Therapeutic Potential of Biologically Reduced Silver Nanoparticles

Hindawi Publishing CorporationJournal of NanoscienceVolume 2013 Article ID 940719 8 pageshttpdxdoiorg1011552013940719

Research ArticleTherapeutic Potential of Biologically Reduced SilverNanoparticles from Actinomycete Cultures

M K Sukanya1 K A Saju1 P K Praseetha2 and G Sakthivel2

1 Department of Biotechnology Noorul Islam College of Arts and Sciences Kumaracoil Kanyakumari District Tamil Nadu India2Department of Nanotechnology Noorul Islam Centre for Higher Education Noorul Islam UniversityKumaracoil Kanyakumari District Tamil Nadu India

Correspondence should be addressed to P K Praseetha crkpkpgmailcom

Received 29 April 2013 Revised 16 August 2013 Accepted 24 August 2013

Academic Editor Kalimuthu Kalishwaralal

Copyright copy 2013 M K Sukanya et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Silver nanoparticles are applied in nanomedicine from time immemorial and are still used as powerful antibiotic and anti-inflammatory agents Antibiotics produced by actinomycetes are popular in almost all the therapeutic measures and this studyhas proven that these microbes are also helpful in the biosynthesis of silver nanoparticles with good surface and size characteristicsSilver can be synthesized by various chemical methodologies and most of them have turned to be toxic This study has beensuccessful in isolating the microbes from polluted environment and subjecting them to the reduction of silver nanoparticlescharacterizing the nanoparticles by UV spectrophotometry and transmission electron microscopy The nanoparticles producedwere tested for their antimicrobial property and the zone of inhibition was greater than those produced by their chemicallysynthesized counterparts Actinomycetes helpful in bioremediating heavy metals are useful for the production of metallicnanoparticles The biosynthesized silver nanoparticles loaded with antibiotics prove to be better in killing the pathogens and haveopened up new areas for developing nanobiotechnological research based on microbial applications

1 Introduction

Human activities are greatly altering ecosystems worldwideat unprecedented rates leading to accelerated loss of biodi-versity [1] and environmental pollution Soil is perhaps themost endangered component of our environment being opento potential contamination by a variety of different pollutantsarising from human activities such as nuclear industrial andactivities agricultural [2 3]There are hundreds of sources ofheavy metal pollution including the coal natural gas paperand chloralkali industries [4] ldquoHeavy metalsrdquo is a generalcollective term which applies to the group ofmetals andmet-alloids with atomic density greater than 4 gcm3 or 5 times ormore greater than water [5ndash7]

Microorganisms in the soil are responsible for nitrogenfixation assimilation and degradation of organic residues torelease nutrients Field studies of metal contaminated soilshave demonstrated that elevated metal loadings can result indecreased microbial community [8ndash11] Actinomycetes pop-ulations are relatively lower than other soil microbes and

contain a predominance of streptomyces that are tolerant toacid conditions [12]

Classically the nanoparticles are produced by physicaland chemical methods [13] But these methods are costlytoxic and nonecofriendly Currently there is a growing needto use environmentally friendly nanoparticles that do notproduce toxic wastes in their process synthesis protocolSilver has gained interest over the years because of its distinc-tive properties such as good conductivity chemical stabilityand catalytic and antibacterial activity [14ndash16] The currentstudy throws light on silver nanoparticles (AgNPs) which arepotentially been exploited in medicine for antibacterial anti-fungal antiviral and anti-inflammatory therapy The presentstudy was carried out to determine the synthesis of AgNPsby actinomycetes from heavy metal-polluted sites exhibitinghigher levels of antibacterial activity against human patho-gens The biosynthesized nanoparticles do have an enhancedtherapeutic potential when compared to their chemically syn-thesized counterparts due to their small size and doping ofnatural antibiotics of actinomycete origin

2 Journal of Nanoscience

2 Material and Methods

21 Sample Collection Soil samples were collected for theisolation of actinomycetes from heavy metal-polluted andnon-polluted areas The heavy metal-polluted sample wascollected from Travancore Titanium Products LtdThiruvan-anthapuram and nonpolluted sample was collected fromNoorul Islam college of Arts and Science Kumaracoil Theheavy metal-polluted and nonpolluted samples were ran-domly named as P and NP respectively

Each soil sample was taken from three different locations(05 km intervals) of particular station from a depth of 10ndash15 cm and mixed evenly From the mixed sample 100 g weretaken as a representative sample of the particular field Thesoil samples were collected using clean dry and sterile poly-thene bags with a sterile spatula The samples were immedi-ately transported to the laboratory and stored in the refriger-ator

