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Morphological alterations in erythrocytes treated with silver nanoparticles biomineralized by marine sediment-derived Bacillus sp. VITSSN01

Morphological alterations in erythrocytes treated with silver nanoparticles biomineralized by... Ann Microbiol (2014) 64:1291–1299 DOI 10.1007/s13213-013-0773-z ORIGINAL ARTICLE Morphological alterations in erythrocytes treated with silver nanoparticles biomineralized by marine sediment-derived Bacillus sp. VITSSN01 Theerthagiri Revathy & Rajamani Saranya & Mangalam Achuthanandan Jayasri & Kumar Saurav & Krish Suthindhiran Received: 7 March 2013 /Accepted: 8 November 2013 /Published online: 5 January 2014 Springer-Verlag Berlin Heidelberg and the University of Milan 2013 Abstract Biomineralization-inspired preparation of nanopar- and transmission electron microscopy. The mean particle size ticles by marine microorganisms is in the limelight of modern was 46 nm. Hemotological toxicity of nanoparticles is very nanotechnology. In recent years, the use of marine microor- severe form and less studied. We therefore checked the syn- ganisms for the synthesis of nanoparticles has been gaining thesized silver nanoparticles for toxicity against erythrocytes importance due to the simplicity and eco-friendliness of the and found that the silver nanoparticles exhibited moderate approach. Here we describe the synthesis of silver nanoparti- hemolytic activity against human erythrocytes, with a half cles using halotolerant Bacillus sp. isolated from the southern maximal effective concentration (EC ) value of 60 μg/ml. coastal waters of India. Our selective and enriched isolation Microscopic studies of the treated erythrocytes showed slight technique resulted in the isolation of a silver nitrate-resistant structural perturbations. The results of our study strongly sug- novel marine Bacillus sp. isolated from sediments collected at gest that marine microorganisms could be a potential source Ennore Port, Chennai, India. The strain was characterized by for the rapid and eco-friendly synthesis of nanoparticles. the polyphasic taxonomic approach, and phenotypic and phy- logenetic analysis identified the strain as Bacillus sp. Keywords Marine microorganisms Bacillus sp. VITSSN01. The resistant strain was further assayed for the . . . VITSSN01 Silver nanoparticles Hemolytic activity synthesis of silver nanoparticles and its biological activity Antimicrobial activity evaluated. Nanoparticles were synthesized under optimized nutritional and cultural conditions with shaking and the pro- duction continuously monitored. The nanoparticles thus pro- Introduction duced were then characterized by atomic force microscopy, X- ray diffraction, Fourier transform-infrared spectrophotometer Nanotechnology is considered to be a modern science involved with the synthesis of different nanoparticles using various physical, chemical and biological methods (Kaushik et al. : : : T. Revathy R. Saranya M. A. Jayasri K. Suthindhiran (*) 2010). The chemical synthesis of nanoparticles is associated Marine Biotechnology and Biomedicine Lab, School of Biosciences with a number of limitations, including contamination from and Technology, VIT University, Vellore, India e-mail: ksuthindhiran@vit.ac.in precursor chemicals, use of toxic solvents, possible generation of hazardous by-products, among others (Kowshik et al. 2003). K. Suthindhiran The need for efficient and inexpensive methods to e-mail: sudhindhira@gmail.com biosynthesize nanoparticles is gaining importance because the K. Saurav physical and chemical processes are costly and tedious. There- Key Laboratory of Marine Bio-Resources Sustainable Utilization, fore, the development of biological methodologies for synthe- Centre for Marine Microbiology, Research Network for Applied sizing nanoparticles is currently receiving increasing attention Microbiology, Guangdong Key Laboratory of Material Medica, in the search to overcome these limitations and to achieve a South China Sea Institute of Oceanology (CAS), Guangzhou, China high yield of nanoparticles at low cost and with non-toxic 1292 Ann Microbiol (2014) 64:1291–1299 byproducts. Many biological resources are known to produce Soil sample analysis nanoparticles either intracellularly and extracellularly (Mann 1996), and the search for cost-effective and rapid synthesis The physicochemical properties of the sediments, such as pH, processes has revealed that microorganisms may provide po- electrical conductivity, nitrogen, phosphorus, potassium, soil tential and alternative sources. In particular, marine bacteria texture, lime status, ferrous, manganese, zinc and copper, were could be used for the production of nanoparticles because they analyzed as described in previous studies (Lindsay and Norvell synthesize the desired nanoparticles using inexpensive sub- 1978; Kowshik et al. 2003) and recorded. The collected sedi- stances, in an eco-friendly, non-toxic environment and within ment samples were also processed and analyzed for trace metal a short time. Further these bacteria synthesize the nanoparticles concentration using atomic absorption spectrometry. extracellularly which reduces downstream processing (Beveridge and Murray 1980;Sadowskietal. 2008) Evaluation of resistance to silver nitrate Marine microorganisms are considered to be a potential source for the rapid and cost-effective production of metal The maximum tolerance concentration of the isolated strain nanoparticles (Nanda and Saravanan 2009). Moreover, among was determined by the well diffusion and broth dilution the various biological systems, marine bacteria are relatively methods with various concentrations of silver nitrate (100– easy to manipulate, and the nanoparticles synthesized by these 4,000 mg/l). bacteria are compatible for use in various medicinal applica- tions due to their low toxicity. Some bacteria can survive and Optimization of nutritional and growth conditions grow even at high metal concentrations and can synthesize the for the resistant strain nanoparticles using their innate defensive mechanisms (Mohanapuria et al. 2008). Although several applications for The nutritional and cultural conditions for the isolated strain producing silver nanoparticles are currently in use, these also were optimized by inoculating the strain onto different culture cause cytotoxicity and genotoxicity to various life forms. medium (nutrient agar, marine agar and starch agar) supple- Therefore, it is also important to assess the toxicity of the mented with sufficient salt concentrations and minerals. The synthesized nanoparticles. Erythrocytes are a good model for effect of various