World Journal of Pharmaceutical Research

Lakhsmi et al. World Journal of Pharmaceutical

SJIF Research
Impact Factor 5.990

Volume 4, Issue 7, 1592-1611. Research Article ISSN 2277– 7105

ISOLATION, SCREENING, IDENTIFICATION,

CHARACTERIZATION AND APPLICATIONS OF GREEN
SYNTHESIZED SILVER NANOPARTICLE FROM MARINE
ACTINOMYCETES-STREPTOMYCES ALTHIOTICUS

Dr.S.Yamini Sudha Lakhsmi*, Haritha Lakhsmi.D and Sharmila.S

Department of Medical Biochemistry, University of Madras, Taramani, Chennai-600113.

ABSTRACT
Article Received on
05 May 2015, Crystalline Silver Nanoparticles have been found to have diagnostic
and therapeutic applications in medical field. In the present study, the
Revised on 30 May 2015,
Accepted on 24 June 2015 marine actinomycetes were isolated from the soil sample collected
from the coastal area and the isolates were screened for antibacterial
activity against the pathogenic bacteria by cross streaking method. The
*Correspondence for
Author potential isolate was selected and it was identified as Streptomyces
Dr.S.Yamini Sudha althioticus by 16SrRNA sequence method. The silver nanoparticles
Lakhsmi were synthesized from the potent isolate Streptomyces althioticus using
Department of Medical
silver nitrate as a reducing agent by biological method. The formation
Biochemistry, University
of silver nanoparticles were indicated by the colour change from
of Madras, Taramani,
Chennai-600113. colourless to reddish brown, characterized by SEM, TEM revealing
the size of nanoparticle to be 50nm having sphere shape. XRD and
FTIR showing the crystalline nature and the functional groups (OH, C- C, NH) present in the
nanoparticle. The antimicrobial study was carried out against B.subtilis, E.coli, S.aureus,
and Proteus vulgaris. The maximum zone of inhibition was showed against B.subtilis around
21mm. These nanoparticles were found to have a significant free radical scavenging activity
and a potent cytotoxic activity against MCF7 cell line invitro.

KEYWORDS: Streptomyces althioticus, SEM, TEM, XRD, FTIR analysis, antimicrobial,

free radical, cytotoxic activities.

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INTRODUCTION
In earth surface, marine environment occupies 71%,[1] and it provides broad scope for the
identification of novel natural drugs,[2] In the marine environment, marine microorganisms
are the eminent source to discover new bioactive compounds. Among marine
microorganisms, the actinomycetes are the major source for the production of new drugs
which is proven by the various earlier researches.[3] Actinomycetes are a group of prokaryotic
gram positive filamentous bacteria which produce two types of branching mycelium, aerial
mycelium and substrate mycelium,[4] Some actinomycetes genera are slow growing
microorganism,[5] They are belongs to the domain bacteria, phylum actinobacteria, subclass
actinobacteridae, order actinomycetales, suborder actinomycineae, family actinomycetaceae.
The member of this order contains high G+C content (>55 mol%) in their DNA.[6] About
two-thirds of naturally occurring antibiotics have been isolated and most of the antibiotics are
isolated from the genus Streptomyces,[7] Since the treatment for several diseases is not yet
identified, there is an urgent need to fulfill the need of antibiotics. In the present study, the
marine actinomycetes are isolated from coastal sand to produce antibiotics. Delivering
therapeutic compound to the target site is a major problem in treatment of many diseases. So
these antibiotics are converted to nanoparticle to make ease of drug delivery. The
engineering, characterization, synthesis, and use of materials and devices of 100 nanometers
or less is called nanotechnology. Nanotechnology is the science of the small and it is often
referred to as the ‗tiny science‘. Nanotechnology represents the design, production and
application of materials at atomic, molecular and macromolecular scales, in order to produce
new nanosized materials,[8] According to the definition from NNI (National Nanotechnology
Initiative), nanoparticles are of nanosized particles that are ranging from 1 to 100 nm in at
least one dimension. Different types of nanoparticles are available but metal-based
nanoparticles have significant role in diagnosis and drug delivery due to its biocompability.
In the metal nanoparticles silver has unique physical, chemical and biological properties and
it also shows bactericidal and fungicidal activity. In the present study, due to above
applications of silver nanoparticles, it is synthesized from the cell free extract of
Streptomyces althioticus by silver nitrate as a reducing agent.

