International Journal of Nanotechnology

and Application (IJNA)

ISSN(P): 2277-4777; ISSN(E): 2278-9391
Vol. 4, Issue 5, Oct 2014, 9-18
TJPRC Pvt. Ltd.

ANTIMICROBIAL ACTIVITY OF EXTRACELLULARLY SYNTHESIZED SILVER

NANOPARTICLES USING SACCHAROPOLYSPORA ERYTHRAEA, A POTENT
ACTINOMYCETE AND ITS EFFICACY AGAINST MOSQUITO LARVAE
PRABHA S. B.1, VIGNESH A2, ELUMALAI D3, KALEENA P. K4 & MURUGESAN K5
1,2,5

Professor, CAS in Botany, University of Madras, Guindy Campus, Chennai, Tamil Nadu, India
3,4

Department of Zoology, Presidency College (Autonomous), Chennai, Tamil Nadu, India

ABSTRACT
The actinomycetes have the ability to produce secondary metabolites. The biosynthesis of silver nanoparticles
using terrestrial actinomycete Saccharopolyspora erythraea is the first and novel report ever reported. Although
actinomycetes are well known for its antibiotic production, it is less exploited in terms of usage for nanoparticles.
The silver nanoparticles are characterized using several analyses such as UV spectrophotometer, XRD, FTIR,
FE-SEM and HR-TEM. The silver surface plasma resonance was observed at 450nm which confirmed the formation of
AgNPs. The XRD pattern confirmed the formation of nanocrystals. The FTIR analysis is involved in the reduction of
silver salts into respective nanoparticles. In FE-SEM with EDX, the average size of the nano particle was found to be 82.13
to 93.86nm and it confirmed the chemical analysis of the fields. The HR-TEM analysis with SAD showed the synthesized
AgNPs are polydispersed in 6 to 26 nm scale which confirmed the formation of AgNPs. The antimicrobial activity of the
ethyl acetate extract of AMP14 amended with that of the SNPs gave good results and showed maximum zone of inhibition
against the five human pathogens namely Bacillus subtilis, Klebsiella pneumoniae, Vibrio cholerae, Salmonella typhi and
Staphylococcus aureus. The ethyl acetate extract of the AMP14 was found to be less toxic than that of the synthesized
silver nanoparticles against the larvae of the Anopheles stephensi and Aedes aegypti the larvicidal activity was the first
report of Saccharopolyspora erythraea.

KEYWORDS: Saccharopolyspora erythraea, Silver Nanoparticle, Larvicidal Activity, Antimicrobial Activity,

HR-TEM, Anopheles stephensi, Aedes aegypti

INTRODUCTION
Mosquitoes are the vectors for various dreadful diseases of mankind and one of the most medically significant
vectors transmitting pathogens and continue to have a devastating impact on human beings and other animals. Among the
3492 species of mosquitoes recorded worldwide, more than a hundred species are capable of transmitting various diseases
to human and other vertebrates (Rueda, 2008) such as malaria, dengue, filariasis, yellow fever, Japanese encephalitis and
chikungunya (Nour et al, 2009).
Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus are the major urban vectors of malaria, dengue
and lymphatic filariasis respectively. About 90% of all malarial deaths in the world occur mostly in Africa and South
Sahara. Aedes aegypti is the principal vector of dengue fever, hemorrhagic dengue fever and is reported to infect more than
hundred million, every year in South East Asia to a lesser extent in more than 110 countries in the tropics. Incidence of
dengue fever has increased in the last few years, and nearly half of the worlds population is now at risk
www.tjprc.org

editor@tjprc.org

10

Prabha S. B, Vignesh A, Elumalai D, Kaleena P. K & Murugesan K

(Elumalai et al, 2013, Rahuman, 2008).