22 Physiochemical Analysis of Soil The physiochemicalcharacteristics such as soil color soil texture pH electricalconductivity (EC) organic carbon (OC) and nutrient con-tent such as nitrogen phosphorus calcium and magnesiumof soil samples were estimated The characteristics of the soilwould definitely influence the type of microbes present in theenvironment and the nutrient requirements for determiningthe growth characteristics of actinomycetes can be estimatedby the same

23 Population Density Study of Actinomycetes Populationdensity studies of actinomycetes were performed by serialdilution method The number of microbial population perunit volume of sample is reduced by serial dilution for isola-tion of individual colonies About 1 gm of soil was transferredto conical flask containing 100mL of sterile distilled waterThis was then homogenized well for few minutes 1mL ofhomogenized sample was pipetted into a test tube labeled as10minus3 dilution This procedure was repeated using sterilepipette for each dilution up to 10minus7 dilution

24 Isolation and Culture of Actinomycetes Isolation of acti-nomycete cultures was done by plating technique in PotatoDextroseAgar (PDA)medium (supplementedwith cyclohex-amide [25 120583gmL] and nalidixic acid [10120583gmL] as antifungaland antibacterial compounds resp) After obtaining satisfac-tory growth (requires about 7ndash14 days) the strains were sub-cultured in PDA slants in order to obtain pure culture Pureisolates were maintained at 4∘C in refrigerator for furtherstudies

25 Colony Characterization of Actinomycetes Actinomyceteisolates were observed using hand lens and the colony mor-phology was recorded with respect to color shape size andnature of the colony

26 Microscopic Characterization Actinomycete isolateswere microscopically characterized by Lactophenol CottonBlue mounting The cell morphology was photographically

recorded with respect to spore chain morphology and myce-lium structure

27 Carbohydrate Fermentation Test Carbohydrate fermen-tation test was performed to confirm the actinomycete cul-tures up to genus level Bushnell Hass Broth was preparedwith sugars such as glucose sucrose lactose arabinose anddextrose An indicator phenol red wasmixed with the brothand final pH was adjusted to 73 The test cultures were inoc-ulated into appropriately labeled medium All the tubes wereincubated at room temperature for 7 days and were checkedfor a positive result of fermentation

28 Starch Hydrolysis Test The starch plate was inoculatedwith the organism to be tested It was incubated at an opti-mum temperature of 48 hours The plate was flooded withiodine and the results were observed Formation of blue colorindicated that there was no hydrolysis while a clear zoneindicated hydrolysis

29 Sequence Analysis of 16S rRNA Genes from Actinomycetes

291 Extraction of Genomic DNA from Actinomycetes It isimportant to taxonomically place the actinomycetes basedupon their genus and species levelsThe 16srRNA sequencingis the most reliable technique for taxonomically positioningthe organisms The actinomycete strain Streptomyces sp II(NP1) was grown in 20mL of actinomycete broth in 100mLconical flask at 37∘C in a rotary shaker (200 rpm) for over-night 15mL of culture was transferred to the eppendorf tubeand centrifuged at 8000 rpm for 2min The supernatant wasdiscarded and drained well onto a tissue paperThe pellet wasresuspended in 400 120583L of sucrose TE buffer To this 32120583L oflysozyme (10mgmL) was added and incubated the tubes at37∘C for 30min 100 120583L of 05M EDTA at pH 8 and 60 120583Lof 10 SDS were added To this 15120583L of proteinase K(20mgsdotmLminus1) was added and incubated the tubes at 50∘C(water bath) for 12 hrsThe tubewas brought to room temper-ature and 250 120583L of equilibrated phenol (equilibrated withTris HCl) was added and mixed well and 250 120583L of chloro-form was added The solution was centrifuged at 10000 rpmfor 10min and extracted twice with phenol chloroform (1 1ratio)The aqueous phasewas extracted once againwith chlo-roform isoamylalcohol (24 1 ratio) and the supernatant wascollected and precipitated with 2 volume of absolute ethanolThe precipitate was centrifuged at 10000 rpm for 10min anddiscarded the supernatant Air dried it completely The pelletwas washed with 70 ethanol and allowed it to dry at roomtemperature After complete drying the pellet was dissolvedin 20ndash50120583L of sterile distilled water and stored at minus20∘C

292 PCR Amplification of 16S rRNA The 16S rRNA genewas amplified from genomic DNA obtained NP1 culturesby PCR with Universal Forward primer-16S rRNA F-51015840-AGAGTTTGA TCCTGGCTCAG-31015840 and Universal Reverseprimer-16S rRNA R-51015840-ACG GCT ACC TTG TTA CGACTT-31015840 The reaction mixture contained 25 to 50 ng of DNAEx Taq PCR buffer 15mM MgCl

