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Nanomed J, Vol. 1, No. 4, Summer 2014 229
Received: Nov. 19, 2013; Accepted: Jan. 22, 2014
Vol. 1, No. 4, Summer 2014, page 229-237
Received: Apr. 22, 2014; Accepted: Jul. 12, 2014
Vol. 1, No. 5, Autumn 2014, page 298-301
Online ISSN 2322-5904
http://nmj.mums.ac.ir
Original Research
Production of gold nanoparticles by Streptomyces djakartensis isolate B-5
Sara Biglari1*
, Gholam Hosein Shahidi2
, Gholam Reza Sharifi3
1
Department of Agricultural Biotechnology, College of Agriculture, Shahid Bahonar University of Kerman, Iran
2
Department of Plant pathology, College of Agriculture, Shahid Bahonar University of Kerman, Iran
3
Department of Agricultural Biotechnology, College of Agriculture, Hormozgan University, Iran
Abstract
Objective(s): Biosynthesis of gold nanoparticles (NGPs) is environmentally safer than
chemical and physical procedures. This method requires no use of toxic solvents and
synthesis of dangerous products and is environmentally safe. In this study, we report the
biosynthesis of NGPs using Streptomyces djakartensis isolate B-5.
Materials and Methods: NGPs were biosynthesized by reducing aqueous gold chloride
solution via a Streptomyces isolate without the need for any additive for protecting
nanoparticles from aggregation. We characterized the responsible Streptomycete; its genome
DNA was isolated, purified and 16S rRNA was amplified by PCR. The amplified isolate was
sequenced; using the BLAST search tool from NCBI, the microorganism was identified to
species level.
Results: Treating chloroauric acid solutions with this bacterium resulted in reduction of gold
ions and formation of stable NGPs. TEM and SEM electro micrographs of NGPs indicated
size range from 2- 25 nm with average of 9.09 nm produced intracellular by the bacterium.
SEM electro micrographs revealed morphology of spores and mycelia. The amplified PCR
fragment of 16S rRNA gene was cloned and sequenced from both sides; it consisted of 741
nucleotides. According to NCBI GenBank, the bacterium had 97.1% homology with
Streptomyces djakartensis strain RT-49. The GenBank accession number for partial 16S
rRNA gene was recorded as JX162550.
Conclusion: Optimized application of such findings may create applications of
Streptomycetes for use as bio-factories in eco-friendly production of NGPs to serve in
demanding industries and related biomedical areas. Research in this area should also focus on
the unlocking the full mechanism of NGPs biosynthesis by Streptomycetes.
Keywords: Bio-factory, Green synthesis, Nanogold, Biosynthesis, rRNA, Streptomyces
*Corresponding author: Sara Biglari, Department of Agricultural Biotechnology, Shahid Bahonar University
of Kerman, Iran.
Tel: +989133415137, Email: biglari.sara@gmail.com
Production of nanogold particles by Streptomyces djakartensis
230 Nanomed J, Vol. 1, No. 4, Summer 2014
Introduction
Metallic nanoparticles are presently
applied in different fields of electronics,
biotechnology, cosmetics and medicine (1,
2). Biomedical applications of nanogold
particles [NGPs] have received great
attention in recent years (3, 4, 5) and have
attracted enormous scientific and
technological interest due to their unique
properties (6, 7). In the recent years,
scientists accelerate the application of
nanoparticles to the prevention,
biodiagnosis (3), drug delivery (8, 9) and
treatment of cancer (10-12).
Nanotechnology represents economic
alternatives for chemical and physical
methods of nanopaticles formation. It
combines biological principles with
physical and chemical procedures to
generate nano-sized particles with specific
functions. Among the biological
organisms, some microorganisms such as
bacteria, fungi, and yeasts have been
exploited for biosynthesis of nanoparticles
(13, 14). Among tested microorganisms,
species of Streptomyces have the most
diversity for production of intracellular
NGPs (15); however, they are becoming
promising BioFactories in this regard (16).
Various methods have been reported over
the last two decades for the synthesis of
Au nanoparticles, who involve reduction
of HAuCl4 with a chemical reducing agent
or some organic compounds (17);
however, green techniques in the
biosynthesis of NGPs would benefit from
the development of clean, nontoxic, and
environmentally acceptable procedures
concerning micro-organisms.
Streptomycetes are grampositive aerobic
bacteria; some of them are capable of
transformation of some heavy metals ions
and reducing them to amorphous or
crystalline metal particles. In this paper we
report the biosynthesis of NGPs by
reducing aqueous gold chloride solution
viaa Streptomyces isolate without the need
for any additive for protecting
nanoparticles from aggregation. We
characterized the responsible
Streptomycete; its genome DNA was
isolated, purified and 16S rRNA was
amplified by PCR. The amplified isolate
was sequenced; using the BLAST search
tool from NCBI, the microorganism was
identified to species level.
Materials and Methods
Soil sampling and isolation of
Streptomyce-tes
Soil samples were randomly collected
from grasslands, orchards and vegetable
fields in Sefteh village in Kerman
Province of Iran. Soil samples were taken
from a depth of 10 to 20 cm below the soil
surface using an open-end soil borer (20
cm in depth, 2.5 cm in diameter) as
described by Lee and Hwang (18). After
air-drying at room temperature for 7 to 10
days, soil samples were sieved with a 0.8
mm mesh and preserved in polyethylene
bags at room temperature. Ten gram of
each soil sample was brought up to final
volume of 100 ml by adding sterile
distilled water. The mixtures were shaken
vigorously for one hour and allowed to
settle for an extra hour. The supernatant
was gently removed and diluted to 10-2
-
10-6
with sterile distilled water. Aliquots of
each of 10-3
- 10-6
dilutions added to
autoclaved CGA at 50° C (1:20 v/v) and
poured to sterile Petri plates. After
solidification, plates incubated at 28° C for
7- 10 days. From day 7, Streptomyces
colonies isolated as pure cultures on CGA,
grown at 28° C for one week and stored at
4° C for further investigations.
