Egypt. J. Exp. Biol. (Bot.

), 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

Bioa ctiv ity Enhancement with M icrobial Silv er Nanoparticl es

Produc ed an d Characteriz ed from Strepto myc es a nd Tr ichoderm a

ABSTRACT:
Two microbial isolates selected from Egyptian
soil were used for biological synthesis of
silver nanoparticles using silver
solution. The selection of these
microorganisms was depending on the ability
of actinomycetes and fungi to AgNPs
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
mg/l, respectively. Description of AgNPs from
Streptomyces clavuligerus supernatant and
Trichoderma harzianum AUMC 5408 mycelia
were carried out. Results of UV-analysis
showed spectrum located between 420 - 440
nm for both culture filtrate and mycelium
respectively characteristics of AgNPs. Also,
the Fourier Transform Infrared Spectroscopy
(FTIR) analysis was studied. The average
size of AgNPs were between range from 2.0
to 4.5 nm in case of Streptomyces
clavuligerus, but 5.33 - 29.46 nm in case of
Trichoderma harzianum AUMC
Enhancement of the antimicrobial activity of
crude antimicrobial compounds with Silver
Nanoparticles was assayed. The results
revealed that addition of AgNPs to the culture
filtrate of Streptomyces clavuligerus and
Trichoderma harzianum AUMC 5408 that have
antimicrobial activity increased the diameter
of inhibition zone between 3-5 mm in both
cases.
KEY WORDS:
Silver Nanoparticles , UV-visible,
analysis, TEM, Antimicrobial activity.
CORRESP ONDENCE:
A m r A b d el - Monei m E l - W as ei f
Botany and Microbiology Dept., Faculty of
Science (Boys), Al -Azhar University, Cairo,
Egypt .
E-mail: Amrelwaseif@yahoo.c om
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
nitrate N ag w a A hm ed A b d al l ah
two
Microbiology Department, Faculty of Science,
Ain Shams University, Cairo, Egypt.
ARTI CLE CODE: 42.02.17
I NTRODUCTI ON :
Nanotechnology is mainly concerned
with the synthesis of nano-materials using
different systems and their applications.
Nowadays, nano-particles are used in the
areas of agriculture, health care, environment
and consumer good s (El-Ghwas and El -
W aseif, 2016) . Nano-particles which
synthesized by chemical and physical
pathways are low yielding, energy intensive,
5408.
difficult to scale up, producing high levels of
hazardous wastes, requires the use of costly
organometallic precursors and yield extremely
expensive materials. Also, the produced
nano-particles exhibit undesirable aggregation
with time. Various microorganisms (bacteria,
yeast, and fungi) are known to synthesize
silver nanoparticles. The produced
nanoparticles have different size and shape.
Among the microorganisms, actinomycetes
producing nano-particles, that need more
studies. Actinomycetes are Gram positive free
living saprophytic bacteria, and it is a major
FTIR source for production of natural metabolites
with different biol ogical activity such as
antibiotics. Also, it plays an important role in
control of infectious diseases, in enzymes,
vitamins, antitumor agents, enzyme inhibitors
and production of novel pharmaceuticals
compounds (Knetsch and Koole, 2011) . Silver
nanoparticles are very important due to their
antimicrobial activity against the multidrug
resistant microorganisms due to their small
size. Therefore, AgNPs are extensively used