2 10mM deoxynucleoside

Journal of Nanoscience 3

triphosphate mixture 50 pmol of each primer and 05U ofEx Taq polymerase PCR conditions consisted of an initialdenaturation at 94∘C for 5min 30 cycles at 94∘C for 1minannealing 58∘C (THB-14) 63∘C (ACT-20) for 1min and 72∘Cfor 1min and final 5min extension at 72∘CThe amplificationproducts were examined by agarose gel electrophoresis andpurified by using a QIA quick PCR clean-up kit with theprotocol suggested by Qiagen Inc

293 Sequencing of 16S rRNAGenes Thecomplete 16S rRNAgene was sequenced by using the PCR products directly assequencing template with the above-mentioned primers Allsequencing reactions were carried out with an ABI 3730automated DNA sequencer (Applied Biosystems MonzaItaly)

294 Sequence and Phylogenetic Analysis Nucleotidesequences were compared to those in the Gene Bank Data-base with the Basic Local Alignment Search Tool (BLAST)algorithm to identify known closely related sequences TheDNA sequences were aligned and phylogenetic tree was con-structed by neighbor joining method

295 Screening of Actinomycetes for Reducing ActivityScreening of actinomycetes was performed using nitratereduction test Nitrate broth was used to determine the abilityof an organism to reduce nitrate (NO

3) to nitrite (NO

2) using

the enzyme nitrate reductase Nitrate broth was prepared andsterilized properly The isolated strains grown in PDA brothwere subculture individually in 50mL nitrate broth in 100mLErlenmeyer flask and incubated at 37∘C for 24ndash48 hours Atthe end of incubation 1mL of alpha naphthylamine reagentand 1mL of sulfanilic acid reagent were added to the testmedium and the color change was observed If there was nochange in color zinc metal dust was sprinkled and observedfor any notable changes

210 Biosynthesis of AgNPs

2101 Production of Actinomycete Biomass Actinomycetecultures are required in considerable amounts for the purposeof biosynthesis Hence it becomes essential to mass-culturethe strains For the production of biomass the actinomycetesstrains such as Streptomyces sp I (P1) Rhodococcus sp (P8)and Streptomyces sp II (NP1) were grown aerobically in acti-nomycetes broth The culture flasks were incubated in roomtemperature at 37∘C After 15 days of incubation period bio-mass was harvested using Whatman filter paper The har-vested biomass was washed with double-distilled water andfilters the biomassThen the filtered biomass was incubated indouble distilled water for a day After incubation the biomasswas separated from layer of water for further studies

2102 Synthesis of AgNPs Actinomycete mat (20 gm) wasobtained from actinomycete broth and used for the synthesisof AgNPs The harvested cell filtrate was suspended in steriledouble distilled water for 1 day After one-day incubationthe cell filtrate (biomass) was obtained by passing it through

Whatman filter paper 20 gm of biomass (fresh weight) wasgrind well using mortar and pestle and mixed with 200mLof Millipore water in a 500mL Erlenmeyer flask and agitatedin the same condition for 72 hour at 37∘C The same wasrepeated for the cell-free filtrate (filtered water) along withexperimental flask (20mL filtered water in 200mL of Mil-lipore water) Biomass and water samples were randomlynamed as B and W

For the synthesis of AgNPs 50mL of 1mM AgNO3solu-

tion was mixed with 50mL of cell filtrate in a 250mL Erlen-meyer flask and agitated at 37∘C in darker condition Thesame was done for the cell-free filtrate (50mL of 1mMAgNO

3solution with 50mL filtered water) Simultaneously

control without silver ions was also run along with the exper-imental flasks Colour change was noted at specific intervals(12 hours and 72 hours)Thenanoparticleswere characterizedbyUV-visible spectroscopy and transmission electronmicro-scope (TEM) analysis

2103 Characterization of Synthesized AgNPs

2104UV-Visible SpectroscopicAnalysis Thereduction of sil-ver ions was confirmed by qualitative testing of supernatantby UV-visible spectrophotometer 1mL of sample superna-tant waswithdrawn after 24 hr and absorbancewasmeasuredby using UV-visible spectrophotometer at a wavelength of400ndash600 nm

2105 Transmission Electron Microscopic Analysis of AgNPsTEM analysis is a technique to examine the specimen struc-ture composition or properties in nanometric levels Thecell-free filtrate of Streptomyces sp II was treated with silvernitrate and then the solution was centrifuged and washedwith distilled water for 4-5 timesThe pellet was collected anddried in hot air oven The dried particles were scanned on aPhilips transmission electron microscopy (TEM) at a magni-fication of 35000X