Culture medium
Casein glycerol agar (CGA) was prepared
from basic ingredients as described by
Dhingra and Sinclair (19) and used as
Streptomyces growth medium.
Excluding the agar from CGA, casein
glycerol (CG) medium was applied for
Streptomyces submerged cultures.
Submerged cultures and preparation of
biomass
CG media used to prepare liquid media.
Isolated Streptomyces inoculated to CG
Biglari S, et al
Nanomed J, Vol. 1, No. 4, Summer 2014 231
media and shaked on rotary shakers at 29°
C and 130 rpm for five days; cultures
filtered through Whatman filter paper No.
1, rinsed twice with distilled water to
remove the media. The resulting biomass
was then used for screening.
Screening for production of NGPs
Portions of biomass of each isolate
removed and divided to two samples in
glass tubes of bench centrifuge. Both
samples centrifuged at 4000 rpm/5 min,
clear supernatants discarded and colorful
biomass resuspended in DDH2O and
subjected to two more cycles of low
centrifugations. Two ml of 10-3
mol of
HAuCl4 added to 0.2 g of biomass pellet
and agitated to uniformly resuspend; while
control received plain DDH2O. Both
samples placed on rotary shaker at 130
rpm and 29° C for 12 – 24 hr. Color
change from yellow to purple red was
indicative of bioactivity of tested
Streptomyces in production of NGPs;
however, NGPs were confirmed with
further analysis as described in coming
sections.
Verification of intra-cellular and/or
extra-cellular production of NGPs
To verify whether NGPs are produced
intra and/or extra-cellular, portion of
colored biomass (treated for 12-24 hr with
HAuCl4) was subjected to low speed
centrifugation at 4000 rpm for 10 min.
Since the supernatant was colorless and
clear, it was not further processed for
presence of NGPs (all particle sizes of
NGPs are colorful as red, purple, violet
and not yellowish). The red violet pellet
was resuspended in DDH2O and subjected
to two more cycles of low speed
centrifugation. The final pellet
resuspended in DDH2O and applied to 0.2
µ filter. The filtrate was colorless clear but
the retained biomass was red violet
indicative of probable production of intra-
cellular presence of NGPs. The Millipore
filter-retained biomass relatively
desiccated under vacuum and used for
further TEM and Flame photometric
analysis.
TEM studies
Streptomyces djakartensis isolate B-5
selected through screening as described at
previous section and used for evaluations
with TEM (Philips CM10, 100kv).
The biomasses of both treated and control
were investigated as described by Mandal,
et al. (20). Carbon and formvar coated
TEM grids were applied with a thin layer
from 12 – 24 hr HAuCl4 treated mycelial
suspension (in DDH2O) and excess
removed by Whatman filter paper No 1.
Electro micrographs with scale bars
prepared from more than 200 GNPs and
used to measure particle size distribution
and mean size values; however, no
Zetasizer was available to do this task with
higher precision.
Flame photometric analysis
The Millipore filter-retained biomass of S.
djakartensis isolate B-5 having red violet
color was further rinsed with large volume
of DDH2O to retain the mycelia and spores
but wash off traces of ionic Au from the
biomass. Filter-retained biomass (0.2 g,
relatively dry) was suspended in 20 ml
DDH2O and sonicated using a sonicator
(NSK dental sonicator, Varios 570 Ipiezo
engine) to rapture the cells, homogenize
the sample and release intracellular NGPs.
From homogenized sample (dilution of 10-
1
), further dilutions of 10-4
, 10-5
and 10-6
made in DDH2O and used to
measure NGP
concentration by a flame photometer
(Varian AA240FS) equipped with gold
lamp. Average ppm concentration revealed
by the instrument used to calculate the
approximate conversion yield (Au ion to
NGPs).
Uv–vis spectroscopy
The produced NGPs also analyzed by use
of uv–vis spectroscopy. In this regard, the
sample which was used in flame
Production of nanogold particles by Streptomyces djakartensis
232 Nanomed J, Vol. 1, No. 4, Summer 2014
photometric analysis (previous section)
was also used for this evaluation. The
spectrum was taken from 400-800 nm
wave lengths with a double beam
spectrophotometer Model ScanDrop 250-
211F075.
STM studies
The criteria of spore surface and myclial
morphology determined for this
Strepyomyces isolate by a STM (Lutz
100A, 20kv) as described by Gehring, et
al., (21).
Extraction of Genomic DNA
The active Streptomyces sp. isolate (earlier
designated as B-5, while isolated) was
propagated in submerged culture of CG
medium in a shaker incubator at 28°C and
130 rpm for 6 days.
The colonies were then harvested using
bench centrifugation at 10000 rpm for 5
min and genomic DNA isolated by CTAB
method as described by Baharlouei, et al.
(22).
Polymerase chain reaction (PCR)
Two sets of primers consist of F16 SS (5’-
ACGGGTGAGTAACACG-3’) and R16
SS (5’-TACCGCGGCTGCTGGCACG-
3’) were designed based on the known
sequences from the family 16S rRNA
genes of Streptomyces spp. The primers
were synthesized in Isogene Company,
Netherlands. A PCR was run which
utilized
melting temperature of 94°C for 5 min,
following 35 cycles of 94°C for 1 min,
55°C for 1 min, 72°C for 1 min and
final extension at 72°C for 7 min.
Electrophoresis of sample in 1% agarose
gel and photography of resolved band
performed as described by Sambrook and
Russell (23).
Cloning of DNA fragment of 16S rRNA
gene of Strepyomyces isolate B-5
The amplified genes fragment was purified
with an AccuPrep® PCR purification kit
(Takapouzist, Iran), ligated into the
pTZ57R/T vector using InsT/A Clone™
PCR cloning kit (Fermentas, Germany).
