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International Journal of ChemTech Research CODEN (USA): IJCRGG, ISSN: 0974-4290, ISSN(Online):2455-9555 Vol.10 No.3, pp 577-582, 2017
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Detection on sliver nanoparticles production by Streptomyces spp. isolated from soil samples in Hilla city
Samer M. Al-Hulu
Al-Qasim Green University, College of Food Sciences, Hilla-Iraq
Abstract : (22) soil samples were collected from Hilla city. Ten isolates of Actinomycetes were recovered. Five isolates were identified as Streptomyces spp. Depending on morphological and biochemical assay. Streptomyces spp. Isolates were tested for sliver nanoparticle production. The results showed that one Streptomyces spp. Isolate have ability for producing of sliver nanoparticle. Silver nanoparticles (Ag NPs) production by Streptomyces spp. was detectedby adding (1mM silver nitrate) to supernatant culture, yellowish- brown colour production after incubation indicate to formation of Silver nanoparticles. Cultural characteristics of Streptomyces spp.4 isolate was aerial mycelium with grey color, yellowish-brown substrate mycelium on yeast malt agar. Atomic force microscopy (AFM) results showed that AgNPs are spherical in shapes with the particle diameter (121.30nm). The surface thickness is 191 nm ,it represents thickness of the film surface roughness. Antimicrobial activity of particle was determined. The results showed that Streptomyces.4with high activity againstE.coli with (14 mm) inhibition zone compared (11mm) against S.aureus ,(8 mm) against C. albicans.
Key words : Streptomycesspp., sliver nanoparticle production,Antimicrobial activity.
Introduction
Nanotechnology research is one of emerging areas of research with its using in science for manufacturing of new compounds at nanolevel 2. Generally Nanoparticles are 0.1–1000 nm in size and made by two methods:top–down and bottom–up 19.Silver nanoparticles have exclusive optically, electric, and thermal featureswhich incorporate into products which range from photovoltaics to biological and chemical sensors 10. Silver nanoparticles have a great importance among metal nanomaterials, because physicochemical properties1.
Streptomycetes are actinomycetes member, which live in natural environment3.It’s a saprophytic organisms which spend the most life cycles as semi dormant spores. In life cycle, Streptomycetes sporesgerminate for producing substrate mycelium, which during maturation fragments into chains of spores8.
Generally, synthesize many different metabolites, antibioticis represent the most known product which used in veterinary and pharmaceutical side 17.Streptomyces spp. positive for gram stain, filament bacteria and produce different compound with having biologically active compounds such ashydrolytic enzymes, antibiotics, and enzyme inhibitors 21.A biologically synthesized AgNPs using Streptomyces sp. VITBT7 isolated from soil was recorded24. These AgNPs showed SPR peak at 420 nm and spherical shape with 20–70 nm in size .AgNPs showed antimicrobial activity against fungal and bacterial pathogens 23.An extracellular synthesizes AgNPs by Streptomyces sp. JAR isolatedfrom the soil samples and with size AgNPs was 68.13 nm, and showed antimicrobial activity against bacterial and fungal pathogens5.
This study aimed for isolation of Streptomyces spp. having ability for producing of sliver nanoparticle and study antibacterial activity for these particle.
Materials and Methods
Isolation of Streptomyces Spp.
(22) Soil samples gathered from Hilla city. Samples treated with calcium carbonateLdehydrate in hot air oven (45oC) for 1 hr. to decreasing the bacteria and mold incidence. Dilution plate technique was used for isolating of Streptomyces spp .on (YMD) agar medium .The pH was made(7.2).The plate incubated at 30oC for ten days 20-25.
Screening for Streptomyces spp with silver nanoparticles production:
Streptomyces was cultivated to Luria Broth medium and incubate at 37ºC on closing rotary shaker (100 rpm for 72 hrs). Broth was centrifuging (7500 rpm for 15 min). Pellet and supernatant were collect separately. Ten ml of 1mM (AgNO3) add into fifty ml of supernatant1.
Characteristics of Streptomyces spp.4 isolate:
Streptomyces cultural characteristics examined on YMD agar,such as aerial mycelium color, substrate myceliumcolor and pigment production by Streptomyces spp.isolate. Morphological characteristics of Streptomyces spp. checked 20.Carbon utilization sources was made 4.
Antibacterial activity for silver nanoparticles:
The biosynthesized activity of AgNPs was tested by well diffusion method. Wells were made in Muller Hinton agar. Plates inoculated with (S.aureus,E.coli, C.albicans) as test pathogens . Fifty microliters of AgNPs were pipetted into each well. After incubation at 37 oC overnight. Inhibition zone diameter was measured in mm 16.