Among 72 countries worldwide more than 1.3 billion people are threatened by lymphatic filariasis, commonly
known as elephantiasis. Over 120 million people are infected currently, with about 40 million disfigured and incapacitated
by the disease (WHO, 2012). Culex mosquitoes are painful and persistent biters and are responsible for filariasis.
The most widely used conventional methods for mosquito control are the chemical control since it produces
immediate control and relatively inexpensive. Chemical control is generally carried out by the indoor residual spraying of
insecticides such as dichloro diphenyl tri-chloro ethane, hexa chlorocyclo hexane, benzene hexa chloride, melathion and
synthetic pyrothroid. The development of resistance against these chemicals in various mosquito populations has been
reported.
One of the approaches for controlling mosquito- borne diseases is the interruption of disease transmission by
either killing or preventing mosquito bite by using repellents or by causing larval mortality in a large scale at the breeding
centers of the vector. The control of mosquito larvae worldwide depends on continued application of organophosphates and
insect growth regulators (Hoffmann et al, 2008).
Synthesis of nanoparticles using plants or microorganisms could potentially eliminate this problem by making the
nanoparticles more bio-compatible. Indeed, over the past several years, plants, algae, fungi, bacteria, and viruses have been
used as an energy-efficient and nontoxic production of metallic nanoparticles (Thakkar et al, 2009).
Silver nanoparticles have been used extensively in the food storages, textile coating, in health industry and it
contains number of environmental applications as anti bacterial and larvicidal agents. The evidence of toxicity of silver is
still not clear inspite of decades of use.
Actinomycetes are screened for its routine bioactive substances for its high commercial value of bioactive
compounds. Approximately two thirds of antibiotics and several medicinal important compounds have been isolated from
actinomycetes.
Some of the secondary metabolites species of actinomycetes produce are avermectins, tetranectin, faerifungin and
macrotetrolides and flavonoids produced by the genera involved are Micromonospora, Actinomadura, Actinoplanes,
Micropolyspora, Nocardiopsis, Oerskonia, Thermonospora, Streptoverticillium and Chainia were found to be toxic for the
mosquitoes (Ando, 1983).
The Gram positive actinomycetes are primarily recognized as the potential source for antibiotics, potential
degraders and organism of academic curiosity. Actinomycetes are less exploited for production of nanoparticles
eventhough they are well exploited for the production of high metabolite value and antibiotics. For the intracellular and
extracellular biosynthesis of nanoparticles, only limited reports are available for actinomycetes.
Silver nanoparticles shows very strong bactericidal activity against Gram positive as well as Gram negative
bacteria including multiresistant strains (Shrivastava et al, 2007). Hence there is a huge scientific progress in the study of
biological application of ZnO and Ag and other metal nanoparticles. Actinomycetes provides a valuable resource for novel
products of industrial interest, including antimicrobial agents.

Impact Factor (JCC): 1.8003

Index Copernicus Value (ICV): 3.0

Antimicrobial Activity of Extracellularly Synthesized Silver Nanoparticles

Using Saccharopolyspora erythraea, a Potent Actinomycete and its Efficacy Against Mosquito Larvae