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440
in dental materials (Kokura et al., 2010),
coating stainless steel in medical devices
(Sheng and Liu, 2011), cosmetics (Birla et al.,
2009), and water treatment (Lara et al.,
2011). Antibacterial activity of AgNPs was
studied in various researches (Hajipour et al.,
2012; Cioffi et al., 2005) . Due to vast
emerging applications of AgNPs in d istinct
fields, there is an increase in searching for
synthesis of AgNPs and there is a pressing
need to increase their yield, for which
optimization of the process is very important
step.
For a long time, the antimicrobial
medications have been utilized t o restrain or
kill microorganisms. However, microbial
resistance to these drugs has developed on a
very large scale over time, greatly reducing
their effectiveness and is an ever -growing
problem (Kim et al., 2007). A study says that,
the drug resistant infections will kill an extra
10 million people a year worldwide more than
currently die from cancer by 2050 unless
action is taken. Therefore, one of the most
promising strategies for overcoming microbial
resistance is the use of nanoparticles.
Nanotechnolog y gave the solution to medicine
because it can find materials in nanoscale
diameter that have an enhanced bioactivity.
The main reason for their importance is the
increased specific surface area of these
nanoparticles in comparison to their volume,
which enables their interaction with bio -
organics present on the viable cell surface
(Kim et al., 2007).
The current approach suggests that
rapid synthesis of nanoparticles from silver
nitrate would be suitable for developing a
biological process for mass scale production
of formulations. The present study focused on
AgNPs production from biological source,
characterization using UV-visible, FTIR
analysis, TEM and role of AgNPs in
improvement of antimicrobial activity of
natural product from Actinomycetes and fun gi.
M ATERI AL AND M ETHODS:
M i croorgani sm s:
Two microorganisms were used in this
study: an actinomycete isolated from Egyptian
soil that was identified as Streptomyces
clavuligerus using molecular techniques
depending on 16s DNA sequence. The other
microorg anism was the soil fungus
Trichoderma harzianum AUMC 5408, which
was purchased from the Egyptian culture
collection of Assiut university Mycological
Centre (AUMC).
P roducti on of Si l ver Nanoparti cl es
(AgNP s):
Aft er 7 days of inc ubati on of
Strept omyces cl av uliger us on st arch ni trat e
medium (g/l): s tarc h 10. 0, p ot assium ni trat e
2. 0, K 2 HP O 4 1. 0, MgS O 4 . 7H 2 O, 0. 5, N aCl 0.5,
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Egypt. J. Exp. Biol. (Bot.), 13(2): 439 – 445 (2017)
FeS O 4 0.01, and C aCO 3 3. 0; t he pH w as
adjust ed t o 7.0 and 5 days f or Tric hoderm a
harzi anum AU MC 5408 on Cz apek D ox
medium (g/l): sucros e 30. 0, K 2 HP O 4 1. 0, KCl
0. 5, N aNO 3 2. 0, Mg SO 4 .7H 2 O 0. 5 and F eS O 4
0. 01; t he pH w as adjust ed t o 7.0 , t he m yc elia
(biomass) were sep arat ed from the c ultur e
broth b y filtrati on, then the m yc elia wer e
washed twic e wit h distilled water under st erile
conditions . T her eaft er, the super natant of
Strept omyces clav uligerus and the m yc elium
of Tric hoderm a harzi anum AU MC 5408 wer e
used f or the s ynt hesis of AgNPs. A sol ution of
silver nitrat e (1 mM) was prepar ed b y
dissolving 0. 017 g of t he comp ound in 100 ml
of distilled wat er. Then, 95 ml of silver ni trat e
soluti on w as added to 5 ml of Strept omyc es
clavuli ger us s uper nat ant and incubated agai n
for 7 d ays at room t emperat ure under dark
conditions w her eas , 100 ml of silver ni trat e
soluti on w as added to about 5. 0 g of
Trichoderma harzianum AU MC 5408 m yc elium
and incubated again for 5 days at 28⁰C under
150 rpm and dark conditions and b oth wer e
observed f or c olour chang ed (El -Ghw as and
El-W aseif, 2016).
Characteri zati on of Si l ver Nanoparti cl es :
Ul travi ol et (UV) spectrum :
The synthesis of AgNPs was confirmed
by ultraviolet (UV) spectrum analys is using:
T80 + UV/VIS Spectrometer, PG Instrument
Ltd. Range: 190 -1000 nm.
FTI R spectrum m easurem ent:
FTIR spectrum was obtained by mixing
with potassium bromide at 1: 100 ratio which
was compressed to a 2-mm semi-transparent
disk for 2 min. spectra over t he wave length
(4000 - 400 cm - 1 ) were recorded using Nexus
670 FTIR spectrophotometer (Iclet Co., USA)
Transm i ssi on El ectron M i croscopy (TEM ):
This study was undertaken to know the
size and shape of AgNPs. The TEM image
was carried out using: Electron prob e micro-
analyser JEOL – JXA 840A, Model Japan.
Thin films of the sample were prepared on a
coated copper grid by just placing a very
small amount of the sample on the grid. Then
the film on the TEM grid was allowed to dry
and the images of AgNPs were taken .