2106 Protein Expression Studies It becomes essential tounderstand the nature of the proteins which assist the reduc-tion of AgNPs by the actinomycete strains In order to com-pare the extracellular protein production and their molecularmass the selected test strains (AgNPs synthesizing and non-synthesizing) were subjected to SDS-Poly Acrylamide GelElectrophoresis [SDS-PAGE] following the method ofLaemmli (1970)Molecular weightmarkers for peptides rang-ing from 1KDa to 205KDa were used as protein markers

2107 Antibacterial Activity of Actinomycetes Strains andAgNPs against Human Pathogens The antimicrobial activityof actinomycete strains and AgNPs against human pathogenssuch as Staphylococcus aureus Klebsiella pneumonia Proteusvulgaris Pseudomonas aeruginosa and Escherichia coli wereobtained from ATCC and measured using disc diffusionmethod Cultures of mentioned pathogens were grown inMuller-Hinton broth (Sigma India) at 27∘C on a rotaryshaker at 200 rpm 20120583L of biosynthesized AgNPs solution


(a) Streptomyces sp 1 (P1) (b) Rhodococcus sp (P8) (c) Streptomyces sp II (NP1)

Figure 1 Colony morphology and microscopic view of actinomycetes


Figure 2 Microscopic observations of cultured actinomycete

was added into each disc After incubation the diameter ofthe inhibition zones was measured

3 Results

Actinomycete strains isolated from the soil samples were col-lected from heavy metal-polluted and nonpolluted soil Theactinomycete isolates were characterized on the basis of col-ony characterizationmacroscopic appearance and biochem-ical test such as carbohydrate and starch hydrolysis test

31 Isolation and Identification of Actinomycetes Four generaof actinomycetes which included 10 different species wereisolated from the heavy metal-polluted and nonpolluted soilsamples The species identified in the nonpolluted soil wereStreptomyces sp I (NP4) Streptomyces sp II (NP1) Strep-tomyces sp III (NP10) Rothia sp (NP5) Actinomadura sp(NP2) and Rhodococcus sp (NP7) In the heavy metal-polluted soil sample four species were identified which wereStreptomyces sp I (P1) Streptomyces sp II (P2) Streptomycessp III (P3) and Rhodococcus sp (P8) The results revealedthat Streptomyces sp was the predominant actinomycete inboth the soil samples Though the actinomycetes belong to

the same genera the species identified from the nonpollutedsoil differed in the morphological and biochemical charac-teristics of the same species of the same genera identifiedfrom the heavy metal-polluted soil The results of the presentinvestigation (Figures 1 and 2) show that all the actinomycetesshowed branched filamentous fragmented separated orlong mycelium arrangement

32 Biosynthesis of AgNPs The color change (brown colour)occurred in the biomass and cell-free extractwhen challengedwith 1mM AgNO

3(Figures 3(a) to 3(d)) in 48 hrs and

attained maximum intensity after 72 hrs The increase inintensity during the period of incubation indicated the for-mation of AgNPs The production of silver nanoparticles ishigher in cell-free filtrates than in biomass samples Controlwithout silver ions showed no change in color when incu-bated under the same conditions

33 Characterization of Silver Nanoparticle by UV-Vis Spec-troscopy The light absorption patterns of the cell filtrate andcell-free filtrate of actinomycetes were monitored after 72hours in the range of 290ndash600 nm by using UV-visible spec-trophotometer UV-visible absorption spectra of AgNPs are


(a) Control (without AgNO3) (b) At the beginning of the reaction

(c) Cell filtrate after 48 hours (d) Cell-free filtrate after 72 hours

Figure 3 Biosynthesis of AgNPs by actinomycete strains

shown in Figure 4 Biosynthesis of AgNPs from cellfree fil-trate of actinomycete showed an intense peak at 450 nm forthe sample WNP1 at wavelength range of 400ndash500 nm

34 Transmission Electron Microscopic Studies (TEM) of Syn-thesized AgNPs TEM image of biosynthesized AgNPs wasobtained by using cell-free filtrate of Streptomyces sp II afterreacting with silver nitrate The size of the particles rangedfrom 5 to 40 nm The nanoparticles were observed to bespherical The observations can be made from Figure 5

35 Protein Expression Studies Polyacrylamide gel Electro-phoresis was used to assess the compounds produced duringbiosynthesis of AgNPs Separate protein bands were visual-ized for the actinomycete strains that produce nanoparticleson staining the gel which was absent in the actinomycetestrains lacking the reduction activity Few bands were notedwith molecular weights 20 and 30 kDa Figure 6 explains thedetails

