Biosynthesis and Characterization of Silver Nanoparticles produced by Phormidium ambiguum and Desertilum tharense

The world faces a challenge with pervasion of multidrug resistant bacteria which encouraged the scientists to develop and discover alternative ecofriendly and easy to produce new antibacterial agents. Two Egyptian cyanobacteria were isolated and identified according to 16S rRNA gene sequencing as Phormidium ambiguum and Desertifilum tharense . The sequences were deposited in the GenBank with accession numbers of MW762709 and MW762710 for Desertifilum tharense and Phormidium ambiguum, respectively. These isolates have the ability to produce silver nanoparticles (Ag-NPs) extra- and intracellularly under light and dark conditions. The results of UV-Vis analysis showed promising extracellular Ag-NPs synthesis by Desertifilum tharense and Phormidium ambiguum under light conditions. Therefore, these Ag-NPs were characterized and evaluated for antibacterial and antioxidant activity. TEM, SEM and XRD analyses revealed the spherical crystals with face-centered cubic structures and size range of 6.24 – 11.4 nm and 6.46 – 12.2 nm for Ag-NPs of Desertifilum tharense and Phormidium ambiguum , respectively. XRD and EDX results clearly confirmed the successful synthesis of Ag-NPs in its oxide form or chloride form. The FTIR spectrum data confirmed the presence of hydroxyl and amide groups. Desertifilum tharense Ag-NPs displayed the largest inhibition zone ranged from 9 mm against Micrococcus luteus ATCC 10240 to 25 mm against methicillin resistant S. aureus (MRSA) ATCC 43300. For Phormidium ambiguum Ag-NPs, the inhibition zone diameter was in a range of 9 – 18 mm. The Ag-NPs of Phormidium ambiguum exhibited the highest scavenging activity of 48.7% comparing with that of Desertifilum tharense which displayed 43.753%.


Introduction
Nanotechnology is a sonorous area of science, engineering, and technology concerning the design and manufacture of materials at the nanoscale. The products of (Tamura et al., 2004). The analysis involved 13 nucleotide sequences of which 2 sequences of 16S rRNA gene amplified from cyanobacterial isolates while 11 sequences representing the most similar hits were obtained from the NCBI gene bank database. Evolutionary analyses were conducted in MEGA5 software.

Biosynthesis of silver nanoparticles (Ag-NPs) by cyanobacteria
The cyanobacterial isolates were screened for synthesis of Ag-NPs extra-and intracellularly under light and dark conditions. According to the method of Patel et al. (2015), after 15 days incubation, the broth cyanobacterial cultures were centrifuged (Universal 16r, Hettich) at 6000 rpm/10 min at 10°C.
For extracellular synthesis of Ag-NPs, the supernatant was added to AgNo3 solution to reach the final concentration of 1 mM AgNO3. To evaluate the synthesis of Ag-NPs intracellularly, the harvested biomass was washed twice at least with sterile distilled water, kept at -20°C overnight to facilitate complete lysis of cells (Mahamuni et al., 2019), then 0.5 gram of wet weight biomass was suspended in 10 ml of 1mM AgNO3 solution, pH 7. Both experiments were incubated at 30°C/48 h, either under direct light provided from fluorescent white lamps or in dark conditions provided through wrapping the tubes with aluminum foil. As a control, the AgNO3 solution, supernatant and 0.5 gram of wet weight biomass suspended in 10 ml of sterile water were incubated under the same conditions.

Characterization of Ag-NPs
To apply some characterization tests, the dried nanoparticles are used. To prepare the dried Ag-NPs synthesized by isolated cyanobacteria, the Ag-NPs suspension obtained after incubation for 72 h was centrifuged at 6000 rpm for 15 min at 10°C. The pellet was washed 3 times with sterile distilled water, spread in Petri plates and dried at 30°C for 24 h. The dried nanoparticles were scraped by a scalpel, harvested, weighted and stored in sterile microtube for quantitative estimation (Hamida et al., 2020).
The following characterization tests were applied to confirm the formation of Ag-NPs by isolated cyanobacteria.

Visual Color Change Test
Formation of silver nanoparticles was detected by the visual color change from pale yellow to brownish color within the incubation time.

