Colloids and Surfaces A: Physicochem. Eng.

Aspects 462 (2014) 264–270

Contents lists available at ScienceDirect

Colloids and Surfaces A: Physicochemical and

Engineering Aspects
journal homepage: www.elsevier.com/locate/colsurfa

Exploitation of anaerobic enriched mixed bacteria (AEMB) for the

silver and gold nanoparticles synthesis
K. Siva Kumar a , G. Kumar b , E. Prokhorov a,∗ , G. Luna-Bárcenas a , G. Buitron b ,
V.G. Khanna c , I.C. Sanchez d
a
Centro de Investigación y de Estudios Avanzados del IPN, Querétaro 76230, Qro., Mexico
b
Academic Unit Juriquilla, Institut of Engineering, Universidad Nacional Autonoma de Mexico, Querétaro, 76230, Mexico
c
Department of Biotechnology, College of Engineering, Daegu University, Kyoungsan, Kyoungbook 712-714, Republic of Korea
d
Department of Chemical Engineering, The University of Texas at Austin, Austin, TX 78712, USA

h i g h l i g h t s g r a p h i c a l a b s t r a c t

• Biosynthesis of silver and gold

nanoparticles.
• Anaerobic enriched mixed bacteria.
• Spherical nanoparticles with the size
of 5–65 nm.
• Amine groups or carboxalate groups
reduce and stabilized of nanoparti-
cles.

a r t i c l e i n f o a b s t r a c t

Article history: We report a simple and efficient extracellular biosynthesis of silver and gold nanoparticles (Ag and
Received 21 May 2014 AuNPs) using anaerobic enriched mixed bacteria (AEMB) for the first time. Biosynthesized silver and gold
Received in revised form 4 September 2014 nanoparticles were characterized by UV–vis spectroscopy, transmission electron microscopy (TEM), X-
Accepted 11 September 2014
ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). The results demonstrated that
Available online 20 September 2014
as-prepared nanoparticles are spherical in shape with the size of 5–65 nm in range. For gold nanoparti-
cles, 2–7 particles were self-assembled into 1D chain-like structure. FTIR results evidenced interaction
Keywords:
between the nanoparticle’s surface and the reductive groups. Additionally, the dominant bacterial species
Silver and gold nanoparticles
Biogenic synthesis
in the enriched mixed culture have been identified via PCR-DGGE and DNA sequencing analysis. It was
Anaerobic enriched mixed bacteria found that major bands belong to Klebsiella pneumoniae, Lactobacillus amylotrophicious and Salmonella
Self-assembly enterica, which were responsible for the rapid reduction of silver and gold nanoparticles. This simple and
green protocol could be used to prepare large-scale and economically viable synthesis of other metallic
nanoparticles.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction

In recent years metal nanoparticles have gained consider-

∗ Corresponding author at: CINVESTAV del IPN, Unidad Querétaro, Libramiento
Norponiente 2000, Fracc. Real de Juriquilla, Querétaro 76230, Mexico.
Tel.: +52 442 2 11 99 21; fax: +52 442 2 11 99 38.
E-mail address: prokhorov@qro.cinvestav.mx (E. Prokhorov).
able interest in various interdisciplinary field of nanotechnology
because of their unique properties and practical applications [1].
Among metal nanoparticles, gold and silver nanoparticles are the
most common ones used in various areas due to their poten-
tial applications in medicine, catalysis, electronics, optics and

