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Bio-fabricated metal nanoparticles are generally bio-compatible, inexpensive, and eco-friendly and therefore, are used preferably in medical and material science research and industries. Considering the importance of bio-fabricated materials, we isolated, characterized and identified bacterial strain OS4 as Stenotrophomonas maltophilia using 16S rDNA gene sequence analysis. This Gram negative bacterial strain significantly reduced hexavalent chromium when grown at neutral pH. Subsequently, the supernatant of log phase grown culture of strain OS4 reduced silver nitrate at room temperature. The nanoparticles generated using strain OS4 were characterized by UV-visible, Nanophox particle size analyzer, XRD, SEM and FT-IR spectroscopy. The nanoparticles had an absorption peak of 428 nm; a characteristic of silver nanoparticles. They were cubical in shape with an average particle size of 93 nm. The FT-IR analysis suggests that the biological moieties especially protein component may have caused the reduction of silver nitrate. The XRD spectra exhibited the characteristic Bragg peaks of 111, 200, 220 and 311 facets of the face centred cubic of nanoparticles suggesting that these nanoparticles are crystalline in nature. Bio-fabricated silver nanoparticles also exhibited significant bactericidal activity against medically important bacteria such as S. aureus, E. coli and S. marcescens.
Keywords: Bio-reduction, Chromium, Stenotrophomonas maltophilia, Bio-fabrication, antimicrobial activity
Scientists belonging to diverse disciplines are working hard to find suitable, eco-friendly and inexpensive methods for producing nanomaterials with broad spectrum activity. The nanotechnology and its resulting product 'nanomaterials' synthesized so far have shown some profound effect on human health, economic growth and the environment. The development of techniques for the controlled production of nanoparticles of well-defined size, shape and composition, to be used in biomedical sciences, optics and electronics, is however, still a major challenge . Therefore, realizing the undeniable magnitude of this technology and its consequential impact on human health and environment, various governments and private sectors at global level has significantly increased their funding in vital sectors like medicine, manufacturing, energy and transportation. Following this, the material science research in recent times has achieved considerable importance due to the unique property of nanoparticle size and shape . Also, the inherent characteristics of nanoparticles such as high surface to volume ratios and quantum confinement result in materials that are qualitatively different from their bulk counterparts . Generally, nanoparticles are synthesized by different physico-chemical methods [4, 5, 6], which are however, expensive and environment disruptive. Therefore, interest is growing every day to develop inexpensive, clean, non-toxic and eco-friendly strategies for the synthesis of nanoparticles. In this regard, the advent of fabrication of bio-based advanced nanomaterials has provided solutions to the problems, For example, fabricated materials are now being used as/in (i) catalysts in chemical reactions (ii) antibacterial agents [8, 9] (iii) biosensors  (iv) cancer therapy  and (v) bio-control agents  etc. Recently, eco-friendly synthetic chemistry approaches have however, involved some biological systems such as yeast , fungi , bacteria  and plant extracts  for the synthesis of nanoparticles. Some of the most commonly used microorganisms for developing microbes based silver nanoparticles include fungi like Fusarium  and Aspergillus ; bacteria such as Enterobacteria , Pseudomonas , Bacillus , and Geobacter . Mechanistically, the ability of bacterial strain to reduce nitrate  has been exploited in the reduction of silver nitrate in to elemental nanomaterial .
Considering the significance of bio-based fabricated nanomaterials, the present study was designed to find bacterial strain originating from the heavy metal contaminated sites and to characterize bacterial strain both biochemically and molecularly. The bacterial strain was further tested for its nitrate and chromium reducing ability. The bacterial strain was also used to synthesize silver nanoparticles at room temperature in the absence of any reducing agent. The resulting nanoparticles were subsequently characterized using some of the standard analytical techniques like, UV-visible, Nanoparticle size Analyzer, SEM, XRD and FT-IR spectroscopy. Finally, the antibacterial activity of synthesized nanoparticles was detected using both Gram-positive and Gram-negative bacteria.
