Department of Microbiology, School of Life sciences
Bharathidasan University, Tiruchirappalli- 620 024
Tamilnadu, India.
Corresponding author: nthaju2002@yahoo.com
Abstract:
The colloidal silver nanoparticles were synthesized by reducing silver nitrate
(AgNO
3
) solution with the freshwater cyanobacterium, Oscillatoria earlei NTDM03 at room temperature for 28 days. The interaction between the cyanobacteria and silver nitrate forms colloidal silver nanoparticles in the range of 200nm. Cyanobacteria were releases organic biomolecules which enhance the reduction of silver nitrate and precipitate them on cell surface as colloidal silver.
Keywords : Oscillatoria sp , colloidal silver, nanoparticles, protein shell, FTIR.
Introduction:
One key aspects of nanotechnology concerns the development of reliable experimental protocols for the synthesis of nanomaterials over a range of chemical composition size and high monodispersity. Colloidal silver have been used in the

manufacturer of photographic films, cosmetics, preservatives, antibacterial coatings, dietary supplements, also used in the nanoelectronics such as non-linear optical properties
(Karavanskii et al ., 2004, Efras et al . 2003) and microelecronics such as electro analysis of cytochrome c (Lin et al ., 2008). Recent development in nanotechnology has opened up possibilities for new and revolutionary approaches for the assembly and analysis of molecular structure and devices that are a few to tens of nanometric in size.
The synthesis and assembly of nanoparticles would benefit from the development of clean, non-toxic and environmentally acceptable “ green chemistry
” procedures, probably involving wide range of micro organisms such as bacteria, fungi, actinomycetes and cyanobacteria. Formation of extracellular and intracellular silver nanoparticles by cyanobacteria such as Plectonema boyanum UTEX 485 (Lengke et al ., 2007), Spirulina platensis (Govindraju et al., 2008), Lyngbya majuscule (Chakraborty, et al.
2008) when interaction with aqueous silver nitrate.
Nanoparticles obtained by this method show some degree of selectivity in attachment to the cell wall of bacteria demonstrating potential application in biological labeling (Warad et al ., 2004). Silver nanoparticles are used in photographic reaction, catalysis, chemical analysis and silver metal and its compound have been known to have strong inhibitory effect on bacteria as well as a broad spectrum of antimicrobial activities. Silver ions work against bacteria in a number of ways; they interact with thiol groups of enzyme and proteins that are important for the bacterial respiration and the transport of important substances across the cell membrane and within the cell (Cho et al.
2005). Silver ions are bound to the bacterial cell wall, altering the function of the bacterial cell membrane (Percival et al. 2005). The silver metal and its compounds were the effective preventing infection of the wound (Wright,

et al.
1999). Synthesis of nanosized silver substituted hydroxyapatite would have antibacterial activity for the implant surgery (Rameshbabu et al., 2007).
Silver metal was slowly changed to silver ions under our physiological system and interact with bacterial cells, thus silver ions will not to be high enough to cause human cells damage. Silver nanoparticles have a high specific surface area and a high fraction of surface atoms that lead to high antimicrobial activity compared to bulk silver metal (Cho et al .,
2005).
In the present study, a fresh water cyanobacterium, Oscillatoria earlei NTDM03 was isolated from the eutrophic pond in Tiruvarambur at Tiruchirappalli and selected for the synthesis of colloidal silver nanoparticles; since cyanobacteria are efficient in rapid biosorption of metals, low production cost, ease in separation of compounds from the culture media.
Materials and Methods:
The filamentous fresh water cyanobacterium, Oscillatoria earlei NTDM03 was grown in batch culture in BG-11 medium. The pH of the medium was adjusted to 8.0 using a 1m
NaOH solution before experimentation; the culture was transferred and grown for approximately 6-8 weeks to reach a stationary growth phase. It was then centrifuged and washed three times with sterile distilled water, deionized water to remove the salts and trace metals from the medium before being used for the experiments.
The experiments were conducted to examine the role of cyanobacteria in the synthesis of silver nanoparticles from aqueous solution of AgNO
3
[HiMedia]. The experiments were conducted using washed cyanobacteria and without the presence of the growth medium. To