http://dx.doi.org/10.1016/j.colsurfa.2014.09.021
0927-7757/© 2014 Elsevier B.V. All rights reserved.
 K. Siva Kumar et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 462 (2014) 264–270
biosensors [2–5]. Different innovative approaches have been devel-
oped to synthesize silver and gold nanoparticles particularly
chemical reduction methods using different reducing agents such
as NaBH4 , citrate, and ascorbic acid [6–8]. However, such chemical
reduction processes have its own drawbacks such as environ-
mental toxicity issues, stability and agglomeration effect without
additional stabilizing agent and difficulties in handling chemical
reagents [9]. Recently, alternative methods have been emerged
such as polysaccharides and biopolymers such as cellulose deriva-
tives as reducing and stabilizing agent, but these biopolymers are
relatively slow and inefficient reducing agents leads aggregation of
nanoparticles above critical concentration, difficult to control the
size and shape [10]. Consequently, there is an increasing interest for
the use of greener environmentally friendly biogenic approaches
to produce metal nanoparticles due to their growing success and
nanoparticle ease of formation without any additional reducing
agents. These features will help minimize the problem associated
with environmental toxicity or biological hazards [11,12].
Numerous green synthesis methods have been exploited to
prepare silver and gold nanoparticles such as vitamins, sugars,
plant extract biopolymers and microorganisms as reducing and
capping agents [13,14]. Among these, the use of microorgan-
ism is preferred to synthesize metal nanoparticles because of
ease of handling, large scale production that will detonate poten-
tial applications in various areas of nanotechnology [15]. Several
microorganisms have been utilized intracellularly and extracellu-
larly to produce silver and gold nanoparticles [15,16]. Recently,
Malarkodi et al. [17] reported extracellular biosynthesis of gold
nanoparticles using Klebsiella pneumoniae as the reducing agent.
Similarly, Kalpana et al. showed the production of silver nanoparti-
cles from K. pneumoniae bacterial culture [18]. Korbekandi et al.
reported biosynthesis of silver nanoparticles using Lactobacillus
Casei bacteria [19]. Klaus et al. [20] have shown that Pseudomonas
stutzeri AG259, isolated from a mine were used to synthesize sil-
ver nanoparticles with well-controlled sizes. In addition, Kalathil
et al. reported an extracellular electrochemically active biofilm
obtained from enriched mixed bacteria culture to synthesize sil-
ver and gold nanoparticles in the presence of sodium acetate as
electron donor [21,22]. From the previous reports, silver and gold
nanoparticles were produced by individual bacteria culture; how-
ever there are no reports about the mixed bacterial culture being
used for biosynthesis of silver and gold nanoparticles. The bacterial
species used in this study are opportunistic pathogens, and their
bio safety level is not harmful. Besides, these bacteria are reported
to grow in the low cost medium such as wastewater and cellulosic
wastes.
Previously, anaerobic enriched mixed bacteria (AEMB) were
used in the process of producing hydrogen via dark fermentation
at pilot scale level [23,24]. Recently, AEMB has been used in active
biofilm formation in microbial fuel cells (MFCs) and biosensor based
applications widely. The AEMB play crucial role in MFCs, microbial
electrolysis cells (MECs) and MFC-based biosensors applications
[25]. One important factor that controls the performance of MFCs
is the formation of biofilm from anaerobic enriched mixed bacteria
culture on different electrode surfaces. Recently, the modification
of electrode surfaces by nanomaterials and activation centers on
the carbon or graphene electrodes significantly increase the spe-
cific surface area and electron transfer kinetics [26]. Moreover, by
increasing the specific surface area of electrode and/or electrocat-
alytic activity could increase the bioanode performances [25]. It has
