EFFECT OF NANOPARTICLES ON

MICROORGANISMS

INTRODUCTION
Nanoparticles are defined as particulate dispersions or
solid particles with a size in the range of 10-1000nm. The
drug is dissolved, entrapped, encapsulated or attached to a
nanoparticle matrix. Depending upon the method of
preparation, nanoparticles, nanospheres or nanocapsules
can be obtained.
Over the past few decades, inorganic nanoparticles,
whose structures exhibit significantly novel and improved
physical, chemical, and biological properties, phenomena,
and functionality due to their nanoscale size, have elicited
much interest. Nanophasic and nanostructured materials are
attracting a great deal of attention because of their potential
for achieving specific processes and selectivity, especially in
biological and pharmaceutical applications.
Discoveries in the past decade have demonstrated that
the electromagnetic, optical, and catalytic properties of
noble-metal nano-crystals are strongly influenced by shape
and size. This has motivated an upsurge in research on the
synthesis routes that allow better control of shape and size,
with projected applications in nano-electronics and
spectroscopy.
 Recent studies have demonstrated that specially
formulated metal oxide nanoparticles have good
antibacterial activity, and antimicrobial formulations
comprising nanoparticles could be effective bactericidal
materials.

Antibacterial effect of AgO nanoparticle:

Among inorganic antibacterial agents, silver has been
employed most extensively since ancient times (Silver S,
Phung LT., 1996) to fight infections and control spoilage. The
antibacterial and antiviral actions of silver, silver ion, and
silver compounds have been thoroughly investigated.
However, in minute concentrations, silver is nontoxic to
human cells. The epidemiological history of silver has
established its nontoxicity in normal use. Catalytic oxidation
by metallic silver and reaction with dissolved monovalent
silver ion probably contribute to its bactericidal effect .
Microbes are unlikely to develop resistance against silver, as
they do against conventional and narrow-target antibiotics,
because the metal attacks a broad range of targets in the
organisms, which means that they would have to develop a
host of mutations simultaneously to protect themselves.
Thus, silver ions have been used as an antibacterial
component in dental resin composites, in synthetic zeolites ,
and in coatings of medical devices.
 Elechiguerra and coworkers found that silver
nanoparticles undergo a size-dependent interaction with
human immunodeficiency virus type 1, preferably via
binding to gp120 glycoprotein knobs. The size-dependent
interaction of silver nanoparticles with gram-negative
bacteria has also been reported by the same group.
However, little is known about how the biological activity of
silver nanoparticles changes as the shape of the particles
changes.
For these reason, investigated the shape dependence
of the antibacterial activity of silver nanoparticles against
Escherichia coli. Silver nanoparticles of different shapes were
synthesized by solution phase routes, and their interactions
with E. coli were studied. Energy-filtering transmission
electron microscopy (EFTEM) was used as a complementary
technique to examine the treated cells.
The antimicrobial activity of silver nanoparticles against
E. coli was investigated as a model for Gram-negative
bacteria. Bacteriological tests were performed in Luria-
Bertani (LB) medium on solid agar plates and in liquid
systems supplemented with different concentrations of
nanosized silver particles. These particles were shown to be
an effective bactericide.
Silver nanoparticles exhibit antibacterial properties via
bacterial inactivation and growth inhibition. The mechanism
is not yet completely understood. This work was aimed at
elucidating the effect of silver nanoparticles on inactivation
of Escherichia coli, by studying particle-particle interactions
in aqueous suspensions. Stable, molecularly capped,
positively or negatively charged silver nanoparticles were
mixed at 1 to 60microgmL(-1) with suspended E. coli cells to
examine their effect on inactivation of the bacteria.

Gold nanoparticles with the same surfactant were used

as a control, being of similar size but made up of a
presumably inert metal. Log reduction of 5log(10) and
complete inactivation were obtained with the silver
nanoparticles while the gold nanoparticles did not show any
inactivation ability. The effect of molecularly capped
nanoparticles on E. coli survival was dependent on particle
number. Log reduction of E. coli was associated with the
ratio between the number of nanoparticles and the initial
bacterial cell count. Electrostatic attraction or repulsion
mechanisms in silver nanoparticle - E. coli cell interactions
did not contribute to the inactivation process.