Ultraviolet -Visible (UV-Vis) spectroscopic analysis
In time intervals of 1 and 24 h, 1 ml from each sample was centrifuged at 6000 rpm for 5 min. The absorption measurements in the wavelength range of 300-550 nm were estimated using UV-Vis spectroscopy (Specord 210 plus, Analytic Jena, Germany). The isolates showed an absorption peak in the range between 400 and 450 nm were considered as silver nanoparticle-producing cyanobacteria (Sadowski, 2010).

Scanning electron microscopy (SEM)
After 48h, SEM was used to characterize the surface morphology of nanoparticles at an accelerating voltage of 30 kV. The Ag-NPs solution was centrifuged, and the pellet was allowed to dry. The dried nanoparticles were examined through coated with gold by a coater to prevent building-up the electrical charges (Keskin et al., 2016). Also, Ag-NPs were characterized in a particle suspension.

Transmission electron microscopy (TEM)
TEM gives 1,000 fold higher morphological resolution for both size and shape compared to SEM (Sadowski, 2010). Two-dimensional and high-resolution Ag-NPs images were captured with TEM, Jeol, JEM-1400, Japan. A drop of Ag-NPs suspension was placed on copper-grid carbon coated with 300 mesh palladium and carbon, and allowed to dry. The Ag-NPs morphology was observed by TEM processed at an operating voltage of 80 kV.

X-ray diffraction (XRD) analysis.
The crystal structure of Ag-NPs was characterized using "x-ray-dx pert pro panalytical diffractometer". The powdered Ag-NPs were penetrated by X-rays and scanned across the region of 2θ, from range 0° to 80°.

Fourier transform infrared spectrometer (FT-IR) analysis
The chemistry and variations of functional groups attached to Ag-NPs surface were identified with FT-IR spectrometer (NICOLET 380,China). The dried nanoparticles were mixed with potassium bromide in a ratio of 1:100. The sample of 100 μl was placed in the attenuated total reflectance (BRUKER) analyzer. The silver nanoparticle solution was analyzed by ATR-FT-IR. The IR ray's spectrum is scanned across 4000 -400/cm with diffuse reflectance mode (DRS-800) within 4cm -1 resolution (Sadowski, 2010) (https://www IR Spectrum Table & Chart, Sigma-Aldrich).

Antibacterial efficiency of Ag-NPs
The antibacterial activity of Ag-NPs synthesized by isolated cyanobacteria was assessed applying well diffusion assay (Jena et al., 2013) against 6 bacterial strains including G + and Gbacteria. These bacterial strains were Bacillus cereus ATCC 10876, Methicillin resistant Staphylococcus aureus (MRSA) ATCC 43300, Micrococcus luteus ATCC 10240, Pseudomonas aeruginosa ATCC 9027, Salmonella typhimurium ATCC 14028 and Escherichia coli O157:H7 wild type strain 93111 as a representative enterohaemorrhagic E. coli (EHEC). The broth culture of each bacterial strain was prepared in soyabean casein digest broth (OXOID) with incubation at the optimum temperature/24h. The optical density (OD600) of each culture was adjusted to 0.5±0.1 using the UV/Vis spectrophotometer. Using sterile cotton swabs, each individual culture was spread on the surface of nutrient agar, in which the wells of 10 mm diameter were prepared using a sterile cork borer. Each well was filled with 100 μl either from vancomycin hydrochloride (100 mg/ml) as a positive control, 100 μl Ag-NPs resulting solution (1 mM) or AgNO3 solution (1 mM) or. The plates were incubated for 24 h at the optimum temperature for each microbe. The inhibitory effect of Ag-NPs was estimated through measuring the inhibition zone diameter. Also, the relative inhibition percentage was calculated according to the next formula (Kumar et al., 2010): Relative percentage inhibition of Ag-NPs = (100*(y-x))/z-y as: x: total inhibition area of Ag-NPs; y: total inhibition area of AgNO3 solution 1mM and z: total inhibition area of the standard drug.

Antioxidant activity of Ag-NPs
The capability of cyanobacterial Ag-NPs to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals was assessed through mixing 100 μl of tested Ag-NPs resulting solution with 1 ml from methanolic solution of 0.1 mM DPPH. The mixture was vortexed and incubated at 37°C for 30 min in dark. The absorbance of the samples was measured at 515 nm. The free radical scavenging activity was calculated using the following equation: DPPH scavenging activity (%) (antioxidant activity, %) = ((A0-A1)/A0)*100 where: A0 is the absorbance of the control reaction and A1 is the absorbance of reaction mixture (Hamouda et al., 2017).