been shown that gold and silver nanoparticles have pronounced
electrocatalytic activities, which can be utilized to enhance the MFC
output [25].
The aim of this work is to use the above strategy to promote
in situ reduction of silver and gold nanoparticles mediated by
AEMB culture. Metal nanoparticles can be used in the formation
265
of biofilms containing nanoparticles on the electrode surface. In
contrast to conventional biosynthesis methods, AEMB synthesis
is non-toxic, environmentally friendly and have additional advan-
tages such as easy and rapid preparation, high stability and low cost
for industrial applications. Identification of the dominant bacterial
species present in the mixed cultures was elucidated via PCR-DGGE
and DNA sequencing.
2. Materials and methods
2.1. Preparation of anaerobic enriched mixed culture
The enriched mixed culture was prepared from the swine
manure collected in Central Mexico. Initially, the swine manure
was cultivated in the nutrient medium. The basal nutrient medium
was followed Endo formulation [27] and contained the fol-
lowing ingredients (g/l): NH4 CO3 5.24, NaHCO3 6.72, K2 HPO4
0.125, MgCl2 ·6H2 O 0.1, MnSO4 ·6H2 O 0.015, FeSO4 ·7H2 O 0.025,
CuSO4 ·5H2 O 0.005, CoCl2 ·5H2 O 0.00012. After 3 transfers in the
medium, the enriched mixed culture obtained was used for further
synthesis work. The dominant bacterial population present in the
consortia was revealed by PCR-DGGE.
2.2. PCR-DGGE, DNA sequencing and phylogenetic analysis
Total genomic DNA was isolated using the Blood & Tissue
Genomic DNA Extraction. Miniprep System (Viogene, Taiwan) fol-
lowing the manufacturer’s instructions. The isolated DNA was
confirmed by agarose gel electrophoresis (0.75%) and the samples
were stored at −20 ◦ C for further PCR reactions. The PCR mixtures
(50 ␮l) contained each deoxynucleoside triphosphate at a concen-
tration of 200 mM, 1.5 mM MgCl2 , each primer at a concentration
of 0.2 mM, 1.25 U of Taq DNA polymerase (Promega, Madison, WI)
and the PCR buffer provided with the enzyme. The amplification
consisted of a DNA denaturing step at 94 ◦ C for 5 min, followed
by 30 cycles of denaturation at 94 ◦ C for 1 min, 1 min annealing
at 55 ◦ C for EUB968gc–UNIV1392r and extension at 72 ◦ C for 1 min.
The cycling included a final extension step at 72 ◦ C for 10 min to
ensure full extension of the product. All PCR operations were per-
formed with an automatic thermal cycler iCyclerTM (Bio-Rad, Los
Angeles, CA). PCR products were analyzed by electrophoresis at
100 V for 30 min through 1.5% (w/v) agarose gel. The amplified
PCR products were used for denatured gradient gel electrophore-
sis (DGGE) analysis. The DGGE profile of the PCR-amplified DNA
was obtained following the method mentioned [28,29] using a
DCodeTM Universal Mutation Detection System (BioRad, USA). The
6% (w/v) acrylamide solution was used to cast a gel with denat-
urant gradients ranging from 40% to 60%. Electrophoresis was
conducted in a 1× TAE (Tris/acetic acid/EDTA) buffer solution at
80 V and 60 ◦ C for 12 h. The gels were stained for 10 min with ethid-
ium bromide and visualized under UV radiation. The number of
operational taxonomic units (OTU) for each sample was defined
as the number of DGGE bands. The selected DGGE bands on the
gel was excised with a sterile razor blade, placed in 1.5 mL cen-
trifuge tube and add 50 ␮l of sterile 1× TAE buffer, then incubated
overnight at 4 ◦ C to reclaim the DNA. Additional PCR-DGGE analy-
ses were performed to ensure the purity of reclaimed DNA. Analysis
of targeted DNA sequences was performed in a DNA sequencer
(Tri Biotech, Taiwan). The bioinformatic analysis was carried out
by using the tool BLASTN facility available from NCBI Web site
(http://www.ncbi.nlm.nih.gov/BLAST) to align the partial 16S rDNA
sequences with the reference microorganisms available in the Gen-
Bank database. The procedure has been explained in our previous
reports [30].
266 K. Siva Kumar et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 462 (2014) 264–270