Escherichia coli, XL1blue and plasmid
pTZ57R/T were provided from Cinagen
Co., Iran and Fermentas Co., Germany,
as bacterial host strain and vector in
cloning experiments, respectively.
Results
Soil sampling and isolation of
Streptomyces spp
From the soil samples cultured, over 50
Streptomyces isolated recovered which
used for further evaluations.
Screening for production of NGPs
The isolate, designated as B-5, showed
high performance in production of
NGPs compared to others.
The isolate was further characterized
and identified as Streptomyces
djakartensis. As indicated in Figure 1,
color change from pale yellow (Au ion)
to red violet (production of NGPs) was
indicative of NGPs production by this
isolate.
NGPs size analyses
To assess approximate size properties of
NGPs for S. djakartensis isolate B-5, by
Figure 1. Color change indicative of NGPs
production by Streptomyces strain; the color
change of aqueous solution of HauCl4 to red after
24 h incubation with active biomass of this strain
was indicative of NGPs production. Test tube
containing before (right) and after (left) 24 h
incubation of Streptomyces djakartensis isolate B-
5.
Biglari S, et al
Nanomed J, Vol. 1, No. 4, Summer 2014 233
use of TEM electro micrographs, NGP
sizes measured, particle size distribution
plotted, mean value and standard
deviation
determined statistically using Microsoft
Office Excel 2007 software.
Analysis of particle size distribution
and mean of particle sizes are presented
in Fig. 2.
As indicated in the figure, intracellular
NGPs produced by S. djakartensis isolate
B-5 range from 2- 25 nm with mean size
of 9.09 nm.
These values assessed from evaluating 213
NGPs in electro micrographs.
As indicated earlier, no Zetasizer was
available to measure particle sizes with
higher precision.
Figure 2. NGPs particle size distribution and mean
of particle sizes produced by Streptomyces
djakartensis isolate B-5.
TEM studies
The NGPs were observed within the
cells in TEM electro micrographs.
NGPs production by S. djakartensis
isolate B-5 is indicated in Figure 3.
The approximate sizes of NGPs were
estimated 2- 25 nm. Presence of both
spherical (probably icosahedra) and
triangular NGPs within the cells were
noticeable; however, spherical ones
were dominant. Although some
aggregations were noticeable in the
electro micrographs, but magnification
of the pictures clearly revealed margins
of NGPs in each aggregated mass.
Figure 3. TEM electro micrograph of intracellular
production of NGP by Streptomyces djakartensis
isolate B-5. The approximate sizes of NGPs are 2-
25 nm. Both spherical (probably icosahedra) and
triangular NGPs are noticeable. Some aggregations
are also present which upon magnification margins
of NGPs is recognizable in each aggregated mass.
Flame photometric analysis
The result of flame photometric analysis of
sonicated biomass of S. djakartensis
isolate B-5 (red violet biomass retained on
0.2µ Millipore filter) indicated presence of
gold in the dilutions used. The average
mean concentration of gold from the three
dilutions (10-4
, 10-5
and 10-6
) was
indicative of 12.93 ppm.
Considering the original used
concentration of Au ion in 10-3
mol as 197
ppm, the percentage of recovery calculated
was 6.56%. The low recovery is due to
use of low ratio of original biomass to Au
ion in the experiment; however the
recovery would improve if the proportions
are optimized.
Uv–vis Spectroscopy: The uv–vis spectrum
of NGPs produced by S. djakartensis
isolate B-5 indicated presence of a broad
single peak with maximum absorbance at
530 nm. The spectrum is indicated in
Figure 4.
Production of nanogold particles by Streptomyces djakartensis
234 Nanomed J, Vol. 1, No. 4, Summer 2014
STM studies
STM electro micrograph of Streptomyces
djakartensis isolate B-5 was indicative that
spores form in simple chains and have
smooth surfaces.
Figure 4. UV-Vis Spectrum of nanogold particles
produced by Streptomyces djakartensis isolate B-5.
Presence of a broad single peak with maximum
absorbance at 530 nm is prominent.
No specific sculptures detected on spore
surfaces. STM electro micrograph of this
isolate is indicated in Figure 5. As
indicated in the figure, this strain forms
long straight chains of spores with smooth
surfaces.
Cloning of 16S rRNA genes
Fragment of 16S rRNA gene was cloned
as of ~800 bp from Streptomyces isolate
B-5 via forward and reverse primers of 16
SS using cDNA as the template.
The amplified PCR fragment was cloned
and sequenced from both sides.
The fragment consisted of 741 nucleotides.
The result of amplification of 16S rRNA
gene of Streptomyces isolate B-5 is
indicated in Figure 6.
DNA sequencing
Recombinant plasmid was sequenced in
both directions by extending 16 SS reverse
and forward primers by Automatic DNA
Sequencer (Macrogene, Korea).
The sequence data of Strepyomyces isolate
B-5 was analyzed using the Chromas
software version 1.41.
Nucleotide fragment BLAST-ed by
nucleotide blast program in the NCBI
databank and sequence aligned with
related sequences from other isolates of
Streptomyces by DNAMAN software
package (Lynnon Biosoft, Quebec,
Canada).
Figure 5. STM image of Streptomyces djakartensis
isolate B-5. This strain forms long straight chains
of spores with smooth surfaces.
Figure 6. Amplification of 16S rRNA gene (800
bp) of Streptomyces isolate B-5 (right) from
recombinant pTZ57R/T plasmid (3000 bp, digested
with EcoRI and PstI enzymes) along the DNA size
ladder (left, 100 bp).
Nucleotide sequence homology matrix
and accession number
Homology matrix of the sequence of
partial 16S rRNA gene from Streptomyces
isolate B-5, and sequences of other
families of partial 16S rRNA genes are
represented in Table 1.