2. Materials and Methods
2. 1. 1. Isolation and bacterial characterization
The soil samples were collected in sterile polythene bags (15 - 12 cm2) from the rhizosphere of pea (Pisum sativum) fields located at the outskirts of Ghaziabad, Uttar Pradesh, India. Historically, the agronomic field was irrigated consistently by industrial sewage water of Hindon river. In order to isolate the bacterial strain, a serial dilution assay was carried out in normal saline solution and 10 µL of diluted suspension was spread plated on nutrient agar medium. Plates were incubated at 28±2 °C for three days. A total of 20 bacterial strains were selected and characterized. Biochemical activities included citrate utilization, indole production, methyl red test, nitrate reduction, Voges Proskauer, catalase test, oxidase carbohydrates (dextrose, mannitol and sucrose) utilization, starch hydrolysis, and gelatin liquefaction test .
16S rDNA based identification
Of the total 20 bacterial strains, strain OS4 was identified by 16S rDNA gene sequence analysis. The partial sequencing of 16S rDNA of the strain OS4 was done commercially by Sequencing Service, Macrogen Inc., Seoul, South Korea using universal primers, 518F (50CCAGCAGCCGCGG TAATACG30) and 800R (50TACCAGGGTATCTAATCC30). Later, nucleotide sequence data was deposited in the Gen-Bank, NCBI, sequence database. The online NCBI program nBLAST was used to find out the related sequences with known taxonomic information in the databank at NCBI website (http://www.ncbi.nlm.nih.gov/BLAST) to accurately identify the bacterial strain OS4. Phylogenetic tree was constructed by the neighbour-joining method  of the MEGA 4.1 software programme .
2. 1. 2. Optimization of growth and -hexavalent chromium reduction conditions
The effect of viable bacterial populations and pH on hexavalent chromium (Cr6+) reduction was assessed using nutrient broth (NB) amended with 100 µg ml-1 of Cr6+. The sterilized medium was adjusted to pH 2 to 12 with 1M HCL or 1M NaOH. A-100 µl of exponentially grown culture of S. maltophilia OS4 was inoculated into NB medium containing upto100 µgml-1 of hexavalent chromium and incubated at 35±2 ÌŠC in an orbital shaking incubator at 120 rpm upto 48 h. For Cr6+ reduction, one ml culture from each flask was centrifuged (6000 rpm) for 10 min. at 20 ÌŠC, and Cr6+ in the supernatant was determined by the 1,5-diphenyl carbazide method [28, 29].
2. 1. 3. Medium and growth conditions for supernatant preparation
The bacterial isolate OS4 was inoculated in sterile King`s B base broth medium (pH 7.2) containing glycerol 15ml/l, protease peptone 20g/l, hydrogen phosphate 1.5g/l and magnesium sulphate 7H2O 1.5g/l. Bacteria was allowed to grow at 35±2 -C for 24 h in a 500 ml Erlenmeyer flask with working volume of 300 ml with agitation at 120 rpm on orbital shaking incubator (Remi CIS 24BL, India). Culture medium was then centrifuged at 8000 rpm to obtain cell-free supernatant.
2. 2. Preparation and characterization of silver nanoparticles
2. 2. 1. Ratio optimization of bacterial supernatant and aqueous solution of silver nitrate
One milli molar aqueous solution of freshly prepared silver nitrate (AgNO3) was used to obtain silver nanoparticles. Two ml supernatant extracted from exponentially grown bacterial culture was added to 98 ml of 1mM AgNO3 solution. The reaction mixture was incubated in dark at room temperature (Fig. 3a).
2. 2. 2. UV- VIS and Nanophox spectra analysis
The reduction of silver (Ag+) ions was carefully monitored by measuring the UV-vis spectrum of the reaction medium incubated overnight after diluting a small amount of aliquot prepared in double distilled water. Since silver nanoparticles are soluble in water, the colour changes were observed. A yellowish brown colour formation was noticed during the synthesis phase. The concentration of AgNP produced was measured using a UV-VIS spectrometer (Thermo Spectronic 20D+) between 250 and 600 nm wavelength, using 10-mm-optical-path-length quartz cuvettes. Further analyses of nanoparticles distribution and stability in solution were observed by the Nanophox particle size analyser.