Antibacterial effect of ZnO nanoparticle:

The antimicrobial activity of zinc oxide nanoparticles on

Enterobacter aerogenes was examined. A set of different
concentrations of nanoparticles at 50, 75,100 & 125 µg/ml
were tested on gram negative Enterobacter sp. for their
concentration dependent antimicrobial effects. A number of
other bacteria including E. coli & B. subtilus were also
evaluated against zinc oxide nanoparticles suspension to
check the bacterial growth.
The antimicrobial activity was examined by drawing
growth curves using spectrophotometer assisted absorption
observations. The zinc oxide nanoparticles adsorption on
bacterium surface was visualized by scanning and
transmission electron microscopies. The antimicrobial
activity was further confirmed by disc diffusion method. The
increased concentrations of zinc oxide nanoparticles
effectively checked the bacteria growth and increased the
diameter of inhibition zone in the experiment.
In presence of zinc oxide nanoparticles, Enterobacter
sp. growth was significantly reduced while the zinc oxide
powder showed lower levels of antimicrobial activity when
compared at same dose levels of 50, 75, 100 & 125 µg/ml.
Further studies on a mixture of lower levels of antimicrobial
drugs and zinc oxide nanoparticle formulation are in
progress with a view to effectively curtail the comparatively
large dose regimen of the anti-microbial drugs.
Metal oxide nanoparticles have marked antibacterial
activity. The toxic effect of these nanoparticles, such as
those comprised of ZnO, has been found to occur due to an
interaction of the nanoparticle surface with water, and to
increase with a decrease in particle size. In the present
study, we tested the ability of ZnO nanoparticles to affect
the viability of the pathogenic yeast, Candida albicans
(C. albicans). A concentration-dependent effect of ZnO on
the viability of C. albicans was observed. The minimal
fungicidal concentration of ZnO was found to be 0.1 mg ml − 1
ZnO; this concentration caused an inhibition of over 95% in
the growth of C. albicans. ZnO nanoparticles also inhibited
the growth of C. albicans when it was added at the
logarithmic phase of growth. Addition of histidine (a
quencher of hydroxyl radicals and singlet oxygen) caused
reduction in the effect of ZnO on C. albicans depending on
its concentration. An almost complete elimination of the
antimycotic effect was achieved following addition of 5 mM
of histidine. Exciting the ZnO by visible light increased the
yeast cell death. The effects of histidine suggest the
involvement of reactive oxygen species, including hydroxyl
radicals and singlet oxygen, in cell death. In light of the
above results it appears that metal oxide nanoparticles may
provide a novel family of fungicidal compounds

Nanoparticle metal oxides offer a wide variety of

potential applications in medicine due to the unprecedented
advances in nanobiotechnology research. In this work, the
effect of zinc oxide (ZnO) nanoparticles prepared by
mechano-chemical method on the antibacterial activity of
different antibiotics was evaluated using disk diffusion
method against Staphylococcus aureus and Escherichia coli.
The average size of ZnO nanoparticles was between 20 nm
and 45 nm. Although ZnO nanoparticles (500 microg/disk)
decreased the antibacterial activity of amoxicillin, penicillin
G, and nitrofurantoin in S. aureus, the antibacterial activity
of ciprofloxacin increased in the presence of ZnO
nanoparticles in both test strains. A total of 27% and 22%
increase in inhibition zone areas was observed for
ciprofloxacin in the presence of ZnO nanoparticles in S.
aureus and E. coli, respectively. The enhancing effect of this
nanomaterial on the antibacterial activity of ciprofloxacin
was further investigated at three different contents (500,
1000, and 2000 microg/disk) against various clinical isolates
of S. aureus and E. coli The enhancing effect of ZnO
nanoparticles on the antibacterial activity of ciprofloxacin
was concentration-dependent against all test strains. The
most enhancing activities were observed in the contents of
the 2000 microg/disk.