Cyanobacteria identification by Light microscope examination
Two isolates of cyanobacteria (AM5E and AM6E) were purified and characterized morphologically according to Rippka et al. (1979). The AM5E and AM6E isolates were characterized as filamentous and non heterocystous cyanobacteria ( Fig. 1A -1D). The filaments were unbranched. No gas vacuoles were observed within the cells. The filaments of AM5E were long, thin, straight or slightly curvy, and non-constructed. Also, they were motile and had sharp apical terminals. The filaments may exist in a separate state or in bundles. The filaments of AM6E isolate were thicker, and exist as separate filaments ( Fig. 1C and D). The filaments were slightly motile and had round ends.

Molecular identification and phylogenetic analysis of cyanobacterial isolates
According to 16S rRNA gene sequence of the two isolates, AM5E showed 100% similarity to Desertifilum tharense and Desertifilum dzianese, while AM6E showed 99.6% similarity to Phormidium ambiguum. The phylogenetic relatedness was confirmed in the neighbor joining tree (Fig. 2). Sequences were deposited in the NCBI GenBank as Desertifilum tharense AM5E (accession number: MW762709) and Phormidium ambiguum AM6E (accession number: MW762710).

Characterization of biosynthesized silver nanoparticles (Ag-NPs) by cyanobacteria
Generally, silver nanoparticles are synthesized through two pathways comprising enzymatic and non-enzymatic reduction (Otari et al., 2015). The nitrate reductase enzyme, which involves in enzymatic synthesis of silver nanoparticles, converts nitrate to nitrite and an electron shuttle is induced thus reducing the silver ions to silver nanoparticles. The enzymatic production of silver nanoparticles is fast and non-toxic approach. Conversely, the non-enzymatic synthesis of Ag-NPs depends on chemical reduction of silver atoms using reducing and stabilizing compounds produced by plants or microorganisms.

Visual color change
Reduction of silver atoms into silver nanoparticles extra-and intracellularly using isolated cyanobacteria could be observed visually through the color change from pale yellow to brown. Generally, this process is time dependent as the intensity of brown color is directly proportional to reaction time (Khan et al., 2019). Saifuddin et al. (2009) reported that the changing in color was due to the excitation of surface plasmon vibrations in the resultant nanoparticle. The Ag-NPs produced by cyanobacteria were observed to be highly stable for a variant time ranged from 1to120 h (Govindaraju et al., 2009).
In this study, the brown color was observed after 1hr, either for Desertifilum tharense (Fig. 3) or Phormidium ambiguum (Fig. 4) indicating their ability to synthesize Ag-NPs extra-and intracellularly under both light and dark conditions. For the both isolates, after 48 hours, the darkest brown color was perceived under light conditions for extra-and intracellularly produced Ag-NPs. Comparing the intra-and extracellular biosynthesis of Ag-NPs, the dark brown color was evidence for promising extracellular biosynthesis by Desertifilum tharense or Phormidium ambiguum. Therefore, further characterization testes, antibacterial and antioxidant activity were assessed for Ag-NPs synthesized extracellularly under light conditions.

UV-Vis spectrophotometer analysis
Formation of Ag-NPs detected by visual observation of color change was further confirmed by sharp peaks given in the visible region from UV-Vis spectrum. Ag-NPs   characterization using UV-visible spectrophotometer is considered the most widely technique (Sun et al., 2001;Gowramma et al., 2015). In this study, UV-Vis spectrophotometer analyses were carried out at absorbance spectrum range of 300 -550 nm for Ag-NPs synthesized extracellularly under light conditions. For Desertifilum tharense, Ag-NPs gave the sharp SPR (surface plasmon resonance) peak at 450 nm after one hour (Fig. 5A). With increasing reaction time reached 24h, the peak shifted to shorter wavelength region as two peaks appeared at 410 and 435 nm (Fig. 5B). Kathiraven et al. (2014) reported that when SPR band moves towards shorter wavelength that means a decrease in particle size. This phenomenon was not observed for Ag-NPs synthesized by Phormidium ambiguum as the SPR peak appeared at 450 nm after 1 and 24 hours ( Fig. 6A and 6B).  It was known that Ag-NPs give the plasmon resonance peak in absorption spectrum between 410 -450 nm (Kashyap et al., 2019). Consequently, the results confirmed successful extracellular Ag-NPs synthesis by Desertifilum tharense and Phormidium ambiguum under light conditions.

