Abstract

A simple and eco-friendly method for the synthesis of hybrid bead silver nanoparticles (AgNPs) employing the aqueous extract derived from natural and renewable source namely tropical benthic green seaweed Ulva flexuosa was developed. This route involves the reduction of Ag+ ions anchored onto macro porous methacrylic acid copolymer beads to AgNPs for employing them as antibacterial agents for in vitro water disinfection. The seaweed extract itself acts as a reducing and stabilizing agent and requires no additional surfactant or capping agent for forming the AgNPs. The nanoparticles were analyzed using high-resolution transmission electron microscopy, UV–Vis spectroscopy, Fourier transform infrared spectroscopy, scanning electron microscopy, energy dispersive X-ray analysis and inductively coupled plasma optical emission spectroscopy. The study elucidates that such biologically synthesized AgNPs exhibit potential antibacterial activity against two Gram positive (Bacillus subtilis, Staphylococcus aureus) and two Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacterial strains tested. The bacterial count in treated water was reduced to zero for all the strains. Atomic force microscopy was performed to confirm the pre- and post-state of the bacteria with reference to their treatment with AgNPs. Attributes like facile environment-friendly procedure, stability and high antibacterial potency propel the consideration of these AgNPs as promising antibacterial entities.

ABBREVIATIONS

     
  • AgNPs

    Silver nanoparticles

  •  
  • LB

    Luria Bertani

  •  
  • SEM

    Scanning electron microscopy

  •  
  • EDX

    Energy dispersive X-ray spectroscopy

  •  
  • HRTEM

    High-resolution transmission electron microscopy

  •  
  • FTIR

    Fourier transform infrared spectroscopy

  •  
  • UV

    Ultraviolet spectroscopy

  •  
  • ICP–OES

    Inductively coupled plasma optical emission spectroscopy

  •  
  • AFM

    Atomic force microscopy

  •  
  • ATCC

    American type culture collection

INTRODUCTION

Nanotechnology holds immense potential in advancing water and wastewater treatment to improve treatment efficiency, quality and augment clean water supply through the safe use of unconventional water resources (Qu et al. 2013) with least possible environmental disturbances. Microbial contamination of water resources is a serious concern (Ashbolt 2004) in water disinfection systems. A variety of bacterial species are present in raw water as well as in wastewater. Nanoparticles are receiving worldwide attention due to their extremely minute size and high surface-to-volume ratio, leading to the chemical as well as physical developments in their properties as compared to their bulk counterparts (Iravani 2011; Veisi et al. 2018). Silver nanoparticle applications have been widely studied as disinfectants in medical institutions, and an increasing amount of research has been carried out on their applications in drinking water treatment and distribution systems (Rodriguez et al. 2008; Kumar & Raza 2009; Zhao et al. 2010; Alsabagh et al. 2015; Deng et al. 2017). Silver-based nanomaterial has a mechanism to disrupt bacterial metabolic processes (Cui et al. 2013), increase the cytoplasmic membrane permeability (Morones et al. 2005) and interact with DNA (Li et al. 2011) etc. and ultimately cause lysis of the cell wall. Among silver associated materials, silver nanoparticles are the most important because they have attracted much attention owing to their unique shape-dependent optical, electrical, chemical as well as antibacterial properties. Many protocols have been established for the physical as well as the chemical synthesis of silver nanoparticles (Donga et al. 2014; Xu et al. 2014; Lu et al. 2017; Okafor et al. 2017). However, these methods are energy as well as capital intensive (Dahoumane et al. 2016). Moreover, they employ toxic chemicals and solvents in the synthesis procedure and later on synthetic additives or capping agents, thus impeding their applications in clinical as well as biomedical fields (Akhtar et al. 2013). The need for the advancement of a clean, green, eco-friendly, efficient and cost-effective route to synthesize nanoparticles therefore attracts researchers globally to turn towards ‘green’ chemistry and bioprocesses (Jain et al. 2011; Dahoumane et al. 2017).