11

MATERIALS AND METHODS

Sample Collection
The present investigation was carried out by collection of soil samples from three different districts such as
Kancheepuram, Villupuram and Dharmapuri. Tamil Nadu State, India. The collected samples were carefully stored in
polythene bags and transported to the laboratory for further assay.
Isolation of Actinomycetes
Isolation of actinomycetes was performed by serial dilution and plating technique using Starch Casein Agar
(SCA) medium. Ten grams of soil sample were taken in 95ml of water and placed in a shaker for 2 hours. The preparation
is designated as the master dilution. From the master dilution, actinomycetes were isolated by serial dilution technique and
cultured on SCA.
Screening of actinomycetes for Antibacterial Efficacy
The selected actinomycetes were screened for antibacterial efficacy by agar well diffusion method. The five
human pathogens namely Bacillus subtilis, Klebsiella pneumoniae, Vibrio cholerae, Salmonella typhi and Staphylococcus
aureus were obtained from Microbial Type Culture Collection (MTCC), Chandigarh, India.
Preparation of S. erythraea Ethyl Extract and Synthesis of Ag NPs
Five mL of this bacteria S. erythraea ethyl acetate extract was added to 95 mL of 1 mm aqueous silver nitrate
solution for the reduction of Ag+ ions. The effect of temperature on the synthesis rate of AgNPs was studied by incubation
at 37 C for 24 h. The AgNPs solution, thus obtained was purified by centrifugation twice at 10,000 g. for 15 min at 4 C.
A control setup was also maintained without S. erythraea ethyl acetate extract and color intensity of the extracts was
measured.
Characterization of Silver Nanoparticles
UV- visible spectra were recorded as a function of the reaction time on UV- 160V, spectrophotometer, Shimadzu,
Japan. The studies on size, morphology and composite of the nanoparticles were performed by means of HR TEM, FE
SEM and EDX. The purified AgNPs were examined for the presence of biomolecules using FTIR analysis. The spectrum
obtained from the dried sample was recorded on FTIR spectrum in the diffuse reflectange mode at a resolute on of 4 cm-1
in KBr pellets. Crystalline AgNPs were determined by X-ray diffraction analysis. Briefly, the biosynthesized AgNPs were
laid onto glass substrate on Phillips PW 1830 instrument operating at a voltage of 40 kv and current of 30 mA with Cu K
1 radiation.
Larvicidal Bioassay
The larvae of A. stephensi and A. aegypti were collected from rice field and stagnant water areas of Kanchipuram
and identified in The Zonal Entomological Research Centre, Vellore, and Tamil Nadu to start the colony. The larvae were
reared in the laboratory according to the method of (Kamaraj et al. 2009). One gram of ethyl acetate extract was first
dissolved in 100 ml of distilled water (stock solution). From the stock solution, 100 mg/L was prepared with dechlorinated
tap water for bioassay test of ethyl acetate extract. The larvicidal activity was assessed by the procedure of WHO (1996)
with modifications. For bioassay test, larvae were taken in five batches of 20 in 199 mL of water and 1.0 ml of ethyl
acetate extract concentration. The control was set up with dechlorinated tap water. The numbers of dead larvae were
www.tjprc.org

editor@tjprc.org

12

Prabha S. B, Vignesh A, Elumalai D, Kaleena P. K & Murugesan K

counted after 24 h of exposure, and the percentage of mortality was reported from the average of five replicates.
The experimental media in which 100% mortality of larvae occurs were alone selected for doseresponse bioassay.
Synthesized AgNPs toxicity test was performed by placing 20 mosquito larvae into 200 ml of sterilized double distilled
water with nanoparticles. The nanoparticle solutions were diluted using double distilled water as a solvent according to the
desired concentrations (10, 8, 6, 4 and 2 mg/l). Each test included a set of control groups (silver nitrate and distilled water)
with five replicates for each individual concentration. Mortality was assessed after 24 h to determine the acute toxicities
against fourth instar larvae of A. stephensi and A. aegypti . Twenty mosquito larvae were placed into 250 ml glass beakers
and set in an environmental chamber at 25 C with a 16:8 h light/dark cycle. Each beaker containing the mosquito larvae
distilled water was spiked with solutions of AgNPs in order to achieve target nominal concentrations of 10, 8, 6, 4 and 2
mg/l with a final volume of 200 ml. A negative control (silver nitrate) was used in all experiments and all conditions were
tested in five replicates. In order to compare the mortality of synthesized AgNPs to that of dissolved Ag released the
mosquito larvae were exposed to a range of dissolved Ag concentrations so as to cover the range released from all doses of
AgNPs. To avoid settling of particles especially at higher doses, all treatments were sonicated for an additional 5 min prior
to the addition of mosquito larvae. This additional sonication appeared to significantly decrease the settling of particles.
Dose Response Bioassay
Based on the preliminary screening results, crude bacteria ethyl acetate extract of S. erythrae and synthesized
AgNPs were subjected to doseresponse bioassay for larvicidal activity against the larvae of A. stephensi and A. aegypti.
Different concentrations ranging from 6.25 to 100.0 mg/l (aqueous extract) and 2.0 to 10.0 mg/l (synthesized AgNPs and
silver nitrate) were prepared for larvicidal activity. The numbers of dead larvae were counted after 24 h of exposure,
and the percentage mortality was recorded from the average of five replicates.
Data Analysis
The average larval mortality data were subjected to probit analysis for calculating LC50, LC90 and other statistics
at 95 % fiducial limits of upper confidence limit and lower confidence limit, and chi-square values were calculated using
the software developed by Reddy et al. (1992). Results with p < 0.05 were considered to be statistically significant. Mean
percent larval mortality data were subjected to analysis of variance and compared with Duncans multiple range tests to
determine any differences between plant species and within species and concentrations (SPSS, 11.5). Prior to analysis,
mortality in treatments was corrected with that in controls using Abbott formula (Abbott, 1925).

