Beruflich Dokumente
Kultur Dokumente
), 13(2): 439 – 445 (2017) © Th e Eg yptian Society of Exp erimen tal Biolog y
DOI: 10.5455/eg yjebb.20171108083304
RESEARCH ART ICLE
A m r A b d el - Monei m E l - W as ei f
N ev i n A hm ed Ib r ahi m
S hi m aa A b d E l - A z i z A hm ed
N ag w a A hm ed A b d al l ah
ABSTRACT:
Two microbial isolates selected from Egyptian N ev i n A hm ed Ib r ahi m
soil were used for biological synthesis of S hi m aa A b d E l - A z i z A hm ed
silver nanoparticles using silver nitrate N ag w a A hm ed A b d al l ah
solution. The selection of these two
microorganisms was depending on the ability Microbiology Department, Faculty of Science,
of actinomycetes and fungi to AgNPs
Ain Shams University, Cairo, Egypt.
synthesis and produc tion of antimicrobial
compounds. Results recorded that the dry
weight of AgNPs produced from selected
Streptomyces clavuligerus and Trichoderma
harzianum AUMC 5408 were 66.5 and 11.0
ARTI CLE CODE: 42.02.17
mg/l, respectively. Description of AgNPs from
Streptomyces clavuligerus supernatant and
Trichoderma harzianum AUMC 5408 mycelia I NTRODUCTI ON :
were carried out. Results of UV-analysis
Nanotechnology is mainly concerned
showed spectrum located between 420 - 440
nm for both culture filtrate and mycelium with the synthesis of nano-materials using
respectively characteristics of AgNPs. Also, different systems and their applications.
Nowadays, nano-particles are used in the
the Fourier Transform Infrared Spectroscopy
areas of agriculture, health care, environment
(FTIR) analysis was studied. The average
and consumer good s (El-Ghwas and El -
size of AgNPs were between range from 2.0
to 4.5 nm in case of Streptomyces W aseif, 2016) . Nano-particles which
clavuligerus, but 5.33 - 29.46 nm in case of synthesized by chemical and physical
pathways are low yielding, energy intensive,
Trichoderma harzianum AUMC 5408.
difficult to scale up, producing high levels of
Enhancement of the antimicrobial activity of
hazardous wastes, requires the use of costly
crude antimicrobial compounds with Silver
organometallic precursors and yield extremely
Nanoparticles was assayed. The results
expensive materials. Also, the produced
revealed that addition of AgNPs to the culture
nano-particles exhibit undesirable aggregation
filtrate of Streptomyces clavuligerus and
with time. Various microorganisms (bacteria,
Trichoderma harzianum AUMC 5408 that have
yeast, and fungi) are known to synthesize
antimicrobial activity increased the diameter
of inhibition zone between 3-5 mm in both silver nanoparticles. The produced
nanoparticles have different size and shape.
cases.
Among the microorganisms, actinomycetes
producing nano-particles, that need more
KEY WORDS: studies. Actinomycetes are Gram positive free
living saprophytic bacteria, and it is a major
Silver Nanoparticles , UV-visible, FTIR source for production of natural metabolites
analysis, TEM, Antimicrobial activity. with different biol ogical activity such as
antibiotics. Also, it plays an important role in
control of infectious diseases, in enzymes,
CORRESP ONDENCE:
vitamins, antitumor agents, enzyme inhibitors
A m r A b d el - Monei m E l - W as ei f and production of novel pharmaceuticals
Botany and Microbiology Dept., Faculty of compounds (Knetsch and Koole, 2011) . Silver
Science (Boys), Al -Azhar University, Cairo, nanoparticles are very important due to their
Egypt . antimicrobial activity against the multidrug
E-mail: Amrelwaseif@yahoo.c om resistant microorganisms due to their small
size. Therefore, AgNPs are extensively used
extract, 5.0; NaCl, 5.0 and agar, 20.0; the pH metal nanoparticles (Chan and Don, 2012). In
was adjusted to 7. Used for growth of the present study, the supernatant of
bacterial strains and Sabouraud dextrose agar Streptomyces clavuligerus and the mycelium
(SDA) for pathogenic fungi. of Trichoderma harzianum AUMC 5408 were
Bi oacti vi ty assay: used for the synthesis of AgNPs. After
incubation at appropriate conditions, colour
To produce the crude antimicrobial
change was observed confirming the
compounds by Streptomyces clavuligerus, it
formation of silver nanoparticles. The weight
was grown in 250 ml conical flask of starch
and pH of AgNPs p roduced from
nitrate medium 150 rpm at 30 ⁰ C for 7 days.