Biglari S, et al
Nanomed J, Vol. 1, No. 4, Summer 2014 235
Table 1. Homology matrix of the sequence of partial 16S rRNA gene from Streptomyces isolate B-5 and partial
sequences of related species.
B-5: Streptomyces djakartensis isolate B-5
Homology matrix of 11 sequences
sequence of B5 100%
Streptomyces djakartensis strain RT-49 97.1% 100%
Streptomyces enissocaesilis strain RT-46 97.1% 100.0% 100%
Streptomyces geysiriensis strain RT-63 97.1% 100.0% 100.0% 100%
Streptomyces olivaceus strain RT-54 97.1% 100.0% 100.0% 100.0% 100%
Streptomyces plicatus strain RT-57 97.1% 100.0% 100.0% 100.0% 100.0% 100%
Streptomyces rochei strain T170 97.1% 99.6% 99.6% 99.6% 99.6% 99.6% 100%
Streptomyces rochei strain Lac3 97.1% 99.5% 99.5% 99.5% 99.5% 99.5% 99.4% 100%
Streptomyces avidinii strain SU4 97.1% 98.2% 98.2% 98.2% 98.2% 98.2% 99.3% 99.3% 100%
Rhodococcus sp. SH05-06 87.9% 89.5% 89.5% 89.5% 89.5% 89.5% 88.9% 88.7% 88.6% 100%
Thermomonospora sp. YIM 75085 84.7% 84.4% 84.4% 84.3% 84.4% 84.4% 85.5% 85.7% 83.3% 82.7% 100%
As indicated in the table, Streptomyces
isolate B-5 has 97.1% homology with
Streptomyces djakartensis. The GenBank
accession number for partial 16S rRNA
gene of Streptomyces isolate B-5 was
recorded as JX162550.
Discussion
The area of microbial biosynthesis of
NGPs is a relatively new. The mechanism
of biosynthesis has not been fully
elucidated in the literature; however,
according to Kitching (24) Au+3
ions
switch on oxidative stress response genes.
Possible methylation step reduces Au+3
to
Au+1
, then NADH (or NADPH) dependant
proteins (e.g. ATPase, 3-glucan binding
protein and glycer-aldehydes 3phosphate
dehydrogenase) in the cell wall and
cytoplasmic regions reduce the Au+1
to
Au0
.
The neutral Au forms NGPs, which in turn
are stabilized by proteins, most likely
different from the proteins involved in the
synthesis.
Streptomycetes are characterized by
complex secondary metabolites [3]. These
diverse characteristics plus their simple
growth needs has made them to receive
high attention in screening for NGPs
among microorganisms; however, they
produce over two-thirds of the clinically
useful antibiotics of natural origin (25).
Colony morphology, SEM electro
micrographs and sequencing of 16sRNA
of our tested microorganism indicated that
it belongs to the genus Streptomyces. Its
genome DNA was isolated, purified and
16S rRNA was amplified by PCR. The
amplified isolate was sequenced; using the
BLAST search tool from NCBI the
microorganism was identified.
Streptomyces isolate B-5 had 97.1%
homology with S. djakartensis. Since it is
the first time that its ability in production
of gold nanoparticles is
investigated, it was not feasible to deduce
NGPs metabolic pathways from the
genetic analysis and even phylogenic tree
of their closely related species.
Streptomyces djakartensis isolate B-5
examined in this study proved its ability to
produce gold nanoparticles from an
aqueous solution containing HAuCl4.
The results clearly showed that NGPs
formed by reduction of Au (III) ions by the
bacterium. Evaluations of the electro
Production of nanogold particles by Streptomyces djakartensis
236 Nanomed J, Vol. 1, No. 4, Summer 2014
micrograph of intracellular biosynthesized
NGPs indicated that they ranged in size
from 2 to 25 nm with average size of 9.09
nm.
The results of our investigation and similar
studies may create valuable applications of
Streptomycetes as bio-factories for eco-
friendly production of NGPs to serve in
demanding industries and relative
biomedical areas. For optimization,
research in this area should focus on
unlocking the full mechanism of NGPs
biosynthesis by S. djakartensis isolate B-5.
We would like to postulate that biological
synthesis of NGPs is worth because of
eco-friendly, environ-mentally acceptable
solvent system, and lack of toxic
chemicals involved in this green
biosynthesis method; however more resea-
rch is needed to optimize the procedure.
Conclusion
Optimized application of such findings
may create applications of Streptomycetes
for use as bio-factories in eco-friendly
production of NGPs to serve in demanding
industries and related biomedical areas.
Research in this area should also focus on
the unlocking the full mechanism of NGPs
biosynthesis by Streptomycetes.
Acknowledgements
This study performed partially under the
grant No. 91003706 provided by Iran
National Science Foundation (INSF).