36 Antibacterial Activity of AgNPs and Actinomycetes Strainsagainst Human Pathogens The antibacterial activity of acti-nomycetes was investigated against various pathogenic bac-teria of Gram-positive (Staphylococcus aureus) and Gram-negative (E coli Pseudomonas aeruginosa Klebsiella pneumo-nia and Proteus vulgaris) strains using disc diffusion method(Table 1) Out of the three actinomycete isolates Streptomycessp I (P1) exhibited maximum antibacterial activity againstPseudomonas aeruginosa (10mm)The details are depicted inTable 1 and Figure 7(a) to 7(e)

The antibacterial activity of AgNPs was investigatedagainst various pathogenic bacteria The highest antimicro-bial activity was observed against Pseudomonas aeruginosafollowed by Staphylococcus aureus and Klebsiella pneumoniaThe antibacterial activity of the bionanoparticles for Pseudo-monas aerogenosa was the maximum (26mm) followedby Staphylococcus aureus (23mm) Klebsiella Pneumonia(21mm) and E coli (19mm) Compared to the commercialantibiotics like kanamycin which produces inhibition zonesin the order of 22mm 18mm 21mm and 16mm for the path-ogens silver nanoparticles exhibited better killing efficiency


Table 1 Antibacterial activity of AgNPs against pathogenic bacteria

Sl no Strains Diameter of zone of inhibition (mm)E coli Staphylococcus aureus Pseudomonas aeruginosa Klebsiella pneumoniae Proteus vulgaris

1 BP1 19 20 20 17 mdash2 BP8 16 21 26 20 mdash3 BNP1 16 20 24 21 mdash4 WP1 mdash 21 18 mdash mdash5 WP8 mdash 23 18 18 mdash6 WNP1 mdash 20 22 19 97 Control mdash mdash mdash mdash mdash

300 400 500 60000

01

02

03

04

05

06

07

08

Abso

rban

ce

Wavelength (nm)

WNP1WP1WP8

Figure 4 UV-visible spectrum of AgNPs synthesized by differentactinomycete strains

This test shows that silver nanoparticles are nearly 7085 and 60 effective compared to Kanamycin and Arith-romycin respectively

4 Discussions and Conclusion

The actinomycete species that showed positive result innitrate reduction test were screened and selected for the pro-duction of silver nanoparticles Streptomyces sp I Strepto-myces sp II and Rhodococcus sp were further selected for thebiosynthesis of AgNPs

The results revealed that Streptomyces sp was the pre-dominant actinomycete in both soil samples Though theactinomycetes belong to the same genera the species identi-fied from the nonpolluted soil differed in the morphologicaland biochemical characteristics of the same species of thesame genera identified from the heavy metal-polluted soilThe results of the present investigation show that all theactinomycetes showed branched filamentous fragmentedseparated or long mycelium arrangement which falls in linewith the findings of [17] The identification of actinomyceteswas carried out using the references [18ndash20] The strainisolated Streptomyces sp II (NP1) was confirmed using 16S

Figure 5 Size measurement of silver nanoparticles by transmissionelectron microscopy

202 kDa

NP2 NP1

116 kDa

66kDa

45kDa

201 kDa144kDa65kDa

Marker

Figure 6 Protein bands produced by the actinomycete strains

rRNA sequence analysis Based on 16srRNA sequence andphylogenetic analysis Streptomyces sp II (NP1) isolated fromnonpolluted soil was confirmed as Streptomyces lomondensis

UV studies with WNP1 sample showed an intense peakat 450 nm which proves the presence of AgNPs [21] Theproduction of silver nanoparticles was further confirmed byobtaining a TEM image The size of particles ranged from 65to 80 nm The nanoparticles were observed to be spherical[22] The study also revealed that specific proteins weresynthesized by the actinomycetes when they were involvedin the production of nanoparticles The molecular weight of


(a) (b) (c)

(d) (e)

Figure 7 Inhibition zones produced by biosynthesized AgNPs against different bacterial strains

the protein bands revealed a similarity to heat shock proteinsThese are stress-related proteins which assist in the reductionof silver nanoparticles

Streptomyces sp was highly susceptible to Gram-positivebacteria and showed moderate activity against Gram-nega-tive bacteria [23]Rhodococcus sp also produced antibacterialactivity against pathogenic bacteria [24] Many researchers[25] used Escherichia coli as a model for Gram-negative bac-teria and proved that AgNPs may be used as an antimicrobialagent

As the microbes are well adapted to heavy metal degra-dation they are capable of reducing silver nitrate to AgNPsSilver nanoparticles by nature do have an antibacterial prop-erty These nanoparticles synthesized by actinomycetes areloaded with antibiotic substances during biosynthesis Thusthe antibiotic effect of biosynthesized silver nanoparticles isbetter than silver nanoparticles produced by chemical meth-ods