Table 1
Affiliation of DGGE fragments determined by their 16S rDNA and isolated microorganisms.

Sequence No. Family Closest match Homology (%) Sequence length (bp)

1 Gamma proteobacteria Klebsiella pneumoniae (Accession No. NR 074913) 99 418

2 Firmicutes Lactobacillus amylotrophicious (Accession No. NR 042511) 98 416
3 Gamma proteobacteria Salmonella enterica (Accession No. NR 074935) 99 414

2.3. Bio-synthesis of Au and Ag nanoparticles

In a typical synthesis procedure, 2 mL of 0.2 mM AgNO3 pre-

cursor solution were added to 10 mL of freshly prepared anaerobic
enriched mixed culture. The pH of the AEMB has been kept as neu-
tral (7.0), since the neutral pH is supportive for the growth and
maintains as active bacterial culture. The solution mixture was
allowed for sparing by N2 gas for 5 min to achieve strict anaerobic
environment. The final solution mixture closed tightly and magnet-
ically stirred at 35 ◦ C. Important to note, the mixture were stirred
without any exposure to light. Within 30 min the transparent col-
loidal suspension turn into light yellow in color and the reaction
completes in 2–3 h by obtaining stable dark yellow color colloidal
suspension. AuNPs were prepared in a similar fashion by adding
2 mL of 5 mM HAuCl4 solution to 10 mL of freshly prepared anaer-
obic enriched mixed culture. After the addition of HAuCl4 solution,
the solution mixture was allowed for sparing N2 gas for 5 min and
then magnetically stirred at 35 ◦ C under dark environment for 1 h.
The color of the final product turns into dark pink color
suggesting formation of gold nanoparticles. Upon completion of
the reaction, the nanoparticles were isolated by centrifugation
at 2500 rpm for 20 min and subsequently washed with copious
amount of di-ionized water to remove the excess surface bounded
bacteria culture filtrate. Further confirmation of formation and size
of the final product was analyzed by UV–vis and TEM analysis.