2. 2. 3. FTIR spectra analysis
Fourier Transform IR (Perkin Elmer) spectrometer was used to ascertain the biological moieties involvement in particle synthesis. In order to remove any free biomass residue, the residual solution after the reaction was centrifuged at 8000 rpm for 30 min. and the resulting pellets was mixed in 20 ml sterile double distilled water and cyclomixed for 10 min. on vortex mixer. Thereafter, the centrifugation and re-dispersing process was repeated three times. The FT-IR spectrum of the silver nanoparticles was recorded on Thermo-Nicolet Nexus 670 spectrometer using KBr pellets and the spectra were collected at a resolution of 4 cm−1 in the wave number region of 400-4000 cm−1.
2. 2. 4. X-ray Diffraction and FESEM analysis
X-ray Diffraction analysis (XRD) was carried out using Rigaku Miniflex X-ray diffractometere with Cu-Kα radiations (λ=0.15406 nm) in 2θ range from 200 to 800. Furthermore, morphology of the AgNPs was examined by field emission scanning electron microscopy (HITACHI SU6600 FESEM). The sheet of the samples were prepared on a carbon coated copper grid by dropping a tiny amount of the sample and then allowed to dry prior to measurements.
2. 3. Antibacterial assay
The bio-fabricated silver nanoparticles were tested for bactericidal activity by agar well-diffusion method against both Gram positive Staphylococcus aureus and Gram negative Escherichia coli and Serratia marcescens. The pure culture of each bacterium was sub-cultured in nutrient broth medium. Each bacterial strain was spread uniformly onto the individual plates by using sterile glass rod spreader. Wells of 8mm diameter were punched into nutrient agar plates using gel puncture. By using a micropipette, nanoparticle (12.5, 25 and 50 µg) suspension was poured into each well on all plates. Plates were then incubated at 35±2 ÌŠC for 48h and the level of zone of inhibition of bacterial growth was measured.
3. Results and discussion
3. 1. 1. Characterization of bacterial strain
In the present study, hexavalant chromium resistant bacterium was isolated from industrial effluents contaminated soil. Of the 20 bacterial isolates, strain OS4 was selected especially due to its ability to tolerate high level of most toxic form of chromium and was characterized morphologically and biochemically (Table1). Strain OS4 grew well on nutrient agar (NA) plates amended with 1200 µg K2 Cr2 O7 /ml. The chromium resistant bacterial strain was found to be Gram-negative, rod shaped and produced green pigments on NA plates. The freshly grown cultures showed a positive reaction for citrate utilization, nitrate reduction, catalase, and could hydrolyze starch and gelatin, but were negative for other biochemical tests (Table 1). On the basis of the characteristics observed for strain OS4 and compared with those listed in Bergey`s Manual of Determinative Bacteriology , strain OS4 was presumptively identified as Stenotrophomonas sp. In order to further validate strain OS4 and to identify the bacterial species, it was subjected to 16S rDNA sequence analysis. The sequence of 16S rDNA of strain OS4 was submitted to Gen-Bank (Gen-Bank accession number JN247637). A similar search was performed by using the BLAST program that indicated a close genetic relatedness of strain OS4 with the rDNA sequence of S. maltophilia (16S: 99% similarity with the reference sequence HQ185400.1) in NCBI database. Such a higher identical value confirmed the strain OS4 to be Stenotrophomonas maltophilia. A phylogenetic tree constructed by MEGA4 software based on 16S rDNA partial sequence is presented in Figure 1. Microorganisms in general have been found to survive in metal contaminated environment  and this property of metal tolerance by microbes have been/being exploited well in the bioremediation strategies to clean up contaminated sites . Some of the strategies adopted by bacterial populations to protect themselves from the nuisance of metal toxicity includes- (i) restriction of metal entry in to the cell either by reduced uptake/active efflux or by the formation of complexes outside the cell (ii) avoidance and sequestrations and (iii) enzymatic reduction of free ions in the cytosol. Despite these mechanisms, the information on the impact of hexavalent chromium on bacterial populations inhabiting contaminated environment is contradictory. As an example, the Gram positive Bacillus strains tolerated chromium up to the concentration of 500 (PSB1), 400 (PSB7), and 550 µg ml-1 (PSB10), respectively, when grown on chromium amended NA plates  while other Gram-positive bacterium Bacillus sphaericus isolated from serpentine soil could tolerate 800 µg ml-1 Cr (VI) . The differential response of bacterial cells even within the same group is likely due to the variation in the compositions of medium used or variable growth conditions . However, whatever have been the reasons; the bacterial strain isolated in this study exhibited a high level of tolerance to hexavalent chromium which could be an advantage while using this strain under chromium stressed soils.