initiate the experiment 5mL of the silver solution (~560mg/l) was added to 5mL of washed cyanobacteria culture. The experiment were conducted at 25 o C for 28 days after the incubation period with silver solution and maintained in the dark, and cyanobacteria populations were measured with time. Abiotic control experiments consisting of ~560mg/l in silver were conducted using AgNO
3
solution without inoculation of cyanobacteria (Lenkge et al ., 2007)
Aqueous silver nitrate solution (10 -3 M) taken in a clean conical flask inoculated with cyanobacteria. The reduction of the Ag
+
ions by test cyanobacterium in the solution was studied by using the aqueous component and measuring the UV - Vis Spectrum of solutions.
The UV - Visible spectra of this solution was recorded in Lambda 35 in absorbance mode from 300 to 900nm.
The experimental system after incubation of AgNO
3
solution to form slime sediment
(brown color), which is used for the characterization analysis by using FTIR (Fourier
Transform Infra Red) spectroscopy. The silver nanoparticles prepared as described above were analyzed by Spectrum RX1. The FTIR spectrum was taken in the mid IR region of 400-
4000 cm
-1
. The spectrum was recorded using ATR (Attenuated Total Reflectance) technique.
The sample was directly placed in the Potassium bromide crystal and the spectrum was recorded in the transmittance mode.
A drop of aqueous solution containing the silver nanomaterials (brown color) was placed on the sterile stainless steel coupon and it air dried to applied gold sputtering. These plates were examined at different magnifications [10,000X, 40,000X] by scanning electron microscope (Model – JEOL INSTRUMENT JSM-5610). The natures of elements were identified by EDS.

Results and Discussion:
On addition of AgNO
3 to the Oscillatoria earlei NTDM03 cultures, the soluble silver was completely precipitated within 28 days. A grayish–black color silver precipitate on cyanobacteria was observed (Fig 1.). The test cyanobacterial cultures were killed within several hours. In abiotic experiments using AgNO
3 total soluble silver concentration were constant until completion of experiment. Figure 2 shown that the UV- vis spectra in 450nm is due to the plamon resonance of silver nanoparticles (Fig 2a.) and low wavelength region recorded from the reaction medium exhibited an absorption band at 265nm and it was attributed to aromatic amino acids of proteins. It is well known that the absorption band at
265nm arises due to electronic excitations in tryptophan and tyrosine residues in the proteins
(Duran et al., 2005). This observation indicates the release of protein into solution by O. earlei NTDM03 (Fig 2b.) The nature of strong peak located between 240-260nm corresponds to the surface plasmon resonance of reducing protein.
The FTIR spectrum recorded from the film of silver nanoparticles, formed after 28 days of incubation with the cyanobacteria. The bands seen at 3280 cm
−1
and 2924 cm
−1
were assigned to the stretching vibrations of primary and secondary amines respectively. The two bands observed at 1379 cm
−1
and 1033 cm
−1
can be assigned to the C–N stretching vibrations of aromatic and aliphatic amines, respectively. Vigneshwaran et al ., (2007) reported that the presence of protein as the stabilizing agent surrounding the silver nanoparticles. It is reported

that the proteins can bind to nanoparticles either through free amine groups or cysteine residues in the proteins (Gole et al ., 2001; Mandal et al., 2005) and via the electrostatic attraction of negatively charged carboxylate groups in enzymes present in the cell wall of mycelia (Sastry et al ., 2003) and therefore, stabilization of the silver nanoparticles by protein is a possibility. Rosen et al (2003) suggested that enzyme like silver reductase and the amino acids (present in proteins/enzymes) such as aspartic acid and tryptophan (Mandal et al .,
2005) have been reported in various microbial systems responsible for the reduction of metal ions. Sastry et al ., (2003) reported that fungi and actinomycetes are the important sources of enzymes that can catalyze specific reactions leading to inorganic nanoparticles.
The silver nanoparticles synthesized using Spirulina platensis showed strong bands at
1,651, 1,545 and 1,241 cm
-1
. These bands correspond to the primary, secondary and tertiary amide bands of polypeptide and proteins (Govindraju et al.
, 2008). Report on various species of cyanobacteria and algae having the ability to adsorb and accumulated heavy metal (Gadd,
1990; Kuyucak et al ., 1990; Hameed, 2005; Mohamed, 2001) and indeed polysaccharide in the cyanobacterial cells and soluble polymer around the cells facilitate binding of metal ions
(Philippis et al., 2001). The metal binding site in cyanobacteria is through the formation of metallothioneins that bind metal ions as metal thiolate. It has already been reported that the silver nanoparticles can be stabilized and are well functional groups, such as carboxyl
(Chapman et al ., 2001 and Abe et al ., 1998), hydroxyl (Temgire et al ., 2004). Amido (Yin et al ., 2003) and thiol (Liu et al ., 2002) groups, except the steric effect arising from the large molecular structures of the organic modifier (protein) on the surface of the particles (Fig 3.)
The overall peaks from FTIR observation confirm the presence of protein in the samples of silver nanoparticles. It can be assumed that nitrate reducing bacteria to produce