Antibacterial effect of MgO nanoparticles

Nanocrystals of common metal oxides such as

MgO, CaO,ZnO, TiO2, Al2O3, and Fe2O3, have been shown
to be highly efficient and active adsorbents for many toxic
chemicals including air pollutants, chemical warfare agents,
and acidic gases. In most cases, destructive adsorption takes
place on the surface of the nanocrystals, so that the
adsorbate is chemically dismantled and thereby made
nontoxic. In particular, aerogel-prepared nanocrystalline
MgO has been shown to have a small average particle size (4
nm), high surface area ( 500 m2/g) and high reactivity.

The use of these nanocrystalline metal oxides is

limited under conditions where liquid water or water vapor is
present due to their tendency to adsorb water, and thereby
be partially deactivated toward adsorption of the target
pollutants. Although the target pollutants are usually
subjected to conversion reactions in the presence of water
as well, relatively large amounts of water can reduce the
amount of adsorption of the target adsorbate decreasing the
efficiency of the destructive adsorbent.

Activated carbon is made up of mainly graphitic

structures, which exhibit a less polar surface, and the
tendency to adsorb water is lower compared with
nanocrystalline metal oxide surfaces. Therefore, it was
hypothesized that if the metal oxide particles could be
coated with a porous carbon, the water problem might be
minimized, while still allowing destructive sorption of target
pollutants, perhaps, by stepwise adsorption on carbon
followed by migration to the nanocrystalline oxide surface.
REVIEW OF LITRETURE

In the world of emerging nanotechnology,one of the

primary concerns is the potential environmental impact of
nanoparticles. An efficient way to estmate nanotoxicity is to
monitor the response of bacteria exposed to nickel oxide,
cobalt oxide zinc oxide, copper oxide, iron oxide, titanium
oxide, iron oxide against model organism , Escherichia coli.
The toxicity of these metal oxide nanoparticles was tested
usin two methods: culturing in liquid media containing
nanoparticles and electrospraying the nanoparticles directly
onto the bacterial surface. Aqueous exposure mimics the
natural interaction between microbial species as
nanoparticles diffuse in the environment. On the other hand,
the electrospray technique allows direct interaction between
the nanoparticles and cells. This exposure method grants
insight into how “nano” associated properties from metal
nanoparticles affect the environment(Angela k.
Horst,biochemistry ,clarke college,2009)

Drinking water purification

 With the evolution of human civilization, our
understanding of pure drinking water underwent dramatic
changes. In early civilizations, the commonly practiced
measure for purity was the taste of the water. Water was
recognized as a symbol for the origin of life and for its
medicinal value; it was not designated as a carrier of
diseases. In the 17th century Anton van Leeuwenhoek’s
discovery of the microscope started to change the
perception of purity: we were empowered to see beyond the
suspended particles e.g the tiny material particles to the
microorganisms. Following the discoveries of Louis
Pasteur(study of microorganism based diseases) and John
Snow(linking of cholera spread in London with the quality of
water). Our understanding of pure drinking water was
changed(M.N. Baker, J.M. Taras, America Water Works
Association, Denver, 1981)

A number of chemical and biological

contaminants have endangered the quality of drinking water.
An overview of important events during last 200 years in
the area of drinking water purification. Realizing the
molecular nature of contamination in drinking water
significant progress has been made to utilize the chemistry
of nanomaterials for water purification.Three major type of
contaminants available in water are halogenated organics
including pesticides, heavy metals and microorganisms(T.
Pradeep, Anshup,2009).