Streptomyces clavuligerus and Trichoderma
Also, other antimicrobial compounds were
harzianum AUMC 5408 were tabled in table 1.
produced by Trichoderma harzianum AUMC
Results noted that Streptomyces clavuligerus
5408 using 250 ml conical flask of under 150
has superior than Trichoderma harzianum
rpm at 28 ⁰ C for 5 days.
AUMC 5408 Thus, the need for clean, cost
The antimicrobial activity was effective, eco-friendly and bi ocompatible
investigated by using agar well diffusion synthesis of metal nanoparticles encouraged
method as follow: 20.0 ml of the media the investigator to exploit the biological
(Nutrient agar medium for bacterial strain and sources as nano factories. Chemical
SDA for pathogenic fungi at 28 - 30 ⁰ C was synthesis of nano-particles resulting in some
inoculated with 20.0 µl of the prepared test toxic chemical species adsorbed on the
microorg anisms' suspensions and poured in surface that could have effects in m edical
9.0 cm diameter plates and mixed well and applications (Singh et al., 2012a; Chladek et
allow to solidify. After solidification, holes of al., 2011) .
9.0 mm diameter were made in the agar plate Table 1. Production of Silver Nanoparticles (AgNPs)
by the aid of a sterile Cork -borer. For each
sample, duplicate holes were made and the n Weight of AgNPs Final
Microorganisms
(mg/L) pH
100.0 µl of the culture filtrate was poured in
the prepared holes using an automatic Streptomyces clavuligerus 66.5 7.4
micropipette. The Petri -dishes were kept in a Trichoderma harzianum AUMC 5408 11.0 8.0
refrigerator for one hour to permit
homogenous diffusion of the antimicrobial Characteri zati on of Si l ver Nanoparti cl es:
agent before growth of the test Ul travi ol et (UV) spectrum :
microorganism s, and then the plates were The biosynthesis of Ag + in the culture
incubated at 37 ◦ C for 24 hours for Gram filtrate of Streptomyces clavuligerus and
positive and Gram -negative bacteria and at mycelium of Trichoderma sp. were detected
28 ⁰ C for 72 hours for pathogenic fungi. The by the reaction mixture at regular intervals by
antimicrobial activities were determined by using UV spectroscopy. The UV-Vis spectrum
measuring the diameter of inhibition zone of AgNPs showed sharp narrow absorption
(Oluwafemi and Debiri, 2008). spectrum located between 420 - 440 nm for
both culture filtrate (CF) and mycelium
RESULTS AND DI SCUSSI ON : respectively (Figs 1 & 2). Several reports
stated that, the biosynthesis of Ag + happened
Production of Silver Nanoparticles (AgNPs):
due to the electron shuttle quinines and
The primary detection of silver reducing agents such as enzymes (Navin et
nanoparticles production can be indicated al., 2011). Biological method of synthesis of
clearly by the colour change of the treated nanoparticles exhibit strong absorption of
AgNO 3 solution from colourless into reddish electromagnetic waves in the visible range
brown due to reduction of silver ions in the due to their optical resonant property, called
solution to nano-sized silver atoms Surface Plasmon Resonance (SPR) due to
(nanoparticles) and accordingly the excitation excitation of surface Plasmon vibrations.
of surface plasmon vibrations arises in this
Fig. 2. UV-Vis spectra absorbance of synthesis AgNPs by mycelia of Trichoderma harzianum 5408.