3. 2. 1. Chromium reduction assay
The effect of pH on chromium reduction by exponentially grown bacterial strain OS4 was variable (Fig. 2).The bacterial strain grew well at pH 7 and could remove hexvalent chromium maximally by 91% after 48h growth. However, with increase or decrease in pH, there was a corresponding decrease in bacterial growth which subsequently affected the reduction of hexavalent chromium very negatively. (Fig. 2). For example, a maximum decrease (100%) in chromium reduction by strain OS4 was observed at pH 2 compared to those recorded at pH 7. In a similar study, Wani et al.  have also observed a variable effect of pH on chromium reduction by Bacillus sp. grown in nutrient broth treated differently with varying concentrations of hexavalent chromium. The chromium reducing ability of strain OS4 thus suggests that this strain might have enzyme chromium reductase which possibly led to the reduction of chromium, as also reported by Farrel and Ronallo .
3. 3. Characterization of biosynthesized nanoparticles
The colour of reaction mixture changed from colourless to yellowish brown in 30 min. when two millilitre supernatant prepared from strain OS4 was added to 1mM 98 ml solution of AgNO3. The intensity of colour further increased with increasing incubation periods (Fig. 3a, b). Formation of AgNPs using 1mM solution of AgNO3 was confirmed by UV-visible spectral analysis. In the UV-vis absorption spectrum, a strong and broad peak, located at about 428 nm, was observed for nanoparticles synthesized using the bacterial supernatant (Fig. 4a, 4b). Similar UV-VIS spectrophotometric peak formation is well documented for various metal nanoparticles with size ranging from 2 to 100 nm [35, 36].
Later on, the particle size distribution curve of silver nanoparticles was determined and is presented in Fig. 4. The average size of the colloidal silver nanoparticles synthesized was found to be 93 mm. The size patterns recorded in this study are in good agreement with the data observed under XRD, UV-VIS spectroscopy and scanning electron microscopy (SEM) studies. Furthermore, particle size analyzer was also used to check the size and stability of Ag nanoparticles in suspension. It was found that there was no change in the particle size distribution with time suggesting that the size of Ag nanoparticles were stable. In a similar study, the biogenic nanoparticles size and distribution was analysed by Chauhan et al. . The XRD analysis confirms the formation of single phase cubic silver nanoparticles (Fig. 5). All the peaks matched well with the standard JCPDS card No. 04-0783 of cubic silver nanoparticles which correlates well with FESEM results (Fig. 6). Average particle size calculated from XRD data was found to be 93 nm which correlated well with the results obtained by particle size analyzer. Furthermore the FESEM image of the synthesized silver nanoparticles was determined (Fig. 7). The FESEM results validate the formation of cubic nanoparticles capped with its bio-moieties. This indicates the reduction of silver ions to elemental silver. The synthesized nanoparticles were stable in solution over a period of three months time at room temperature.