Atomic force microscopy for silver nanoparticles:
Silver nanoparticles topography examined by AFM (Model AA2000 , made in USA) University of Babylon, College of Science, physical department Thin film for sample prepareon a glass slide (100 µL of the sample) was taken and dropped onto the slide, and drying for 5 min. 9.
Results and Discussion
Isolation of Streptomyces spp. isolates:
(22) soil samples gathered from different places in Hilla town. Ten of these were identified as Actinomycetes spp.Out of these five Streptomyces spp. isolates were identified as Streptomyces spp..All isolates positive forgram stain with aerial mycelium grey in color and yellowish- green substrate mycelium when cultivated on yeast malt extract agar.Actinomycetes are positive for gramexhibit a filament growth like fungi. Its aerobic and wide spread in nature13. Streptomyces are, gram positive, aerobic actinomycetes which form branch, substrate mycelium, aerial hyphae that differentiate tostrings of spores, with LL-diaminopimelic acid andno major characteristic sugars in whole-organism hydrolysates12 and having DNA rich in guanine plus cytosine14.
Screening for biosynthesis of silver nanoparticles:
Five isolates of Streptomyces spp. were checked for production of nanoparticle. One Streptomyces spp. isolate have ability for sliver nanoparticle production .Streptomyces spp.4 supernatant was pale-yellow in color before the addition of silver ions, after that changed to yellowish-brown at the end of the reaction with silver ions (Figure 1).
A
B
Figure (1): Streptomyces spp.4Supernatant mixed with 1Mm AgNO3 .A: Atfirst of incubation (a pale yellow color),B: after 72 hour of incubation ( a yellowish- brown color).
Yellowish-brown color which appearin the flasks mixed with silver nitrate was a indication forformation of silver nano particle 22by reducing of Ag+ ions and formation of surface plasmon resonance in the reaction mixture, no colour change appeared in culture filtrates without silver nitrate15- 26. The Nanoparticlessynthesis by actinomycetes has many importance e.g. they are safe to handle, easily available, and possess variable metabolites that may help in reduction. Moreover, these particles have unnumbered applications6.
The cultural features ofStreptomyces spp.4
The features of Streptomyces spp.4culturewas examined.It was grampositive and aerial myceliumgrey in color when grown on yeast malt extract agar, it was unable melanin producing on tyrosine broth medium. It diagnosed as Streptomycesdepending on morphology and sugar fermentation (Table1).
Table (1):Morphology and biochemical test of Streptomyces spp.4
Results |
Streptomyces spp.4 |
+ |
gram stain |
grey |
aerial mycelium |
yellow-green |
substrate mycelium |
- |
Melanin producing |
+ |
Earthy odor |
- |
Indole |
+ |
Methyl red production |
- |
Vogesproskauer |
+ |
Citrate utilization |
Sugar fermentation |
|
+ |
glucose |
- |
sucrose
|
+ |
mannitol |
- |
ribose |
Atomic force Microscope (AFM) of sliver nanoparticles:
The 3D images results of AFM indicate to formation of homogeneous distribution of sliver nanoparticles and spherical in shape. The image topography of AgNps indicates the formation of nanoparticles with agglomeration.
Figure(2):3D picture of Atomic force Microscope (AFM) of sliver nanoparticles synthesized by Streptomyces spp.4.
The surface thickness is 191 nm ,itillustrate thickness of film surface roughness, which account for the highest crystalline granular tops on the surface. The regularity in the grown film. The granules with a vertical arrangement on the crystalaxis and equal heights.
Avg. Diameter:121.30 nm
Figure (3):Atomic force Microscope image with nanoparticles size distribution of biological synthesized AgNPs synthesized by Streptomyces spp.4.
Figure (3)shows on the granular aggregates distribution on film surface. Average diameter for sliver nanoparticle producing by Streptomyces spp.4 equal 121.30nm. Faghri Zonooz and Salouti 7 showed that Streptomyces spp.producing sliver nanoparticle with spherical in shape and size ranged (10-100 nm). Subashini23, record that a biologically synthesized AgNPs using Streptomyces spp. VITBT7 isolated from soil samples. These AgNPs having spherical shape and the size is (20–70) nm in range.
Antimicrobial activity:
Antimicrobial activity for sliver nanoparticle produced by Streptomyces spp.4 was tested by well diffusion method. Results showed that Streptomyces spp.4 have higher activity against E.coli with inhibition
zone (14)mm compared with (11mm) against S.aureus and (8) mm against candida albicans. Silver nanoparticle display a broad bactericidal against gram positive and gram negative bacteria and multi resistant strains 18. Antibacterial activity of silver nanoparticles was detected 15.Kamel et al.11record antimicrobial activity of silver nanoparticles.
Figure (4):Antimicrobial Activity of Silver Nanoparticles against test pathogens
References:
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