carbon sources, nitrogen sources, tempera- such toxicity assessments because the results are a direct ture, pH and salt concentrations on the growth of the isolate measurement of the toxicity of the tested compound. The were also evaluated. in vitro hemolytic assay evaluates erythrocyte lysis by mea- suring hemoglobin release upon the treatment of compound. Taxonomy Here we report the identification and characterization of Bacillus sp. VITSSN01 isolated from a marine sediment The strain was morphologically characterized by examining sample collected at Ennore Port, southeastern coast of India. the cells after Gram staining by bright field microscopy at We determined that this bacterium synthesizes silver nanopar- 100× magnification (Olympus, Japan). The DNAwas isolated ticles that show moderate cytotoxicity against human erythro- using the HipurA Bacterial DNA Isolation and Purification kit cytes and perturb the erythrocyte membrane, leading to chang- (HiMedia, India) and amplified by PCR using the Medox es in morphology (discoid to echinocytic shape). Master Mix kit (Medox, India) according to the instructions of the manufacturer. The primers (27F and 1492R) and the PCR conditions were adapted from Lane (1991)and Weisburg Materials and methods et al. (1991). The design of the sequencing primers and the methodology for the sequencing were adapted from previous Sample collection and isolation of bacteria reports (Magarvey et al. 2004). The 16s rRNA gene sequence was analyzed for its similarity and homology with existing Sediment samples were collected at a depth of 20 cm under sequences available in the databank of National Center for aseptic conditions from Ennore Port, Chennai, southeastern Biotechnology (NCBI) using BLAST search. The DNA se- coast of India, and the samples were transported to the labo- quences were aligned using ClustalW software (Saitou and ratory and processed immediately. For the isolation of the Nei 1987). The phylogenetic tree was constructed using strain, the culture medium was prepared by using marine agar MEGA (ver. 3.1) software (Kumar et al. 2001). The bootstrap supplemented with soil extract (25 %) and sea water (25 %), values of 1,000 replicates were obtained. and the samples were serially diluted and inoculated. The plates were incubated at 28 °C for 1 week. Discrete colonies Synthesis of silver nanoparticles were isolated and repeatedly subcultured to obtain pure cul- tures. The isolates were maintained in slant culture at 4 °C as Marine broth containing 0.3 mM silver nitrate was prepared well as in glycerol stock at −40 °C. and the isolated strain was inoculated into this medium. Ann Microbiol (2014) 64:1291–1299 1293 Control media were prepared without silver nitrate and micro- In-vitro hemolytic activity bial culture. The culture flasks were incubated for 7 days in an orbital shaker at room temperature. After incubation, the In vitro hemolytic activity was performed as described earlier culture broth was homogenized and the supernatant lyophi- (Suthindhiran and Kannabiran 2009). Human erythrocytes +ve lized and analyzed for the presence of silver nanoparticles were obtained from the peripheral blood (O )of healthy (Rumov et al. 2009). volunteers. The blood was used within 24 h after bleeding and washed three times in nine volumes of sterile 0.9 % saline solution. After each washing, cells were centrifuged at 150 g Characterization of silver nanoparticles for 5 min and the supernatant discarded. The final pellet was diluted 1:9 (v/v) in sterile Dulbecco’s phosphate buffer saline The bioreduction of metal nanoparticles was monitored and (pH 7) containing 0.5 mm boric acid and 1 mM calcium the absorbance spectrum of silver nanoparticles was measured chloride. The hemolytic activity of the silver nanoparticles between 300 and 800 nm using UV-Visible spectroscopy was tested under in vitro conditions in 96 well plates (Systronics, Japan). The synthesized nanoparticles were also (Malagoli 2007). To each well, 100 μl of 0.85 % sodium analyzed and identified by atomic force microscopy (AFM) chloride solution containing 10 mM calcium chloride was (Olympus, Japan), X-ray diffraction (XRD; Bruker, Germa- added. The first well served as negative control and contained ny), Fourier transform infrared spectroscopy (FT-IR; only water; all other wells contained 100 μlof synthesized Schimadzu, Japan) and transmission electron microscopy silver nanoparticles at various concentrations (5–500 μg/ml), (TEM) (model 3010; JOEL, Japan). The lyophilized silver with the exception of the last well which served as the positive nanoparticles for XRD analysis and its pattern were recorded control and contained 20 μl of 0.1 % of Triton X-100 dis- between the range of 10 and 80 wave numbers at 2θ at solved in 0.85 % of saline. Each well was then inoculated with different intensities (Mandal et al. 2006). The lyophilized 100 μl of a 2 % suspension of human erythrocytes in 0.85 % samples were used for TEM. The samples were placed direct- ly on copper grids and air dried. TEM measurements were Table 1 Physicochemical properties of sediment samples collected from performed on a JOEL 3010 electron microscope (Prakasham Ennore Port, Chennai, India et al. 2012). Physicochemical properties of sediment samples Measured value Nutrients Nitrate 0.5 Phosphorous 97.2 Potassium 208 Calcium 3348 Magnesium 525 Sulphur 900.6 Sodium 973 Zinc 0.69 Manganese 7.52 Iron 24.99 Copper 0.49 Boron NA Parameters pH 8.25 Electrical conductivity (ms/cm) 5.24 Organic matter (%) 0.50 Cation exchange capacity (Meq/100 g) 25.33 Ca saturation (%) 2.06 K saturation (%) 64.69 Mg saturation (%) 16.91 Na saturation (%) 16.35 NA, Not available Fig. 1 Sampling site, Ennore Port at Chennai, located on the southeast- ern coast of India The unit for nutrients is parts per million (ppm) 1294 Ann Microbiol (2014) 64:1291–1299 saline containing calcium chloride (10 mM). After 30 min of et al. 1992). In this study we demonstrated the biological incubation at room temperature, the contents were centri- synthesis of silver nanoparticles by a newly isolated marine fuged, and the absorbance of the supernatant was determined derived strain, Bacillus sp. VITSSN01 and its toxicity to at 540 nm as a measure of liberated hemoglobin. The tests human erythrocytes. were carried out in triplicate, and the values were expressed as The strain was isolated from a sediment sample collected at mean ± standard deviation. Ennore Port, southern coast of India (Fig. 1). Ennore Port is located on the Coromandel Coast in the Bay of Bengal Sea Morphological alterations Table 2 Biochemical The morphological changes in the treated and control eryth- Test Result