Scanning and Transmission electron microscopy
Scanning electron microscopy (SEM) micrographs indicated the presence of Ag-NPs produced by Desertifilum tharense (Fig. 7A and 7B) and Phormidium ambiguum ( Fig. 8A and 8B) as irregular polydisperse clusters of particles.
The Ag-NPs were further characterized for shape and size determination by transmission electron microscopy (TEM). The TEM micrographs (7C) and (8C) showed spherical shape of Ag-NPs produced by Desertifilum tharense and Phormidium ambiguum,  respectively with good particles dispersal without agglomeration. Also, TEM micrographs displayed the size of particles in a range of 6.24 -11.7 nm and 6.46 -12.2 nm for Ag-NPs produced by Desertifilum tharense and Phormidium ambiguum, correspondingly. The SPR peak at 435 nm of Ag-NPs synthesized by Desertifilum tharense confirmed the spherical shape of these particles as reported by Govindaraju et al. (2009). Generally, the cyanobacterial silver nanoparticles are found in different shapes. Patel et al. (2015) studied the shape of Ag-NPs produced by different cyanobacterial strains. They found that the Ag-NPs produced by Anabaena sp. and Limnothrix sp. appeared as elongated particles, while Ag-NPs of Coelastrum sp. and Botryococcus braunii appeared with spherical shape. The particles formed by Synechocystis sp. were irregular clusters.
The size of Ag-NPs produced by Desertifilum tharense and Phormidium ambiguum was in a range reported by Singh et al. (2014). In other studies, the size of particles produced by cyanobacteria was larger. Rashed et al. (2018) produced Ag-NPs with an average size of 60 nm from Convolvulus arvensis extract. The average size of Ag-NPs from Chlorella sp. was 90.6 nm (Kashyap et al., 2019). Some cyanobacterial cultures synthesized silver particles with a size larger than 100 nm. Keskin et al. (2016) and Kashyap et al. (2019) produced particles with average size of 140 nm from Synechococcus sp., and size of 136.2 and 241.8 nm from Scenedesmus vacuolatus and Lyngbya putealis, respectively.

X-ray diffraction (XRD) analysis
X-ray diffraction analysis (XRD) is a technique used to determine the crystallographic structure of a material through irradiating it with X-rays and measuring the intensities and A C B B C A scattering angles of the X-rays that leave the material. XRD pattern was investigated in the range of 0° to 80° at diffraction angle of Pos (°2Th.).
it was noticed that there are additional peaks at 54.82° and 57.5° in the diffractogram of Desertifilum tharense Ag-NPs, and at 54.78° and 57.47° in that of Phormidium ambiguum Ag-NPs. These peaks could be attributed to the presence of unreduced AgNO3 as stated by Mehta et al., (2017).

EDX analysis
The purity, elemental constituents and relative abundance of Ag-NPs synthesized by Desertifilum tharense and Phormidium ambiguum were analyzed by means of Energy Dispersive X-ray (EDX) as presented in Figures (10A) and (10B), respectively.
XRD and EDX results clearly confirm the successful synthesis of Ag-NPs in its oxide form or chloride form.

Transform Infrared Spectroscopy (FTIR)
The structure of organic compounds on the surface of AgNPs was investigated using FTIR Spectroscopy.
The FTIR spectrum of Ag-NPs synthesized by Desertifilum tharense and Phormidium ambiguum specified peaks around 3450, 2065, 1634 and 428 cm -1 (Fig. 11). Strong absorption peak at 3450 cm -1 assigned to O-H stretching vibration of polysaccharides, and N-H stretching vibration of proteins. This indicates the polysaccharides and proteins found in extracellular extract may play role in synthesis of Ag-NPs (Hamida et al., 2020;Mahiuddin et al., 2020). Also, the polysaccharides and proteins, as organic capping agents, may contribute in particles stabilization. Jena et al. (2013) reported that the capping peptide can bind to nanoparticles by free amino groups or by different residues inside the proteins. The peak around 1634 cm -1 attributed to stretching vibration of C=O in amides (Mahiuddin et al., 2020). The peak at 428 cm -1 is related to binding Ag-NPs with oxygen from hydroxyl groups (AL-Katib et al., 2015).

