INTRODUCTION:

Due to the outbreak of the infectious diseases caused by different pathogenic bacteria and the development of antibiotic resistance the pharmaceutical companies and the researchers are searching for new antibacterial agents (Morones et al. 2005 and Kim et al. 2007). Bionanotechnology has emerged up as integration between biotechnology and nanotechnology for developing biosynthetic and environmental friendly technology for synthesis of nanomaterials. Nanoparticles are clusters of atoms in the size range of 1-100 nm. The metallic nanoparticles are most promising as they show good antibacterial properties due to their large surface area to volume ratio, which is coming up as the current interest in the researchers due to the growing microbial resistance against metal ions, antibiotics and the development of resistant strains (Gong et al. 2007).

Different types of nanomaterials like copper, zinc, titanium (Retchkiman schabes et al. 2006), magnesium, gold (Gu et al. 2003),alginate(Ahemd et al. 2005) and silver have come up but, silver nanoparticles have proved to be most effective as it has good antimicrobial efficacy against bacteria, viruses and other eukaryotic microorganisms (Gong et al. 2007). At the same time the biologically synthesized silver nanoparticles has many applications includes catalysts in chemical reactions, biolabelling, electrical batteries(Peto et al. 2002) and optical receptors (Krolikowska et al. 2003 and Joerger et al. 2001)

Numerous organisms have been found to synthesize nanoparticles. Biological production systems are of special interest due to their effectiveness and flexibility (Nithya et al. 2011). Microbial source to produce the silver nanoparticles shows the great interest towards the precipitation of nanoparticles due to its metabolic activity. Ofcourse the precipitation of nanoparticles in external environment of a cell, it shows the extracellular activity of organism. There are few reports published in literature on the biosynthesis of silver nanoparticles using fungal as source (Husseiny et al. 2007). The use of bacterial strain in the biomanufacturing process has the advantage that ease of handling than the fungal sources (Jose Elechiguerra et al. 2005; Beveridge and Murray, 1980).

Considering the growing technological demand for ecofriendly and development of reliable process for the synthesis of silver nanoparticles, the present work was undertaken. In this present work, microbial production of silver nanoparticles by using Proteus mirabilis and its antimicrobial activity against various pathogenic microorganisms was investigated. This research work implies the different medium composition for production of silver nanoparticles and characterization of particles done by UV-VIS spectrometer and SEM.

MATERIALS AND METHODS:

Source of microorganism

The bacterial strain Proteus mirabilis was obtained from the infected tomato sample. The obtained pure culture was maintained in nutrient agar medium (HiMedia, Mumbai, India) slant at 27°C as well as subcultured from time to time to regulate its viability in the laboratory during the study period.

Production of biomass

The bacterial strains Proteus mirabilis were cultured, to produce the biomass for biosynthesis in two different liquid broth namely nutrient broth medium and Xylose lysine deoxycholate broth (XLD) medium. The culture flasks were incubated on an orbital shaker at 27°C and agitated at 220 rpm. The biomass was harvested after 36 hours (hrs) of growth and centrifuged at 12000 rpm for 10 minutes. The supernatant material was collected for the further reaction to synthesis of nanoparticles.

Synthesis of silver nanoparticles

In a typical synthesis of silver nanoparticles extracellularly, 50mL aqueous solution of 1mM Silver nitrate (AgNO3) was treated with 50mL of Proteus mirabilis bio-mass supernatant solution in 150mL Erlenmeyer flask (pH adjusted to 7-8). The whole mixture was put into a shaker at 40°C (200 rpm) for 5 days and maintained in the dark. Control experiments were conducted with uninoculated media, to check for the role of bacteria in the synthesis of nanoparticles (Kannan Natarajan et al. 2010).

Characterization of silver nanoparticles

The reduction of silver ions was monitored by measuring the UV-VIS spectrum of the reaction medium at 24hrs time interval by drawing 1cm³ of the sample and the absorbance was recorded at a resolution of 0.5nm at 200-700nm using UV-VIS spectrophotometer (Elico, UV-VIS SL 191). The bacterial filtrate embedded with silver nanoparticle was dried under vacuum and subjected to SEM studies (EVO 50). Particle size analyzing experiments were carried out by means of laser diffractometry (CILAS 1064 Particle Size analyzer).

Bacterial susceptibility to nanosilver

The antimicrobial activity of isolated microbial silver based nanoparticles pellet were tested by standard well cutting method. The test bacteria such as Staphylococcus aureus, Pseudomonas species, Proteus vulgaris and Bacillus subtilis were included in this study to assess the susceptibility pattern of the compounds. 100μl of the diluted components were loaded on marked wells with the help of micropipette and the plates were incubated at 37^◦C for 24 hrs for observing inhibition rate.

RESULTS:

The bacterial strain of Proteus mirabilis was inoculated into different basal medium such as XLD and Nutrient Broth medium. Although the growth in Nutrient and XLD broth medium resulted same amount of biomass, but the synthesis of silver nanoparticles was maximum in XLD medium than the nutrient broth were shown in the figure 1.