MATERIALS AND METHODS

SAMPLE COLLECTION
Marine sediments were collected from marina the coastal area of Tamil nadu at 10 m length
and 5 m depth with sterile spatula in sterilize polythene bags. The samples were closed tightly

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transported to the laboratory aseptically and stored in the refrigerator at 4°C until further
use.[9]

ISOLATION OF ACTINOMYCETES
Collected soil samples were treated with 1% calcium carbonate and air dried for 3 to 4 days
under in vitro lab condition to increase the number of actinomycetes propagules in the
samples,[9] Isolation of actinomycetes was performed by soil dilution plate technique,[10] The
pretreated soil sample was serially diluted in a sterile water from 10-1 to 10 -10 dilutions.[11]
The medium used for the isolation and cultivation of marine actinomycetes was starch casein
agar medium. From each suitable dilution, 0.1 ml was taken and spread evenly with sterile L-
shaped glass rod and kept for incubation at 30ºc for 7 days. After incubation period the
developed colonies were individually purified by streak plate method and then subcultured to
ensure for their purity. Pure culture was transferred on slants and preserved at 4ºC for further
analysis.[12]

SCREENING FOR ANTIMICROBIAL ACTIVITY

The antimicrobial activity was studied preliminary by cross streak method against bacteria.
The test organisms used are Bacillus subtilis, Staphylococcus aureus, Escherichia coli,
Proteus vulgaris. After the preliminary test of the isolates for their antimicrobial activities the
most active isolate was selected for further study. The most active isolate was subjected to
16s rRNA sequencing to identify the gene and species.

SIVER NANOPARTICLE SYNTHESIS

PREPARATION OF CELL FREE MICROBIAL EXTRACT
The liquid medium, ISP-2 broth was taken in Erlenmeyer flask for bulk production. ISP-2
media containing Peptone - 5.0 g/l, Yeast extract - 3.0 g/l, Malt extract - 3.0 g/l, Dextrose -
10.0 g/l, Distilled water - 1 lit and pH 7.2. The isolated actinomycetes were inoculated into
250 ml of ISP-2 broth culture, incubated at 30°C for 7 days under shake flask condition. After
the incubation period was complete, the culture was centrifuged at 5000 rpm for 30 min and
the supernatant was used for the biosynthesis of AgNPs,[13] The collected supernatant (pH
7.0) was added separately to the reaction vessel containing silver nitrate at a concentration of
1 mM (1% (v/v)) and incubated on an orbital shaker in the dark condition for 72 hrs at 30° C.
The reaction was carried out in the dark after the addition of the AgNO3 and color change
appeared transparent.

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SEM (SCANNING ELECTRON MICROSCOPE)

SEM is a most widespread analytical instrument available in analytical laboratories destined
to characterize physical properties such as morphology, shape, size or size distribution of
materials at the microscale and nanoscale.

Sample preparation
Silver nanoparticle synthesized from Streptomyeces althioticus was allowed to dry
completely and ground well. Since the specimen was at high vaccum, fixation was usually
performed by incubation in a solution of a buffered chemical fixative gluteraldehyde. The dry
specimen was mounted on a specimen stub using an adhesive epoxy resin or electrically
conductive double-sided adhesive tape and sputter coated with gold palladium alloy before
examination in the microscope.

TRANSMISSION ELECTRON MICROSCOPY (TEM)

The TEM can yield information such as particle size, size distribution and morphology of the
nanoparticles. TEM images can also be used to judge whether good dispersion has been
achieved or whether agglomeration is present in the system. TEM analysis of synthesized
silver nanoparticles was prepared by drop-coating biosynthesized nanoparticles solution on
carbon-coated copper TEM grids (400 μm × 40 μm mesh size). Samples were dried and kept
under vacuum in desiccators before loading on to a specimen holder. Characterization of the
sample was done using TEM (JEOL MODEL 1200 EX INSTRUMENT (80kV).