Acknowledgments

The authors deeply acknowledge Noorul Islam University forsupport extended to perform the current study They thankthe Department of Biotechnology Rajiv Gandhi Centrefor Biotechnology Thiruvananthapuram for analyzing datausing TEM

References

[1] P H Raven ldquoScience sustainability and the human prospectrdquoScience vol 297 pp 954ndash958 2002

[2] O Morton-Bermea E Hernandez Alvarez I Gaso and NSegovia ldquoHeavy metal concentrations in surface soils fromMexico Cityrdquo Bulletin of Environmental Contamination andToxicology vol 68 no 3 pp 383ndash388 2002

[3] M R Kalantari M Shokrzadeh A G Ebadi C Mohammadi-zadeh M I Choudhary and A-U Atta-ur-Rahman ldquoSoil pol-lution by heavy metals and remediation (Mazandaran-Iran)rdquoJournal of Applied Sciences vol 6 no 9 pp 2110ndash2116 2006

[4] D GMcDonald andA F Grandt ldquoLimestone- Lime Treatmentof AcidMineDrainage-Full Scalerdquo EPA Project Summary EPA-600S7-81-033 1981

[5] J O Nriagu ldquoA global assessment of natural sources of atmo-spheric trace metalsrdquoNature vol 338 no 6210 pp 47ndash49 1989

[6] J R Garbarino H Hayes D Roth R Antweider T I Brintonand H Taylor Contaminants in the Mississippi River U SGeological Survey Circular 1133 Virginia Va USA 1995

[7] S J Hawkes ldquoWhat is a ldquoHeavy Metalrdquordquo Journal of ChemicalEducation vol 74 no 11 p 1374 1997

[8] M J Jordan andM P Lechevalier ldquoEffects of zinc smelter emis-sions on forest soil microflorardquo Canadian Journal of Microbiol-ogy vol 21 no 11 pp 1855ndash1865 1975

[9] P C Brookes and S P McGrath ldquoEffects of metal toxicity onthe size of the soil microbial biomassrdquo Journal of Soil Sciencevol 35 no 2 pp 341ndash346 1984


[10] KChander andPC Brookes ldquoEffects of heavymetals frompastapplications of sewage sludge onmicrobial biomass and organicmatter accumulation in a sandy loam and silty loam UK soilrdquoSoil Biology and Biochemistry vol 23 no 10 pp 927ndash932 1991

[11] A Konopka T Zakharova M Bischoff L Oliver C Nakatsuand R F Turco ldquoMicrobial biomass and activity in lead-con-taminated soilrdquo Applied and Environmental Microbiology vol65 no 5 pp 2256ndash2259 1999

[12] F L Davies and S T Williams ldquoStudies on the ecology ofactinomycetes in soil IThe occurrence and distribution of acti-nomycetes in a pine forest soilrdquo Soil Biology and Biochemistryvol 2 no 4 pp 227ndash238 1970

[13] Z-J Jiang C-Y Liu and L-W Sun ldquoCatalytic properties of sil-ver nanoparticles supported on silica spheresrdquo Journal of Phys-ical Chemistry B vol 109 no 5 pp 1730ndash1735 2005

[14] P Mukherjee A Ahmad D Mandal et al ldquoFungus-mediatedsynthesis of silver nanoparticles and their immobilization inthemycelial matrix a novel biological approach to nanoparticlesynthesisrdquo Nano Letters vol 1 no 10 pp 515ndash519 2001

[15] I Sondi and B Salopek-Sondi ldquoSilver nanoparticles as antimi-crobial agent a case study on E coli as a model for Gram-negative bacteriardquo Journal of Colloid and Interface Science vol275 no 1 pp 177ndash182 2004

[16] X Chen and H J Schluesener ldquoNanosilver a nanoproduct inmedical applicationrdquo Toxicology Letters vol 176 no 1 pp 1ndash122008

[17] M Buee P E Courty D Mignot and J Garbaye ldquoSoil nicheeffect on species diversity and catabolic activities in an ectomy-corrhizal fungal communityrdquo Soil Biology and Biochemistry vol39 no 8 pp 1947ndash1955 2007

[18] R E Buchanan and N E Gibbons Bergeyrsquos Manual of Determi-native Bacteriology 8th edition 1974

[19] L Sembiring Selective isolation and characterisation of strepto-mycetes associated with the rhizosphere of the tropical legumeParaserianthes falcataria (L) Nielsen [PhD thesis] University ofNewcastle Tyne UK 2003