Antibacterial activity
Currently, using nanoparticles as antimicrobial agents are gaining excessive interest. Generally, the nanoparticles are evaluated as antimicrobials alone or in combination with antibiotics. The nanoparticles can increase antimicrobial effect of some antibiotics against G+ and G-bacteria (Li et al., 2011).
In this study, 6 bacterial pathogens were selected as indicator microorganisms for screening the antibacterial activity of Ag-NPs synthesized extracellularly under light conditions by the two isolated cyanobacteria. The results were compared with vancomycin hydrochloride (100 mg/ml) and AgNO3 (1 mM) for evaluating the relative percentage inhibition of Ag-NPs.
For Phormidium ambiguum Ag-NPs, the inhibition zone diameter was in a range of 9 -18 mm. Maximum relative percentage inhibition of 86.6% was displayed against Salmonella typhimureum ATCC 14028, followed by E. coli O157:H7 wild type strain 93111 (81.7%) and P. aeruginosa ATCC 9027 (75.1%). The antibacterial effect of silver nanoparticles is size, charge and shape dependent. The great antibacterial activity of Desertifilum tharense Ag-NPs against tested pathogenic bacteria could be attributed to their particle size (6.24 -11.7 nm) which was smaller than that of Phormidium ambiguum Ag-NPs (6.46 -12.2 nm). This is in accord with the findings of Ivask et al. (2014) Patel et al. (2015 who found that the antibacterial activity of Ag-NPs is inversely proportional with their particle size as smaller size means larger surface area which facilitates the interaction between nanoparticles and microbial cells. The particles with size of 10 nm or smaller can easily penetrate the bacterial cell wall and interact with the cytosol biomolecules. It was known that the antibacterial activity of Ag-NPs is not attributed only to the small size but also to their positive charge. Generally, positively charged particles electrostatically bind to lipopolysaccharides on the outer membrane of Gbacteria or lipoteichoic acids on the surfaces of G + bacteria which facilitate the cell penetration (Glinel et al., 2012). Rajeshkumar et al. (2014) and Otari et al. (2015) found that spherical shape with small size of Ag-NPs increase the contact area that ensuring the elimination of bacterial growth.
In general, the mode of action of Ag-NPs as antibacterial mainly includes loss the outer membrane integrity in Gbacteria, and permeabilization of the bacteria cell membrane through formation of pores across it resulting in releasing the cellular material and causing cell death ( (Raffi et al., 2008 andDehkordi et al., 2011). On the other hand, the pores formed in the cell membrane facilitate the Ag-NPs inflowing into the cell to combine with proteins containing sulfur and DNA causing DNA and enzymes damage, and leading to blocking vital metabolic processes (Hamouda et al., 2019).

Free radical scavenging potential of Ag-NPs
The results confirmed the highest scavenging activity of cyanobacterial Ag-NPs comparing with cyanobacterial extracellular extract. Silver nanoparticles of Phormidium ambiguum exhibited the highest scavenging activity of 48.7% comparing with that of Desertifilum tharense which displayed 43.753% (Table 2).

Conclusion
In this study, Egyptian cyanobacterial isolates of Desertifilum tharense and Phormidium ambiguum are found to be potent for the green synthesis of Ag-NPs extra-and intracellularly under light and dark conditions. They can produce smaller spherical particles with face-centered cubic structures. Presence of amides and hydroxyl groups indicate that proteins and polysaccharides could be considered as important factors in the Ag-NPs biosynthesis. Generally, the characterization assay showed that a Novel Desertifilum tharense cyanobacteria have superior power in green synthesis of AgNPs. The current findings indicate that Ag-NPs may be potent antibacterial agents against different pathogenic bacteria and could be used as alternatives to antibiotics. Further studies are recommended to define the optimal conditions for Ag-NPs biosynthesis. Also, more biological characterizations and in vivo experiments are required to establish the real potential for their application in medical and food sectors.

Availability of data and materials:
The data supporting this article are available in our Figures and Tables. The data sets analyzed in the present study are available from the corresponding author upon reasonable request. Ethics approval and consent to participate: Not applicable. The manuscript does not involve any animals, humans, human data, human tissue or plants.

Consent for publication:
Not applicable. The manuscript does not contain any individual person's data in any form.

Funding:
The authors didn't have any financial support. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. This research is a part of requirements for obtaining Ph.D. degree.