Promising upshot of silver nanoparticles primed from Gracilaria crassa against bacterial pathogens

Background The study on newer antimicrobial agent from metal based nano materials has augmented in recent years for the management of multidrug resistance microorganisms. In our present investigation, we synthesized silver nanoparticles (AgNP’s) from red algae, Gracilaria crassa as beginning material which effectively condensed the silver ions to silver nanoparticles with less price tag and no risk. Methods Silver nanoparticles were prepared by simple reaction of 1 mM AgNO3 with G. crassa extracts at room temperature. The fabricated AgNP’s were subjected for characterization and screened against various microorganisms for antibacterial activity. Results UV–Vis spectroscopy (200–800 nm), XRD, FESEM and EDAX, were performed for AgNP’s. UV–Vis spectroscopy demonstrated the absorption edge at 443 nm and EDAX pattern is purely due to the particle size and face centered cubic (fcc) symmetry of nanoparticles. Average size lays at 122.7 nm and zeta potential was found to be −34.9 mV. The antibacterial outcome of synthesized AgNP’s (at the dose of 20 and 40 µg/ml) was evaluated against Escherichia coli, Proteus mirabilis, Bacillus subtilis and Pseudomonas aeruginosa. The mechanism of synthesized AgNP’s bactericidal bustle is discussed in terms of interaction with the cell membrane of bacteria. The activity was found to be sky-scraping in a dose dependent manner. Conclusion Thus, environmental friendly, cost effective, non hazardous stable nanoparticles were prepared by green synthesis using red algae, G. crassa. Synthesized G. crassa AgNP’s were in acceptable size and shape. Further, it elicits better bactericidal activity against microorganism. This will assure the out put of superior antibacterial formulation for near future. Graphical Abstract


Background
From ancient, handling of microbial infection is an exigent task for microbiologists. Countless drugs have been found to be successful, unfortunately, it leads to mount of drug resistance against particular pathogens with an outlook of stern issues in concern with public wellbeing [1]. Technical community is animatedly trying to expand groundbreaking concepts in drug delivery by challenging the new microbial agent with superior mode of action by its effectual target on the cell membrane or neither on intracellular targets [2,3]. In 21st century, nanotechnology has become inevitable, not because of only claim and also by the way of synthesis. Two way synthesis like physical and chemical methods have several sizeable challenges like technically protracted, expensive and ecological hazardous defects [4]. The current art of exploration in research is heading towards green synthesis of high-yield, squat in cost, non-hazardous and eco-friendly metallic nanoparticles by plants and microorganisms [5,6]. Due to hefty surface area, high reactivity and surface plasmon resonance, these nanoparticles were tailored for definite application by scheming into unambiguous size, shape and morphology leading to exhibit its broad spectrum of activity against multi drug resistance microorganisms [7-10]. A quest for an environmentally sustainable synthesis process has led to a few biomimetic approaches like applying natal principles in the formation of nanoparticles. Among several, bioreduction is the prime and widely practiced functional method in synthesizing the nano materials [11,12]. Noble metal nanoparticles such as silver, gold, which are geared up by plant extracts, algae, bacteria and fungi are broadly applied in drug delivery systems [13], electronics [14,15], biosensors [16], cancer therapeutics and antimicrobial agents [17][18][19]. For three decades, exploration of marine algae has been far above the ground for the search of new and effective natural origin medicines, because it posses elevated quantity of concealed bioactive essence. Several of such compounds, including carbohydrates, alkaloids, steroids, phenols, saponins and flavonoids, etc. [20][21][22]. These multifaceted compounds exhibit a wide range of industrial and biotechnological applications [23]. An extent, these bio-molecules play decisive role in reduction of metal ions and generate the stable eco friendly nanoparticles. Further, AgNP's fetches superior antibacterial activity by interacting with thiol clusters present in bacterial cell by cliping its replication. Literature strappingly supports that these Ag + ions unyoke the respiratory chains and collapse the proton motive forces across the cytoplasmic membrane of bacteria [24]. Gracilaria crassa (G. crassa), a well-known red algae, having potential secondary metabolites [25]. In harmony to the above information, the present study was intended to prepare and typify silver nanoparticles from G. crassa, further to explore its antimicrobial activities against highly resistance microbial inhabitants.

Seaweed collection and extraction
Gracilaria crassa was collected along the coast of Mandapam (9°16′58.9″N 79°18′53.6″E), Rameswaram, Tamilnadu, India. The freshly collected algal material was rinsed with seawater followed by deionized water to get rid of extra impurities. The samples were kept in shade for 15 days drying. The algal material was ground to powder and uphold stockpile by placing at −4°C for further studies. The G. crassa extract was prepared in a conical flask by taking 2.5 g in 100 mL of deionized water. It was heated for 45 min at 50°C and positioned in an orbital shaker for 24 h in order to conquer the maximum extraction of compounds. The Extracts were filtered through whatman No. 1 filter paper and stored in refrigerator for further studies [26].