FTI R spectrum m easurem ent: Transm i ssi on El ectron M i croscopy (TEM ):
FTIR measurements were carried out to The results of transmission electron
identify possible interaction between silver microscope (TEM) showed completely
and protein molecules, which may be difference between AgNPs shape and size
responsible for synthesis, stabilization and from Streptomyces clavuligerus and
well dispersed silver nanoparticles in the Trichoderma sp.
reaction mixture (Navin et al., 2011). FTIR The TEM results of Streptom yces
spectral analysis showed array of absorbance clavuligerus showed that, the AgNPs in the
bands in 400cm - 1 - 4000 cm - 1 . As shown for reaction mixture had a uniform spherical
both supernatant Streptomyces clavuligerus shape and showed varying sizes as observed
and Trichoderma sp. mycelium respectively. in figure 3. Under magnification of 50 nm the
There is a peak at 3445.21 and 3438.46 cm - 1 size of AgNPs were ranging from 2.0 to 4.5
for both supernatant and mycelium nm, under magnification of 100 nm t he sizes
respectively, which is assigned to the primary were 1.25 to 10.3 nm.
amines (N-H stretch group) and its intensity
The TEM image of Trichoderma sp.
showed large amounts of AgNPs were formed.
AgNPs at figure 4 showed average size of
This due to the presences of amide group in
5.33 - 29.46 nm under magnification of 200
proteins which has strong ability to bind metal
and 500 nm. About shape, the optic and clear
indicating that the proteins as capping agent
rounded and oval nanoparticles of AgNPs
to prevent agglomeration and there by
were detected; also, s eparated and
stabilizing the nanoparticles (Zayed et al.,
conjugated nanoparticles were showed. It is
2012). Also, there is a peak at 1638.23 and
worth mentioning that no separately silver
1644.98 cm -1 for both supernatant and
was shown, indicating the strong interaction
mycelium respectively which is assigned to
of Trichoderma sp.
primary amines (N -H bond).
Several researchers discussed the AUMC 5408 with AgNPs was assayed to
effect of AgNPs on the microorganisms and determine the minimum inhibition
proved that, the activity of AgNPs against the concentration (MIC) of growth. Results
pathogen was due to a response of the obtained MIC at conc. of 3.12, 12.5, and
surface of AgNPs with water which led to 6.25% which recorded 17, 14 and 13 mm of
formation of hoisted levels of receptive inhibition zone diameter for Escherichia coli
oxygen species, to be specific hydroxyl NCTC 10416, Staphylococcus aureus ATCC
radicals and thus actuate as oxidative anxiety. 29213 and Candida albicans ATCC 10231,
Also, a presentation of microorganisms with respectively.
AgNPs results in an expanded cell disguise of
the nanoparticles and microbial cell harm CONCLUSI ON:
(Singh et al., 2012b) . The pres ent st udy c oncludes t hat silver
The antimicrobial activity was reported nanop articles w ere eff ectiv el y s ynthesiz ed by
to be due to the penetration of AgNPs into the Strept omyces clav uligerus and Trichoderm a
bacteria, damaging the cell memb rane and harzi anum AU MC 5408 isol at ed from Eg yptian
release of cell contents (Panacek et al., soil. T his was c onfirmed b y usi ng diff erent
2006). On the other hand, the release of Ag+ tec hniques. Mor eover, t he rec ov ered Silver
ions from the nanoparticles may be nanop articles when mi xed with cr ude
contributed to antibacterial and antifungal antimicrobial c ompounds produc ed b y thes e
properties of AgNPs (Kim et al., 2009; Li et microorganisms show maxim um z one of
al., 2010). inhibition agains t E scherichi a c oli NCTC
Also, results proved that, all 10416, S taphyloc occ us aureus ATCC 29213
concentrations of Streptomyces clavuligerus and C andida albic ans ATCC 10231. T his
AgNPs mixed with its culture filtrate have result sugges ts t hat bios ynthesiz ed silver
antimicrobial activity against the pathogenic nanop articles ar e highl y ant agonistic i n natur e
tested strains under study. The zone of and they showed high antimic robial ac tivit y
inhibition was decreased by decreasing the when combined wit h thes e antimicrobial
concentration of AgNPs and the mi nimum biomolec ules becaus e silver nanoparticl es
inhibition concentration (MIC) of growth was allowed them to easil y i nt eract with other
obtained at conc. of 3.12% which recorded particles and i ncreased c yt otoxi cit y agains t
22, 16, and 19 mm of inhibition zone diameter the pat hogens . Thus, t hey may hav e pot ential
for Escherichia coli NCTC 10416, in treatm ent of diseas es c aused b y thes e
Staphylococcus aureus ATCC 29213 and pathogens and in dev eloping antimicrobial
Candida albicans ATCC 10231, respectively. agents agai nst drug resist ance pat hog ens .