RESULTS AND DISCUSSIONS

UV- vis Analysis of Saccharopolyspora erythrae
Silver nanoparticles were synthesized using ethyl acetate extract of S. erythraea and within five seconds, of
incubation period, the colour change was observed by visual observation. Ethyl acetate extract of S. erythraea without
AgNO3 did not show any color change (Figure 2). The aqueous silver nitrate solution turned to brown within five seconds
and the intensity increased in direct proportion to incubation period. The absorption spectrum of S. erythraea ethyl acetate
extract at different wavelength range from 200 to 800 nm and the silver surface plasma resonance observed at 450 nm
confirmed the formation of AgNPs. Chemical synthesis resulted in the formation of brown colour which results from
absorption by colloidal silver nanoparticles in the visible (380- 450 nm) range of the electromagnetic spectrum. The colour
formation was mainly due to the surface plasmon resonance of deposited silver nanoparticles and silver nanoparticles
exhibit striking colors due to excitation of surface plasm on vibrations in the particles (Kapoor, 1998).
Impact Factor (JCC): 1.8003

Index Copernicus Value (ICV): 3.0

13

Antimicrobial Activity of Extracellularly Synthesized Silver Nanoparticles

Using Saccharopolyspora erythraea, a Potent Actinomycete and its Efficacy Against Mosquito Larvae

XRD Pattern of Synthesized Silver Nanoparticles

The XRD pattern compared with the standard confirmed spectrum of silver particles formed in the present study
were shown in (Figure 2) the form of nanocrystals as evidenced by the peaks at 2 values of 27.71o, 32.16o, 46.15o, 54.70o
and 57.68o corresponding to 210, 122, 231, 142, and 241 facets of the face centered cubic crystal structure, which is a
characteristic of nano crystallites, the prominent peaks corresponding to 210 brags reflection. X-ray diffraction (XRD)
pattern show in tense Braggs reflection that can be indexed on the basis of the fcc structure of silver (Mandal, 2005).
FTIR Analysis
FTIR analysis was performed to identify the biomolecules localized on the surface and responsible for the
reduction of silver salts into the respective nanoparticles. FTIR band intensities in different regions of the spectrum for the
synthesized Ag NPs were analyzed (Figure 3) and the spectra showed the respective peaks and functional groups of 2922
cm-1 (C-H, stretch, alkanes), 1753 cm-1 ( C=O, stretch carboxylic acid), 1633 cm-1 (N-H bend 1 amines), 1107 cm-1
( C-N, stretch aliphatic amines). Silver nanoparticles were probably attached to the nitrogen of amine and the amide groups
and oxygen of carbonyl groups. The overall observations confirmed the presence of protein in the sample of silver
nanoparticles. It is reported earlier that proteins could bind to nano particles either through free amine groups or cystiene
residues (Csaki et al, 2002).
FE- SEM Analysis with EDX
The FE-SEM images of nanoparticles having prominently cubic structure and the magnified SEM image
confirmed that the silver nanocubes are in well resolved cubic structure with soft surface and with sharp edges with an
average size of 82.13 to 93.86 nm. The EDX attachment with the FE-SEM was known to provide information on the
chemical analysis of the fields that are being investigated or the composition at specific locations (Figure 4).
Representative profile of the spot EDX was obtained by focusing on AgNPs. In this micrograph, silver nanoparticles in the
size range 50-100 nm were observed (Minaeian, 2008).
HR- TEM Analysis with SAED
HR-TEM images revealed that the synthesized silver nanoparticles are polydispersed in 6 to 26 nm scale and they
were spherical in shape with varying size (Figure 5). However, in contrast to many other reports of metal-resistant bacteria,
for which efflux of toxic ions is the main detoxification mechanism (Gupta et al, 1999), majority of the accumulated silver
is deposited as seed like particles between the outer membrane and the plasma membrane, the size of the deposits small
(515 nm) compared to previous studies using P. stutzeri Ag259 (3546 nm), which may be caused by different cell
growth and metal incubation conditions (Haefeli et al, 1984).
Larvicidal Activity of Ethyl Acetate Extract of S. erythraea and Synthesized Silver Nanoparticles
In the present study, range of concentrations of S. erythraea (100, 50, 25, 12.5 and 6.5 mg/L) and synthesized
AgNPs (10, 8, 6, 4 and 2 mg/L) were tested against 4th instar of A. stephensi, and A. aegypti. The LC50 and LC90 along with
upper and lower confidence limit values and regression equations of A. stephensi, and A. aegypti against ethyl acetate
extract of S. erythraea and the synthesized Ag NPs are presented in (Table 1).
The ethyl acetate extract of S. erythraea were less toxic than the synthesized silver nanoparticles against three
tested mosquito species. The LC50 and LC90 values of ethyl acetate extract of S. erythraea treated against fourth instar
www.tjprc.org