and cultural characteris- rocytes were determined as described by Kondo and tics of Bacillus sp. +ve Tomizawa (1968). A blood sample (O ) was collected from Gram stain Gram-negative rod VITSSNO1 isolated healthy human donors and centrifuged at 600 g for 10 min at from marine sediment Motility Motile 4 °C. About 1 ml of erythrocyte suspension was placed in Colony color Sandal polystyrene tubes containing buffer and different concentra- Starch hydrolysis + tions of silver nanoparticles (0.1 and 1 mM). The sample was Casein hydrolysis + incubated at room temperature for 30 min and the cells the Gelatin hydrolysis + observed under the light microscope (100× magnification). Urea hydrolysis + Carbon sources (1 % w/v) D-Glucose + Results and discussion D-Sucrose + D-Fructose + Nanobiotechnology is an emerging field which deals with the Lactose − production and application of nanoparticles in biology and D-Galactose − medicine. The development of cost-effective and environmen- Glycerol − tally friendly approaches for synthesizing metal nanoparticles Mannose − using biological sources has recently become a primary focus D-Xylose + of researchers (Rumov et al. 2009). Biological sources are D-Raffinose − regarded as potential biofactories for the production of metal Mannitol + nanoparticles (Narayanan and Sakthivel 2011). Among the Nitrogen sources (1 % w/v) various potential nanoparticle-producing biological systems, Peptone + a bacterium is relatively easy to manipulate and handle and is Yeast extract + applicable for the large-scale production of nanoparticles. The Urea + microbial synthesis of metal nanoparticles depends upon lo- Ammonia + calization of the reductive components of their cells (Slawson Physiological conditions Temperature (°C) 15 − 25 − 30 + 37 + 45 − pH 5 − 7.2 + 7.4 + 7.6 + 8 − NaCl concentration (%) 1+ 3+ Fig. 2 Morphology of Gram-stained Bacillus sp., VITSSN01 strain 6 − observed under optical microscopy (1000× magnification) Ann Microbiol (2014) 64:1291–1299 1295 Fig 3 Phylogenetic tree showing the position of VITSS01 with other Bacillus sp. Number at nodes of tree Percentage level of bootstrap support and is bounded by the Korttalaiyar River, Ennore creek (Table 2). To optimize the cultural characteristics and media (backwater) and the Bay of Bengal. This zone comprises composition, we performed a systematic study in which we lagoons with salt marshes and backwaters. The soil texture evaluated the suitability of a number of carbon and nitrogen appears to be sandy loam. The physicochemical parameters of sources. Bacillus sp., VITSSN01 was found to be capable of the sediment samples are given in Table 1. Physiochemical utilizing different carbon sources, such as glucose, fructose, analysis of soil sediment from this region shows the presence xylose, mannitol and mannose. Better growth was observed if of many mineral ions, including sodium, potassium, magne- yeast extract and peptone were used the sole nitrogen sources, sium, manganese, zinc, phosphorous, copper and nitrate. The and good growth was seen when urea and ammonia were used pH of the sediment was 8.25. In order to mimic the same as inorganic nitrogen sources. Maximum growth was obtained environmental conditions, we supplemented the media with soil extract and seawater. The strain showed maximal growth S2M-1 on medium supplemented with sea water and soil extracts and Abs cultivated at 37 °C, pH 7.2. This modified method yielded 3.5 good results and growth appeared to be abundant. The bacterial cells were found to be aerobic, motile, non- 3.0 acid-fast, Gram-positive rods (Fig. 2). The cultural, morpho- 2.5 logical and biochemical characterization indicates that the 2.0 strain belongs to the genus Bacillus, and it was subsequently 1.5 designated VITSSN01. The colonies were circular, convex 1.0 and easily emulsifiable; they were slightly pinkish on marine agar and yellowish on nutrient agar. The strain tested positive 0.5 for catalase, cytochrome oxidase, gelatinase, casein, urea and 0.0 nm starch hydrolysis. Methyl red and citrate tests were positive, 300 400 500 600 700 800 and the triple sugar iron agar slants showed acid butt (yellow) Fig. 4 UV-Visible absorbance spectrum of silver nanoparticles synthe- and alkali slant (red) along with hydrogen sulphide production sized by Bacillus sp. VITSSN01 1296 Ann Microbiol (2014) 64:1291–1299 Fig. 5 Atomic force microscopy images denoting the aggregation of silver nanoparticles synthesized by Bacillus sp. VITSSN01 when glucose, sucrose and fructose were used as carbon nitrogen sources. To evaluate these optimal growth conditions sources and peptone, yeast extract, urea and ammonia as we cultured the strain under different physiological Fig. 6 X-ray diffraction (XRD) (a) 2000 (b) B B pattern of silver nanoparticles synthesized by Bacillus sp. VITSSN01. a XRD pattern measured in the presence of silver nitrate, b XRD pattern of silver nanoparticles showed a peak of sharp intensity between 30° and 35° θ and between 40° and 45° θ 0 102030405060708090 2θ (degree) Intensity Ann Microbiol (2014) 64:1291–1299 1297 100.0 Fig. 7 Fourier transform-infrared 95 PS-098 spectroscopy spectrum of biosynthesized silver nanoparticles 1139 986 %T 40 2034 931 35 1232 2136 1214 10 3631 0.0 4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.0 cm-1 conditions. Optimal growth was observed at 37 °C and pH the Plasmon ambience excitation of the silver nanoparticles 7.2. The strain grew well between 1 and 3 % salt concentration (Wiley et al. 2006). (NaCl) and showed moderate and sometimes low growth at a Spectroscopic analysis of the bioreduction of metal nano- 6 % salt concentration. The isolated strain was found to be particles showed that the UV-Visible absorbance spectrum resistant towards silver nitrate and when cultured under dark peaked at 420 nm (Fig. 4). The size, morphology and conditions was found to be involved in the extracellular syn- agglomerization of silver nanoparticles were analyzed using thesis of silver nanoparticles. AFM and the images are shown in Fig. 5. Particle size was The BLAST search of 16S rRNA gene sequences of the found to be 46 nm. The XRD pattern of the silver nanoparti- strain showed maximum identity (97 %) with Bacillus sp. cles is shown in Fig. 6. The XRD pattern was measured for SP09-202 (GQ35242), and the phylogenetic tree was con- both control and with the presence of nanoparticles. The structed with bootstrap values. Based on the molecular taxon- powdered nanoparticles showed peaks of sharp intensity be- omy and phylogeny analysis, the strain was identified as a tween 30° and 35° θ and between 40° and 45° θ. The FT-IR novel Bacillus sp. and designated