Several attempts have been made to synthesize different types of nanoparticles using algae as a biological route (Liu et al. 2005; Singaravelu et al. 2007; Xie et al. 2007a, 2007b; Abboud et al. 2014; El-Kassas et al. 2016). Researchers have also attempted synthesizing silver nanoparticles (AgNPs) utilizing different marine macro algae (Govindaraju et al. 2009; Azizi et al. 2013; Aboelfetoh et al. 2017; Ballesteros et al. 2017; Minhas et al. 2018). However, the utilization of this untapped marine resource in the synthesis of AgNPs for practical applications is yet to be pursued meticulously. Studies for the exploration of various natural sources need to be taken further in order to progress the prospects for greener synthesis and to develop an improved protocol for the synthesis and practical application of such products (Wang et al. 2014). A majority of the reports on the synthesis of AgNPs with a biological origin are in liquid-based/suspension systems and not on anchored AgNPs stabilized using a solid support. Therefore, synthesizing AgNPs through an environmentally friendly, non-toxic and hazard-free route using marine macro alga, stabilizing them by anchoring them on solid macro porous resin beads and applying them as an effective water disinfectant is our novel approach in this study. Immobilization of the AgNPs onto a solid support will ensure their stability for effective disinfection of water. Immobilized nanoparticles could turn out to be promising tactics for practical antibacterial applications like disinfection.

Macro algal species covered under Ulvales (Chlorophyta) are commonly encountered worldwide. To date, this biomass has represented quite low-value addition and its utility has been limited to composting (Maze et al. 1993; Badescu et al. 2017), food consumption (Dubigeon et al. 1997; Pirian et al. 2018) and methane production (Brand & Morand 1997; Costa et al. 2012). Therefore, the utilization of this algal biomass with respect to its biological assets, abundance and most importantly its renewability for the purpose of creating nanoparticles that can act as water disinfectants has been attempted.

EXPERIMENTAL

Materials

All the chemicals, solvents and reagents used were of analytical reagent grade or better and were used as received. Luria Bertani (LB) Agar and LB Broth were purchased from HiMedia (Mumbai, India). All the glass containers were rinsed with deionized water obtained from a Q-GARD, Milli-Q system Millipore, Germany (resistivity ¢18 MV cm) and dried in an oven prior to use.

Bacterial cultures

All the bacterial strains used were of the American Type Culture Collection (ATCC) procured from HiMedia (Mumbai, India). The strains used were Escherichia coli (ATCC 8739), Pseudomonas aeruginosa (ATCC 9027), Staphylococcus aureus (ATCC 25923) and Bacillus subtilis (ATCC 6051).

Synthesis of cross-linked methacrylic acid copolymer beads

The synthesis of methacrylic acid copolymer beads was done by following the method reported by Gangadharan et al. (2010). Briefly, a monomer mixture containing divinyl benzene methacrylic acid, toluene and benzoyl peroxide (1% w/w) was prepared and added to the preheated suspension medium, maintained at a temperature of 70 °C, in a three-neck round-bottom flask. The complete reaction mixture was slowly heated to 80 °C under reflux condition for further 5 h with constant stirring. The copolymer beads separated out in the form of white opaque beads.

Preparation of aqueous macro algal extract

Green macro alga (Ulva flexuosa) was collected from Pingleshwar coast, (N 23° 3′ 53.8″, E 68° 48′ 3.08″), Kachchh District, Gulf of Kachchh, India. Briefly, the seaweed was cleaned thoroughly using surface sterilization with autoclaved seawater followed by deionized water in order to remove the extraneous materials adhered onto the surface. The material was shade dried for a period of 2 weeks and powdered with mixer grinder and meshed. To 1 g of this powder, 100 mL MQ water was added and heated at 80 °C for 30 minutes. The extract was further subjected to cooling and then filtered using 0.45 μm membrane. Freshly prepared extract in this manner was then used for all the studies.

Synthesis of silver nanoparticles bound copolymer beads and its stability check

The copolymer beads were equilibrated with 0.01 M AgNO3 for 16 h on a mechanical shaker. The equilibrated beads were washed with Milli-Q water to remove excess silver ions adhering to the copolymer beads. These copolymer beads were subjected to reduction by freshly prepared aqueous seaweed extract (1% conc.) under sonication at an ambient condition with a high-density ultrasonic 6 mm probe (Labsonic/Ultrasonic Processor, Sonopros-10,000MP) immersed directly in the mixture of copolymer beads and seaweed extract. The reaction time during the ultrasonic irradiation was 10 minutes. This step was undertaken as ultrasound which is considered as a green extraction procedure (Chemat et al. 2012; Rombaut et al. 2014) that would facilitate the breakdown of glycosidic linkages of the seaweed polysaccharides. The mixed solution was incubated for 24 h at 120 rpm at 25 °C. After 24 h, the beads turned brown indicating the formation of silver nanoparticles that was also confirmed by high-resolution transmission electron microscopy (HRTEM) analysis. The stability of these AgNPs was tested by placing them in aqueous condition at neutral pH for a period of 7 days (168 h). The absorbance spectra of the hybrid bead AgNPs did not show much variation thereby indicating the stability of these particles. These beads were then dried at room temperature (25 °C) and subjected to further characterization.