nitrate reductase enzyme (protein). This reduces to silver nitrate solution form silver nanoparticles. The silver ions were reduced in the presence of nitrate reductase, leading to the formation of a stable silver hydrosol 10-25 nm in dm. and stabilized by the capping peptide.
Scanning electron microscopic studies showed that the accumulation of silver nanoparticles at cell surfaces of Oscillatoria earlei NTDM03. Small agglomerated silver nanoparticles with size ranging from 100nm to 200nm (extracellularly) were also precipitated in solution and were also deposited at cell surfaces (Fig 4a.) The cyanobacterial cells were encrusted with silver nanoparticles and separation of some filaments into their constituent cells were still observed. The nanoparticles were not in direct contact even within the aggregates, indicating stabilization of the nanoparticles by capping agent. This corroborates with the previous observation by Ahmed et al (2007) on Fusarium oxysporum . The results of energy dispersive spectroscopy (EDS) study showed the presence of elemental silver signal which conforms the synthesis of silver nanoparticles by Oscillatoria earlei NTDM03 (Fig
4b).
Alternative synthetic technology for nanoparticels involves controlled precipitation of nanoparticles from precursors dissolved in a solution. Various thermodynamic factors as well as van der Waal’s forces induce particle growth and agglomeration resulting in bigger particles that settle down over time (Dutta, 2004). Syntheses of gold and silver nanoparticles by wet chemical synthesis proceed by a reductions reaction followed by subsequent particle growth by ostwalt ripening and finally stop in the growth process. The protein molecule made up of different functional group in amino acid sequences such as amino, carboxyl, sulfate groups present in the cyanobacterial protein favor the formation of extremely smallsized silver nanoparticulates with narrow particle size distribution and hydroxyl and sulfonic

groups are beneficial to synthesis of silver nanoparticle with a slightly larger particle size in a weak reducing environment.
The present study showed the biosynthesis of colloidal silver nanoparticles from aqueous silver nitrate interacted with freshwater cyanobacterium, Oscillatoria earlei
NTDM03 at room temperature for 28 days. The colloidal silver nanoparticles synthesized and precipitated on the surface of cyanobacteria in nanoscale level (200nm). In abiotic experiment precipitation of silver was not observed during the course of study.
References:
1.
Abe, K., Hanada, T., Yoshida, Y., Tanigaki, N., Takiguchi, H., Nagasawa, H.,
Nakamoto, M., Yamaguchi, T., and K. Yase., “Two-dimensional array of silver nanoparticles”, Thin Solid Films.
327: 524-527, 1998.
2.
Ahmad, R.S., Minaeian, S., Shahverdi, H. R., Jamalifar, H., and A. A. Nohi., “Rapid synthesis of silver nanoparticles using culture supernatants of electro bacteria: a novel biological approach”, Process Biochemistry, 42 : 919-923, 2007.
3.
Chakraborty, N., Banerjee, A., Lahiri, S., Panda, A., Ghosh, A. N and R. Pal.,
“Biorecovery of gold using cyanobacterial and a eukaryotic alga with special reference to nanogold formation- a novel phenomenon”, J. appl. Phycol. 21 (1):142-
152, 2009.
4.
Chapman, R., Mulvaney, P., “Electro-optical properties of silver nanoparticle thin films”, Chem. Phys. Lett ., 349 : 358-362, 2001.

5.
Cho, K. H., Park, J. E., Osaka, T., and S. G. Park., “The study of antibacterial activity and preservative effects of nanosilver ingredients”, Electrochimica Acta, 51 : 956-
960, 2005.
6.
Duran, N., Marcato, P., Alves, L.O, Souza, G. I., and E. Esposito., Mechanistic aspect of biosynthesis of silver nanoparticles by several Fusarium oxysporum strains”,
Journal of Nanobiotechnol.
3: 1-7. 2005.
7.
Dutta, J., H. Hofmann., “Self-organization of colloidal nanoparticles”, Ency. of
Nanosci and Nanotechnol, 9 :617-640, 2004.
8.
Efros, A.L and Rosen, “The electronic structures of semiconductor nanocrystals”,
Annu. Rev. Mater. Sci. 30 : 475-521, 2006.
9.
Gadd G.M., “Heavy metal accumulation by bacteria and other microorganisms”,
Experientia. 46 : 834-840, 1990.
10.
Gole A, Dash C, Ramakrishnan V, Sainkar S. R, Mandale A B, Rao M, and M.
Sastry., “Pepsin-Gold colloid conjugate: Preparation, Characterization, and
Enzymatic activity”. Langumir. 17 : 1674- 1679, 2001.
11.
Govindaraju, K., Khaleel Basha, S., Ganesh kumar, V and G. Singaravelu.., “Silver, gold and bimetallic nanoparticles production using single-cell protein ( Spirulina platensis ) Geitler”, J. Mater Sci. 43 : 5115-5122, 2008.
12.
Hameed, A and S. Hasnain, “Cultural characteristics of chromium resistant unicellular cyanobacteria isolated from local environment in Pakistan”, Chin J
Oceanol Limnol .
23 : 433-441, 2005.