as VITSSN01. A neighbor- pattern of silver nanoparticles was measured in the transmit- joining tree based on 16S rRNA gene sequences showed that tance range between 450 and 4,500 wave numbers, and the the isolate occupies a distinct phylogenetic position within the radiation that includes representatives of the Bacillus family (Fig. 3). The number at the nodes of the tree indicates the percentage level of bootstrap support. The score bar represents one nucleotide substitution per 100 nucleotides. The phyloge- netic tree based on the maximum parsimony method also showed the separate clade for the VITSSN01 (tree not shown). The rRNA gene sequence was submitted to GenBank under accession number JX094863. We determined that Bacillus sp. VITSSN01 was capable of the extracellular synthesis of silver nanoparticles. The strain was found to be resistant towards silver nitrate, reducing the silver nitrate to form silver ions under dark conditions. The color of the culture broth changed from yellow to brown after the synthesis of silver nanoparticles, and the pH of the broth increased slightly to 8. This color change is indicative of the reduction of silver nitrate to silver ions. The yellowish–brown Fig. 8 Transmission electron microscopy images of silver nanoparticles color of the silver nanoparticles in aqueous solution is due to synthesized by Bacillus sp. VITSSN01 1298 Ann Microbiol (2014) 64:1291–1299 −1 presence of wave numbers 1,650, 1,290, 2,036, 1,214 cm bacterial strains is very advantageous in terms of cost, time revealed the presence of silver nanoparticles (Fig. 7). The and the environment. There have been reports on the synthesis of study of TEM images revealed silver nanoparticles synthe- silver nanoparticles by a few Bacillus sp. Bacillus licheniformis sized by marine Bacillus sp. VITSS01 as dark spots on the has been reported to have synthesized silver nanoparticles of image (Fig. 8). 50 nm size (Kalimuthu et al. 2008; Kalishwaralal et al. 2008), Marine microorganisms have been found to be a potential and the culture supernatant of Bacillus subtilis has been reported source of metal nanoparticles. The commercial synthesis of to have synthesized silver nanoparticles of 5–60 nm (Saifuddin metal nanoparticles through the use of non-pathogenic et al. 2009). Pugazhenthiran et al. (2009) reported the synthesis of silver nanoparticles (5–15 nm) using Bacillus sp., and Ganesh Babu and Gunasekaran (2009) reported the synthesis (a) Erythrocytes without Ag NPs (4- and 5-nm nanoparticles) mediated by B. cereus.Inour study the marine Bacillus sp., VITSS01 was found to produce silver nanoparticles of 46 nm. Further, our TEM studies indi- cate that the silver nanoparticles are synthesized in the peri- plasmic space of the cell when Bacillus sp. VITSS01 is cultured with silver nitrate. Pugazhenthiran et al. (2009)re- ported that silver nanoparticles accumulated at the periplasmic space of silver nitrate-resistant Bacillus cells when cultured in presence of silver nitrate (. Hemotological toxicity is a very serious toxicity and its effects are less studied (Suwalsky et al. 2010). To evaluate the toxicity of biosynthesized silver nanoparticles, we used human erythrocytes as model cells. The silver nanoparticles (b) Erythrocytes treated with 0.1mM Ag NPs induced hemolysis on the tested erythrocytes with a half max- imal effective concentration (EC ) value of 60 μg/ml. Com- plete hemolysis was obtained with 20 μl of Triton-X 100 (0.1 %) and 1-h incubation (Table 3). Normal human blood cells under physiological conditions have a disc shape and are 8 μm in diameter. In our assay, changes in the shape of the human erythrocytes were observed after incubation with silver nanoparticles: the morphology was altered and the normal discoid shape of the cells changed to echinocytic form (Fig. 9). The extent of the change in shape was found to depend on the concentration of the silver nanoparticles: at 0.1 mM silver nanoparticles, moderate changes occurred in erythrocyte shape; at 1 mM, the intensity of these changes increased and (c) Erythrocytes treated with 1mM Ag NPs. strong structural perturbations were observed. Bilayer couple hypothesis states that the inner and outer layer of the erythro- cyte membrane is affected due to the insertion of some foreign Table 3 Hemolytic ac- Sample (μg) Percentage inhibition tivity of silver nanoparti- cles synthesized by the 60 46.33 marine Bacillus sp. VITSSNO1 against hu- 65 58.33 man erythrocytes 70 63.62 75 68.33 80 68.77 The experiments were 85 69.21 carried out in triplicate, 90 70.39 and the results are shown 95 73.04 as the mean value. Tri- Fig. 9 Morphology of red blood cells affected by silver nanoparticles. a ton-X 100 was used as a Erythrocytes without silver nanoparticles (Ag NPs). b Erythrocytes treat- 100 87.04 positive control ed with 0.1 mM Ag NPs. c Erythrocytes treated with 1 mM Ag NPs Ann Microbiol (2014) 64:1291–1299 1299 2+ Malagoli D (2007) A full length protocol to test hemolytic activity of species (Iglic et al. 1998). Similarly, Cd alters the molecular palytoxin on human erythrocytes. Invertebr Surviv J 4:92–94 structure of the lipid bilayer of erythrocytes (Suwalsky et al. Mandal D, Bolander Debabrata Mukhopadhyay ME, Sarkar G, 2004), titanium induces structural perturbations in erythrocytes Mukherjee P (2006) The use of microorganisms for the formation 2+ (Suwalsky et al. 2005)and Zn causes structural changes in of metal nanoparticles. Appl Microbiol Biotechnol 68:485–492 Mann S (1996) Biomimetic materials chemistry. VCH, New York, pp 1–40 cell membranes (Suwalsky et al. 2009). Our study shows that Mohanapuria P, Rana NK, Sudesh Kumar Y (2008) Biosynthesis of the silver nanoparticles induce the structural alterations in nanoparticles: technological concepts and future applications. J human erythrocytes. Nanoparticle Res 10:507–517 The results of this study indicate that marine microorgan- Nanda A, Saravanan M (2009) Biosynthesis of silver nanoparticles from Staphylococcus aureus and its antimicrobial activity against MRSA isms could provide a potential and alternate source for the AND MRSE. Nanomedicine: NBM 5:452–456 synthesis of nano-sized metal particles. Detailed and periodi- Narayanan KB, Sakthivel N (2011) Green synthesis of biogenic metal cal investigation of the Indian marine environment would nanoparticles by terrestrial and aquatic phototrophic and heterotro- result in the isolation of novel microorganisms which can be phic eukaryotes and biocompatible agents. Adv Colloid Interf Sci 169:59–79 used for studying and furthering our understanding of the Prakasham RS, Sudheer Kumar B, Sudheer Kumar Y, Girija Shankar G biomineralization and biosynthesis of nanoparticles. (2012) Characterization of silver nanoparticles synthesized isolate Streptomyces albidoflavus. J Microbiol Biotechnol 22:614–621 Acknowledgments The study was supported by an institutional grant. 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Morphological alterations in erythrocytes treated with silver nanoparticles biomineralized by marine sediment-derived Bacillus sp. VITSSN01

