no effect on these enzymes [16].
Benko et al. (2012) reported the toxicity of Selenium containing compounds in mice by
treating mice with different selenium compounds and concentrations. As a result, they reported
that maximum accumulation of Selenium happened in the liver and spleen of mice [17]. Chiba et
al. (1987) suggested that Se and Sn have maximum accumulation in the bone, liver, and spleen of
mice [18].
Shakibaei et al. (2010) have produced intracellular selenium nanoparticles using bacillus
sp. Msh 1, an isolate from the Caspian sea, with 142-255 nm in Size but transmission electron
microscopy (TEM) revealed that spherical nanoparticles with 80-200 nm that had maximum
absorbance at 450-500 nm in Uv/ Vis analysis and confirmed by XRD analysis [19]. Debieux et
al. (2011) have produced spherical (150 nm in Size) nanoparticles using Thauera selenates and
confirmed it by TEM analysis [20].
Dhanjal and Cameotra (2010) isolated Bacillus cereus CM100B from soil that was able to
produce intra- and extra-cellular selenium nanoparticles with a Size of 150-200 nm based on
TEM analysis. These selenium nanoparticles gave maximum absorbance at 590 nm [21].
Lee et al. (2007) reported that Shewanella sp. HN41 is able to reduce Selenium (IV)
during respiration and produce selenium nanoparticles with 164-181 nm in Size [22]. Yadav et
al. (2008) and Anand et al. (2005) reported the appearance of red color after 12 hours of bacterial
culture in a broth containing Selenium and regarded it as a reason for Selenium (0) production
[23].
In the study of Lorti et al. (1992), it was reported that Pseudomonas stutzeri was able to
tolerate 2.53 mM of sodium selenite [24]. Kessi et al. (1999) reported the maximum to tolerate
Selenium for Rhodospirillum rubrum as 1.5 mM [25]. Kashiwa et al. (2000) reported that
Bacillus sp. was able to reduce 20 mM of selenate to selenite and two mM of selenite to
elemental Selenium, but the reduction rate of selenate was higher [26]. Roux et al. (2001)
reported that the Ralstonia metallidurans could tolerate 6mM selenite and reduce it to Selenium
(0) [27].
Zahir et al. (2003) isolated Enterobacter tailored from rice farm drainage that was able to
grow at 500-5000 micrograms per liter of selenate in such a manner that reduced 81-94% of it
during five days [28].
Saeedeh Pouri et al. (2018) isolated Bacillus cereus BIPCO4, which produced selenium
nanoparticles and can tolerate 37.5 mM of sodium selenate [29].
The structural properties of SeNP are determined and characterized by studying
parameters like Size, shape, etc., for analyzing the structural properties, and the following
methods are employed UV visible spectral analysis, X-ray diffraction analysis(XRD), Fourier
transforms resonance spectroscopy [FTIR] analysis, dynamic light scattering (DLS) analysis,
transmission and scanning electron microscopy. UV spectroscopy determines the "absorption
maximum of nanoparticles depending on the concentration of the precursor and another
component of the reaction mixture [30].
A microbial source, Bacillus cereus mediated synthesis of selenium nanoparticles showed
absorption maximum at 590 nm. At the same time, nanoparticles synthesized from lemon leaf
extract exhibited maximum absorption at 395 nm [31].
The biologically synthesized SeNP had a different absorption maximum than the
chemically synthesized one due to its low and variable band gap energy [32].
Selenium-enriched Lactobacillus has been reported as an immune stimulatory agent that
can be used to increase the lifespan of cancer-bearing animals [33].
Ren et al. (2013) demonstrated the antitumor action of hyaluronic acid-capped SeNPs in
the Heps tumor mice model [34].
FTIR is used to analyze the surface interaction between synthesized nanoparticles with
other molecules that take part in the synthesis and stabilization of nanoparticles. The DLS
technique was used to measure the hydrodynamic diameters of synthesized nanoparticles. XRD
and X-ray photoelectron spectroscopy is generally used for characterizing the composition of
selenium nanoparticles [35].
SEM and transmission electron microscopy (TEM) with selected electron diffraction
(SAED) are well-known techniques to determine the structure, morphology, and Size of prepared
nanoparticles [36].
Webster (2011) used BSA to stabilize the SeNP, and there observed an increased size of
particles of about 100 nm, while these particles without the coating were 40-60 nm [37].
SeNP increases in the concentration of the stabilizer result in a reduction in the
concentration of the stabilizer, resulting in a reduction in the size of the nanoparticles from 130
nm to 48 nm, also aiding in uniformity [38].
METHODOLOGY
1. Enrichment of Bacteria
2. Cultivation of Bacteria
3. Production of selenium nanoparticle
4. Identification of Bacteria