[20] M George A Anjumol G George and A A M Hatha ldquoDis-tribution and bioactive potential of soil actinomycetes fromdifferent ecological habitatsrdquo Journal of Microbiology Researchvol 6 no 10 pp 2265ndash2271 2011

[21] PMulvaney ldquoSurface plasmon spectroscopy of nanosizedmetalparticlesrdquo Langmuir vol 12 no 3 pp 788ndash800 1996

[22] N Y Tsibakhashvili E I Kirkesali D T Pataraya et al ldquoMicro-bial synthesis of silver nanoparticles by Streptomyces glaucusand Spirulina platensisrdquo Advanced Science Letters vol 4 no 11-12 pp 3408ndash3417 2011

[23] K J Narayana P Prabhakar M Vijayalakshmi Y Venkates-warlu and P S Krishna ldquoBiological activity of phenylpropionicacid isolated from a terrestrial Streptomycetesrdquo Polish Journal ofMicrobiology vol 56 no 3 pp 191ndash197 2007

[24] L S Singh I Baruah and T C Bora ldquoActinomycetes of Loktakhabitat isolation and screening for antimicrobial activitiesrdquoBiotechnology vol 5 no 2 pp 217ndash221 2006

[25] Q L Feng JWaGQChenK ZCui TMKim and JOKimldquoAntimicrobial activity of silver nanoparticles against bacterialspeciesrdquo Journal of Biomedical Materials Research B no 52 pp662ndash668 2003

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of



CeramicsJournal of


CompositesJournal of

NanoparticlesJournal of



International Journal of

Biomaterials


NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



MetallurgyJournal of


BioMed Research International

MaterialsJournal of


Nano

materials


Journal ofNanomaterials


2 Material and Methods

21 Sample Collection Soil samples were collected for theisolation of actinomycetes from heavy metal-polluted andnon-polluted areas The heavy metal-polluted sample wascollected from Travancore Titanium Products LtdThiruvan-anthapuram and nonpolluted sample was collected fromNoorul Islam college of Arts and Science Kumaracoil Theheavy metal-polluted and nonpolluted samples were ran-domly named as P and NP respectively

Each soil sample was taken from three different locations(05 km intervals) of particular station from a depth of 10ndash15 cm and mixed evenly From the mixed sample 100 g weretaken as a representative sample of the particular field Thesoil samples were collected using clean dry and sterile poly-thene bags with a sterile spatula The samples were immedi-ately transported to the laboratory and stored in the refriger-ator

22 Physiochemical Analysis of Soil The physiochemicalcharacteristics such as soil color soil texture pH electricalconductivity (EC) organic carbon (OC) and nutrient con-tent such as nitrogen phosphorus calcium and magnesiumof soil samples were estimated The characteristics of the soilwould definitely influence the type of microbes present in theenvironment and the nutrient requirements for determiningthe growth characteristics of actinomycetes can be estimatedby the same

23 Population Density Study of Actinomycetes Populationdensity studies of actinomycetes were performed by serialdilution method The number of microbial population perunit volume of sample is reduced by serial dilution for isola-tion of individual colonies About 1 gm of soil was transferredto conical flask containing 100mL of sterile distilled waterThis was then homogenized well for few minutes 1mL ofhomogenized sample was pipetted into a test tube labeled as10minus3 dilution This procedure was repeated using sterilepipette for each dilution up to 10minus7 dilution

24 Isolation and Culture of Actinomycetes Isolation of acti-nomycete cultures was done by plating technique in PotatoDextroseAgar (PDA)medium (supplementedwith cyclohex-amide [25 120583gmL] and nalidixic acid [10120583gmL] as antifungaland antibacterial compounds resp) After obtaining satisfac-tory growth (requires about 7ndash14 days) the strains were sub-cultured in PDA slants in order to obtain pure culture Pureisolates were maintained at 4∘C in refrigerator for furtherstudies

25 Colony Characterization of Actinomycetes Actinomyceteisolates were observed using hand lens and the colony mor-phology was recorded with respect to color shape size andnature of the colony

26 Microscopic Characterization Actinomycete isolateswere microscopically characterized by Lactophenol CottonBlue mounting The cell morphology was photographically

recorded with respect to spore chain morphology and myce-lium structure

27 Carbohydrate Fermentation Test Carbohydrate fermen-tation test was performed to confirm the actinomycete cul-tures up to genus level Bushnell Hass Broth was preparedwith sugars such as glucose sucrose lactose arabinose anddextrose An indicator phenol red wasmixed with the brothand final pH was adjusted to 73 The test cultures were inoc-ulated into appropriately labeled medium All the tubes wereincubated at room temperature for 7 days and were checkedfor a positive result of fermentation