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Publisher
Springer Journals
Copyright
Copyright © 2013 by Springer-Verlag Berlin Heidelberg and the University of Milan
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
ISSN
1590-4261
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1869-2044
DOI
10.1007/s13213-013-0773-z
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Abstract

Ann Microbiol (2014) 64:1291–1299 DOI 10.1007/s13213-013-0773-z ORIGINAL ARTICLE Morphological alterations in erythrocytes treated with silver nanoparticles biomineralized by marine sediment-derived Bacillus sp. VITSSN01 Theerthagiri Revathy & Rajamani Saranya & Mangalam Achuthanandan Jayasri & Kumar Saurav & Krish Suthindhiran Received: 7 March 2013 /Accepted: 8 November 2013 /Published online: 5 January 2014 Springer-Verlag Berlin Heidelberg and the University of Milan 2013 Abstract Biomineralization-inspired preparation of nanopar- and transmission electron microscopy. The mean particle size ticles by marine microorganisms is in the limelight of modern was 46 nm. Hemotological toxicity of nanoparticles is very nanotechnology. In recent years, the use of marine microor- severe form and less studied. We therefore checked the syn- ganisms for the synthesis of nanoparticles has been gaining thesized silver nanoparticles for toxicity against erythrocytes importance due to the simplicity and eco-friendliness of the and found that the silver nanoparticles exhibited moderate approach. Here we describe the synthesis of silver nanoparti- hemolytic activity against human erythrocytes, with a half cles using halotolerant Bacillus sp. isolated from the southern maximal effective concentration (EC ) value of 60 μg/ml. coastal waters of India. Our selective and enriched isolation Microscopic studies of the treated erythrocytes showed slight technique resulted in the isolation of a silver nitrate-resistant structural perturbations. The results of our study strongly sug- novel marine Bacillus sp. isolated from sediments collected at gest that marine microorganisms could be a potential source Ennore Port, Chennai, India. The strain was characterized by for the rapid and eco-friendly synthesis of nanoparticles. the polyphasic taxonomic approach, and phenotypic and phy- logenetic analysis identified the strain as Bacillus sp. Keywords Marine microorganisms Bacillus sp. VITSSN01. The resistant strain was further assayed for the . . . VITSSN01 Silver nanoparticles Hemolytic activity synthesis of silver nanoparticles and its biological activity Antimicrobial activity evaluated. Nanoparticles were synthesized under optimized nutritional and cultural conditions with shaking and the pro- duction continuously monitored. The nanoparticles thus pro- Introduction duced were then characterized by atomic force microscopy, X- ray diffraction, Fourier transform-infrared spectrophotometer Nanotechnology is considered to be a modern science involved with the synthesis of different nanoparticles using various physical, chemical and biological methods (Kaushik et al. : : : T. Revathy R. Saranya M. A. Jayasri K. Suthindhiran (*) 2010). The chemical synthesis of nanoparticles is associated Marine Biotechnology and Biomedicine Lab, School of Biosciences with a number of limitations, including contamination from and Technology, VIT University, Vellore, India e-mail: ksuthindhiran@vit.ac.in precursor chemicals, use of toxic solvents, possible generation of hazardous by-products, among others (Kowshik et al. 2003). K. Suthindhiran The need for efficient and inexpensive methods to e-mail: sudhindhira@gmail.com biosynthesize nanoparticles is gaining importance because the K. Saurav physical and chemical processes are costly and tedious. There- Key Laboratory of Marine Bio-Resources Sustainable Utilization, fore, the development of biological methodologies for synthe- Centre for Marine Microbiology, Research Network for Applied sizing nanoparticles is currently receiving increasing attention Microbiology, Guangdong Key Laboratory of Material Medica, in the search to overcome these limitations and to achieve a South China Sea Institute of Oceanology (CAS), Guangzhou, China high yield of nanoparticles at low cost and with non-toxic 1292 Ann Microbiol (2014) 64:1291–1299 byproducts. Many biological resources are known to produce Soil sample analysis nanoparticles either intracellularly and extracellularly (Mann 1996), and the search for cost-effective and rapid synthesis The physicochemical properties of the sediments, such as pH, processes has revealed that microorganisms may provide po- electrical conductivity, nitrogen, phosphorus, potassium, soil tential and alternative sources. In particular, marine bacteria texture, lime status, ferrous, manganese, zinc and copper, were could be used for the production of nanoparticles because they analyzed as described in previous studies (Lindsay and Norvell synthesize the desired nanoparticles using inexpensive sub- 1978; Kowshik et al. 2003) and recorded. The collected sedi- stances, in an eco-friendly, non-toxic environment and within ment samples were also processed and analyzed for trace metal a short time. Further these bacteria synthesize the nanoparticles concentration using atomic absorption spectrometry. extracellularly which reduces downstream processing (Beveridge and Murray 1980;Sadowskietal. 