Antioxidant and Antibacterial Efficacy of Silver Nanoparticles Synthesized from Extract of Filamentous Cyanobacterium Scytonema sp

Contemporary algal based metallic nanoparticles with magnified biological potential have gained considerable attention. In the present study, silver nanoparticles (AgNPs) were synthesised by using cell-free extracts of a filamentous cyanobacterium Scytonema sp., isolated from the rooftop of Acharya Narendra Deo Kisan P.G. College, Babhnan, Gonda. The Scytonema sp. was harvested during the late exponential growth phase to advance the efficiency of nanoparticle synthesis. The distinct change in color and the emergence of a specific surface plasmon resonance (SPR) peak at 410 nm in the UV-visible spectrum was evidenced of AgNPs synthesis after 60 h of incubation. The antioxidant potential of AgNPs, including DPPH and ABTS radical scavenging methods, revealed quite similar pattern and significantly higher activity (about two folds higher than that of crude extract at 80 μm/mL concentration). Furthermore, the AgNPs exhibited pronounced, dose-dependent antibacterial activity against Escherichia coli (E. coli). The findings of present study point up the Scytonema sp. as an effective natural resource for the green synthesis of AgNPs with promising antioxidant and antibacterial utilization in biomedical and ecological spheres.

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DPPH radical scavenging (%): 20 vs 40 vs 80Close ×

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Introduction

Nanoparticles are materials that range from 1 to 100 nm in size. Recently, metallic nanoparticles have attracted considerable attention due to their wide range of applications in diverse fields (Pathak et al., 2019a, 2019b; Yiblet and Mintessnot; 2025) [15,16,26] . Despite novel physicochemical properties, metallic nanoparticles may pose potential health hazards to living organisms, including humans, especially when synthesized using conventional chemical and physical methods. Therefore, the growing concerns within the scientific community have led to the development of non-toxic, ecofriendly biosynthetic approaches that employ renewable resources and require lower energy input (Pathak et al., 2019a, 2019b; Bhardwaj et al., 2024) [15,16,4] . Among these, green-synthesized silver nanoparticles (AgNPs) have emerged as potent agents exhibiting antimicrobial, antifungal, antibacterial, anticancer, antioxidant, and anti-inflammatory activities (Bhardwaj and Naraian; 2021; Takci et al., 2023; Li et al., 2024) [3,24,9] . Cyanobacteria are a better biological candidate for nanoparticles synthesis because of having enhanced growth rate and biomass productivity (Pathak et al., 2019a, 2019b; Bhardwaj and Naraian; 2021) [15,16,3] . Use of cyanobacterial extracts for the nanoparticles biosynthesis is being widely practiced these days, as the extraction of nanoparticles formed inside the cell is a very costly and complex process. The cell extracts of the cyanobacterium Anabaena doliolum were utilized for the synthesis of AgNPs (Singh et al., 2014) [21] . Husain et al. investigated the biosynthesis of AgNPs from 30 different cyanobacterial strains (Husain et al., 2015) [7] . Sonker et al. (Sonker et al., 2017) [23] synthesized AgNPs utilizing the cell extract of the cyanobacterium Nostoc sp. strain HKAR-2 isolated from hot springs. Genus Scytonema has tremendous potential for nitrogen fixation and rich source of various biologically active compounds such as phycobilins, antibiotics, mycosporine-like amino acids (MAAs), scytonemin, toxins, etc. (Nowruzi et al., 2020) [11] . During the synthesis of AgNPs, these compounds act as reducing and stabilizing agents. In the present investigation, we used the cell extract of the rooftop-dwelling cyanobacterium Scytonema sp. for the synthesis of AgNPs. We also attempted to screen the in vitro antioxidant and antibacterial activity of the biosynthesized AgNPs.

Materials and methods

Materials and methods

Collection of Scytonema sp. and growth condition

The cyanobacterium Scytonema sp. was collected and purified from rooftop of Acharya Narendra Deo Kisan P.G College, Babhnan, Gonda and grown in BG-11 -medium (without nitrogen sources) (Rippka et al., 1979) [19] under axenic conditions, at temperature 26 ± 2 °C, light intensity 40-50 µmol photons m⁻² s⁻¹ and photoperiod 14:10 h (light/dark cycle).

Growth measurement

Growth of cyanobacteria was monitored by determining the changes in chlorophyll a (Chl a) content after desired time intervals. The Chl a was extracted in methanol. The recovered cyanobacterial sample was kept in 100% methanol overnight at 4 °C and its content was determined by method reported by Porra (Porra., 2002) [17] after observing absorption at 665.2 nm and 652 nm.

Chl a (μg/ml) = 16.29*A665.2 -8.54*A652 Where, A is the optical density (O.D.) at 665.2 and 652 nm, respectively.

Cell extract preparation and green synthesis of AgNPs

Cells of Scytonema sp. were harvested during the late exponential (log) phase. The collected cells were centrifuged (Remi mini centrifuge RM-02) at 6000 rpm for 15 minutes and the pellet was washed with double distilled water (DDW) and sonicated using ultrasonic vibration at 30% amplitude for 15 minutes. The homogenate was exposed to centrifugation (3 times), the supernatant was diluted through a series of dilutions with 1 mM AgNO3, and the reaction mixtures (10 mL) were incubated at 37 0 C for 60 h. Only the sample containing Scytonema cell extract served as the positive control. The formation of AgNPs was monitored visually at an interval of 12 h by observing the change in color of the reaction mixture.