Bio-fabrication of silver nanoparticles by the bacterial supernatant depends on the biological functional groups present on the bacterial surface. And hence, in order to understand better the kind of information about the functional groups involved in the capping or reduction process, FT-IR analysis of the AgNP was done. The FTIR spectra of AgNP in the range of 1000-4000 cm-1 were taken just to ascertain the presence of functional groups that could possibly be involved in the process (Fig. 8). The bands characterization including hydroxyl and amine group peaks were assigned at 3376 cm-1, alkyl and CHO had a broad band ranging between 2967-2851 cm-1, C=O of amide groups at 1644 cm-1, COO- of the carboxylate groups appeared at 1584 and 1544 cm-1, the band located at 1238, 1398 and 1740 cm-1 represented COO- anions where as those located at 726 cm-1 was assigned SO3- groups. The IR spectra revealed the role of proteins and enzymes involved in capping and reduction of silver ions in the formation of nanoparticles. Similarly, biological functional group involved in the capping of biogenic silver nanoparticles was observed by Kumar and Mamidyala . The band at 1650 cm-1 arises due to carbonyl stretch and -N-H stretch vibrations in the amide linkages, clearly indicating the presence of protein/peptide on the surface that appears to be acting as a capping/stabilizing agent [38, 39].
3. 4. Antibacterial activity
After characterizing the nanoparticles, the newly synthesized bio-fabricated AgNPs were tested for their antibacterial activity. Interestingly, the bio-fabricated AgNPs exhibited significant antibacterial activity against both Gram-negative (S. marcescens and E. coli) and Gram-positive (S. aureus) bacteria grown in nutrient agar medium treated with different concentrations of nanoparticles. The antibacterial activity expressed in terms of zone of inhibition was quite visible on nutrient agar plates (Fig. 8 a, b, c). A maximum zone of inhibition was recorded for S. marcescens (25 mm), E. coli (23 mm) and S. aureus (22 mm) when 50 µg AgNPs was loaded into 8 mm agar well. Generally, the antibacterial activity of silver nanoparticles increased considerably with corresponding increase in concentrations ranging from 12.5 µg to 50 µg. For instance, when the concentration of nanoparticles was increased from 12.5 to 50 µg per well, the growth of bacterial strains namely E. coli, S. aureus and S. marcescens was declined significantly by 91, 69 and 66% respectively (Fig. 9). The bactericidal property of these nanoparticles suggested that these particles were more diffusible in the growth medium which in turn allowed greater interaction between bacterial cells and each nanoparticle. Similar bactericidal impact of some other silver ions on microbial communities is known. For example, in many cases it has been proposed that ionic silver strongly interacts with thiol groups of vital enzymes and inactivates them. Also, there are evidence suggesting that DNA replication is halted when bacterial cells are exposed to silver ions (Yang et al 2009) . Even-though, we could not pinpoint the exact site where nanoparticles could affect the bacterial cells but it was very clear that Gram negative bacterial strains were most susceptible to nanoparticles for reasons yet not clearly defined.
The bacterial culture S. maltophilia isolated and well characterized in this study exhibited chromium reducing ability; a characteristics that could help in cleanup of chromium contaminated environment. Further, the culture supernatant of S. maltophilia strain OS4 was used for synthesis of silver nanoparticles using silver nitrate at room temperature. It is proposed that the reduction of the silver ions may be due to the protein component contributed by the enzyme reductase, since chromium reduction is the specific property of this strain. The UV-visible spectrum showed a surface Plasmon resonance peak at around 428 nm, which is characteristic of silver nanoparticles. The silver nanoparticles formed were cubical in shape with an average particle size of 93 nm, crystalline in nature, and the particle surface was anionic; these properties were confirmed by Nanophox, SEM, XRD and FTIR analysis. The silver nanoparticles generated here also showed good antibacterial activity against both Gram-positive and Gram-negative bacteria. Thus, the bacterial strain OS4 used in this study is likely to provide many fold benefits such as (i) may be helpful in curing chromium polluted sites (ii) could be used to generate bio-fabricated silver nanoparticles and (iii) could be effective in the management of infections and diseases.