ournal of applied physics nanotechnology · citation: suparna roy, anantharaman p (). biosynthesis...

Journal of Applied Physics & Nanotechnology

www.scientonline.org J Appl Phys NanotechnolVolume 1 • Issue 1 • 002

Research Article

Biosynthesis of Silver Nanoparticles by Sargassum Ilicifolium (Turner) C. Agardh with their Antimicrobial Activity and Potential for

Seed Germination

Suparna Roy* and Anantharaman PCentre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, Tamilnadu, India

IntroductionNanoparticles are peculiar in their physical and chemical properties in comparison

to bulk compounds. So, Nano scale research gain the attention in the modern field of material science and research groups. In various fields, the novel applications of Nanoparticles are gradually improving and became as emerging needs. But chemical originated silver Nanoparticles had been reported for its toxic nature on the vegetation. So, to know the toxic level and mechanism, chemical originated silver Nanoparticles had been investigated for its effect on various seed germination such as Wang, et al. (2015) [1] reported that chemically originated silver Nanoparticles and Silver Nitrate (AgNO3) had a significant effect on seed germination of Radish (Raphanus sativus L.). The effect of chemical Fe and Ag Nanoparticles on seed germination had been evaluated for flax (Linum usitatissimum L, cv. Electra), ryegrass (Lolium perenne L, cv. Tove), and two-rowed barley (Hordeum vulgare L, cv. Annabell) in combination of soil compositions by Sayed El-Temsah et al, and the effect of Ag Nanoparticles also had been reported to evaluate its effect on seed germination of Vigna radiata [2]. The effect of chemical originated nano silver on seed germination and seedling growth in fenugreek seeds was investigated by Hojjat [3].

The majority of previous reports showed that chemical originated Ag Nanoparticles had a toxic effect on seed germination and vegetation. So, eco-friendly and phyto-friendly biosynthesized silver Nanoparticles may negotiate and replace the toxic

*Corresponding Author: Suparna Roy, Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, Tamilnadu, India, Email: [email protected]

This article was published in the following Scient Open Access Journal:Journal of Applied Physics & NanotechnologyReceived December 30, 2017; Accepted January 10, 2018; Published January 25, 2018

AbstractChemical originated Nanoparticles are recognized for their utility in various fields such

as pharmaceuticals, medicine and also as bio-fertilizer, but Nanoparticles also had been reported for its toxic effect on the environment and human, so reasonably Nanoparticles are under safety concern. So, biosynthesis of silver Nanoparticles is now a leading interest in research with evaluation of their phyto-friendly nature. The aqueous extract of brown seaweed (Sargassum ilicifolium) had been used for the synthesis of silver Nanoparticles. The synthesized silver Nanoparticles were characterized using UV-visible spectroscopy, Fourier Transform Infrared (FT-IR) Spectroscopy and the morphological structure was characterized with Scanning Electron Microscopy. The antibacterial activity of green synthesized silver Nanoparticles was assayed against six human pathogenic bacteria which showed that biosynthesized silver Nanoparticles had high inhibitory activity against Escherichia coli (0.83 ± 0.05cm), Pseudomonas aeruginosa (0.53 ± 0.05 cm), Proteus mirabilis (0.46 ± 0.05cm) and Klebsiella pneumoniae (0.4 ± 0.1cm) in comparison to antibiotic Chloramphenicol (5mg/ml). The phyto-toxicity of biosynthesized silver Nanoparticles was investigated by studying its effect on seed germination of Abelmoschus esculentus and Raphanus sativus var. longipinnatus. The seed germination test showed that the biosynthesized silver Nanoparticles had quite good promoting effect on germination of both seeds. The seed germination was 60 percent in case of Abelmoschus esculentus and seed treated with water also had 60 percent seed germination but in case of Raphanus seed, the seed germination was 100 percent in comparison to normal water treated seeds which was 60 percent. This study clearly confirmed that biosynthesized silver Nanoparticles had accelerating effect on both seed germination and consequently indicated that biosynthesized silver Nanoparticles is environmentally safe and can be used as nano-bio-fertilizer after further long duration treatment with plant growth and crop yield.