editor@tjprc.org

14

Prabha S. B, Vignesh A, Elumalai D, Kaleena P. K & Murugesan K

larvae of A. stephensi (LC50 = 32.99; LC90 = 94.07 mg/L) and A. aegypti (LC50 = 37.26; LC90 = 114.00 mg/L). While LC50
and LC90 values of synthesized AgNPs treated against fourth instars larvae of A. stephensi (LC50 = 3.89; LC90 = 11.82
mg/L) an A. aegypti (LC50 = 4.02; LC90 = 12.64 mg/L) respectively. The larvicidal potential of hexane, chloroform, ethyl
acetate, acetone, methanol, and aqueous leaf extracts of N. nucifera and synthesized silver nanoparticles using aqueous leaf
extract against the fourth instar larvae of A. subpictus and C. quinquefasciatus have been tested (Santhoshkumar et al,
2011). These silver nanoparticles have been tested against dengue and malarial vector. There is no data available on the
efficacy of silver nanoparticles synthesized using bacteria against mosquito larvae. The larvicidal potential of silver
nanoparticles synthesized using fungus C. lunatus A. aegypti and A. stephenesi has been tested (Salunkhe et al, 2011).
Antimicrobial Efficacy of Saccharopolyspora erythraea
Screening of the isolated actinomycetes amended with silver nitrate for their antibacterial activity against five
human bacterial pathogens was done using well diffusion method. Among all the isolates which are amended with silver
nitrate screened the maximum inhibition zone was observed in Staphylococcus aureus compared with the other four human
bacterial pathogens such as Bacillus subtilis, Klebsiella pneumoniae, Vibrio cholerae, and Salmonella typhi.

CONCLUSIONS
Although, nanoparticles are being synthesized using chemical and physical methods, however the adverse effects
of thee methods sought for the discovery of novel sustainable methods. In the present study, to the best of our knowledge, a
novel terrestrial actinomycete strain of Saccharopolyspora erythraea which can synthesize silver nanoparticles
concomitantly in their respective solutions. These results may open up a novel path for research communities to identify
the particular protein from this potential actinomycete which is responsible for nanoparticle synthesis in extracellular
secretion.
Further

the

larvicidal

efficacy

against.

Anopheles

stephensi,

Aedes

aegypti

AgNPs

synthesized

Saccharopolyspora erythraea and found to have an immediate impact on mosquito control. Therefore, Saccharopolyspora
erythraea may be a more rapid and environmentally friendly approach for vector control other than current approaches.

Figure 1: UV- VIS Absorption Spectra of Silvernanoparticle Synthesized from

Saccharopolyspora erythraea at 1mm Silver Nitrate

Impact Factor (JCC): 1.8003

Index Copernicus Value (ICV): 3.0

15

Antimicrobial Activity of Extracellularly Synthesized Silver Nanoparticles

Using Saccharopolyspora erythraea, a Potent Actinomycete and its Efficacy Against Mosquito Larvae

Figure 2: XRD Patterns of AgNPs Synthesized Using Ethyl Acetate Extract of Saccharopolyspora erythraea
100.0
95