Removal of pesticides with noble

nanoparticles
The discovery of Zero valent iron catalyzes the degradation
of halogenated aliphatics. carbon tetrachloride induces
corrosion in the metals,in a way similar to air and water. The
concept of corrosion was held true ;metal surface is oxidized
to metal chloride,degree of corrosion differs with metals as
protective layers are formed on some metal surfaces and
reactivity is dependent on the reduction
potential(T.Senzaki,Y.Kumgai,Kogyo Yosui 1988). The
reaction product was foun to be metalchloride and partially
dehalogenated organic product and then after iron
nanoparticles for enhanced degradation of pesticides
became popular.
• In the similar way the use of other reactive metals such
as magnesium,tin and zinc was attempted to study the
degradation of
halocarbons(T.Boronina,K.J.Klabunde,Sci.Technol 29
1995)
• It was to be remembered that in the case of extremely
reactive metals,two competing process happen: metals
will catalyze the dehalogenation of organic compounds
 and metal being thermodynamically unstablr in
water,will react to form the corresponding metal
hydroxide. It was found that in case of magnesium-
second process dominates; in case of tin ,carbon dioxide
is the dominant product;in case of zinc, CH4 is the
dominant product.
• The reaction of noble nanoparticles was studied with
widely used pesticides such as endosulfan(A.S Nair, R.T
Tom,
T.Pradeep,J.Environ.2003),malathion(A.S.Nair,T.Pradeep,
J.Nanosci 2007)
chlorpyrifos(A.S.Nair,T.Pradeep,J.Nanosci). the
interaction of pesticides with gold and silver
nanoparticles .showed different spectroscopic
behavior(A.S Nair, R.T Tom, T.Pradeep,J.Environ.2003).

Effect of nanoparticles against environmental

soil microbes

Nanotechnology has attracted global attention

because nanoparticles (NP) have properties unique from
their bulk equivalents. NP of Ag, CuO and ZnO are being
used industrially for several purposes including amendments
to textiles, cosmetics, sprays, plastics and paints (Mueller
NC, Nowack 2008) . A common feature of these three NP is
their antimicrobial activity ( Yoon KY, Hoon Byeon J, Park JH,
Hwang J 2007). The antimicrobial activity of NP largely has
been studied with human pathogenic bacteria, mainly
Escherichia coli and Staphylococcus aureus. Nano-Ag is
inhibitory to E. Coli (Yoon KY, Hoon Byeon J, Park JH, Hwang J
2007) and S. Aureus (Kim JS, Kuk E, Yu KN, Kim JH, Park SJ,
Lee HJ, Kim SH, Park YK, Park YH, Hwang CY, Kim YK, Lee YS,
Jeong DH, Cho MH 2007) . These microbes also are sensitive
to nano-CuO and nano-ZnO (Heinlaan M, Ivask A, Blinova I,
Dubourguier HC, Kahru A, Jones N, Ray B, Ranjit KT, Manna
AC 2008).