Keywords: Seaweed, Silver nanoparticles, Seed germination, Antibacterial activity

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Citation: Suparna Roy, Anantharaman P (2018). Biosynthesis of Silver Nanoparticles by Sargassum Ilicifolium (Turner) C. Agardh with their Antimicrobial Activity and Potential for Seed Germination

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effect of chemical originated silver Nanoparticles. Seaweeds had been reported as raw material for green synthesis of silver Nanoparticles such as Caulerpa racemosa [4], Turbinaria conoides [5] and some seaweed was evaluated for their potential for seed germination [6-11] which revealed that seaweed synthesized silver Nanoparticles was not toxic to seed germination but it was promoting the seed germination in comparison to normal water treatment as well as seaweed extract and seaweed liquid fertilizer. Silver nanoparticles were also reported to their application in medicine to reduce the infection in burn treatment and prevent the adherence of dental bacterial colonization [12,13]. The seaweed synthesized silver Nanoparticles had been reported for their antibacterial activity such Padina tetrastromatica [14], and Turbinaria conoides [15].

Though, some previous research was done on antibacterial activity of seaweeds mediated silver Nanoparticles but many seaweed species are yet not explored in this aspect. Therefore, the focus of the present study is on antibacterial activity of biosynthesized silver Nanoparticles by Sargassum ilicifolium aqueous extract. So far we know, there are very few reports on the effect of biosynthesized silver Nanoparticles on seed germination, so we evaluated the potential of seaweed synthesized silver Nanoparticles on seed germination to prove its phyto-friendly nature and future use of bio-Nano-fertilizers.

Materials and MethodsSynthesis of silver nanoparticles

Seaweed extracts preparation: The seaweed (Figure 1) had been collected from Vadakkadu Rameshwaram, southeast coast of India and identified with CMFRI taxonomy key and other available taxonomic key. After repeatedly washed, the cleaned fresh seaweed was cut into small pieces and small piece of seaweed of 20 g was pasted as fine paste with mortar and pestle and the seaweed paste was dissolved in 100 ml of distilled water and boiled for 15 minutes at 60 ̊Cat water baths. The seaweed extract was kept at room temperature for cooling, and the cold seaweed extract was filtered with whatman filter paper and the extract was used for synthesis of silver Nanoparticles.

Synthesis of Ag-Nanoparticles: The aqueous 1mM solution of Silver Nitrate (AgNO3) was prepared with Silver nitrate (AgNO3) powder. For typical biosynthesis of Silver Nanoparticles, 10 ml of the aqueous extract of seaweed was added to the 90 ml aqueous solution of Silver Nitrate (AgNO3) in 250 ml conical flask and

kept at room temperature for 72 hours within mechanical shaker at 120 rpm with continuous shaking. The colour change of the solution indicated the biosynthesis of Silver Nanoparticles [16].

Preparation of Seaweed extract: The aqueous seaweed extract was prepared according to the extraction of seaweed liquid fertilizer (1:20) after an 90 minutes, of boiling, then seaweed solution was kept at room temperature for complete cooling and the cold seaweed solution was filtered with Whatman filter paper. The seaweed extract and seaweed liquid fertilizer was used to study its effect on seed germination of Abelmoschus esculentus and Raphanus sativus var. longipinnatus seed.

Characterization of Biosynthesized Silver NanoparticlesUV-Vis spectrophotometer

After 72 hours of biosynthesis, for characterization and confirmation of biosynthesis of silver Nanoparticles, the solution was scanned from 300-700 nm with UV-Vis Spectrophotometer (UV-2600 SHIMADZU).