SNP

90
85
80
75
70
65
60
55
%T

3648

50
45
40
35
30
25
2922
2852

20
15

1733

1633

10

1384

1107

5
0.0
4000.0

3600

3200

2800

2400

2000

1800
cm-1

1600

1400

1200

1000

800

600

450.0

Figure 3: FTIR Spectrum of Synthesized AgNPs Using Ethyl Acetate Extract of Saccharopolyspora erythraea

Figure 4: FE-SEM and EDX Image of AgNPs Synthesized by Saccharopolyspora erythraea

Figure 5: Transmission Electron Microscopic Image Showing Synthesized

AgNPs from Saccharopolyspora erythraea

www.tjprc.org

editor@tjprc.org

16

Prabha S. B, Vignesh A, Elumalai D, Kaleena P. K & Murugesan K

Table 1: Larvicidal Activity of Broth S. erythraea and Synthesized AgNPs of S. erythraea against Fourth in Star
Larvae of Anopheles stephensi and Aedes aegypti

REFERENCES
1.

Kapoor, S; (1998), Preparation, characterization and surface modification of silver particles. Langmuir,
14; 1021-1025

2.

Csaki, A, Moller, A, Fritzsche, W. (2002). Gold nanoparticles as novel label for DNA diagnostics. Expert Rev.
Mol. Diag. 2(2); 187-193.

3.

Gupta A, Matsui K, Lo JF, Silver S (1999) Molecular basis for resistance to silver cations in Salmonella. Nat Med
5:183188. doi:10.1038/5545

4.

Minaeian, S, Shahverdi, A. R, Nohi, A. S. Shahverdi, H. R, 2008. Extracellular biosynthesis of silver

nanoparticles by some bacteria. J. Sci. I. A. U (JSIAU), Vol 17, No. 66, 2008.

5.

S. Mandal, S. Phadtare and M. Sastry. Interfacing biology with nano particles. Current Applied Physics, Vol. 5,
No. 2; 2005. Pp. 118-127.

6.

Mitsuiki, S, M. Sakai, Y. Moriyama, M. Goto and Furukawa, K. 2002. Purification and some properties of
Keratinolytic enzyme from an alkaliphilic Nocardiopsis sp. Biosci. Biotechnol. Biochem. 66: 164-167.

7.

Shrivastava, S, T. Bera, A. Roy, G. Singh, P. Ramachandrarao and Dash, D. 2007. Characterization of enhanced
antibacterial effects of novel silver nanoparticles. Nanotechnol, 18:9-12

8.

T. Santhoshkumar, A. A. Rahuman, G. Rajakumar, S. Ma- rimuthu, A. Bagavan, C. Jayaseelan, A. A. Zahir,

G. Elan- go and C. Kamaraj, Synthesis of Silver Nanoparticles Using Nelumbo nucifera Leaf Extract and Its
Larvicidal Activity against Malaria and Filariasis Vectors, Parasi-tology Research, Vol. 108, No. 3, 2011,
pp. 693-702.

Impact Factor (JCC): 1.8003

Index Copernicus Value (ICV): 3.0

Antimicrobial Activity of Extracellularly Synthesized Silver Nanoparticles

Using Saccharopolyspora erythraea, a Potent Actinomycete and its Efficacy Against Mosquito Larvae

9.

17

Roe, D, B. Karandikar, N. Bonn Savage, B. Gibbins and Roullet, J.B. 2008. Antimicrobial surface
functionalization of plastic catheters by silver nanoparticles. J. Antimicro. Chemo. 61: 869-76.

10. R. B. Salunkhe, S. V. Patil, C. D. Patil and B. K. Salunkhe, Larvicidal Potential of Silver Nanoparticles
Synthesized Using Fungus Cochliobolus lunatus against Aedes aegypti (Linnaeus, 1762) and Anopheles stephensi
Liston (Diptera; Culicidae), Parasitology Research, Vol. 109, No. 3, 2011, pp. 823-831.
11. W. S. Abbott, A Method of Computing the Effectiveness of an Insecticide, Journal of Economical Entomology,
Vol. 18, No. 1, 1925, pp. 265-266.
12. Zhang, L, Y. Jiang, M. Povey and York, D. 2007. Investigation into the antimicrobial behavior of suspension of
ZnO nanoparticles (ZnO nanofluids). J. Nanoparti. Res. 9: 479-489.

www.tjprc.org

editor@tjprc.org