1. Enrichment of Bacteria:

All six sample of soil were collected from the different site in college campus and made
suspension with 10 ml of D/W in sterile glass test tubes. First of all Heat treatment was
given to each soil sample at 62.50C Temperature for 30 minutes. Then bacteria were
isolated on Nutrient agar plates using streaking method. Well isolated big and opaque
colonies were selected to further proceed for exploitation of Gram Positive to produce Se
nanoparticle.

2. Cultivation of Bacteria:
To increase the adaptation of bacteria for Se metal a few Nutrient broth tubes with
selenium concentration in increasing amount 0.5mM, 1.0mM, 1.5mM, 2mM, 2.5mM and
3mM concentration were inoculated with isolated colony.

3. Production of Selenium nano-particles by Bacteria:

It was started with preparation of several Selenium salt dilutions like 0.5mM, 0.5mM,
1mM, 1.5mM, 2mM, 2.5mM and 3mM. Each dilution of selenium salt added into Triptic
soya Broth. The Triptic soya broth was then inoculated with inoculums of cultivation
tubes of various soil samples. All broths were kept for 2-3 days for production of
Selenium nano particles.

4. Isolation and Identification of Bacteria producing Se nanoparticles:

Prepare media for isolation of Selenium nanoparticle producing bacteria. From TSB
Broth take single loopful and streak on TSA plate. Incubate at 370C for 3 to 4 days. Make
a suspension from TSA plate and inoculate each biochemical media. Incubate t 370C for
24 hours. After this do Gram staining and Spore staining for identification of bacteria.
After second day, note down the result and characteristics
OBJECTIVES
● To perform the Enrichment technique from different soil samples and determine the
microorganism to produce selenium nanoparticles.
● To determine selenium nanoparticles-producing bacteria perform the cultivation method
of bacteria and then obtain a few that produce selenium nanoparticles.
● To the production of selenium nanoparticles by bacteria in tryptic soya broth.
● To perform Gram's staining and spore staining methods to identify bacteria-producing Se
nanoparticles. Here are Bacillus coagulans, Bacillus brevis, Bacillus alvei, Bacillus
sphaericus, Bacillus fastidiosus.

REFERENCES

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nouveaux" [Letter from Mr. Berzelius to Mr. Berthollet on two new metals]. . 7: 199–206
3. S.patai and Z.rappaport,the chemistry of organic selenium and tellurium
Compounds, "the chemistry of Triple Bonded Functional Group" Volume 192, 1978

4. Fordyce, Fiona (2007). "Selenium Geochemistry and Health"  AMBIO: A Journal of the

Human Environment. 36: 94–97
5. Wessjohann, Ludger A.; Schneider, Alex; Abbas, Muhammad; Brandt, Wolfgang (2007).
"Selenium in chemistry and biochemistry in comparison to sulfur". Biological
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9. Mazokopakis, E.E., Papadakis, J.A.; Papadomanolaki,M.G., et al. (2007). “Effects of 12
months treatment with L-selenomethionine in serum anti-TPO Levels in Patients with
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