Algae mediated green fabrication of silver nanoparticles and examination of its antifungal activity against clinical pathogens

Algae extract has the great efficiency to synthesize the silver nanoparticles as a green route. Brown seaweed mediates the synthesis of silver nanomaterials using extract of Sargassum longifolium. For the improved production of silver nanomaterials, some kinetic studies such as time incubation and pH were studied in this work. 10 mL of algal extract was added into the 1 mM AgNO3 aqueous solution. The pH and reaction time range were changed and the absorbance was taken for the characterization of the nanoparticles at various time intervals, and the high pH level shows the increased absorbance due to the increased nanoparticles synthesis. The synthesized silver nanoparticles were characterized by Scanning Electron Microscope (SEM) showing that the shape of the material is spherical, and X-Ray Diffraction value obtained from range of (1 1 1) confirmed synthesized silver nanoparticles in crystalline nature. TEM measurement shows spherical shape of nanoparticles. The Fourier Transmittance Infrared spectrum (FT-IR) confirms the presence of biocomponent in the algae extract which was responsible for the nanoparticles synthesis. The effect of the algal mediated silver nanoparticles against the pathogenic fungi Aspergillus fumigatus, Candida albicans, and Fusarium sp. S. longifolium mediated synthesized silver nanoparticles shows cheap and single step synthesis process and it has high activity against fungus. This green process gives the greater potential biomedical applications of silver nanoparticles.

1. Introduction

Seaweeds are the natural and renewable living resources in the marine ecosystem and they are consumed for food, feed, and medicine. Seaweeds contain more than 60 elements, macro- and micronutrients, proteins, carbohydrates, vitamins, and aminoacids [1]. Seaweeds are the sources for extracting industrial products such as phycocolloids: carrageenan, alginates, and agar [2, 3]. Sargassum is a big family of marine brown algae and it has a broad application field. Most of the seaweeds have the antibacterial activity against pathogenic bacteria like Vibrio parahaemolyticus, Salmonella sp., Shewanella sp., Escherichia coli, Klebsiella pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, and Proteus mirabilis [4], antibiotic resistant postoperative infectious pathogens [5], and also used as antitumor compounds [6]. Moreover, seaweeds play an important role in adsorption of heavy metals like lead, copper, zinc, and manganese [7].

In the 21st century, nanotechnology is the newly emerging multidisciplinary research area with synthesis of nanosized materials [8]. Nanotechnology is the manipulation and production of materials ranging in size from 1 to 100 nanometer scale [9]. The nanoparticles can play a topmost role in the field of nanomedicines such as health care and medicine diagnostic and screening purposes, drug delivery systems, antisense and gene therapy applications, and tissue engineering and expectations of nanorobots configuration [10]. Many methods adopted in the field of synthesis of nanoparticles are chemical and physical. Nowadays, biological method of nanoparticles synthesis is a vast growing technique in the field of nanotechnology [11]. The biological sources had the more quantity of trouble-free protocols and when applied for the human health associated field, it is easy to approach for maintain aseptic environment during the synthesis process of nanoparticles [12]. Recently, biological materials such as bacteria, fungi, plant, and algae were used to synthesis of nanoparticles. Some are Enterobacteria [13], Aspergillus fumigatus [14], Coriander leaf [15], and Sargassum wightii [16] were used to synthesise of nanoparticles. Among the biological materials, algae is called as “bionanofactories” because the live and dead dried biomass was used for synthesis of metallic nanoparticles. It is low cost and environmentally effective, macroscopic structured material, and has the distinct advantage due to its high metal uptake capacity [17]. The rate and size of the nanoparticles were controlled by optimizing the parameters such as pH, temperature, substrate concentration, and incubation time [18].

This study reported that the exposure time and pH play an important role in the controlling of nanoparticles synthesis by using the S. longifolium algae extract. Nanoparticles synthesis was characterized by UV-vis spectroscopy, crystalline, and morphological structure which were characterized by XRD, SEM, and TEM. The antifungal activity of silver nanoparticles against pathogenic fungus was studied as well.

2. Materials and Methods 2.1. Chemicals

Analytical grade chemicals are silver nitrate, sodium hydroxide, and hydrochloric acid used for preparation of silver nanoparticles (NPs) and role of pH on NPs synthesis. Agar agar, Rose Bengal agar, and Sabouraud Dextrose agar were used for assessment of antifungal activity. All the chemicals and media were purchased from HiMedia (Mumbai, India).