Anti m i crobi al acti vi ty :
Test m i croorgani sm s:
The antimicrobial activity was done
using various pathogenic microorganisms
such as Escherichia coli NCTC 10416 as
model for Gram -negative bacteria;
Staphylococcus aureus ATCC 29213 as model
for Gram -positive bacteria, Candida albicans
ATCC 10231 as model for pathogenic fungi.
Anti m i crobi al acti vi ty m edi a:
The media used for the antimicrobial
activity of the strains under study have the
following compositions (g/l): - Nutrient agar
medium: - D-glucose, 5.0; peptone, 5.0; meat
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El-Waseif et al., Bioactivity Enhancement with Microbial Silver Nanoparticles Produced from Streptomyces and Trichoderma
extract, 5.0; NaCl, 5.0 and agar, 20.0; the pH
was adjusted to 7. Used for growth of
bacterial strains and Sabouraud dextrose agar
(SDA) for pathogenic fungi.
Bi oacti vi ty assay:
To produce the crude antimicrobial
compounds by Streptomyces clavuligerus, it
was grown in 250 ml conical flask of starch
nitrate medium 150 rpm at 30 ⁰ C for 7 days.
Also, other antimicrobial compounds were
produced by Trichoderma harzianum AUMC
5408 using 250 ml conical flask of under 150
rpm at 28 ⁰ C for 5 days.
The antimicrobial activity was
investigated by using agar well diffusion
method as follow: 20.0 ml of the media
(Nutrient agar medium for bacterial strain and
SDA for pathogenic fungi at 28 - 30 ⁰ C was
inoculated with 20.0 µl of the prepared test
microorg anisms' suspensions and poured in
9.0 cm diameter plates and mixed well and
allow to solidify. After solidification, holes of
9.0 mm diameter were made in the agar plate
by the aid of a sterile Cork -borer. For each
sample, duplicate holes were made and the n
100.0 µl of the culture filtrate was poured in
the prepared holes using an automatic
micropipette. The Petri -dishes were kept in a
refrigerator for one hour to permit
homogenous diffusion of the antimicrobial
agent before growth of the test
microorganism s, and then the plates were
incubated at 37 ◦ C for 24 hours for Gram
positive and Gram -negative bacteria and at
28 ⁰ C for 72 hours for pathogenic fungi. The
antimicrobial activities were determined by
measuring the diameter of inhibition zone
(Oluwafemi and Debiri, 2008).
RESULTS AND DI SCUSSI ON :
Production of Silver Nanoparticles (AgNPs):
The primary detection of silver
nanoparticles production can be indicated
clearly by the colour change of the treated
AgNO 3 solution from colourless into reddish
brown due to reduction of silver ions in the
solution to nano-sized silver atoms
(nanoparticles) and accordingly the excitation
of surface plasmon vibrations arises in this
Fig. 1. UV-Vis spectra absorbance of synthesis AgNPs by supernatant Streptomyces clavuligerus.
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441
metal nanoparticles (Chan and Don, 2012). In
the present study, the supernatant of
Streptomyces clavuligerus and the mycelium
of Trichoderma harzianum AUMC 5408 were
used for the synthesis of AgNPs. After
incubation at appropriate conditions, colour
change was observed confirming the
formation of silver nanoparticles. The weight
and pH of AgNPs p roduced from
Streptomyces clavuligerus and Trichoderma
harzianum AUMC 5408 were tabled in table 1.
Results noted that Streptomyces clavuligerus
has superior than Trichoderma harzianum
AUMC 5408 Thus, the need for clean, cost
effective, eco-friendly and bi ocompatible
synthesis of metal nanoparticles encouraged
the investigator to exploit the biological
sources as nano factories. Chemical
synthesis of nano-particles resulting in some
toxic chemical species adsorbed on the
surface that could have effects in m edical
applications (Singh et al., 2012a; Chladek et
al., 2011) .
Table 1. Production of Silver Nanoparticles (AgNPs)
Weight of AgNPs Final
Microorganisms
(mg/L) pH
Streptomyces clavuligerus 66.5 7.4
Trichoderma harzianum AUMC 5408 11.0 8.0
Characteri zati on of Si l ver Nanoparti cl es:
Ul travi ol et (UV) spectrum :
The biosynthesis of Ag + in the culture
filtrate of Streptomyces clavuligerus and
mycelium of Trichoderma sp. were detected
by the reaction mixture at regular intervals by
using UV spectroscopy. The UV-Vis spectrum
of AgNPs showed sharp narrow absorption
spectrum located between 420 - 440 nm for
both culture filtrate (CF) and mycelium
respectively (Figs 1 & 2). Several reports
stated that, the biosynthesis of Ag + happened
due to the electron shuttle quinines and
reducing agents such as enzymes (Navin et
al., 2011). Biological method of synthesis of
nanoparticles exhibit strong absorption of
electromagnetic waves in the visible range
due to their optical resonant property, called
Surface Plasmon Resonance (SPR) due to
excitation of surface Plasmon vibrations.
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442 Egypt. J. Exp. Biol. (Bot.), 13(2): 439 – 445 (2017)