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Production of Nanogold Particles by Streptomyces Bacterium

  • 1. Nanomed J, Vol. 1, No. 4, Summer 2014 229 Received: Nov. 19, 2013; Accepted: Jan. 22, 2014 Vol. 1, No. 4, Summer 2014, page 229-237 Received: Apr. 22, 2014; Accepted: Jul. 12, 2014 Vol. 1, No. 5, Autumn 2014, page 298-301 Online ISSN 2322-5904 http://nmj.mums.ac.ir Original Research Production of gold nanoparticles by Streptomyces djakartensis isolate B-5 Sara Biglari1* , Gholam Hosein Shahidi2 , Gholam Reza Sharifi3 1 Department of Agricultural Biotechnology, College of Agriculture, Shahid Bahonar University of Kerman, Iran 2 Department of Plant pathology, College of Agriculture, Shahid Bahonar University of Kerman, Iran 3 Department of Agricultural Biotechnology, College of Agriculture, Hormozgan University, Iran Abstract Objective(s): Biosynthesis of gold nanoparticles (NGPs) is environmentally safer than chemical and physical procedures. This method requires no use of toxic solvents and synthesis of dangerous products and is environmentally safe. In this study, we report the biosynthesis of NGPs using Streptomyces djakartensis isolate B-5. Materials and Methods: NGPs were biosynthesized by reducing aqueous gold chloride solution via a Streptomyces isolate without the need for any additive for protecting nanoparticles from aggregation. We characterized the responsible Streptomycete; its genome DNA was isolated, purified and 16S rRNA was amplified by PCR. The amplified isolate was sequenced; using the BLAST search tool from NCBI, the microorganism was identified to species level. Results: Treating chloroauric acid solutions with this bacterium resulted in reduction of gold ions and formation of stable NGPs. TEM and SEM electro micrographs of NGPs indicated size range from 2- 25 nm with average of 9.09 nm produced intracellular by the bacterium. SEM electro micrographs revealed morphology of spores and mycelia. The amplified PCR fragment of 16S rRNA gene was cloned and sequenced from both sides; it consisted of 741 nucleotides. According to NCBI GenBank, the bacterium had 97.1% homology with Streptomyces djakartensis strain RT-49. The GenBank accession number for partial 16S rRNA gene was recorded as JX162550. Conclusion: Optimized application of such findings may create applications of Streptomycetes for use as bio-factories in eco-friendly production of NGPs to serve in demanding industries and related biomedical areas. Research in this area should also focus on the unlocking the full mechanism of NGPs biosynthesis by Streptomycetes. Keywords: Bio-factory, Green synthesis, Nanogold, Biosynthesis, rRNA, Streptomyces *Corresponding author: Sara Biglari, Department of Agricultural Biotechnology, Shahid Bahonar University of Kerman, Iran. Tel: +989133415137, Email: biglari.sara@gmail.com
  • 2. Production of nanogold particles by Streptomyces djakartensis 230 Nanomed J, Vol. 1, No. 4, Summer 2014 Introduction Metallic nanoparticles are presently applied in different fields of electronics, biotechnology, cosmetics and medicine (1, 2). Biomedical applications of nanogold particles [NGPs] have received great attention in recent years (3, 4, 5) and have attracted enormous scientific and technological interest due to their unique properties (6, 7). In the recent years, scientists accelerate the application of nanoparticles to the prevention, biodiagnosis (3), drug delivery (8, 9) and treatment of cancer (10-12). Nanotechnology represents economic alternatives for chemical and physical methods of nanopaticles formation. It combines biological principles with physical and chemical procedures to generate nano-sized particles with specific functions. Among the biological organisms, some microorganisms such as bacteria, fungi, and yeasts have been exploited for biosynthesis of nanoparticles (13, 14). Among tested microorganisms, species of Streptomyces have the most diversity for production of intracellular NGPs (15); however, they are becoming promising BioFactories in this regard (16). Various methods have been reported over the last two decades for the synthesis of Au nanoparticles, who involve reduction of HAuCl4 with a chemical reducing agent or some organic compounds (17); however, green techniques in the biosynthesis of NGPs would benefit from the development of clean, nontoxic, and environmentally acceptable procedures concerning micro-organisms. Streptomycetes are grampositive aerobic bacteria; some of them are capable of transformation of some heavy metals ions and reducing them to amorphous or crystalline metal particles. In this paper we report the biosynthesis of NGPs by reducing aqueous gold chloride solution viaa Streptomyces isolate without the need for any additive for protecting nanoparticles from aggregation. We characterized the responsible Streptomycete; its genome DNA was isolated, purified and 16S rRNA was amplified by PCR. The amplified isolate was sequenced; using the BLAST search tool from NCBI, the microorganism was identified to species level. Materials and Methods Soil sampling and isolation of Streptomyce-tes Soil samples were randomly collected from grasslands, orchards and vegetable fields in Sefteh village in Kerman Province of Iran. Soil samples were taken from a depth of 10 to 20 cm below the soil surface using an open-end soil borer (20 cm in depth, 2.5 cm in diameter) as described by Lee and Hwang (18). After air-drying at room temperature for 7 to 10 days, soil samples were sieved with a 0.8 mm mesh and preserved in polyethylene bags at room temperature. Ten gram of each soil sample was brought up to final volume of 100 ml by adding sterile distilled water. The mixtures were shaken vigorously for one hour and allowed to settle for an extra hour. The supernatant was gently removed and diluted to 10-2 - 10-6 with sterile distilled water. Aliquots of each of 10-3 - 10-6 dilutions added to autoclaved CGA at 50° C (1:20 v/v) and poured to sterile Petri plates. After solidification, plates incubated at 28° C for 7- 10 days. From day 7, Streptomyces colonies isolated as pure cultures on CGA, grown at 28° C for one week and stored at 4° C for further investigations. Culture medium Casein glycerol agar (CGA) was prepared from basic ingredients as described by Dhingra and Sinclair (19) and used as Streptomyces growth medium. Excluding the agar from CGA, casein glycerol (CG) medium was applied for Streptomyces submerged cultures. Submerged cultures and preparation of biomass CG media used to prepare liquid media. Isolated Streptomyces inoculated to CG