2.2. Collection and Preparation of Algal Extract

The brown algae Sargassum longifolium was collected from the Tuticorin coastal area, Tamilnadu, India. The marine brown seaweed was thoroughly washed with fresh water and distilled water to remove the salt minerals and metallic compounds on the surface of the seaweed. Clean seaweed was dried at a shady place for ten days. The dried leaves were ground into fine powder. 1 gm of algal powder was mixed with 100 mL of distilled water in the 250 mL Erlenmeyer flask and boiled at 60°C for 10 min. The boiled extract was filtered through Whatman No. 1 filter paper, collected the supernatant, and stored at 4°C for nanoparticles synthesis.

2.3. Green Synthesis of Silver Nanoparticles

Typically, 10 mL of pure algal extract solution was mixed with aqueous solution of 90 mL of 1 mM silver nitrate (AgNO3) solution and kept in room temperature with constant stirring at 120 rpm. A color change of the solution was noted by visual inspection and UV-vis spectroscopy at different time and wavelength confirming the synthesis of silver nanoparticles.

2.4. Effect of pH

The role of pH in the synthesis of silver nanoparticles was carried out by altering the pH of algal extract. The pH range was varied from 6.2, 6.8, 7.8 and 8.4 by using analytical graded 0.1 N sodium hydroxide and 0.1 N hydrochloric acid standard solutions. The influence of pH on the synthesis process was analyzed by UV-vis spectrophotometer in the wavelength range of 380–600 nm.

2.5. Purification and Characterization of Synthesized Silver Nanoparticles

The bioreduction of silver ions in aqueous solution using algae extract was monitored by double beam UV-vis spectrophotometer at different wavelengths from 320 to 700 nm (Perkin Elmer, Singapore). Green synthesized silver nanoparticles were purified by distilled water by repeated centrifugation at 10,000 rpm for 15 min. Crystalline nature of the purified silver nanoparticles was analyzed by XRD (Bruker, Germany, model: D8Advance) and particle morphology was characterized by Scanning Electron Microscope (Hitachi, Model: S-3400N). The functional biomolecules such as carboxyl groups present in the seaweed responsible for the silver nanoparticles formation were characterized by FT-IR (BrukerOptik GmbH Model No.—Tensor 27). The dried silver nanoparticles were compressed with KBr into thin pellets and measured at the wavelength range from 4000 to 400 cm−1.

2.6. Antifungal Assay of Silver Nanoparticles 2.6.1. Clinical Fungal Pathogens

The three fungal pathogenic strains used in the present study were isolated from clinical samples and identified from Microlabs, Vellore District, India, which were Aspergillus fumigatus, Candida albicans, and Fusarium sp.

2.6.2. Assay of Antifungal Activity

The antifungal activity of green synthesized silver nanoparticles against various fungal strains was assayed by Agar well diffusion method. The fungicidal effect of the silver nanoparticles could be assessed by the formation of zone around the well. 100 mL of sterilized Sabouraud Dextrose Agar medium was poured into three sterilized Petri dishes. The fungal strains were grown in Rose Bengal agar and their spores were mixed into the 10 mL sterile distilled water and swapped on the agar. Three wells of 5 mm diameter were prepared and loaded with silver nanoparticles at different concentrations (30, 60, and 90 μL). The plates loaded with the fungal and silver nanoparticles were incubated at 37°C. The antifungal activities against the fungal strains were confirmed by forming the zone around the wells and measured after 24 hrs of incubation. The zone of inhibition was expressed in mm in diameter. The experiments were repeated three times to find the standard deviation and standard error.

3. Results and Discussion 3.1. Visual and UV-Vis Spectrophotometer Analysis

Reduction of silver ions to silver nanoparticles was visually identified by color change from yellow to brown in the aqueous solution of reaction mixture at 1 hr incubation time (Figure 1). Brown formation occurred due to the oscillation of free electrons in the reaction mixture. The color change depended on the incubation time. The deep brown color for silver nanoparticles was attained at 32 hr indicating that the increasing of color intensity is directly proportion to the time of incubation. Furthermore, the nanoparticles formation by the algal extract was confirmed by UV-vis spectroscopy at different wavelengths. Similarly, Chandran et al. [19] synthesized silver nanoparticles using Aloe vera extract taking 24 hr of reaction time in the presence of ammonia which enhances the nanoparticles formation.