Fourier Transform Infrared (FT-IR) spectroscopyThe biosynthesized silver Nanoparticles were centrifuged

at 5000 rpm for 30 minutes to precipitate the pellet at the bottom. The pellet was collected and air dried to make powder at room temperature for further use. The biosynthesized silver Nanoparticles powder was mixed with Potassium bromide (KBr) and made a pellet for use in Perkin Elmer FTIR model 2000 to identify the functional groups which were responsible for the green synthesis of silver Nanoparticles.

Scanning Electron Microscopy The dry powder of biosynthesized silver Nanoparticles was

sprayed on gold coated copper grid to make the thin film on grid and images were taken with JEOL JSM-5610LV Scanning Electron Microscope for morphological identification of biosynthesized silver Nanoparticles.

Antimicrobial activityAntibacterial activity of biosynthesized silver Nanoparticles

using an aqueous extract of Sargassum ilicifolium was assayed by the agar disc diffusion method against six human pathogenic bacteria such as Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Enterococcus faecalis and Proteus mirabilis which were collected from the Department of Medical Microbiology, Raja Muthiah Medical College, Annamalai University. The bacterial cultures were freshly prepared in nutrient broth, which was sub-cultured from pure culture. After 24 hours of culture, each bacterial culture was inoculated into the agar plates and kept for 24 hours. The market available Chloramphenicol antibiotic was used as positive control. The 500mg powder Chloramphenicol was dissolved in 100 ml autoclaved distilled water to make a concentration of 5 mg/ml. The 1 mm Silver Nitrate (AgNo3) solution was used as negative control. The 20µl of seaweed synthesized silver Nanoparticles solution, the solution of Silver Nitrate and antibiotic were given to sterile paper discs and the discs were placed on bacterial plates. After 24 hours of incubation, the zones of inhibition were measured in triplicates from three different plates.

Figure 1. Sargassum ilicifolium (Turner) C. Agardh, Division - Phaeophyta, Class - Phaeophyceae Family - Sargassaceae (09°19’.700’’N and 079°19’.072’’E).





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Seed germination testThe seeds of Abelmoschus esculentus and Raphanus sativus

var. longipinnatus were sterilized with 5% Sodium hypochlorite solution for 15 minutes [17]. The sterile seeds were dipped in biosynthesized silver Nanoparticles solution, normal water, 1 mm AgNO3 solution, seaweed extract, and seaweed bio-fertilizer for overnight. In sterile Petri plates, sterile filter papers were placed and 5 ml silver Nanoparticles solution, normal water, 1 mm AgNO3 solution seaweed extract and seaweed liquid bio-fertilizer were added to the filter paper, and then treated seeds were placed on filter paper within Petri plates. Then, Petri plates were covered and kept at room temperature. After 12 hours germination halted and the germination percentage, mean germination time, germination index, relative root elongation, relative seed germination and germination rate were estimated. Germination parameters were calculated using the following equations [11,18-20].

Relative root elongation (E) = (Mean root length with NPs)/ (Mean root length with control) ×100

Relative seed germination= (Seeds germinated with NPs)/ (Seeds germinated with control) ×100

Germination index (GI) = (Relative seed germination) × (Relative root elongation)/100

Germination Percentage (GP %) = (Gf/n) × 100 where Gf is the total number of germinated seeds at the end of the experiment and n are the total number of the seed used in the test.

Mean Germination Time (MGT) =ΣNiDi/n where Ni is the number of germinated seeds until the ith day and Di is number of days from the start of experiment until the ith counting and n is the total number of germinated seeds.

Germination Rate (GR) = Σ Ni/ Σ Ti Ni, where Ni is the number of newly germinated seeds at time Ti.

GR = (a/1) + (b-a/2) + (c-b/3) +.....+ (n-n-1/N) (6)

Statistical AnalysisMeans and standard deviation was estimated from the triplicates

measurement of zone of inhibition for antibacterial activity.

Results & DiscussionsSynthesis of silver nanoparticles

The mixing of seaweed aqueous solution with Silver Nitrate (1mM) solution produced reddish brown colour in comparison to control, the Silver Nitrate solution (AgNO3) and the aqueous seaweed solution which suggested the formation of Ag-NPs by reduction of the aqueous Ag+ (Figure 2). Due to the surface Plasmon vibrations among the produced Silver Nanoparticles, the colour change occurred [21].