  • 3. Biglari S, et al Nanomed J, Vol. 1, No. 4, Summer 2014 231 media and shaked on rotary shakers at 29° C and 130 rpm for five days; cultures filtered through Whatman filter paper No. 1, rinsed twice with distilled water to remove the media. The resulting biomass was then used for screening. Screening for production of NGPs Portions of biomass of each isolate removed and divided to two samples in glass tubes of bench centrifuge. Both samples centrifuged at 4000 rpm/5 min, clear supernatants discarded and colorful biomass resuspended in DDH2O and subjected to two more cycles of low centrifugations. Two ml of 10-3 mol of HAuCl4 added to 0.2 g of biomass pellet and agitated to uniformly resuspend; while control received plain DDH2O. Both samples placed on rotary shaker at 130 rpm and 29° C for 12 – 24 hr. Color change from yellow to purple red was indicative of bioactivity of tested Streptomyces in production of NGPs; however, NGPs were confirmed with further analysis as described in coming sections. Verification of intra-cellular and/or extra-cellular production of NGPs To verify whether NGPs are produced intra and/or extra-cellular, portion of colored biomass (treated for 12-24 hr with HAuCl4) was subjected to low speed centrifugation at 4000 rpm for 10 min. Since the supernatant was colorless and clear, it was not further processed for presence of NGPs (all particle sizes of NGPs are colorful as red, purple, violet and not yellowish). The red violet pellet was resuspended in DDH2O and subjected to two more cycles of low speed centrifugation. The final pellet resuspended in DDH2O and applied to 0.2 µ filter. The filtrate was colorless clear but the retained biomass was red violet indicative of probable production of intra- cellular presence of NGPs. The Millipore filter-retained biomass relatively desiccated under vacuum and used for further TEM and Flame photometric analysis. TEM studies Streptomyces djakartensis isolate B-5 selected through screening as described at previous section and used for evaluations with TEM (Philips CM10, 100kv). The biomasses of both treated and control were investigated as described by Mandal, et al. (20). Carbon and formvar coated TEM grids were applied with a thin layer from 12 – 24 hr HAuCl4 treated mycelial suspension (in DDH2O) and excess removed by Whatman filter paper No 1. Electro micrographs with scale bars prepared from more than 200 GNPs and used to measure particle size distribution and mean size values; however, no Zetasizer was available to do this task with higher precision. Flame photometric analysis The Millipore filter-retained biomass of S. djakartensis isolate B-5 having red violet color was further rinsed with large volume of DDH2O to retain the mycelia and spores but wash off traces of ionic Au from the biomass. Filter-retained biomass (0.2 g, relatively dry) was suspended in 20 ml DDH2O and sonicated using a sonicator (NSK dental sonicator, Varios 570 Ipiezo engine) to rapture the cells, homogenize the sample and release intracellular NGPs. From homogenized sample (dilution of 10- 1 ), further dilutions of 10-4 , 10-5 and 10-6 made in DDH2O and used to measure NGP concentration by a flame photometer (Varian AA240FS) equipped with gold lamp. Average ppm concentration revealed by the instrument used to calculate the approximate conversion yield (Au ion to NGPs). Uv–vis spectroscopy The produced NGPs also analyzed by use of uv–vis spectroscopy. In this regard, the sample which was used in flame
  • 4. Production of nanogold particles by Streptomyces djakartensis 232 Nanomed J, Vol. 1, No. 4, Summer 2014 photometric analysis (previous section) was also used for this evaluation. The spectrum was taken from 400-800 nm wave lengths with a double beam spectrophotometer Model ScanDrop 250- 211F075. STM studies The criteria of spore surface and myclial morphology determined for this Strepyomyces isolate by a STM (Lutz 100A, 20kv) as described by Gehring, et al., (21). Extraction of Genomic DNA The active Streptomyces sp. isolate (earlier designated as B-5, while isolated) was propagated in submerged culture of CG medium in a shaker incubator at 28°C and 130 rpm for 6 days. The colonies were then harvested using bench centrifugation at 10000 rpm for 5 min and genomic DNA isolated by CTAB method as described by Baharlouei, et al. (22). Polymerase chain reaction (PCR) Two sets of primers consist of F16 SS (5’- ACGGGTGAGTAACACG-3’) and R16 SS (5’-TACCGCGGCTGCTGGCACG- 3’) were designed based on the known sequences from the family 16S rRNA genes of Streptomyces spp. The primers were synthesized in Isogene Company, Netherlands. A PCR was run which utilized melting temperature of 94°C for 5 min, following 35 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 1 min and final extension at 72°C for 7 min. Electrophoresis of sample in 1% agarose gel and photography of resolved band performed as described by Sambrook and Russell (23). Cloning of DNA fragment of 16S rRNA gene of Strepyomyces isolate B-5 The amplified genes fragment was purified with an AccuPrep® PCR purification kit (Takapouzist, Iran), ligated into the pTZ57R/T vector using InsT/A Clone™ PCR cloning kit (Fermentas, Germany). Escherichia coli, XL1blue and plasmid pTZ57R/T were provided from Cinagen Co., Iran and Fermentas Co., Germany, as bacterial host strain and vector in cloning experiments, respectively. Results Soil sampling and isolation of Streptomyces spp From the soil samples cultured, over 50 Streptomyces isolated recovered which used for further evaluations. Screening for production of NGPs The isolate, designated as B-5, showed high performance in production of NGPs compared to others. The isolate was further characterized and identified as Streptomyces djakartensis. As indicated in Figure 1, color change from pale yellow (Au ion) to red violet (production of NGPs) was indicative of NGPs production by this isolate. NGPs size analyses To assess approximate size properties of NGPs for S. djakartensis isolate B-5, by Figure 1. Color change indicative of NGPs production by Streptomyces strain; the color change of aqueous solution of HauCl4 to red after 24 h incubation with active biomass of this strain was indicative of NGPs production. Test tube containing before (right) and after (left) 24 h incubation of Streptomyces djakartensis isolate B- 5.
