Vol.1, No.2, 129-135 (2009) Natural Science
Copyright © 2009 SciRes. OPEN ACCESS
ZnO Nanoparticles: Synthesis and Adsorption Study
K. Prasad1, Anal K. Jha2
1University Department of Physics, T.M. Bhagalpur University, Bhagalpur - 812 007, India; *k.prasad65@gmail.com
2University Department of Chemistry, T.M. Bhagalpur University, Bhagalpur - 812 007, India
Received 21 July 2009; revised 27 July 2009; accepted 30 July 2009.
A low-cost, green and reproducible probiotic
microbe (Lactobacillus sporogens) mediated
biosynthesis of ZnO nanoparticles is reported.
The synthesis is performed akin to room tem-
perature in five replicate samples. X-ray and
transmission electron microscopy analyses are
performed to ascertain the formation of ZnO
nanoparticles. Rietveld analysis to the X-ray
data indicated that ZnO nanoparticles have
hexagonal unit cell structure. Individual
nanoparticles having the size of 5-15 nm are
found. A possible involved mechanism for the
synthesis of ZnO nanoparticles has been pro-
posed. The H2S adsorption characteristic of ZnO
nanoparticles has also been assayed.
Keywords: ZnO Nanoparticle; Biosynthesis;
Nanobiotechnology; Eco-friendly; H2S Adsorption
Nature by dint of its diversity provides exponential pos-
sibilities in terms of endearing adaptability of its con-
stituent cohorts. Both bacteria and fungi make such an
exciting category of microorganisms having naturally
bestowed property of reducing/oxidizing metal ions into
metallic/oxide nanoparticles thereby functioning as
‘mini’ nano-factories. [1,2] It is indeed their chemical
constitutions (or metabolic status) which provide them
strength to withstand such environmentally diverse
habitats. The non-pathogenic, gram positive, mesophilic
facultative anaerobe Lactobacillus, commonly used for
curdling of milk forms part of the beneficial community
of microbes present in the human intestinal tract.
Zinc oxide (ZnO) is considered to be a technologically
prodigious material having a wide spectrum of applica-
tions such as that of a semiconductor (Eg = 3.37 eV),
magnetic material, electroluminescent material, UV-abs-
orber, piezoelectric sensor and actuator, nanostructure
varistor, field emission displaying material, thermoelec-
tric material, gas sensor, constituent of cosmetics etc.
[3-9] There are several synthesis procedures for the
preparation of ultrafine oxide nanoparticles such as sol-
gel, hydrothermal, solvothermal, flame combustion,
emulsion precipitation, fungus mediated biosynthesis,
etc. [10-16] Each method has its own merits and demer-
its. They are time consuming, capital intensive and re-
quire trained manpower. Besides, the development of
eco-friendly, 'green' synthesis protocols is in line with
the recent RoHS and WEEE legislation stipulated by the
EU. Therefore, an urge to develop green synthesis pro-
tocols which goes in consonance with the above men-
tioned stipulations is need of the hour. Microbes exhibit
a natural capability to adapt to changes in their environ-
ment. Recent research devoted towards the study of in-
teraction between inorganic substances and biological
systems has highlighted its potential application for the
production of nanomaterials with interesting techno-
logical properties. [1,2,17-20] Numerous recent publica-
tions have highlighted the potential for microbes, par-
ticularly bacteria (including thermophilies) and fungi, to
synthesize metallic and/or oxide nanoparticles. [21-35]
The facultative nature of Lactobacilli, offers the poten-
tial to produce nanoparticles under both oxidizing and
reducing conditions. [2,18,35]
No work to the best of author’s acquaintance has so
far been reported regarding the synthesis of ZnO nano-
particles employing Lactobacilli. Lactobacilli strain,
cultured from spores an effort has been taken for synthe-
sizing ZnO nanoparticles (ZnO NPs) in the present work.
We have tried to explore a cost effective, green and read-
ily reproducible approach for the purpose of scaling up
and subsequent downstream processing. An effort to
understand the nano-transformation mechanism of bio-
synthesis has also been made. It is well established that
Hydrogen sulfide (H2S) is a colorless, corrosive and
highly toxic gas, a low concentration of which in air,
brings smell of rotten eggs and it substantially contrib-
utes towards air pollution. [36,37] The potential of ZnO
NPs towards H2S adsorption has also been assayed in the
present study.