NP of Ag, CuO and ZnO are reported to attack

bacterial membranes. Short exposure of E. coli cells to nano-
Ag destabilizes the outer membrane, collapses the plasma
membrane potential and decreases ATP( Lok CN, Ho CM,
Chen R, He QY, Yu WY, Sun H, Tam PK, Chiu JF, Che CM
2006). Pits in E. coli cell walls were observed after nano-Ag
treatment(Gogoi SK, Gopinath P, Paul A, Ramesh A, Ghosh
SS, Chattopadhyay A 2006) and promoted release of green
fluorescent protein from transformed E. coli cells (Gogoi SK,
Gopinath P, Paul A, Ramesh A, Ghosh SS, Chattopadhyay A
2006). Exposure of E. coli to nano-ZnO also causes loss in
membrane integrity (Reddy KM, Feris K, Bell J, Wingett DG,
Hanley C, Punnoose A 2007) Likewise, toxicity of NP of CuO
and ZnO are connected with cell membrane damage
(Heinlaan M, Ivask A, Blinova I, Dubourguier HC, Kahru A
2008).
 NP action may be due in part to their release of
free ions. Heavy metal ions have diverse effects on bacterial
cell function. For Cu ions, the mechanism may involve
oxidative stress (Cioffi N, Torsi L, Ditaranto N, Tantillo G,
Ghibelli L, Sabbatini L, Bleve-Zacheo T, D'Alessio M,
Zambonin PG, Traversa E 2005). The redox cycling of Cu
ions results in depletion of glutathione and affects the
sulfhydryl groups of proteins causing DNA damage and lipid
oxidation (Stohs SJ, Bagchi D 1995). Like Cu, Zn also is an
essential element for cells; levels of Zn above the essential
threshold level inhibit bacterial enzymes including
dehydrogenase (Nweke CO, Alisi CS, Okolo JC, Nwanyanwu
CE 2007) and certain protective enzymes, such as
thiolperoxidase, and glutathione reductase (Nguyen TMP,
Phan TN, Robert EM 2006). Zn inhibition of NADH oxidase is
proposed to impede the respiratory chain of E. Coli (Beard
SJ, Hughes MN, Poole RK 1995) Additionally, loss of
membrane potential is associated with inhibition by Zn ions
at cytochrome c oxidase in Rhodobacter sphaeroides (Mills
DA, Schmidt B, Hiser C, Westley E, Ferguson-Miller S 2002).
Ag ions inactivate proteins with SH groups and prevent the
ability of DNA to replicate (Feng QL, Wu J, Chen GQ, Cui FZ,
Kim TN, Kim JO 2000) . Holt and Bard (Holt KB, Bard AJ
2005) propose that NADH dehydrogenase in the electron
transport chain of E coli is inhibited by Ag ions.
 Extensive use and increasing demand for NP
will lead to their accumulation in the environment, especially
in landfills and their water effluents. Control of pathogenic
microbes by antimicrobial NP is a promising approach to
defeat the multiresistant pathogens such as methicillin-
resistant S. Aureus (Jones N, Ray B, Ranjit KT, Manna AC
2008). However, nontarget effects on the populations of
microbes that play beneficial roles in the environment could
have negative consequences. Many microbes have essential
roles in element cycling, (carbon, sulfur, nitrogen, etc.),
while others degrade pollutants and promote plant growth
(Molina MA, Ramos JL, Espinosa-Urgel M, Van Wees SC, Ent
S, Pieterse CM 2008). Nowack and Bucheli (2007) found little
published information about the release of NP in the
environment in their efforts to model the risk of Ag NP. Novel
and unprecedented sources are likely: recently,
commercially available nano-Ag-treated socks were found to
release Ag upon washing the socks (Benn TM, Westerhoff P
2008). Concern for nontarget effects of environmental
accumulation of Ag has been raised (Ramos-González MI,
Campos MJ, Ramos JL 2005).

The toxicity of NP against environmental microbes has been

little studied. Vibrio fisheri has been used because of its
natural light emitting property in assessment of toxicity and
Bacillus subtilis has been examined as an example of a
spore-forming bacterium (Adams LK, Lyon DY, Alvarez PJ,
Yoon KY, Hoon Byeon J, Park JH, Hwang J, Heinlaan M, Ivask
A, Blinova I, Dubourguier HC, Kahru A 2008). The aim of this
study was to evaluate the antimicrobial activity of nano-Ag,
nano-CuO and nano-ZnO using a biosensor constructed in
Pseudomonas putida KT2440. This pseudomonad is
beneficial in the environment because of its bioremediation
potential and it is a strong root colonizer (Ramos-González
MI, Campos MJ, Ramos JL, Child R, Miller CD, Liang Y,
Narasimham G, Chatterton J, Harrison P, Sims RC, Britt D,
Anderson AJ 2007 The biosensor was constructed to emit
light from luxAB genes under the control of a promoter
containing a single heavy metal binding domain (MTCGHC).
Because the luciferase encoded by luxAB requires FMNH2 as
a substrate, expression from this promoter permits light
output dependent on the energy status of the cells (Koga K,
Harada T, Shimizu H, Tanaka K 2005).

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