  • 5. Biglari S, et al Nanomed J, Vol. 1, No. 4, Summer 2014 233 use of TEM electro micrographs, NGP sizes measured, particle size distribution plotted, mean value and standard deviation determined statistically using Microsoft Office Excel 2007 software. Analysis of particle size distribution and mean of particle sizes are presented in Fig. 2. As indicated in the figure, intracellular NGPs produced by S. djakartensis isolate B-5 range from 2- 25 nm with mean size of 9.09 nm. These values assessed from evaluating 213 NGPs in electro micrographs. As indicated earlier, no Zetasizer was available to measure particle sizes with higher precision. Figure 2. NGPs particle size distribution and mean of particle sizes produced by Streptomyces djakartensis isolate B-5. TEM studies The NGPs were observed within the cells in TEM electro micrographs. NGPs production by S. djakartensis isolate B-5 is indicated in Figure 3. The approximate sizes of NGPs were estimated 2- 25 nm. Presence of both spherical (probably icosahedra) and triangular NGPs within the cells were noticeable; however, spherical ones were dominant. Although some aggregations were noticeable in the electro micrographs, but magnification of the pictures clearly revealed margins of NGPs in each aggregated mass. Figure 3. TEM electro micrograph of intracellular production of NGP by Streptomyces djakartensis isolate B-5. The approximate sizes of NGPs are 2- 25 nm. Both spherical (probably icosahedra) and triangular NGPs are noticeable. Some aggregations are also present which upon magnification margins of NGPs is recognizable in each aggregated mass. Flame photometric analysis The result of flame photometric analysis of sonicated biomass of S. djakartensis isolate B-5 (red violet biomass retained on 0.2µ Millipore filter) indicated presence of gold in the dilutions used. The average mean concentration of gold from the three dilutions (10-4 , 10-5 and 10-6 ) was indicative of 12.93 ppm. Considering the original used concentration of Au ion in 10-3 mol as 197 ppm, the percentage of recovery calculated was 6.56%. The low recovery is due to use of low ratio of original biomass to Au ion in the experiment; however the recovery would improve if the proportions are optimized. Uv–vis Spectroscopy: The uv–vis spectrum of NGPs produced by S. djakartensis isolate B-5 indicated presence of a broad single peak with maximum absorbance at 530 nm. The spectrum is indicated in Figure 4.
  • 6. Production of nanogold particles by Streptomyces djakartensis 234 Nanomed J, Vol. 1, No. 4, Summer 2014 STM studies STM electro micrograph of Streptomyces djakartensis isolate B-5 was indicative that spores form in simple chains and have smooth surfaces. Figure 4. UV-Vis Spectrum of nanogold particles produced by Streptomyces djakartensis isolate B-5. Presence of a broad single peak with maximum absorbance at 530 nm is prominent. No specific sculptures detected on spore surfaces. STM electro micrograph of this isolate is indicated in Figure 5. As indicated in the figure, this strain forms long straight chains of spores with smooth surfaces. Cloning of 16S rRNA genes Fragment of 16S rRNA gene was cloned as of ~800 bp from Streptomyces isolate B-5 via forward and reverse primers of 16 SS using cDNA as the template. The amplified PCR fragment was cloned and sequenced from both sides. The fragment consisted of 741 nucleotides. The result of amplification of 16S rRNA gene of Streptomyces isolate B-5 is indicated in Figure 6. DNA sequencing Recombinant plasmid was sequenced in both directions by extending 16 SS reverse and forward primers by Automatic DNA Sequencer (Macrogene, Korea). The sequence data of Strepyomyces isolate B-5 was analyzed using the Chromas software version 1.41. Nucleotide fragment BLAST-ed by nucleotide blast program in the NCBI databank and sequence aligned with related sequences from other isolates of Streptomyces by DNAMAN software package (Lynnon Biosoft, Quebec, Canada). Figure 5. STM image of Streptomyces djakartensis isolate B-5. This strain forms long straight chains of spores with smooth surfaces. Figure 6. Amplification of 16S rRNA gene (800 bp) of Streptomyces isolate B-5 (right) from recombinant pTZ57R/T plasmid (3000 bp, digested with EcoRI and PstI enzymes) along the DNA size ladder (left, 100 bp). Nucleotide sequence homology matrix and accession number Homology matrix of the sequence of partial 16S rRNA gene from Streptomyces isolate B-5, and sequences of other families of partial 16S rRNA genes are represented in Table 1.
  • 7. Biglari S, et al Nanomed J, Vol. 1, No. 4, Summer 2014 235 Table 1. Homology matrix of the sequence of partial 16S rRNA gene from Streptomyces isolate B-5 and partial sequences of related species. B-5: Streptomyces djakartensis isolate B-5 Homology matrix of 11 sequences sequence of B5 100% Streptomyces djakartensis strain RT-49 97.1% 100% Streptomyces enissocaesilis strain RT-46 97.1% 100.0% 100% Streptomyces geysiriensis strain RT-63 97.1% 100.0% 100.0% 100% Streptomyces olivaceus strain RT-54 97.1% 100.0% 100.0% 100.0% 100% Streptomyces plicatus strain RT-57 97.1% 100.0% 100.0% 100.0% 100.0% 100% Streptomyces rochei strain T170 97.1% 99.6% 99.6% 99.6% 99.6% 99.6% 100% Streptomyces rochei strain Lac3 97.1% 99.5% 99.5% 99.5% 99.5% 99.5% 99.4% 100% Streptomyces avidinii strain SU4 97.1% 98.2% 98.2% 98.2% 98.2% 98.2% 99.3% 99.3% 100% Rhodococcus sp. SH05-06 87.9% 89.5% 89.5% 89.5% 89.5% 89.5% 88.9% 88.7% 88.6% 100% Thermomonospora sp. YIM 75085 84.7% 84.4% 84.4% 84.3% 84.4% 84.4% 85.5% 85.7% 83.3% 82.7% 100% As indicated in the table, Streptomyces isolate B-5 has 97.1% homology with Streptomyces djakartensis. The GenBank accession number for partial 16S rRNA gene of Streptomyces isolate B-5 was recorded as