13.
Karavanskii, V. A., Simakin, A. V., Krasovskii, V. I, and P. V. Ivanchenko., “Nonlinear optical properties of colloidal silver nanoparticles produced by laser ablations in liquids”, Quantum Electron. 34 : 644-648, 2004.
14.
Kuyucak, N., B. Volesky, FL Raton., Heavy metals. CRC press, Boca Raton, 1990.
15.
Lengke, M.F, Fleet, M.E., and G. Southam., “Biosynthesis of Silver Nanoparticles by
Filamentous Cyanobacteria from Silver (I) Nitrate Complexs”, Langmuir . 23 : 2694–
2699, 2007.
16.
Lin, L., Qiu, P., Cao, X., and L. Jin., “Colloidal silver nanoparticles modified electrode and its application to the electro analysis of cytochrome c”, Electrochimica
Acta .
53 : 5368-5372, 2008.
17.
Liu J, Ong W and A. E. Kaifer., “A 'macro cyclic effect' on the formation of capped silver nanoparticles in DMF”, Langmuir. Material. 10 : 5981, 2002.
18.
Mandal S, Phadtare, S and Sastry M., “Interfacing biology with Nanoparticles”,
Current Applied Phys. 5 : 118-127, 2005.
19.
Mohammed, Z.A. “Removal of cadmium and manganese by a non-toxic strain of the freshwater cyanobacterium Gloeothece magna”. Water Res. 33 : 4405-4409, 2001.

20.
Percival, S. L., Bowler, P. G., and D. Russell., “Bacterial resistance to silver in wound care”, Journal of Hospital infection, 60: 1-7, 2005.
21.
Philippis R.D., Sili, C., Paperi, R, and M. Vincenzini., “Assessment of the metal removal capability of two capsulated cyanobacteria, Cyanospira capsulata and
Nostoc PCC7936”, Appl. Phycol. 13 : 293, 2001.
22.
Rameshbabu, N., Sampath kumar, T.S., Prabhakar, T.G., Sastry, V.S., K. Murty, K and K.P.Rao., “Antibacterial nanosized silver substituted hydroxyapatite synthesis and characterization
”,
J Biomed Mater Res. 80 A: 581-591, 2007.
23.
Sastry, M., Ahmad, A., Islam, NI, and R. Kumar., “Biosynthesis of metal nanoparticles using fungi and actinomycete”, Current Sci., 85: 162-170, 2003.
24.
Temgire M.K and S.S. Joshi., “Optical and Structural studies of Silver nanoparticles”,
Rad Phys .
71 : 1039, 2004.
25.
Vigneshwaran, N., Ashtaputre, N.M., Varadarajan, P.V., Nachane, R.P. Paralikar,
K.M. and R.H. Balasubramanya., “Biological synthesis of silver nanoparticles using the fungus Aspergillus flavus., Mat.Lett, 61 : 413-1418, 2007.
26.
Warad H. C., S. C. Ghosh, C. Thanachayanont, J. Dutta and J. G. Hilborn., “Highly luminescent manganese doped ZnS quantum dots for biological labeling”, In: Proc.
Int. Conference on Smart Materials (Smartmat ‘04), Chiang Mai.(Ed.), 2004.
27.
Wright, J. B., Lam, K., Hansen, D., and R.E. Burrell., “Efficacy of topical silver against fungal burn wound pathogens”, American Journal of Infection Control, 27:
344-350, 1999.

28.
Yin, B., Ma, H., Wang, S, and S. Chen., “Electrochemical synthesis of silver nanoparticles under protection of Poly (N- Vinylpyrrolidine)”, J. Phys. Chem. 107 :
8898, 2003.
Figure 1. Microphotography of fresh water cyanobacterium, Oscillatoria earlei NTDM03

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35

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Figure 2 . UV-Vis spectra recorded as a function of reaction of an aqueous solution of silver nitrate with the cyanobacteria filtrate (2a). Low wavelength region recorded from the reaction medium exhibited an absorption band at 265nm (2b).

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Figure 3 . FTIR analysis of O. earlei NTDM03 which shows the presence of protein shell for reduction of silver ions.

4a
4b
Figure 4.
SEM view of whole mount of cyanobacterial cells cultured in the presence of
AgNO
3
at 25 o
C and 28 days; (4a) formation of colloidal silver nanoparticles on the cyanobacterial cells (4b) EDS of the silver nanoparticles was confirmed the presence of elemental silver signal in high percentage.