Characterization of Biosynthesized Silver NanoparticlesUV-Visible spectrophotometer

The biosynthesized Silver Nanoparticles was scanned from 200nm to 700nm.The absorbance peak at 440 to 460 nm indicated the biosynthesized of Silver Nanoparticles (Figure 3).

The only aqueous extract which was used for synthesis of silver Nanoparticles also analysed for FT-IR to identify the functional groups which was present in water extract of the mentioned seaweed which was listed below table 1 and the spectrum is represented at figure 4. The functional groups which are responsible for the synthesis of silver Nanoparticles by a reduction of 1 mm AgNO3 with the aqueous extract of the mentioned seaweed was listed below table 2 and the spectrum is represented at figure 5.

The SEM image (Figure 6) showed the high density, spherical, cubical and hexagonal shaped and well distributed Ag-NPs synthesized from seaweed extract.

Figure 2. Representing (A) the aqueous solution of seaweed extract and (B) Biosynthesized Silver Nanoparticles solution.

Figure 3. Representing the UV-visible spectrum of biosynthesized Silver Nanoparticles by aqueous extract of Sargassum ilicifolium.

Peaks Bonds Functional Groups3649.52 N-H Stretch , C-H Stretch Amide, Alkane2910.13 O-H Stretch, Carboxylic Acid O-H Carboxylic1113.95 C-O Stretch, Ester, Ether538.99 C-Br, C-I Stretch Alkyl halides514.07 C-Br, C-I Stretch Alkyl halides

Table 1. FT-IR peak values of the aqueous extract of Sargassum ilicifolium.




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Effect of biosynthesized Silver Nanoparticles, seaweed extract and seaweed liquid bio-fertilizer on seed ger-mination of Abelmoschus esculentus

The figures 7a-7d showed the effect of biosynthesized Silver Nanoparticles on seed germination of Abelmoschus esculentus.

The relative root elongation (500) and relative seed germination (150) was the highest at 48 hours, for the seeds treated with biosynthesized silver Nanoparticles but germination index which was 750, the highest germination index for the seeds treated with biosynthesized Ag-Nanoparticles at 48 hours of treatment (Figure 7a). The effect of biosynthesized Silver Nanopaerticles and normal water on the seed germination of Abelmoschus esculentus was the same. The maximum germination rate was found at 24 hours for both treatments such as biosynthesized Silver Nanoparticles (SL-AgNPs) treated seeds (0.076) and normal water treated seeds (0.076) and the germination rate was gradually decreased with increased of time (Figure 7d). The mean germination time was gradually increased with increase of time for both seeds which may be due to more effect and reaction of biosynthesized Silver Nanoparticles (Figure 7c). The seed germination percentage of normal water treated seeds were comparatively higher than biosynthesized

Figure 4. Representing the FT-IR spectrum of aqueous extract of Sargassum ilicifolium.

Figure 5. FT-IR spectrum of biosynthesized silver Nanoparticles synthesized by Sargassum ilicifolium aqueous extract.

Peaks Bonds Functional Groups481.04 C-I, C-Br Stretch Alkyl halides

1019.12 C-F Stretch, C-O Bend Alkyl halides, Ether 3448.15 O-H Stretch, H-Bond, N-H Alcohol, Amine, Amide 2920.20 O-H Stretch, Carboxylic Acid O-H Carboxylic2849.30 C-H Bend, C-H Stretch Aldehydes, Alkyl1632.74 C=C Stretch Alkenyl1382.55 N-O Bend, C-F Stretch Nitro , Alkyl halide

Table 2. FT-IR peak values of Sargassum ilicifolium mediated biosynthesized Silver Nanoparticles.