XRD (X RAY DIFFRACTION)

X-ray diffraction (XRD) is the most powerful and routine technique for determining the
structural properties of crystalline solids, the requirement for a single-crystal specimen of
appropriate size and quality imposes a limitation on the scope of this technique.In this
method, a monochromatic X-ray directed onto a sample and the interaction between these
planes of atoms and X-ray lead to diffracted rays being emitted. The characterization of the
purified silver nanoparticles was conducted with an XRD-6000 X-ray diffractometer
(Shimadzu,Japan) operated at a voltage of 40 kV and a current of 30Ma.

FTIR (FOURIER TRANSFORM INFRARED SPECTROMETRY)

A known weight of sample (1g) was taken in a mortar and pestle and ground with 2.5 mg of
dry potassium bromide. The powder so obtained was filled in a 2 mm internal diameter
micro-cup and loaded onto FTIR set at 260 c + or – 10 c. The samples were scanned using

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infra-red in the range of 4000-400 cm-1 using FTIR spectrometer. The spectral data obtained
were compared with the refer reference chart to identify the functional groups present in the
sample.

ANTIBACTERIAL ACTIVITY
Antibacterial activity of silver nanoparticle synthesized from cell free filterate of
Streptomyces althioticus was tested. The tested organisms are Escherichia coli (MTCC443),
Vibrio cholera (MTCC3904), Bacillus subtilis (MTCC441), Staphylococcus aureus
(MTCC96).

FREE RADICAL SCAVENGING ACTIVITY (DPPH ASSAY)

The ability of the AgNPs to annihilate the DPPH radical (1, 1-diphenyl-2-picrylhydrazyl) was
investigated. Stock solution of sample was prepared to the concentration of 1mg/ml.
20,40,60,80,100,120,140,160,180 g of each sample were added to DPPH (0.1%). The
reaction mixture was incubated for 30 min at room temperature and the absorbance (A) was
recorded at 517 nm. The experiment was repeated for three times. BHT (Butylated Hydroxy
Toluene) was used as standard control. The annihilation activity of free radicals was
calculated as % inhibition.

CYTOTOXIC STUDY ON CANCER CELL LINE

MTT ASSAY
MTT is known as (3-(4, 5-diphenyl thiazol-2yl)-2, 5-diphenyl tetrazolium bromide. MTT
assay was first proposed by Mossmane in 1982. MTT is cleaved by mitochondrial
dehydrogenize in viable cells, yielding a measurable purple product formazan. The formazan
production is proportionate to the viable cell number and inversely proportional to the degree
of cytotoxicity. The MTT assay was carried out in a multiwell plate and each well was
washed with MEM (w/o) FBS. And 200 micro liter of MTT conc. of (5mg/ml) was added. It
was incubated for 6-7hrs in 5% CO2 incubator. After incubation 1ml of DMSO was added in
each well and mixed by pipette and leave for 45sec. And it showed the purple color
formation. The suspension is transferred into the cuvette of spectrophotometer and O.D
values were read at 595nm. % of cell viability was calculated using the formula. (OD of
sample/OD of cell control)(y)*100=%cell viability. Graph was plotted using the % of cell
viability at Y-axis and concentration of the sample in X-axis. Cell control and sample was

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included in each assay to compare the full cell viability in cytotoxic and antitumor activity
assessments.

RESULTS AND DISCUSSIONS

SCREENING OF ISOLATED MARINE ACTINOMYCETES
About 10 isolates are obtained from isolation technique was subjected to screening, among
those one isolate shows potent activity against test microorganism. By cross streak method
preliminary screening was done to identify the potent isolate. The growth along the central
streak is that of Actinomycetes. The growth of all the perpendicularly streaked test bacteria
was completely inhibited. From the fig.1 it is clear that the isolate possess high bactericidal
activity.