28 Starch Hydrolysis Test The starch plate was inoculatedwith the organism to be tested It was incubated at an opti-mum temperature of 48 hours The plate was flooded withiodine and the results were observed Formation of blue colorindicated that there was no hydrolysis while a clear zoneindicated hydrolysis

29 Sequence Analysis of 16S rRNA Genes from Actinomycetes

291 Extraction of Genomic DNA from Actinomycetes It isimportant to taxonomically place the actinomycetes basedupon their genus and species levelsThe 16srRNA sequencingis the most reliable technique for taxonomically positioningthe organisms The actinomycete strain Streptomyces sp II(NP1) was grown in 20mL of actinomycete broth in 100mLconical flask at 37∘C in a rotary shaker (200 rpm) for over-night 15mL of culture was transferred to the eppendorf tubeand centrifuged at 8000 rpm for 2min The supernatant wasdiscarded and drained well onto a tissue paperThe pellet wasresuspended in 400 120583L of sucrose TE buffer To this 32120583L oflysozyme (10mgmL) was added and incubated the tubes at37∘C for 30min 100 120583L of 05M EDTA at pH 8 and 60 120583Lof 10 SDS were added To this 15120583L of proteinase K(20mgsdotmLminus1) was added and incubated the tubes at 50∘C(water bath) for 12 hrsThe tubewas brought to room temper-ature and 250 120583L of equilibrated phenol (equilibrated withTris HCl) was added and mixed well and 250 120583L of chloro-form was added The solution was centrifuged at 10000 rpmfor 10min and extracted twice with phenol chloroform (1 1ratio)The aqueous phasewas extracted once againwith chlo-roform isoamylalcohol (24 1 ratio) and the supernatant wascollected and precipitated with 2 volume of absolute ethanolThe precipitate was centrifuged at 10000 rpm for 10min anddiscarded the supernatant Air dried it completely The pelletwas washed with 70 ethanol and allowed it to dry at roomtemperature After complete drying the pellet was dissolvedin 20ndash50120583L of sterile distilled water and stored at minus20∘C

292 PCR Amplification of 16S rRNA The 16S rRNA genewas amplified from genomic DNA obtained NP1 culturesby PCR with Universal Forward primer-16S rRNA F-51015840-AGAGTTTGA TCCTGGCTCAG-31015840 and Universal Reverseprimer-16S rRNA R-51015840-ACG GCT ACC TTG TTA CGACTT-31015840 The reaction mixture contained 25 to 50 ng of DNAEx Taq PCR buffer 15mM MgCl

2 10mM deoxynucleoside






3) to nitrite (NO

2) using





















3 Results





















300 400 500 60000

01

02

03

04

05

06

07

08

Abso

rban

ce

Wavelength (nm)

WNP1WP1WP8







202 kDa

NP2 NP1

116 kDa

66kDa

45kDa

201 kDa144kDa65kDa

Marker





(a) (b) (c)

(d) (e)





Acknowledgments


References


































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials








3) to nitrite (NO

2) using





















3 Results





















300 400 500 60000

01

02

03

04

05

06

07

08

Abso

rban

ce

Wavelength (nm)

WNP1WP1WP8







202 kDa

NP2 NP1

116 kDa

66kDa

45kDa

201 kDa144kDa65kDa

Marker





(a) (b) (c)

(d) (e)





Acknowledgments


References


































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials









3 Results





















300 400 500 60000

01

02

03

04

05

06

07

08

Abso

rban

ce

Wavelength (nm)

WNP1WP1WP8







202 kDa

NP2 NP1

116 kDa

66kDa

45kDa

201 kDa144kDa65kDa

Marker





(a) (b) (c)

(d) (e)





Acknowledgments


References


































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials
















300 400 500 60000

01

02

03

04

05

06

07

08

Abso

rban

ce

Wavelength (nm)

WNP1WP1WP8







202 kDa

NP2 NP1

116 kDa

66kDa

45kDa

201 kDa144kDa65kDa

Marker





(a) (b) (c)

(d) (e)





Acknowledgments


References


































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials







300 400 500 60000

01

02

03

04

05

06

07

08

Abso

rban

ce

Wavelength (nm)

WNP1WP1WP8







202 kDa

NP2 NP1

116 kDa

66kDa

45kDa

201 kDa144kDa65kDa

Marker





(a) (b) (c)

(d) (e)





Acknowledgments


References


































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




(a) (b) (c)

(d) (e)





Acknowledgments


References


































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials










CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



Documents

Therapeutic Potential of Biologically Reduced Silver Nanoparticles