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Ag-Nanoparticles treated seeds at 24 and 96 hours; but the seed germination percentage was equal for both seeds treated with water and biosynthesized Ag-Nanoparticles at 48 hours (Figure 7b). The two types of seaweed extracts such as normal seaweed extract and the seaweed liquid bio-fertilizer had no effect on seed germination of Abelmoschus esculentus. The 1 mm solution of AgNO3 was also used as a negative control to study its effect on seed germination in comparison to normal water, but only 20 percent seed germination occur in case of AgNO3 solution and 60

percent seed germination occurred in the case of seeds treated with normal water (Figure 8). This result proved that 1mM AgNO3 solution was toxic to seed germination of Abelmoschus esculentus. The only one seed of Abelmoschus esculentus was germinated (Figures 8a and 8b). The anatomical study was done for the only one germinated seed which was treated with 1mm AgNO3 revealed that the root tip with root cap was chocked and damaged due to treatment of 1 mm AgNO3 solution, but in case of normal water treatment, the germinated seeds had the normal root with

Figure 6. Representing the SEM images of biosynthesized silver Nanoparticles synthesised by Sargassum ilicifolium aqueous extract.

a b

c d

Figure 7. (a) Seed germination index, (b) Seed germination percentage, (c) Mean seed germination time, (d) Seed germination rate.



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fair root cap and shoot growth was comparatively good. But the normal seaweed aqueous extract and seaweed liquid fertilizer extract had a completely negative effect on seed germination. After one week of treatment, no seed germination occurred in case of the seeds treated with seaweed aqueous extract and seaweed liquid fertilizer of Sargassum ilicifolium (Figures 8c - 8f).

Effect of biosynthesized Silver Nanoparticles, seaweed extract and seaweed liquid bio-fertilizer on seed ger-mination of Raphanus sativus var. longipinnatus

The effect of biosynthesized Silver Nanoparticles on seed germination of Raphanus sativus var. longipinnatus was

Figure 8. Seed germination percentage of Abelmoschus esculentus in different treatments.

a b

Figure 8a. showing the ladies finger seed germination with normal water and 1 mM AgNO3 solution, 8b. showing the ladies finger seed treated with seaweed extract.

c d

e f

Figure 8c and 8d. The root of seedling of normal water treated seed showed plenty of branch root development and the sectional simple microscopic view of normal root with the well-developed vascular system, tissues and its connection with branch roots, (e) and (f) - The root tip treated with 1 mm AgNO3 solution and sectional simple microscopic view of root with the distorted damaged vascular system and tissues.



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comparatively better than the effect of the normal water, seaweed extract and seaweed liquid fertilizer. The seed germination index was the highest at 24 hours (133.33) for seeds treated with biosynthesized Silver Nanoparticles (SL-AgNPs) but the relative seed germination was the highest at 96 hours (200) (Figure 9a). The mean germination time, for biosynthesized Silver Nanoparticles was less than the mean germination time of seed treated with normal water at 24 hours, but the mean germination time was gradually increased with increase of duration of treatment at 48 hours and 96 hours (Figure 9c). It indicated that seeds treated with biosynthesized Silver Nanoparticles germinated faster than seeds treated with normal water, seaweed extract and seaweed liquid bio-fertilizer. The germination percentage for biosynthesized Silver Nanoparticles treated seeds was increased with time in comparison to normal water treated seeds, seaweed extract treated seed and seaweed liquid bio-fertilizer treated seed (Figure 9b). The seed germination percentage was comparatively higher for seeds treated with biosynthesized Silver Nanoparticles to increase of time than normal water treated seeds. The germination percentage reached to the maximum at 96 hours (100). The germination rate was the highest at 48 hours for biosynthesized Silver Nanoparticles treated seeds (Figure 9d). The 1mM Silver Nanoparticles (AgNO3) solution was used

as a negative control to study its effect on seed germination. After one week of observation, there was no seed germination of the seeds which was treated with 1 mm AgNO3 solution that proved the Silver Nitrate solution (AgNO3) had a toxic effect on seed germination of Raphanus sativus var. longipinnatus but the seeds were germinated with normal water treatment (Figure 9e). The seaweed extract and seaweed liquid fertilizer was also used as a positive control to study their effect on seed germination of Raphanus sativus var. longipinnatus (Figure 9g). After one week of treatment, no seed germination occurred for the seeds treated with seaweed extract and seaweed liquid bio-fertilizer (Figure 9f). The root length germinated seeds of radish and ladies finger were measured with standard measuring scale for calculation of seed germination parameters (Figure 10a and b).