16s rRNA sequencing

The 16 S rRNA sequencing analysis of the isolate yielded 1313 base pairs and NCBI BLAST
search analysis showed that the sequence was 99% similar to the sequence of Streptomyces
althioticus strain NR_115392.1. A BLAST analysis carried out through blastn search
through GenBank (http://www.ncbi.nlm.nih.gov) revealed that the strain belongs to the genus
Streptomyces. 16s rRNA sequencing method is used to identify the genus and species of the
organism. From the blast and FASTA sequence, the data confirmed that the new isolate
should be assigned to the genus Streptomyces althioticus. (fig.2a and fig2b)

Extracellular biosynthesis of silver nanoparticles using culture supernatant of

Streptomyces althioticus
Production of silver nanoparticles by the culture supernatants of Streptomyces althioticus
with aqueous silver nitrate solution, 1mM was investigated. Before and after exposure of
silver nitrate to culture supernatant of Streptomyces althioticus was shown in the Fig3. The
appearance of colour change from yellowish colour to brown color shown in the flask (A and
B) clearly indicates the formation of silver nanoparticles in the reaction mixture (Durán,
2005).

Characterization of biosynthesized Ag NPs

Scanning electron microscopy (SEM)
From (Fig.5) the result obtained for the SEM for the silver nanoparticles synthesized from
Streptomyces althioticus was found to be round, spherical, oval aggregates. Baskararao et al.,
2013 reported that the biologically synthesized AgNPs from marine actinomycetes are

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characterized by SEM and confirmed that the shape to be round, spherical with aggregates.
From the above results suggested that the silver nanoparticles were synthesized due to the
action of Streptomyces cell-free extract, which act as good bio-reductant for biosynthesis.

Transmission Electron Microscopy (TEM)

The TEM image (Fig.6) the results revealed that AgNps in the reaction mixture were
spherical in shape. AgNps synthesized by Streptomyces althioticus were found to be 50 nm.
Majority of the AgNps were monodispersed showing exact size as observed under TEM.
Particle size analysis revealed that the produced silver nanoparticles are in the size of 50 nm.
A study was done by Vandana Rathod et al., 2014 reported that AgNps in the reaction
mixture were spherical in shape. AgNps synthesized by Streptomyces sp VDP-5 were found
to be in the range of 30-40nm. Another study done by Vidya and Balagurunathan 2013
reported that the diameter of the AgNps synthesized by Streptomyces sp was found to be 20 –
40 nm. Further analysis of particle size distribution denoted that more than 75% of particles
are in the range of 20-40 nm, suggesting this microbial strain has potential to produce low-
size particles with high surface area.

X-Ray Diffraction (XRD)

The XRD pattern of the silver nitrate treated sample shows four intense peaks in the 2Ө
spectrum ranging from 30 to 80. The exact nature of the biosynthesized silver nanoparticles
formed can be deduced from the XRD spectrum of the sample. The characteristic XRD peaks
2Ө at 38°, 46°, 65° and 74°correspond to the (1 1 1), (2 0 0), (2 2 0) and (311) planes for
silver, respectively. The diffraction peaks indexed at (1 1 1), (2 0 0), and (2 2 0) representing
crystalline structures were well-documented for silver previously by various researchers.The
strong, narrow diffraction peaks shown in (Figure.7) indicate that the product has a
crystalline structure.

Fourier Transform Infrared spectroscopy (FTIR)

FTIR measurements were carried out to identify possible interaction between silver and
protein molecules, which may be responsible for synthesis, stabilization, (capping material)
and well dispersed silver nanoparticles in the reaction mixture,[17] Fig.8 shows the FTIR
spectrum of the freeze-dried powder of silver nanoparticles formed after 24 h of reaction. The
spectral data recorded revealed two types of vibrations (i.e., stretching and bending) in the
wavelength range of 4,000 to 500 Cm-1. It could be evident from the figure the presence of
an amine vibration band at 3,418 Cm-1 representing a primary amine (N-H) stretching, and

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amide (N-H) bending vibration bands at 1635.18 and 1431.14 Cm-1. Furthermore, the FTIR
spectra of biosynthesized silver nanoparticles also revealed peaks at 1372.71 Cm-1 stretching
vibrations of aliphatic C-H bonds. Several C-N stretching vibration peaks were also observed.
In addition, the presence of band at 1163.09 Cm-1 in the FTIR spectra suggested that capping
agent of biosynthesized nanoparticles possesses an aromatic amine groups with specific
signatures of amide linkages between amino acid residues in the proteins in the infrared
region of the electromagnetic spectrum,[18] This type of FTIR spectra supports the presence of
a protein type of compound on the surface of biosynthesized nanoparticles, confirming that
metabolically produced proteins acted as capping agents during production and prevented the
reduced silver particles agglomeration. In fact, carbonyl groups from the amino acid residues
as well as peptides are known for strong silver binding property. These data further indicate
that the isolated marine, Streptomyces althioticus, produces extracellular protein compound
that can bind to synthesized nanoparticles through free amine groups, as well as cysteine
residues present in the protein, and thereby acting as a capping agent during synthesis of
silver nanoparticles. Similar observation was reported by Prakasham et al., 2012 based on
FTIR spectra recorded for S. albidoflavus CNP10 biosynthesized nanoparticles.