Partial characterization of AgNPs by UV-VIS spectroscopy

The absorption spectrum was taken at different time interval (0, 12, 24, 36, 48, and 60 h) by using UV-Vis spectrophotometer (Shimadzu) with the resolution of 1 nm at the wavelength range of 200-800 nm, possessing a scanning speed of 200 nm/minutes.

2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay

Free radical scavenging activity of desired concentrations (e.g., 20, 40 and 80 µg/mL) of Scytonema sp. extract and biosynthesized AgNPs was measured using a conventional DPPH radical scavenging assay (Shen et al., 2010) [20] .

2,2′azino-bis(3-ethylbenzothiazoline)-6-sulfonate (ABTS) radical scavenging assay

The antioxidant activity of Scytonema sp. extract and biosynthesized AgNPs of desired concentrations (20, 40 and 80 µg/mL) was evaluated using the ABTS radical scavenging assay following standard procedures provided by Re et al. (Re et al., 1999) [18] with minor modifications. The reduction of ABTS•⁺ was monitored spectrophotometrically at 734 nm, and scavenging activity was expressed as percentage inhibition.

Antibacterial activity

Antibacterial activity was assessed by the disc diffusion method against Gram negative bacterium E. coli following standard method with slight modification (Bauer et al., 1966) [9] . E. coli was grown in Muller Hinton Agar (MHA) medium at 37 °C. The desired concentration (05 μg/mL and 15 μg/mL) of AgNPs solution was soaked into separate discs on the Petri plates. Ampicillin served as positive control and double distilled water (DDW) was taken as negative control. After 24 h of incubation, the inhibition zones around the discs were measured.

Statistical analysis

The results express the mean values of three replicates of each experiment. One-way analysis of variance was performed for statistical analyses.

Results and Discussion

Growth profile of Scytonema sp. The growth pattern of Scytonema sp., monitored by evaluating Chl a content at regular intervals, showed a typical sigmoidal pattern with lag, exponential, and stationary phases (Fig. 1). The late exponential phase is often associated with enhanced production and release of extracellular biomolecules (Singh et al., 2014, San Keskin et al., 2016) [21,8] . These compounds are useful in biotechnological applications, including the biological synthesis of nanoparticles.

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Growth profile (In vitro) of Scytonema sp. estimated by Chl-a content.

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Partial characterization of AgNPs

The initiation of reduction of AgNO3 solution into AgNPs started after 12 h of the addition of 1 mM AgNO3 solution into the cell extract and completed after 60 h of incubation. The visual color change to dark red, and time-dependent increase in surface plasmon resonance (SPR) intensity at 410 nm confirm the successful extracellular biosynthesis of stable silver nanoparticles by Scytonema sp. extract (Harutyunyan et al., 2025) [5] (Fig. 2).

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UV-VIS absorption spectrum of Scytonema sp. extract based AgNPs at 410 nm after 60 h of incubation.

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DPPH and ABTS assay

The results of antioxidant assay showed a clear concentration-dependent increase in radical scavenging activity of the AgNPs in both (DPPH and ABTS) assays (Table 1), indicating their effective antioxidant capacity. The free radical scavenging activity in DPPH and ABTS assay follow quite similar pattern and was about two folds higher than that of crude extract at 80 μm/mL concentration, though slightly lower than that of ascorbic acid at equivalent concentrations. This observation is consistent with previous reports (Bedlovičová et al., 2020) [2] . [22,14] [12,6,25,13,10] .

Sr. No.	Sample	Concentration(µg mL⁻¹)	DPPH radical scavenging (%)	ABTS radical scavenging (%)
1.	Cell extract of Scytonema sp.	20	13.1 ± 1.4	16.2 ± 2.1
		40	23.6 ± 1.1	27.2 ± 1.2
		80	37.2 ± 1.2	40.1 ± 1.9
2.	AgNPs	20	32.8 ± 1.3	29.2 ± 2.1
		40	51.0 ± 1.0	55.3 ± 1.1
		80	72.1 ± 1.2	72.4 ± 2.0
3.	Ascorbic acid(standard)	20	46.1 ± 1.9	43.2 ± 1.0
		40	63.6 ± 1.2	67.0 ± 1.4
		80	82.3 ± 1.7	79.1 ± 2.3

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In vitro antioxidant activity of biosynthesized AgNPs evaluated by DPPH and ABTS radical scavenging assays at varying

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Conclusion

The present study explores a sustainable step towards the green synthesis of AgNPs using cell-free extracts of the rooftop dwelling cyanobacterium Scytonema sp. The harvesting of Scytonema sp. at late exponential phase enabled effective nanoparticle synthesis and was confirmed by a characteristic SPR peak at 410 nm. The results of antioxidant and antibacterial efficacy of AgNPs provide valuable insight and highlight its potential in the field of pharmaceuticals. These findings explore Scytonema sp. as an eco-friendly natural resource for the synthesis of versatile molecule AgNPs.

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Zone of inhibition exhibited by AgNPs biosynthesized from

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