K. Prasad et al. / Natural Science 1 (2009) 129-135
Copyright © 2009 SciRes. OPEN ACCESS
2.1. Biosynthesis of ZnO Nanoparticles
Pharmaceutical grade Lactic acid Bacillus spore tablets
(SporeLac DS, Sanyko Pharmaceuticals, Japan) were
procured and two tablets were dissolved in 50 mL sterile
distilled water containing standard carbon and nitrogen
source. As per specification, each tablet was capable of
producing 120 million spores of the bacterium. The cul-
ture solution was allowed to incubate on room tempera-
ture overnight. Next day, the presence of Lactobacillus
was confirmed under an optical microscope. The pH of
this source culture solution was observed to be equal to 3.
Now, 10 mL of this source culture was doubled in vol-
ume by mixing equal volume of sterile distilled water
containing nutrients in five different hard glass test tubes.
In yet another tube instead of adding the source culture
solution, sterile distilled water containing nutrients was
pooled and this was treated as control. All these culture
tubes were gently heated on a steam bath and were al-
lowed to incubate overnight in laboratory ambience for
another 24 hours on orbital shaker. Next day, the pH was
taken and found to be in the range of 4-5 in case of cul-
ture solution and 7 in case of control. Small quantity of
NaHCO3 was added in culture solution until it attains pH
6. It was brought to this pH as a lower value delays the
process of transformation. [2] Similarly, a small volume
of distilled water along with carbon and nitrogen source
and NaHCO3 were pipetted in the control tube and the
pH = 8.5 were recorded. Analytical reagent grade Zinc
Chloride (ZnCl2) was taken into use for preparing a so-
lution of 0.25(M) strength at room temperature. Control
solution was prepared by adding 100 mL sterile distilled
water, carbon and nitrogen containing nutrients and the
mild base in known quantitative ratio (5:1:1). To each of
these tubes, 20 mL of Zinc Chloride solution was added.
The pH of the control tube was noted to be 8-9 in 5 dif-
ferent set of experiments. Culture solution containing
tubes including control tube were heated on the steam
bath up to 80°C for 5 to 10 minutes. An appearance of
starch like haziness in solution and white deposition at
the bottom of the tube was perceived as an indication of
commencement of transformation. No such deposition or
haziness was observed in control tube. The tubes were
allowed to incubate in the laboratory ambience for an-
other 9 hours, after which distinctly markable coalescent
white clusters deposited at the bottom of all the tubes
except in control. A remarkable change in pH was ob-
served at this stage (6.0 to 7.5) excluding control (8 to 9).
The chemical reactions which proceed in the culture
medium may be as follows:
(Lact ate) (Pyruvate)
(Glu cose)
 33HCONaNaHCO
23 COOHHCO  
  ClZnZnCl 2
2)(2 OHZnOHZn 
2.2. Characterization
The formation of ZnO NPs was checked by X-ray dif-
fraction (XRD) technique using an X-ray diffractometer
(XPERT-PRO, Pan Analytical) with CuK radiation ( =
1.5406Å) over a wide range of Bragg angles (10 2
80). The XY (2θ vs. intensity) data obtained from this
experiment were plotted with the WinPLOTR program
and the angular positions of the peaks were obtained
with the same program. [38] The dimensions of the unit
cell, hkl values and space group of ZnO NPs were ob-
tained using the DICVOL program in the FullProf 2000
software package and then refinement was carried out
through the profile matching routine of FullProf. [39]
The Bragg peaks were modeled with pseudo-Voigt func-
tion and the background was estimated by linear inter-
polation between selected background points. The crys-
tallite size (D) and the lattice strain of ZnO NPs were
estimated by analyzing the broadening of X-ray diffrac-
tion peaks, using Williamson-Hall approach. [40]
sin)/(2)/(cos 
DK (1)
is diffraction peak width at half intensity
(FWHM) and
is the lattice strain and K is the
Scherrer constant (0.89). The term Kλ/D represents the
Scherrer particle size distribution. TEM micrograph of
ZnO NPs was obtained using Hitachi H-7500 transmis-
sion electron microscope. The specimen was suspended
in distilled water, dispersed ultrasonically to separate
individual particles, and two drops of the suspension
deposited onto holey-carbon coated copper grids.
3.1. Structural and Microstructural Studies
Rietveld refinements on the X-ray (XRD) data were
done on ZnO NPs, selecting the space group P6/mmm.