2.4. Characterization techniques

The formation of AgNPs and AuNPs were monitored by UV–vis

spectroscopy (UV–vis spectrophotometer: Agilent 8453). The mor-
phology of the biosynthesized nanoparticles were analyzed by
JEOL JEM-1010 Transmission Electron Microscope (TEM) operated
at 80 kV. Crystalline structure was determined by X-ray diffrac-
tion and was performed using a Rigaku diffractometer ULTIMA
IV, equipped with the CuK␣ radiation ( = 1.5406 Å). The possible
interactions between nanoparticles surface and the mixed bacterial
culture was performed by Fourier-transform infrared spectroscopy
(FTIR) on a Perkin Elmer spectrophotometer using an ATR accessory
in the range 4000–650 cm−1 .
3. Results and discussion
3.1. Identification of dominant bacterial species in AEMB
It is necessary to know the dominant bacterial species present
in the mixed cultures to draw the mechanism to fully understand
the background of the nanoparticles synthesis. Thus, widely known
PCR-DGGE based 16S rDNA sequence has been employed for the
identification of V3–V6 regions of 424 bp long nucleotide sequence.
Three major bands were detected and they were sequenced and
closely related match search was performed via NCBI database. The
bands representation was shown in Table 1 and Fig. 1.
3.2. Biosynthesis of silver and gold nanoparticles
Novel biosynthesis of silver and gold nanoparticles using AEMB
at room temperature was demonstrated in this report. When the
silver and gold precursor (AgNO3 and HAuCl4 ) were added into
Fig. 1. PCR-DGGE, DNA sequencing and phylogenetic analysis of anaerobic enriched
mixed bacteria culture.
aqueous solution containing enriched mixed bacteria, the reaction
was noticed after a few minutes by noticing the color change in the
reaction mixture. The stable and dark yellow and pink colors have
been observed within 2–3 h suggesting that the complete reduc-
tion of silver and gold salt into AgNPs and AuNPs (inset Fig. 1).
Apparently, reduction of chloroaurate ions was quite rapid in con-
trast to silver ions. On adding aqueous chlorouric acid solution
to the enriched mixed bacteria culture solution, the color of the
reaction mixture changed to ruby red. This change indicates the
formation of gold nanoparticles after ca. 15 min of the reaction.
Moreover, by using the mixed bacteria culture, the nanoparti-
cles can be prepared more rapidly and effective when compared
to the other previously reported microorganism-based synthe-
sis methods. For instance, some reports state the production of
nanoparticles after 24–96 h [31–33]. Additionally, as-prepared sil-
ver nanoparticles exhibit smaller nanoparticles size (5–65 nm) that
are well dispersed. It is noteworthy that three different domi-
nant bacteria species in the mixed culture (identified by PCR-DGGE
and DNA sequencing): K. pneumoniae, Lactobacillus amylotrophi-
cious and Salmonella enterica were encountered previously for the
reduction of metal nanoparticles individually [17–19]. In our syn-
thesis method, three identified bacteria culture take part in the
reduction process, thus the reduction process was quite rapid and
effective.
 K. Siva Kumar et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 462 (2014) 264–270
Fig. 2. UV–vis spectra of bio-synthesized silver and gold nanoparticles colloids. Inset
shows photographs of (a) pure AEMB culture solution, (b) the as-prepared AuNPs
and (c) the as-prepared AgNPs colloidal suspension.
3.3. Characterization of Ag and Au nanoparticles
It is widely known that UV–vis spectroscopy is used to examine
the shape and size of the metal nanoparticles by carefully observ-
ing of the peak position and shape of the peak [34]. Fig. 2 shows
Fig. 3. (a) TEM images of as-prepared silver nanoparticles. (b) and (c) Magnified image of silver nanoparticles. (d) Size distribution of AgNPs average of four different images.
267
the UV–vis absorption spectra of biosynthesized colloidal AgNPs
and AuNPs exhibit the absorption peak at 425 nm and 520 nm.
Both spectra have shown the characteristic absorption profile of
spherical silver and gold nanoparticles regime, respectively [35].
The appearance of a high intensity SPR absorption peak for Au and
AgNPs that suggests the conversion of AgNPs and AuNPs is signifi-
cantly high (ca. 80–90%). In comparison with other microbial based
synthesis, the present synthesis produced high yield [21]. Further-
more, the color of the final solution turns into dark yellow and pink;
these color changes reveal the high concentration of the nanoparti-
cle within the culture medium (inset of Fig. 1). Further confirmation
of shape and size of as-prepared nanoparticles were subjected to
transmission electron microscopy (TEM) imaging.
Transmission electron microscopy images of as-prepared AgNPs
and AuNPs colloidal suspensions are shown in Figs. 3 and 4. It is
observed that silver nanoparticles are spherical and are homoge-
neously dispersed with an average particle size of 5–50 nm. The
particle size distributions are presented in Fig. 3d. Particle size
distribution has been determined using ImageJ software for four
different images obtained from the TEM measurements for Ag and
Au NPs, respectively.