2008) Evaluation of resistance to silver nitrate Marine microorganisms are considered to be a potential source for the rapid and cost-effective production of metal The maximum tolerance concentration of the isolated strain nanoparticles (Nanda and Saravanan 2009). Moreover, among was determined by the well diffusion and broth dilution the various biological systems, marine bacteria are relatively methods with various concentrations of silver nitrate (100– easy to manipulate, and the nanoparticles synthesized by these 4,000 mg/l). bacteria are compatible for use in various medicinal applica- tions due to their low toxicity. Some bacteria can survive and Optimization of nutritional and growth conditions grow even at high metal concentrations and can synthesize the for the resistant strain nanoparticles using their innate defensive mechanisms (Mohanapuria et al. 2008). Although several applications for The nutritional and cultural conditions for the isolated strain producing silver nanoparticles are currently in use, these also were optimized by inoculating the strain onto different culture cause cytotoxicity and genotoxicity to various life forms. medium (nutrient agar, marine agar and starch agar) supple- Therefore, it is also important to assess the toxicity of the mented with sufficient salt concentrations and minerals. The synthesized nanoparticles. Erythrocytes are a good model for effect of various carbon sources, nitrogen sources, tempera- such toxicity assessments because the results are a direct ture, pH and salt concentrations on the growth of the isolate measurement of the toxicity of the tested compound. The were also evaluated. in vitro hemolytic assay evaluates erythrocyte lysis by mea- suring hemoglobin release upon the treatment of compound. Taxonomy Here we report the identification and characterization of Bacillus sp. VITSSN01 isolated from a marine sediment The strain was morphologically characterized by examining sample collected at Ennore Port, southeastern coast of India. the cells after Gram staining by bright field microscopy at We determined that this bacterium synthesizes silver nanopar- 100× magnification (Olympus, Japan). The DNAwas isolated ticles that show moderate cytotoxicity against human erythro- using the HipurA Bacterial DNA Isolation and Purification kit cytes and perturb the erythrocyte membrane, leading to chang- (HiMedia, India) and amplified by PCR using the Medox es in morphology (discoid to echinocytic shape). Master Mix kit (Medox, India) according to the instructions of the manufacturer. The primers (27F and 1492R) and the PCR conditions were adapted from Lane (1991)and Weisburg Materials and methods et al. (1991). The design of the sequencing primers and the methodology for the sequencing were adapted from previous Sample collection and isolation of bacteria reports (Magarvey et al. 2004). The 16s rRNA gene sequence was analyzed for its similarity and homology with existing Sediment samples were collected at a depth of 20 cm under sequences available in the databank of National Center for aseptic conditions from Ennore Port, Chennai, southeastern Biotechnology (NCBI) using BLAST search. The DNA se- coast of India, and the samples were transported to the labo- quences were aligned using ClustalW software (Saitou and ratory and processed immediately. For the isolation of the Nei 1987). The phylogenetic tree was constructed using strain, the culture medium was prepared by using marine agar MEGA (ver. 3.1) software (Kumar et al. 2001). The bootstrap supplemented with soil extract (25 %) and sea water (25 %), values of 1,000 replicates were obtained. and the samples were serially diluted and inoculated. The plates were incubated at 28 °C for 1 week. Discrete colonies Synthesis of silver nanoparticles were isolated and repeatedly subcultured to obtain pure cul- tures. The isolates were maintained in slant culture at 4 °C as Marine broth containing 0.3 mM silver nitrate was prepared well as in glycerol stock at −40 °C. and the isolated strain was inoculated into this medium. Ann Microbiol (2014) 64:1291–1299 1293 Control media were prepared without silver nitrate and micro- In-vitro hemolytic activity bial culture. The culture flasks were incubated for 7 days in an orbital shaker at room temperature. After incubation, the In vitro hemolytic activity was performed as described earlier culture broth was homogenized and the supernatant lyophi- (Suthindhiran and Kannabiran 2009). Human erythrocytes +ve lized and analyzed for the presence of silver nanoparticles were obtained from the peripheral blood (O )of healthy (Rumov et al. 2009). volunteers. The blood was used within 24 h after bleeding and washed three times in nine volumes of sterile 0.9 % saline solution. After each washing, cells were centrifuged at 150 g Characterization of silver nanoparticles for 5 min and the supernatant discarded. The final pellet was diluted 1:9 (v/v) in sterile Dulbecco’s phosphate buffer saline The bioreduction of metal nanoparticles was monitored and (pH 7) containing 0.5 mm boric acid and 1 mM calcium the absorbance spectrum of silver nanoparticles was measured chloride. The hemolytic activity of the silver nanoparticles between 300 and 800 nm using UV-Visible spectroscopy was tested under in vitro conditions in 96 well plates (Systronics, Japan). The synthesized nanoparticles were also (Malagoli 2007). To each well, 100 μl of 0.85 % sodium analyzed and identified by atomic force microscopy (AFM) chloride solution containing 10 mM calcium chloride was (Olympus, Japan), X-ray diffraction (XRD; Bruker, Germa- added. The first well served as negative control and contained ny), Fourier transform infrared spectroscopy (FT-IR; only water; all other wells contained 100 μlof synthesized Schimadzu, Japan) and transmission electron microscopy silver nanoparticles at various concentrations (5–500 μg/ml), (TEM) (model 3010; JOEL, Japan). The lyophilized silver with the exception of the last well which served as the positive nanoparticles for XRD analysis and its pattern were recorded control and contained 20 μl of 0.1 % of Triton X-100 dis- between the range of 10 and 80 wave numbers at 2θ at solved in 0.85 % of saline. Each well was then inoculated with different intensities (Mandal et al. 2006). The lyophilized 100 μl of a 2 % suspension of human erythrocytes in 0.85 % samples were used for TEM. The samples were placed direct- ly on copper grids and air dried. TEM measurements were Table 1 Physicochemical properties of sediment samples collected from performed on a JOEL 3010 electron microscope (Prakasham Ennore Port, Chennai, India et al. 2012). Physicochemical properties of sediment samples Measured value Nutrients Nitrate 0.5 Phosphorous 97.2 Potassium 208 Calcium 3348 Magnesium 525 Sulphur 900.6 Sodium 973 Zinc 0.69 Manganese 7.52 Iron 24.99 Copper 0.49 Boron NA Parameters pH 8.25 Electrical conductivity (ms/cm) 5.24 Organic matter (%) 0.50 Cation exchange capacity (Meq/100 g) 25.33 Ca saturation (%) 2.06 K saturation (%) 64.69 Mg saturation (%) 16.91 Na saturation (%) 16.35 NA, Not available Fig. 1 Sampling site, Ennore Port at Chennai, located on the southeast- ern coast of India The unit for nutrients is parts per million (ppm) 1294 Ann Microbiol (2014) 64:1291–1299 saline containing calcium chloride (10 mM). After 30 min of et al. 1992). In this study we demonstrated the biological incubation at room temperature, the contents were centri- synthesis of silver nanoparticles by a newly isolated marine fuged, and the absorbance of the supernatant was determined