Figure 1 depicts the observed, calculated and difference
XRD profiles for ZnO NPs after final cycle of refine-
ment. It can be seen that the profiles for observed and
calculated one are perfectly matching. The value of χ2
comes out to be equal to 3.16, which may be considered
to be very good for estimations. The profile fitting pro-
cedure adopted was minimizing the χ2 function. [41] The
XRD analyses indicated that ZnO NPs has a hexagonal
unit cell. The crystal data and refinement factors of ZnO
K. Prasad et al. / Natural Science 1 (2009) 129-135
Copyright © 2009 SciRes. OPEN ACCESS
Figure 1. Rietveld refined pattern of ZnO NPs in the space group P6/mmm. Symbols repre-
sent the observed data points and the solid lines their Rietveld fit. Inset: Williamson-Hall plot
for ZnO NPs.
Table 1. The crystal data and refinement factors of ZnO NPs obtained from X-ray powder diffraction data.
Parameters Results Description of parameters
Crystal System
Space group
a (Å)
b (Å)
c (Å)
α (°)
β (°)
γ (°)
Rp (profile factor) = 100[Σ|yi-yic|/Σ|yi|], where yi is the observed intensity and yic is the
calculated intensity at the ith step.
Rwp (weighted profile factor) = 100[Σωi|yi-yic|2/Σωi(yi)2]1/2, where 2
/1 ii
and 2
is variance of the observation.
Rexp (expected weighted profile factor) = 100[(n-p)/Σωi(yi)2]1/2, where n and p are the
number of profile points and refined parameters, respectively.
RB (Bragg factor) = 100[Σ|Iobs-Icalc|/Σ|Iobs|], where Iobs is the observed integrated inten-
sity and Ical c is the calculated integrated intensity.
RF (crystallographic RF factor) = 100[Σ|Fobs-Fcalc|/Σ|Fobs|], where F is the structure
factor, F = (I/L), where L is Lorentz polarization factor.
χ2 = Σωi(yi-yic)2.
d (Durbin–Watson statistics) = Σ{[ωi(yi-yic)-ωi-1(yi-1-yic-1)]2}/Σ[ωi(yi-yic)]2.
QD = expected d.
S (goodness of fit) = (Rwp/Rexp).
NPs obtained from XRD data are depicted in Table 1.
The lattice parameter as obtained for ZnO NPs is in good
agreement with the literature report (PCPDF No.
#89-0510). Inset Figure 1 illustrates the William-
son-Hall plot for ZnO NPs. A linear least square fitting
cosθ–sinθ data yielded the values of average crys-
tallite size and lattice strain respectively to be 11 nm and
0.0035. The low value of lattice strain might be due to
the fact that the procedure adopted in the synthesis of
nanoparticles is natural (biosynthetic) one.
K. Prasad et al. / Natural Science 1 (2009) 129-135
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Figure 2. TEM photograph of ZnO NPs. Inset: ZnO NPs.
Figure 2 shows the TEM micrograph of ZnO NPs
(inset Figure 2) being formed using Lactobacillus strain.
The micrograph clearly illustrates the nanoparticles with
tubules and other irregular forms having the sizes of
5-15 nm. The measurement of size was carried along the
diameter of the particles. The difference in particle size
is possibly due to the fact that the nanoparticles are be-
ing formed at different times. It is found that the size of
the ZnO NPs estimated using TEM analysis to be in
fairly good agreement with the size estimated by the
Williamson-Hall approach.
3.2. Adsorption Study
Figure 3 shows the experimental setup to assess the ad-
sorption capacity of synthesized ZnO NPs as well as
bulk ZnO. Freshly prepared H2S was allowed to pass
through the equal quantities of bulk ZnO and ZnO NPs
(5 gm each) for a fixed span of time (30 min.) and flow
of gas was suitably regulated. The degree of absorption
was assessed directly through the change in colour of
lead acetate solution (from clear solution to black). It
was observed that the presence of bulk ZnO blackens the
solution (due to formation of lead sulfide) within 5 min-
utes, while ZnO NPs does the same in 25 min. The ex-
periment was pursued as five replicates and each gave
approximately the same result. This happens due to the
fact that nanoparticles have large surface to volume ratio
and hence a high surface activity and these features
might have led to better degree of adsorption of H2S in
comparison to its bulk counterpart. H2S absorption by
ZnO proceeds according to the reaction: ZnO + H2S
ZnS + H2O that results into formation of inert Zinc sul-
Lactobacilli cells are prokaryotes in terms of cellular
organization. They are gram positive (a thick pepti-
doglycan cell wall) bacteria showing facultative anaero-
bic properties, which probably make them suitable can-
didate microorganism for biosynthesis of metal as well
as oxide nanoparticle. Like most of the bacteria, they
have a negative electro-kinetic potential; which readily
attracts the cations and this step probably acts as a trig-
ger of the procedure of biosynthesis. Earlier, such a pos-
sibility of biosorption and bioreduction had been re-
ported in case of silver iodide by the Lactobacillus sp.