On the other hand, the mixture of
culture filtrate of Trichoderma harzianum
REFERENCES:
Birla SS, Tiwari VV, Gade AK, Ingle AP, Yadav Hajipour MJ, Fromm KM, Ashkarran AA, Jimenez de
AP, Rai MK. 2009. Fabrication of sil ver Aberasturi D, de Larramendi IR, Rojo
nanoparticles by Phoma glomerata and its T, Serpooshan V, Parak WJ, Mahmoudi M.
combined effect against Escherichi a coli, 2012. Antib acterial properties of
Pseudomonas aerugi nosa and Staphylococcus nanoparticles. Trends Biotechnol., 30(10):
aureus. Lett. Appl. Microbiol., 48(2): 173– 499-511.
179. Kim KJ, Sung WS, Suh BK, Moon SK, Choi JS, Kim
Chan YS, Don MM. 2012. Characterization of Ag JG, Lee DG. 2009. Antifungal activity and
nanoparticles produced by white-rot fungi and mode of action of sil ver nanoparticles on
its in vitro antimicrob ial activities. Int. Arabic J. Candida albicans. BioMetals, 22(2): 235–242.
Antimicrob. Agents, 2(3): 1-8. Kim JS, Kuk E, Yu KN, Kim JH, Park SJ, Lee
Chladek G, Mertas A, Barszczewska-Rybarek HJ, Kim SH, Park YK, Park YH, Hwang
I, Nalewajek T, Zmudzki J, Król CY, Kim YK, Lee YS, Jeong DH, Cho MH.
W, Lukaszczyk J. 2011. Antifungal activity of 2007. Antibacterial effects of sil ver
denture soft lining material modified by sil ver nanoparticles. Nanomedicine, 3(1): 95 -10.
nanoparticles: a pilot study. Int. J. Mol. Sci., Knetsch MLW, Koole LH. 2011. New strategies in the
12(7): 4735–4744. development of antimicrobial coatings: the
Cioffi N, Ditaranto N, Torsi L, Picca RA, De Giglio example of increasing usage of sil ver and
E, Sabbatini L, Novello L, Tantillo G, Bleve- sil ver nanoparticl es. Polymers, 3(1): 340 –366.
Zacheo T, Zambonin PG. 2005. Synthesis, Kokura S, Handa O, Takagi T, Ishikawa T, Naito
analytical characterization and bioactivity of Y, Yoshikawa T. 2010. Sil ver nanoparticl es as
Ag and Cu nanoparticles embedded in poly- a safe preservative for use in
vinyl-methyl-ketone films. Anal. Bioanal. cosmetics. Nanomedicine, 6(4): 570–574.
Chem., 382(8): 1912-1918.
Lara HH, Garza-Treviño EN, Ixtepan-Turrent L, Singh
El-Ghwas DE, El-Waseif AA. 2016. The synthesis of DK. 2011. Silver nanoparticles are broad -
sil ver nanoparticals from Streptomyces sp. spectrum bactericidal and virucidal
with antimicrobial activity. Int. J. Pharm Tech compounds. J. Nanobiotechnol., 9: 30.
Res., 9(4): 179-186.
Li WR, Xie XB, Shi QS, Zeng HY, Ou -Yang YS, Chen Sheng Z, Liu Y. 2011. Effects of sil ver nanoparticl es
YB. 2010. Antib acterial activity and on wast ewater biofilms. Water Res., 45(18):
mechanism of sil ver nanop arti cles on 6039–6050.