JX162550. Discussion The area of microbial biosynthesis of NGPs is a relatively new. The mechanism of biosynthesis has not been fully elucidated in the literature; however, according to Kitching (24) Au+3 ions switch on oxidative stress response genes. Possible methylation step reduces Au+3 to Au+1 , then NADH (or NADPH) dependant proteins (e.g. ATPase, 3-glucan binding protein and glycer-aldehydes 3phosphate dehydrogenase) in the cell wall and cytoplasmic regions reduce the Au+1 to Au0 . The neutral Au forms NGPs, which in turn are stabilized by proteins, most likely different from the proteins involved in the synthesis. Streptomycetes are characterized by complex secondary metabolites [3]. These diverse characteristics plus their simple growth needs has made them to receive high attention in screening for NGPs among microorganisms; however, they produce over two-thirds of the clinically useful antibiotics of natural origin (25). Colony morphology, SEM electro micrographs and sequencing of 16sRNA of our tested microorganism indicated that it belongs to the genus Streptomyces. Its genome DNA was isolated, purified and 16S rRNA was amplified by PCR. The amplified isolate was sequenced; using the BLAST search tool from NCBI the microorganism was identified. Streptomyces isolate B-5 had 97.1% homology with S. djakartensis. Since it is the first time that its ability in production of gold nanoparticles is investigated, it was not feasible to deduce NGPs metabolic pathways from the genetic analysis and even phylogenic tree of their closely related species. Streptomyces djakartensis isolate B-5 examined in this study proved its ability to produce gold nanoparticles from an aqueous solution containing HAuCl4. The results clearly showed that NGPs formed by reduction of Au (III) ions by the bacterium. Evaluations of the electro
  • 8. Production of nanogold particles by Streptomyces djakartensis 236 Nanomed J, Vol. 1, No. 4, Summer 2014 micrograph of intracellular biosynthesized NGPs indicated that they ranged in size from 2 to 25 nm with average size of 9.09 nm. The results of our investigation and similar studies may create valuable applications of Streptomycetes as bio-factories for eco- friendly production of NGPs to serve in demanding industries and relative biomedical areas. For optimization, research in this area should focus on unlocking the full mechanism of NGPs biosynthesis by S. djakartensis isolate B-5. We would like to postulate that biological synthesis of NGPs is worth because of eco-friendly, environ-mentally acceptable solvent system, and lack of toxic chemicals involved in this green biosynthesis method; however more resea- rch is needed to optimize the procedure. Conclusion Optimized application of such findings may create applications of Streptomycetes for use as bio-factories in eco-friendly production of NGPs to serve in demanding industries and related biomedical areas. Research in this area should also focus on the unlocking the full mechanism of NGPs biosynthesis by Streptomycetes. Acknowledgements This study performed partially under the grant No. 91003706 provided by Iran National Science Foundation (INSF). References 1. Kohler JM, Csaki A, Reichert R, Straube W, Fritzche W. Selective labeling of oligonucleotide monolayers by metallic nanobeads for fast optical readout of DNA-chips. Sens Actuators B Chem. 2001; 76(1-3): 166-172. 2. Schatz GC, Lazarides AA, Kelly KL, Jensen TR. Optical properties of metal nanoparticles aggregates important in biosensors. J Mol Struct (Theochem). 2000; 529(1-3): 59-63. 3. Rosi NL, Mirkin CA. Nanostructures in biodiagnostics. Chem Rev. 2005; 105: 1547-1562. 4. Ferrari M. Cancer nanotechnology: opportunities and challenges. Nat Rev Cancer. 2005; 5(3): 161-171. 5. Murphy CJ, Gole AM, Stone JW, Sisco PN, Alkilany AM, Goldsmith EC, et al. Gold nanoparticles in biology: beyond toxicity to cellular imaging. Acc Chem Res. 2008; 41(12): 1721-1730. 6. Liu YL, Shipton MK, Ryan J, Kaufman ED, Franzen S, Feldheim DL. Synthesis, stability, and cellular internalization of gold nanoparticles containing mixed peptide-poly (ethylene glycol) monolayers. Anal Chem. 2007; 79(6): 2221–2229. 7. Khan JA, Pillai B, Das TK, Singh Y, Maiti S. Molecular effects of uptake of gold nanoparticles in HeLa cells. ChemBioChem. 2007; 8(11): 1237-1240. 8. Han G, Ghosh P, Rotello VM. Functionalized gold nanoparticles for drug delivery. Nanomedicine (Lond). 2007; 2(1): 113-123. 9. Sarin H, Kanevsky AS, Wu H, Brimacombe KR, Fung SH, Sousa AA, et al. Effective transvascular delivery of nanoparticles across the blood-brain tumor barrier into malignant glioma cells. J Transl Med. 2008; 6: 1-15. 10. Raoof M, Corr SJ, Kaluarachchi WD, Massey KL, Briggs K, Zhu C, et al. Stability of antibody-conjugated gold nanoparticles in the endolysosomal nanoenvironment: implications for noninvasive radiofrequency-based cancer therapy. Nanomedicine. 2012; 8(7): 1096- 1105. 11. Eghtedari M, Liopo AV, Copland JA, Oraevslty AA, Motamedi M. Engineering of hetero-functional gold nanorods for the in vivo molecular targeting of breast cancer cells. Nano Lett. 2009; 9(1): 287- 291. 12. Bonjar LS. "Nanogold detoxifying machine" to remove idle nanogold particles from blood stream of cancer patients treated with antibody-nanogold therapeutics. Med Hypotheses. 2013; 80(5): 601-605. 13. Sadowski Z. Biosynthesis and Application of Silver and Gold Nanoparticles In: Perez DP, editor. Silver Nanoparticles. China: InTech; 2010. 14. Newman D K, Kolter R. A role for excreted quinones in extracellular electron transfer. Nature. 2000; 405(6782): 94-97. 15. Sapkal MR, Deshmukh AM. Biosynthesis of Gold Nanoparticles by Streptomyces species. Res J Biotech. 2008; 3(2): 36-39.
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