A09*. [42] The mesophilic, non-pathogenic and faculta-
tively anaerobic microbe like Lactobacillus has robust
metabolic capabilities. Addition of simple carbohydrates
into the culture medium tends to lower the value of oxi-
dation-reduction potential (or the Eh value). The oxida-
tion-reduction potential expresses the quantitative char-
acter of degree of aerobiosis having a designated unit
expressed as rH2 (the negative logarithm of the partial
pressure of gaseous hydrogen). By controlling rH2 of the
nutrient medium, conditions can be engineered for the
growth of anaerobes in the presence of oxygen by low-
ering the rH2, and also by cultivating the aerobes in an-
aerobic conditions by increasing the rH2 of the medium.
Figure 3. Experimental set up to study adsorption of H2S by ZnO NPs.
K. Prasad et al. / Natural Science 1 (2009) 129-135
Copyright © 2009 SciRes. OPEN ACCESS
ZnO + H
(In culture solution )
pH - Dependent
membrane bound
[O] [O]
(Lactobacillus Cell)
Partial pressure of gaseous H
)i.e. Eh - dependent upon
available carbon source
(in Culture Solution)
Low pH
Oxidase activity
Low r-H
i.e. HighEh
High Oxidation potential
(In culture solution )
Figure 4. Schematic showing the mechanism for the biosynthesis of ZnO NPs.
Composition of nutrient media, therefore; plays a piv-
otal role in biosynthesis of metallic and/or oxide
nanoparticles which is done in the present investigation.
Energy yielding material – suitable carbohydrate (which
controls the value of rH2), the ionic status of the medium
pH and overall oxidation-reduction potential (Eh) of the
culture medium, all these factors cumulatively negotiate
the synthesis of ZnO nanoparticles in the presence of
Lactobacillus strain. Taking use of the above mentioned
facts, our group had earlier reported synthesis of metallic
cadmium [18], silver [17,34,43] as well as antimony
oxide [1,44] and titanium dioxide. [2] A mildly acidic
pH also activates the membrane bound oxidoreductases
and makes the requisite ambience for an oxide nanopar-
ticle synthesis as illustrated in Figure 4. Therefore, com-
pared to other techniques, the present procedure is less
expensive more reproducible, emphatically non-toxic
and a truly green approach.
A large quantity of hydrogen sulfide is liberated in gas
and petroleum industries and has been considered as a
major pollutant. Besides, according to the international
environmental regulations, H2S contained in the acid
gases should be effectively removed before its release to
atmosphere. Its characteristic odor could easily be per-
ceived in a dilution of 0.002 mg /L in ambient air. Intake
of higher concentrations, could lead to the collapse from
respiratory failure. For the purpose of protection, the
concentration should be reduced to less than 15 ppm. [45]
Pollution of underground aquifers has been a prevalent
problem in the areas adjoining oil and gas reserves,
which miserably affects the health of nearby inhabitants.
Use of ZnO NPs produced using present green and low
cost protocol based devices could prove to be an effec-
tive step towards mitigation of the menace.
The present biosynthesis method is a green low cost ap-
proach, capable of producing ZnO NPs nearby room
temperature. The synthesis of ZnO NPs might have re-
sulted due to variation in the level of rH2 or pH, which
activates the pH sensitive oxido-reductases. ZnO
nanoparticles could be effective in controlling the pollu-
tion generated due to H2S in air as well as underground
aquifers. However, bright possibility exists with regard
to the development of different products/devices in order
to get rid of the menace of different forms of air and/or
water pollution such as face masks, water filters,
de-odorizing cakes and screens. Cosmetic industries can
bank upon this product in order to synthesize sunscreen
lotions etc., which would be done in the immediate fu-
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