Escherichia coli. Appl. Microbiol. Biotechnol., Singh G, Joyce EM, Beddow J, Mason TJ. 2012a.
85(4): 1115–1122. Evaluation of antibacterial activity of ZnO
Navin J, Arpit B, Sonali M, Tarfdar JC, Jitendra P. nanoparticals coated sonochemically on to
2011. Extracellular biosynthesis and textile fabrics. J. Microb iol. Biotechnol. Food
characterization of sil ver nanoparticles using Sci., 2(1): 106-120.
Aspergillus flavus NJP08; A mechanism Singh S, Kumar P, Gopalan N, Shrivastava B, Kuhad
perspective. Nanoscale, 3(2): 635-641. RC, Chaudhary HS. 2012b. Isolation and
Oluwafemi F, Debiri F. 2008. Antimicrobial effect of partial characterization of Actinomycetes with
Phyllanthus amarus and Parqueti na antimicrobial activity against multidrug
nigrescens on Salmonella typhi. Afr. J. Biom. resistant bacteria. Asian Pac. J. Trop.
Res., 11(2): 215-219. Biomed., 2(2): S1147-S1150.
Panacek A, Kvítek L, Prucek R, Kol ar M, Vecerova Zayed MF, Eisa WH, Shabaka AA. 2012. Malva
R, Pizúrova N, Sharma VK, Nevecna T, Zboril parviflora extract assisted green synthesis of
R. 2006. Sil ver colloid nanoparticles: sil ver nanoparticl es Spectrochim. Acta Mol.
synthesis, characterization, and their Biomol. Spectrosc., 98: 423-428.
antibacterial activity. J. Phys. Chem. B,
110(33): 16248–16253.
تعزيز النشاط الحيوي بجسيمات النانوية الفضية الميكروبية التي تنتج وتتوصف من
Streptomyces and Trichoderma
** نجوى أحمد عبد هللا،** شيماء عبد العزيز أحمد،** نيفين أحمد إبراهيم،*عمرو عبد المنعم الوصيف
مصر، القاهرة، جامعة االزهر،) كلية العلوم (بنين،*قسم النبات والميكروبيولوجي
مصر، القاهرة، جامعة عين شمس، كلية العلوم،**قسم الميكروبيولوجي
جسيمات الفضة النانوية كما تم دراستها باستخدام تحليل تم استخدام اثنين من العزالت الميكروبية المختارة
وكان متوسط حجم.الطيف باألشعة تحت الحمراء من التربة المصرية إلنتاج الفضة النانوية من مصدر بيولوجي
نانومتر في حالة4.5 إلى2.0 جسيمات الفضة النانوية من وكان ا ختيار اثنين من.باستخدام محلول نترات الفضة
نانومتر29.46- 5.33 ولكن، Streptomyces clavuligerus الكائنات الدقيقة اعتمادا على قدرة أكتينوميستس
وقد تم.Trichoderma harzianum AUMC 5408 في حالة والفطريات لتخليق جسيمات الفضة النانوية وإنتاج المركبات
تعزيز تعزيز النشاط المضاد للميكروبات من المركبات سجلت النتائج أن الوزن الجاف.المضادة للميكروبات
وأظهرت.المضادة للميكروبات الخام مع الفضة النانوية Streptomyces جسيمات الفضة النانوية المنتجة من
النتائج أن إضافة جسيمات الفضة النانوية لرشيح من clavuligerus Trichoderma harzianum AUMC 5408
Streptomyces clavuligerus and Trichoderma توصيف. لتر على التوالي/ ملغم11.0 و66.5 كانت
التي لها نشاط مضادharzianum AUMC 5408 وأظهرت نتائج تحليل.جسيمات الفضة النانوية نفذت
ملم في كلتا5- 3 للميكروبات زيادة قطر منطقة التثبيط بين نانومتر440-420 األشعة فوق البنفسجية الطيف تقع بين
. الحالتين خصائص.لكل من الرشيح والغزل الفطري على التوالي