ANTIMICROBIAL ACTIVITY OF SYNTHESIZED NANOPARTICLE FROM

Streptomyces althioticus
The result obtained in this study indicates that biologically synthesized AgNPs possess
tremendous antimicrobial properties. The antimicrobial activity was observed against
Staphylococcus aureus, Bacillus subtilis, Vibrio cholera, among these bacteria the least zone
of inhibition was noticed against Escherichia coli and highest was seen against Bacillus
subtilis. Silver has been used for its well known antimicrobial properties, the advances in
generating Ag-NPs have made possible a revival of the use of silver as a powerful
bactericide.

FREE RADICAL SCAVENGING ACTIVITY OF SYNTHESIZED NANOPARTICLE

The free radical scavenging activity of synthesized silver nanoparticle Streptomyces
althioticus increases with increase in silver nanoparticle concentration. In the present study
the figure 9 indicates the maximum rate of inhibition at 150µg.

CYTOTOXICITY OF AGNPS ON MCF7 CELL LINES USING MTT ASSAY

In the present study, MTT assay was studied on MCF cell line which is treated with
synthesized silver nanoparticles of Streptomyces althioticus. The figure MTT assay results

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confirming the in vitro cytotoxicity of AgNPs against MCF cell lines. The results of the
present study are similar to that of the work done by Panchanathan Manivasagan et al., 2013.
After exposure to AgNPs for 24 hrs, the MCF cell shows the significant morphological
changes, which are characteristic features of apoptotic cells, such as loss of membrane
integrity, cell shrinkage, and reduced cell density.[20]

Images for results

Figure.1-depicts the primary screening of potent marine actinomycetes againsmicrobes.

Before incubation (a) After incubation (b)

Figure.2 shows the synthesis of silver nanoparticles from the cell free extract of
Streptomyces althioticus

Figure. 3 Green synthesized silver nanoparticle

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Figure 4 depicts the SEM image of silver nanoparticles from Streptomyces althioticus

Figure 5 shows the TEM image of silver nanoparticles from Streptomyces althioticus

Figure.6 The XRD image of Green synthesized silver nanoparticles from Streptomyces
althioticus

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Figure.7 shows the FTIR image of Green synthesized silver nanoparticles from
Streptomyces althioticus

Table 1 shows Zone of inhibition of the Ag-NPs synthesized from Streptomyces althioticus
with 1mM concentrations of AgNO3.
zone of zone of
S.no TEST ORGANISM
inhibition(mm) inhibition(mm)
1. Staphylococcus aureus 10 18
2. Bacillus subtilis 12 21
3. Proteus vulgaris 9 17
4. Escherichia coli 8 15

Figure.8 depicts the Radical Scavenging Activity of silver nanoparticles synthesized

from marine actinomycetes – Streptomyces althioticus

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Figure.9 Evaluation of Cytotoxicity of AgNPs on MCF7 cell line using MTT assay.

Figure: 2a &2b: FASTA and BLAST analysis

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Fig:2 FASTARun Ended: 2015/3/19Signal G:1542 A:1434 C:1751

Analysis 8:58:35 T:1339
Lane: 65 Base spacing: 15.13223 1313 bases in 16300 scans

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CONCLUSION
In the present study, the identified species was streptomyces althioticus is a good source for
synthesizing silver nanoparticle. The silver nanoparticles exhibited a tremendous potential
antibacterial activity against common human pathogenic bacteria and it also proved that it

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posses both free radical scavenging activity and anticancerous activity. In future, the
compound responsible for these activities could be isolate and converted into nanoparticles
and even extend the study on human cell line. This study concludes that the biosynthesized
AgNPs from actinobacteria can be a prominent source for the development of various
nanomedicines.

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