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).

Fig. 3. TEM of AgNPs produced by Streptomyces clavuligerus

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El-Waseif et al., Bioactivity Enhancement with Microbial Silver Nanoparticles Produced from Streptomyces and Trichoderma 443

Fig. 4. TEM of AgNPs produced by Trichoderma harzianum 5408.

Enhancem ent of antimi crobi al acti vity of The effect of antimicrobial compound
crude antimi crobi al com pound wi th Silver produced in the culture filtrates of
Nanoparti cl es: Streptomyces clavuligerus and Trichoderma
harzianum AUMC 5408, separately and mixed
The biosynthesis of antimicrobial compound
with AgNPs were illustrated in tables 2 & 3.
through Streptomyces clavuligerus and
The results revealed that addition of AgNPs to
Trichoderma harzianum AUMC 5408 was carried
the culture filtrate (CF) of Streptomyces
out using the previously mentioned media.
clavuligerus and Trichoderma harzianum
The well diffusion experiment was AUMC 5408, that have antimicrobial activity
carried out against Escherichia coli NCTC increased the diameter of inhibition zone
10416 as model for Gram -negative bacteria; between 3 - 5 mm in both cases.
Staphylococcus aureus ATCC 29213 as model
for Gram -positive bacteria, Candida albicans
ATCC 10231 as model for pathogenic fungi .
Table 2. Antimicrobial activity of the antimicrobial compound with AgNPs obtained from Streptomyces clavuligerus and MIC
*Diameter of inhibition zone (mm) against
Streptomyces clavuligerus
Escherichia coli Staphylococcus aureus Candida albicans
Crude C.F. 22.0 20.0 22.0
Crude C.F. + AgNPs 27.0 25.0 27.0
Crude C.F. + AgNPs dilution
50% 25.0 19.0 20.0
25% 22.0 17.0 20.0
12.5% 22.0 17.0 20.0
6.25% 22.0 16.0 19.0
3.12% 22.0 16.0 19.0

*Well diameter 9 mm (CF) culture filtrate

Table 3. Antimicrobial activity of the antimicrobial compound with AgNPs obtained from Trichoderma and MIC
*Diameter of inhibition zone (mm) against
Trichoderma sp.
Escherichia coli Staphylococcus aureus Candida albicans
Crude C.F. 22.0 18.0 24.0
Crude C.F. + AgNPs 25.0 21.0 27.0
Crude C.F. + AgNPs dilutions
50% 24.0 16.0 14.0
25% 23.0 15.0 14.0
12.5% 19.0 14.0 13.0
6.25% 18.0 0.0 13.0
3.12% 17.0 0.0 0.0

*Well diameter 9 mm (CF) culture filtrate

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444
Several researchers discussed the
effect of AgNPs on the microorganisms and
proved that, the activity of AgNPs against the
pathogen was due to a response of the
surface of AgNPs with water which led to
formation of hoisted levels of receptive
oxygen species, to be specific hydroxyl
radicals and thus actuate as oxidative anxiety.
Also, a presentation of microorganisms with
AgNPs results in an expanded cell disguise of
the nanoparticles and microbial cell harm
(Singh et al., 2012b) .
The antimicrobial activity was reported
to be due to the penetration of AgNPs into the
bacteria, damaging the cell memb rane and
release of cell contents (Panacek et al.,
2006). On the other hand, the release of Ag+
ions from the nanoparticles may be
contributed to antibacterial and antifungal
properties of AgNPs (Kim et al., 2009; Li et
al., 2010).
Also, results proved that, all
concentrations of Streptomyces clavuligerus
AgNPs mixed with its culture filtrate have
antimicrobial activity against the pathogenic
tested strains under study. The zone of
inhibition was decreased by decreasing the
concentration of AgNPs and the mi nimum
inhibition concentration (MIC) of growth was
obtained at conc. of 3.12% which recorded
22, 16, and 19 mm of inhibition zone diameter
for Escherichia coli NCTC 10416,
Staphylococcus aureus ATCC 29213 and
Candida albicans ATCC 10231, respectively.
On the other hand, the mixture of
culture filtrate of Trichoderma harzianum
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The pres ent st udy c oncludes t hat silver
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‫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

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