Synthesis of silver nanoparticles
AgNP's were synthesized by adopting the method proposed by Sathishkumar et al. with simple modification [27]. 5 ml of algal extract was added in to 95 ml of 1 mM aqueous silver nitrate solution, in 250 ml conical flasks and kept at 30°C in a shaker for overnight to facilitate absolute reduction. The samples were monitored periodically for its color intensity to confirm the formation of AgNP's.

Characterization of silver nanoparticles UV-Vis spectral analysis
The reduction in pure silver ions was recorded by measuring the UV-Vis spectrum of the synthesized AgNP's of G. crassa at room temperature with a Perkin Elmer Lambda 25 UV-Vis spectrometer at the wavelength of 200-800 nm [28].

Particle size and zeta potential studies
Particle size and zeta potential experiments for G. crassa AgNP's were carried out by using a HORIBA Instruments (Singapore) Pvt Ltd, Singapore.

Powder x-ray diffraction (XRD) analysis
The silver nanoparticles were alienated by repeated centrifugation at 12,000 rpm for 10 min followed by redispersion of AgNP's into deionized water for three times. The supernatant was transferred in microwave for drying. X-ray diffraction (XRD) measurement of the AgNP's was carried out using Rigaku smart lab instrument (Japan), function at voltage of 40 kV, 30 mA current with Cu Kα1 radiations.

FESEM-EDAX studies
After careful UV-Vis spectroscopical analysis of synthesized nanoparticles, diameter of nanoparticles were further confirmed by Field emission scanning electron microscopy-energy dispersive X-ray analysis (FESEM-EDAX) by using SUPRA 55-CARL ZEISS, Germany.

UV-Vis spectral analysis
The formation of AgNP's was defined by color transformation [30] from pale yellow to dark brown (Fig. 1). The color change is due to the excitation of the surface plasmon resonance (SPR) (Fig. 2) which elicits λ max of 443 nm.

Particle size and zeta potential
Particle size determination of synthesized AgNP's was revealed underneath by intensity. Laser diffractions exposed by obtained AgNP's were in polydisperse concoction with average size of 122.7 nm (Fig. 3). The zeta potential endows stability of nanoparticles and surface charge. The zeta potential was found to be −34.9 mV. Earlier reports strongly supports, when the zeta potential value positions between −30 and −50 mV, it specifies good stability of nano particles [31]. The high negative value (Fig. 4) substantiates the repulsion between the particles and thereby increases the stability of the AgNP's.

FESEM and energy dispersive X-ray analysis
Field emission scanning electron microscopy investigation was further confirmed the size of silver nanoparticles synthesized from G. crassa. The size (diameter) of the nanoparticles ranges between 60 and 200 nm and the shapes were spherical and some are irregular (Fig. 6). The outcome of FESEM reports were overlapped with earlier reports [33,34]. The energy dispersive X-ray analysis (EDAX) depicts strong indication in the silver region, which authenticate formation of silver nanoparticles  ( Fig. 7). The optical absorption peak at 3 kV, which attributed to metallic silver nanocrystallites owing to surface plasmon resonance [35,36].

Antibacterial assay
Antibacterial activity by agar well diffusion technique was recorded after 24 h incubation of culture plates.
The AgNP's demonstrate excellent antibacterial activity against all tested microorganisms (Fig. 8). AgNP's showed high spectrum of activity against E. coli and P. mirabilis at the concentrations of 20 and 40 µg/ml when compared with standard (Fig. 9). The significant zone of inhibition was exerted due to effect of AgNP's on biochemical process of the bacterial cell by interacting thiol and amino groups of proteins and nucleic acids of cell wall [37][38][39]. Further, this could lead to interaction between nanoparticles and microorganism which results in triggering the discharge of highly reactive oxygen species (ROS), mostly hydroxyl radicals and singlet oxygen [40][41][42]. This augments the deposition of nanoparticles on the bacterial cell surface results large accumulation of silver nanoparticles causing disruption of cellular functions.

Conclusion
In summary, the bio-reduction of aqueous silver ions to silver nanoparticles (AgNP's) was successfully done using marine red algae, G. crassa in trouble-free, economy and ecofriendly manner. The average size of silver nanoparticles was found to be 122.7 nm with high stability of −34.9 mV. Further characterization by UV-Vis spectroscopy, FESEM, EDAX confirm formation nanoparticles which are virtually spherical in shape. XRD divulges fcc structural confirmation. The proved antibacterial potential will lend a hand to develop a powerful antibacterial formulation in near future as biomedical remedies. Hence, authors robustly propose this green synthesis of nanoparticles can be extended to the wide range of applications.