The primary conformation of synthesis of nanoparticles in the medium it was found that aqueous silver ions when exposed to bacterial extract were reduced in solution, there by leading to the formation of silver hydrosol. The bacterial biomass were pale yellow in colour before the addition of silver ions and this changed to dark brownish colour, suggested the formation of silver nanoparticle. Figure 2 depicts a series of typical UV-VIS spectra of the reaction solution recorded at intervals of 24hrs. Under normal p^H6.0 the change in light absorption profile of the medium and change in intensity of the brown color during long term incubation (36hrs), it showed an increased absorbance with increasing time of incubation at characteristic surface plasmon resonance absorption band at 420nm. SEM micrographs showed formation of well-dispersed silver nanoparticles were shown in the figure 3. The average particle size analyzed from the SEM image is observed to be 35nm calculated from the laser diffraction particle size analyzer.

The antibacterial activities of the synthesized silver particles have been investigated against Staphylococcus aureus, Pseudomonas species, Proteus vulgaris and Bacillus subtilis. Silver nanoparticles synthesized from Proteus mirabilis showed very strong inhibitory action against Staphylococcus aureus (30mm zone of inhibition) followed by Pseudomonas species (26mm zone of inhibition), Proteus vulgaris (19mm zone of inhibition), Bacillus subtilis (21mm zone of inhibition) were shown in the figure 4.

Figure 1. Growth of biomass production and Silver nanoparticle synthesis from Proteus mirabilis

Figure 2. UV-VIS absorption spectra of silver nanoparticles synthesized by Proteus mirabilis culture.

Figure 3. SEM micrograph of Silver nanoparticles produced by

Proteus mirabilis

Figure 4. Antimicrobial activity of Silver nanoparticles

DISCUSSION:

Generally, UV-VIS spectroscopy can be used to examine size and shape of the controlled nano particles in aqueous suspense. The UV-VIS absorption showed increasing colour intensity with increased time intervals and this might be due to the production of the silver nanoparticles (Shrivastava and Dash, 2010), Suggested that the shoulder at 370 nm corresponded to the transverse plasmon vibration in silver nanoparticles, whereas the peak at 440 nm due to excitation of longitudinal plasmon vibrations. In the present study the peak value was observed at 420nm.

Scanning electron microscopy has provided further insight into the morphology and size details of the synthesized nanoparticle. Our investigation revealed that nanoparticles are in polydispersed mixture, with the various sizes range from 35 nanometers. Our result has similarity with reported that produced nanoparticles size varies nearly 100 nm and also the solution possesses polydispersed (Haefeli et al. 1984 and Mullaicharam, 2011). Since Klabunde and co-workers demonstrated that reactive metal oxide nanoparticles show excellent bactericidal effects (Stoimenov et al. 2002). Previous result suggests that synthesis of silver nanoparticles from the bacteria Proteus species is effectively against Salmonella typhi and Streptococcus epidermidis (Kannan Natarajan et al. 2010). Several authors have accomplished the biosynthesis of metal nanoparticles using biomass obtained from unicellular organisms like bacteria (Shahverdi et al. 2007), fungi (Varshney et al. 2009) and marine algae(Devina Merin et al. 2010) as well as extracts of plants, e.g. Euphorbia hirta (Elumalai et al. 2010),Catharanthus roseus (Mukunthan et al. 2011),Shorea tumbuggaia (Venkateswarlu et al. 2010), Diopyroskaki (Song et al. 2009) and Moringa oleifera(Prasad and Elumalai, 2011) were effective against various human pathogenic bacteria. The present study silver nanoparticles synthesized from Proteus mirabilis shows maximum zone of inhibition against various pathogenic organisms such as S. aureus and Pseudomonas species. It has been known for a long time that silver ions and silver compounds are highly toxic to most bacteria (Slawson et al. 1992 and Spadaro et al. 1974). Inhibitory action of silver ions on microorganisms show that upon silver ion treatment DNA loses its replication ability and expression of ribosomal subunit proteins as well as some other cellular proteins and enzymes essential to ATP production becomes inactivated (Yamanaka et al. 2005). Finally, this study shows that silver nanoparticles have excellent antibacterial activity against Pathogenic microorganism.

CONCLUSION:

Thus, our result summarize that Proteus mirabilis are capable of producing silver nanoparticles and they are quite stable in solution. The formed silver nanoparticles showed considerable antimicrobial activity against pathogenic microorganisms. This biosynthesis silver nanoparticles prove to be potential candidates for medical applications where antimicrobial activity is essential. Further investigations in the field can lead to the improvement of the medical methods for the treatment of microbial infections.

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Received on 10.03.2013 Accepted on 22.03.2013

Research J. Science and Tech 5(2): April- June, 2013 page 235-238