derived strain, Bacillus sp. VITSSN01 and its toxicity to at 540 nm as a measure of liberated hemoglobin. The tests human erythrocytes. were carried out in triplicate, and the values were expressed as The strain was isolated from a sediment sample collected at mean ± standard deviation. Ennore Port, southern coast of India (Fig. 1). Ennore Port is located on the Coromandel Coast in the Bay of Bengal Sea Morphological alterations Table 2 Biochemical The morphological changes in the treated and control eryth- Test Result and cultural characteris- rocytes were determined as described by Kondo and tics of Bacillus sp. +ve Tomizawa (1968). A blood sample (O ) was collected from Gram stain Gram-negative rod VITSSNO1 isolated healthy human donors and centrifuged at 600 g for 10 min at from marine sediment Motility Motile 4 °C. About 1 ml of erythrocyte suspension was placed in Colony color Sandal polystyrene tubes containing buffer and different concentra- Starch hydrolysis + tions of silver nanoparticles (0.1 and 1 mM). The sample was Casein hydrolysis + incubated at room temperature for 30 min and the cells the Gelatin hydrolysis + observed under the light microscope (100× magnification). Urea hydrolysis + Carbon sources (1 % w/v) D-Glucose + Results and discussion D-Sucrose + D-Fructose + Nanobiotechnology is an emerging field which deals with the Lactose − production and application of nanoparticles in biology and D-Galactose − medicine. The development of cost-effective and environmen- Glycerol − tally friendly approaches for synthesizing metal nanoparticles Mannose − using biological sources has recently become a primary focus D-Xylose + of researchers (Rumov et al. 2009). Biological sources are D-Raffinose − regarded as potential biofactories for the production of metal Mannitol + nanoparticles (Narayanan and Sakthivel 2011). Among the Nitrogen sources (1 % w/v) various potential nanoparticle-producing biological systems, Peptone + a bacterium is relatively easy to manipulate and handle and is Yeast extract + applicable for the large-scale production of nanoparticles. The Urea + microbial synthesis of metal nanoparticles depends upon lo- Ammonia + calization of the reductive components of their cells (Slawson Physiological conditions Temperature (°C) 15 − 25 − 30 + 37 + 45 − pH 5 − 7.2 + 7.4 + 7.6 + 8 − NaCl concentration (%) 1+ 3+ Fig. 2 Morphology of Gram-stained Bacillus sp., VITSSN01 strain 6 − observed under optical microscopy (1000× magnification) Ann Microbiol (2014) 64:1291–1299 1295 Fig 3 Phylogenetic tree showing the position of VITSS01 with other Bacillus sp. Number at nodes of tree Percentage level of bootstrap support and is bounded by the Korttalaiyar River, Ennore creek (Table 2). To optimize the cultural characteristics and media (backwater) and the Bay of Bengal. This zone comprises composition, we performed a systematic study in which we lagoons with salt marshes and backwaters. The soil texture evaluated the suitability of a number of carbon and nitrogen appears to be sandy loam. The physicochemical parameters of sources. Bacillus sp., VITSSN01 was found to be capable of the sediment samples are given in Table 1. Physiochemical utilizing different carbon sources, such as glucose, fructose, analysis of soil sediment from this region shows the presence xylose, mannitol and mannose. Better growth was observed if of many mineral ions, including sodium, potassium, magne- yeast extract and peptone were used the sole nitrogen sources, sium, manganese, zinc, phosphorous, copper and nitrate. The and good growth was seen when urea and ammonia were used pH of the sediment was 8.25. In order to mimic the same as inorganic nitrogen sources. Maximum growth was obtained environmental conditions, we supplemented the media with soil extract and seawater. The strain showed maximal growth S2M-1 on medium supplemented with sea water and soil extracts and Abs cultivated at 37 °C, pH 7.2. This modified method yielded 3.5 good results and growth appeared to be abundant. The bacterial cells were found to be aerobic, motile, non- 3.0 acid-fast, Gram-positive rods (Fig. 2). The cultural, morpho- 2.5 logical and biochemical characterization indicates that the 2.0 strain belongs to the genus Bacillus, and it was subsequently 1.5 designated VITSSN01. The colonies were circular, convex 1.0 and easily emulsifiable; they were slightly pinkish on marine agar and yellowish on nutrient agar. The strain tested positive 0.5 for catalase, cytochrome oxidase, gelatinase, casein, urea and 0.0 nm starch hydrolysis. Methyl red and citrate tests were positive, 300 400 500 600 700 800 and the triple sugar iron agar slants showed acid butt (yellow) Fig. 4 UV-Visible absorbance spectrum of silver nanoparticles synthe- and alkali slant (red) along with hydrogen sulphide production sized by Bacillus sp. VITSSN01 1296 Ann Microbiol (2014) 64:1291–1299 Fig. 5 Atomic force microscopy images denoting the aggregation of silver nanoparticles synthesized by Bacillus sp. VITSSN01 when glucose, sucrose and fructose were used as carbon nitrogen sources. To evaluate these optimal growth conditions sources and peptone, yeast extract, urea and ammonia as we cultured the strain under different physiological Fig. 6 X-ray diffraction (XRD) (a) 2000 (b) B B pattern of silver nanoparticles synthesized by Bacillus sp. VITSSN01. a XRD pattern measured in the presence of silver nitrate, b XRD pattern of silver nanoparticles showed a peak of sharp intensity between 30° and 35° θ and between 40° and 45° θ 0 102030405060708090 2θ (degree) Intensity Ann Microbiol (2014) 64:1291–1299 1297 100.0 Fig. 7 Fourier transform-infrared 95 PS-098 spectroscopy spectrum of biosynthesized silver nanoparticles 1139 986 %T 40 2034 931 35 1232 2136 1214 10 3631 0.0 4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.0 cm-1 conditions. Optimal growth was observed at 37 °C and pH the Plasmon ambience excitation of the silver nanoparticles 7.2. The strain grew well between 1 and 3 % salt concentration (Wiley et al. 2006). (NaCl) and showed moderate and sometimes low growth at a Spectroscopic analysis of the bioreduction of metal nano- 6 % salt concentration. The isolated strain was found to be particles showed that the UV-Visible absorbance spectrum resistant towards silver nitrate and when cultured under dark peaked at 420 nm (Fig. 4). The size, morphology and conditions was found to be involved in the extracellular syn- agglomerization of silver nanoparticles were analyzed using thesis of silver nanoparticles. AFM and the images are shown in Fig. 5. Particle size was The BLAST search of 16S rRNA gene sequences of the found to be 46 nm. The XRD