TEM image in Fig. 4a was taken from the colloidal suspen-
sion of AuNP’s which shows the AuNPs are spherical in shape
with an average particle size of 10–60 nm. It is likely that these
particles spontaneously assemble into 1D (2–7 particles). It is note-
worthy that such self-assembled structures are not produced in
case of AgNP’s (Fig. 4). Such 1D self-assembly structures may be
268 K. Siva Kumar et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 462 (2014) 264–270
Fig. 4. TEM images of (a) the as-prepared gold nanoparticles. (b) Size distribution of AuNPs average of four different images.
(111)
a) AgnP's
b) AunP's
(200)
Intensity (a.u)
b) (220)
(311)
a)
40 50 60 70
2θ (degree)
Fig. 5. XRD spectra of as-prepared silver and gold nanoparticles.
the result of electrostatic attraction between positively charged
amino groups in the protein and the negatively charged sulfur of
gold nanoparticles [36]. Similar self-assembly structures have been
observed for protein monolayer capped gold nanoparticles previ-
ously [37].
The crystalline nature of biosynthesized AgNPs and AuNPs were
confirmed by X-ray diffraction (XRD) analysis. Fig. 5 shows the
XRD patterns of dried powder samples of biosynthesized silver
and gold nanoparticles. Typical XRD patterns of AgNPs show peaks
at 2 values of 38◦ , 44.2◦ , 64.3◦ and 77.7◦ corresponding to the
(1 1 1), (2 0 0), (2 2 0) and (3 1 1) planes of face centered cubic (fcc)
for Ag◦ [38]. Similarly, AuNPs showed peaks at 2 values of 38.2◦ ,
44.5◦ , 64.7◦ and 77.6◦ corresponding to the (1 1 1), (2 0 0), (2 2 0)
and (3 1 1) planes of face centered cubic (fcc) for Au◦ [39]. XRD
pattern showed that silver and gold nanoparticles are essentially
presented in crystalline in nature. The crystalline size of silver and
gold nanoparticles d was estimated from XRD measurements using
well known Scherer formula. The calculated value of d was about
of 35 ± 9.4 nm and 32.3 ± 5.5 nm for silver and gold nanoparticles
respectively, which is close to TEM measurements.
To gain insight into the specific interactions between nanopar-
ticle surface and the active bacterial species, FTIR measurement
has been carried out. Fig. 6 shows two representative FTIR spec-
tra of synthesized AgNPs and AuNPs that exhibit strong bands
95
a) AgnP's
90 b b) AunP's
Transmittance(%) 85
80
75
70 3286 2930
1235
1062
65 1657 1539
4000 3500 3000 2500 2000 1500 1000 500
80
Wavenumber (cm-1)
Fig. 6. FTIR spectra of dried samples: (a) AgNPs and (b) AuNPs.
at 1657 cm−1 , 1539 cm−1 , 1235 cm−1 and 1062 cm−1 . The most
prominent bands at 1657 cm−1 and 1539 cm−1 correspond to the
characteristic vibrations of amide I (C O stretching vibration) and
amide II vibrations of the protein [40]. Other bands at 1235 cm−1
and 1062 cm−1 can be assigned to the C N stretching vibrations of
NH bending of peptide linkage [17]. From FTIR measurement, one
can conclude that AgNPs and AuNPs could bind to free amino or car-
boxylate groups in the protein; these groups may be responsible for
the reduction and stabilization of nanoparticles. Such interaction of
silver and gold nanoparticles with the amine and carboxyl groups is
consistent with the previous reports [17,41,42]. Additionally, TEM
analysis revealed the attachment of silver and gold nanoparticles
with bacteria and formation of stabilization layer on the AgNPs and
AuNPs surface (see Fig. 3a).
In the literature different mechanisms have been proposed for
reduction of metal nanoparticles using different bacterial cultures.
Most of the previous investigations showed that NADH dependent
electron shuttle enzymatic metal reduction process is responsible
for bio-reduction of nanoparticles [43–45]. Other studies demon-
strated that nitrate reductase enzyme system can be responsible
for formation of silver nanoparticles from silver ions [46].
Recently, Ramanathan et al. experimentally demonstrated the
possible mechanism of extracellular bio-reduction of silver and
copper nanoparticles [47,48]. They found that the silver binding
gene in the silver-resistant bacteria is responsible for the reduction
 K. Siva Kumar et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 462 (2014) 264–270 269

Fig. 7. Schematic of proposed mechanism of Ag+ /Au3+ ions reduction and Ag/AuNPs formation.

and stabilization of AgNPs via sulphur containing amino acids Acknowledgements

(cysteine/methionine) in the protein. However, the exact reduction
mechanism of metal ions (Ag+ , Au3+ ) into metal nanoparticles This work was partially supported by CONACYT of Mexico. The
either extracellular or intracellular way is poorly understood. authors are grateful to R.A. Mauricio Sánchez for assistance in FTIR
In our present study based upon FTIR and TEM observation, the measurements, M.A. Martin Adelaido for XRD measurements and
possible mechanism involved in the bio-reduction of Ag and AuNPs Ma. Lourdes Palma Tirado (Campus UNAM Juriquilla, Qro) for TEM
is shown in the scheme (Fig. 7). The first step involves the uptake Measurements.
of Ag+ /Au3+ and formation of complex formation with the AEMB.
The second step, the NADH-dependent reductase which may have
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