Antibacterial activitySeaweed synthesized Silver Nanoparticles had the maximum

inhibitory activity against Escherichia coli (0.83 ± 0.05 cm), followed by Pseudomonas aeruginosa (0.53 ± 0.05 cm), and Proteus mirabilis (0.46 ± 0.05 cm). The zone of inhibition of Ag-Nanoparticles for mentioning four pathogens was greater than the inhibitory activity of antibiotic Chloramphenicol (5mg/ml) and AgNO3 (1mM) which had very less antibacterial activity in

a b

c d

Figure 9. (a) to (d) Representing the effect of biosynthesized Silver Nanoparticles and the aqueous extract of Sargassum ilicifolium on seed germination of Raphanus sativus var. longipinnatus.



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comparison to these four pathogens. But for two pathogens such as Staphylococcus aureus (0.31 ± 0.02cm) and Enterococcus faecalis (0.35 ± 0.05 cm) the zone of inhibition of antibiotic (5mg/ml) was greater than biosynthesized Silver Nanoparticles solution. The zone of inhibition of seaweed mediated Nanoparticles for Staphylococcus aureus and Enterococcus faecalis was (0.15 ± 0.00 cm) and (0.2 ± 0.03cm) (Figure 11).

ConclusionsIt can be concluded from this study, that the biosynthesized

Silver Nanoparticles had a significant effect on seed germination of Abelmoschus esculentus. For Abelmoschus esculentus seeds, the effect of Silver Nanoparticles on seed germination was as equal as normal water, but biosynthesized Silver Nanoparticles had a better effect on seed germination of Raphanus sativus var. longipinnatus than seed germination with treatment of seaweed extract and

Figure 9. (g) Representing seed germination percentage of Raphanus sativus var. longipinnatus in different treatments.

a b

Figure 10. (a) the seedling of raddish and Figure 10 (b) Seedling of ladies finger after treatment with seaweed synthesized silver Nanoparticles (SL) from Sargassum ilicifolium.

seaweed liquid fertilizer. The 1 mm AgNO3 solution had been proved to be toxic to the germination of Abelmoschus esculentus seeds consequently one seed germinated and it was again dried and damaged after 3 days due to distortion and vascular damage which was shown in the sectional view of root. In case of seeds treated with seaweed extract and seaweed liquid fertilizer, no seed germination occurred for Abelmoschus esculentus seeds. The seed germination of Raphanus sativus var. longipinnatus was excellent with the treatment of biosynthesized Silver Nanoparticles where a germination percentage was 100 at 96 hours in comparison to normal water, which indicated that the biosynthesized Silver Nanoparticles had high promoting effect on seed germination promoting of Raphanus sativus var. longipinnatus seed. The seaweed extract, seaweed liquid fertilizer and 1 mm Silver Nitrate solution (AgNO3) was proved completely toxic to seed germination of Raphanus sativus var. longipinnatus. For the

e f

Figure 9. (e) showed the radish seed germination with normal water and 1 mM AgNO3 solution, (f) showed the seaweed extract treated seeds of radish.


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antibacterial activity, the biosynthesized Silver Nanoparticles had a significant zone of inhibition against Escherichia coli, Pseudomonas aeruginosa and Proteus mirabilis in comparison to positive control, the solution of antibiotic Chloramphenicol.

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Figure 11. Showing the zone of inhibition of biosynthesized Silver Nanoparticles synthesized by the Sargassum ilicifolium aqueous extract.

Copyright: © 2018 Suparna Roy, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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