pattern of the silver nanoparti- strain showed maximum identity (97 %) with Bacillus sp. cles is shown in Fig. 6. The XRD pattern was measured for SP09-202 (GQ35242), and the phylogenetic tree was con- both control and with the presence of nanoparticles. The structed with bootstrap values. Based on the molecular taxon- powdered nanoparticles showed peaks of sharp intensity be- omy and phylogeny analysis, the strain was identified as a tween 30° and 35° θ and between 40° and 45° θ. The FT-IR novel Bacillus sp. and designated as VITSSN01. A neighbor- pattern of silver nanoparticles was measured in the transmit- joining tree based on 16S rRNA gene sequences showed that tance range between 450 and 4,500 wave numbers, and the the isolate occupies a distinct phylogenetic position within the radiation that includes representatives of the Bacillus family (Fig. 3). The number at the nodes of the tree indicates the percentage level of bootstrap support. The score bar represents one nucleotide substitution per 100 nucleotides. The phyloge- netic tree based on the maximum parsimony method also showed the separate clade for the VITSSN01 (tree not shown). The rRNA gene sequence was submitted to GenBank under accession number JX094863. We determined that Bacillus sp. VITSSN01 was capable of the extracellular synthesis of silver nanoparticles. The strain was found to be resistant towards silver nitrate, reducing the silver nitrate to form silver ions under dark conditions. The color of the culture broth changed from yellow to brown after the synthesis of silver nanoparticles, and the pH of the broth increased slightly to 8. This color change is indicative of the reduction of silver nitrate to silver ions. The yellowish–brown Fig. 8 Transmission electron microscopy images of silver nanoparticles color of the silver nanoparticles in aqueous solution is due to synthesized by Bacillus sp. VITSSN01 1298 Ann Microbiol (2014) 64:1291–1299 −1 presence of wave numbers 1,650, 1,290, 2,036, 1,214 cm bacterial strains is very advantageous in terms of cost, time revealed the presence of silver nanoparticles (Fig. 7). The and the environment. There have been reports on the synthesis of study of TEM images revealed silver nanoparticles synthe- silver nanoparticles by a few Bacillus sp. Bacillus licheniformis sized by marine Bacillus sp. VITSS01 as dark spots on the has been reported to have synthesized silver nanoparticles of image (Fig. 8). 50 nm size (Kalimuthu et al. 2008; Kalishwaralal et al. 2008), Marine microorganisms have been found to be a potential and the culture supernatant of Bacillus subtilis has been reported source of metal nanoparticles. The commercial synthesis of to have synthesized silver nanoparticles of 5–60 nm (Saifuddin metal nanoparticles through the use of non-pathogenic et al. 2009). Pugazhenthiran et al. (2009) reported the synthesis of silver nanoparticles (5–15 nm) using Bacillus sp., and Ganesh Babu and Gunasekaran (2009) reported the synthesis (a) Erythrocytes without Ag NPs (4- and 5-nm nanoparticles) mediated by B. cereus.Inour study the marine Bacillus sp., VITSS01 was found to produce silver nanoparticles of 46 nm. Further, our TEM studies indi- cate that the silver nanoparticles are synthesized in the peri- plasmic space of the cell when Bacillus sp. VITSS01 is cultured with silver nitrate. Pugazhenthiran et al. (2009)re- ported that silver nanoparticles accumulated at the periplasmic space of silver nitrate-resistant Bacillus cells when cultured in presence of silver nitrate (. Hemotological toxicity is a very serious toxicity and its effects are less studied (Suwalsky et al. 2010). To evaluate the toxicity of biosynthesized silver nanoparticles, we used human erythrocytes as model cells. The silver nanoparticles (b) Erythrocytes treated with 0.1mM Ag NPs induced hemolysis on the tested erythrocytes with a half max- imal effective concentration (EC ) value of 60 μg/ml. Com- plete hemolysis was obtained with 20 μl of Triton-X 100 (0.1 %) and 1-h incubation (Table 3). Normal human blood cells under physiological conditions have a disc shape and are 8 μm in diameter. In our assay, changes in the shape of the human erythrocytes were observed after incubation with silver nanoparticles: the morphology was altered and the normal discoid shape of the cells changed to echinocytic form (Fig. 9). The extent of the change in shape was found to depend on the concentration of the silver nanoparticles: at 0.1 mM silver nanoparticles, moderate changes occurred in erythrocyte shape; at 1 mM, the intensity of these changes increased and (c) Erythrocytes treated with 1mM Ag NPs. strong structural perturbations were observed. Bilayer couple hypothesis states that the inner and outer layer of the erythro- cyte membrane is affected due to the insertion of some foreign Table 3 Hemolytic ac- Sample (μg) Percentage inhibition tivity of silver nanoparti- cles synthesized by the 60 46.33 marine Bacillus sp. VITSSNO1 against hu- 65 58.33 man erythrocytes 70 63.62 75 68.33 80 68.77 The experiments were 85 69.21 carried out in triplicate, 90 70.39 and the results are shown 95 73.04 as the mean value. Tri- Fig. 9 Morphology of red blood cells affected by silver nanoparticles. a ton-X 100 was used as a Erythrocytes without silver nanoparticles (Ag NPs). b Erythrocytes treat- 100 87.04 positive control ed with 0.1 mM Ag NPs. c Erythrocytes treated with 1 mM Ag NPs Ann Microbiol (2014) 64:1291–1299 1299 2+ Malagoli D (2007) A full length protocol to test hemolytic activity of species (Iglic et al. 1998). Similarly, Cd alters the molecular palytoxin on human erythrocytes. Invertebr Surviv J 4:92–94 structure of the lipid bilayer of erythrocytes (Suwalsky et al. 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Nanomedicine: NBM 5:452–456 synthesis of nano-sized metal particles. Detailed and periodi- Narayanan KB, Sakthivel N (2011) Green synthesis of biogenic metal cal investigation of the Indian marine environment would nanoparticles by terrestrial and aquatic phototrophic and heterotro- result in the isolation of novel microorganisms which can be phic eukaryotes and biocompatible agents. Adv Colloid Interf Sci 169:59–79 used for studying and furthering our understanding of the Prakasham RS, Sudheer Kumar B, Sudheer Kumar Y, Girija Shankar G biomineralization and biosynthesis of nanoparticles. (2012) Characterization of silver nanoparticles synthesized isolate Streptomyces albidoflavus. J Microbiol Biotechnol 22:614–621 Acknowledgments The study was supported by an institutional grant. 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Published: Jan 5, 2014

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