INSTRUMENTAL CHARACTERIZATION

The AgNPs bound copolymer beads were investigated by field emission scanning electron microscopy (FE-SEM). These images were obtained using a JEOL JSM- 7100F at an acceleration voltage of 5 kV, and energy dispersive X-ray (EDX) analysis was performed using INCA energy (Oxford Instruments Analytical Ltd, UK) coupled with an EDX facility. HRTEM (JEOL JEM 2100, Japan) was used to characterize the microstructure of the nanoparticles. Fourier transform infrared (FTIR) spectroscopy was performed using a PerkinElmer Spectrum GX FT-IR System. UV–Vis absorption spectrum of the sample was carried out using a Shimadzu UV–VIS–NIR Spectrophotometer (Model UV-3600). Atomic force microscopy (AFM) analysis and imaging were performed using NT-MDT Ntegra Aura model (Moscow, Russia). Bacterial sample preparation for AFM analysis and imaging was performed according to Suresh et al. (2010). Stereomicroscopy was performed to check the AgNPs surface for any crack or rupture. The detail is given in the supplementary document (Figure S1), available with the online version of this paper.

BACTERICIDAL EFFECT OF AgNPS

Spread plate test

The spread plate test was used to measure the antibacterial effect of the copolymer beads containing silver nanoparticles against the selected bacterial strains. The bacteria were cultured in LB broth at 30 °C. 100 μL of 24 h old bacterial culture was spread onto the LB agar plates. Copolymer beads containing silver nanoparticles were placed over the LB agar plates. The copolymer beads without silver nanoparticles were used as the blank. All the plates were incubated at 37 °C for 24 h to measure the zone of inhibition.

Test tube test

All the sterile test tubes were filled with 20 mL of autoclaved water. Bacterial cultures, namely (E. coli: 24 × 106 CFU/mL, P. aeruginosa: 19 × 106 CFU/mL, B. subtilis: 20 × 106 CFU/mL and S. aureus: 18 × 106 CFU/mL) were added to these test tubes. 100 mg (in the dry state) of copolymer beads containing silver nanoparticles were added to the test tubes containing E. coli, P. aeruginosa, B. subtilis and S. aureus bacterial suspensions separately and closed with a cotton stopper in order to avoid contamination. Similarly, 200 mg and 300 mg (in the dry state) of copolymer beads loaded with silver nanoparticles were tested to quantify the number of copolymer beads required to inhibit the bacterial growth completely. All the tubes were kept on the shaker for studying the growth inhibitory effect on the bacterial cultures. 100 μL of supernatant fractions were withdrawn after 2 h, 4 h, 6 h and 8 h of time intervals in order to investigate the influence of contact time in killing the bacterial cells. The supernatant fractions withdrawn were plated directly without any further dilution to obtain the bacterial colony count. Copolymer beads without AgNPs were used as blank in the experiment.

Kinetic study of silver release

The quantity of Ag+ released in water was studied as a function of time (Taglietti et al. 2014). The experimental set up contained a series of 25 mL flasks containing 0.1 g of hybrid bead AgNPs with 30 mL of deionized (DI) water. The flasks were further subjected to shaking at room temperature. Sample aliquots were withdrawn at different time intervals, namely 0 h, 6 h, 12 h, 18 h, 24 h, 48 h, 72 h and 96 h for the analysis of silver ions. The concentration of silver ions in the filtrates was assessed using inductively coupled plasma optical emission spectroscopy (ICP-OES). Silver nanoparticle coated beads were ground into fine powder and 0.2 g of this powder was dissolved in a mixture of HF and HNO3. After the complete dissolution of the powder, it was filtered. The samples containing silver ions eluted from the polymeric beads were diluted up to 50 mL with Milli-Q water prior to quantification. The final concentration was quantified using a set of single-element external calibration standards to enable measurements in the 0–1 ppm concentration range.

RESULTS AND DISCUSSION

Field emission scanning electron microscopy/energy dispersive X-ray analysis

The morphology of copolymer beads bound with AgNPs and their elemental composition were examined with Field emission scanning electron microscopy (FE-SEM) and EDX analysis, respectively (Figure 1). The copolymer beads were examined for their surface characteristics and no cracking was observed on the surface of the copolymer beads (Figure 1(a) and 1(b)) at a scale of 100 μm with a magnification of 180× and 95× respectively. The AgNPs distributed on the surface of the copolymer beads were observed to have uneven size and shape distribution at a scale of 1 μm with a magnification of 5,000× (Figure 1(c)). The distribution of the AgNPs onto the surface of the beads is clearly evident as previously reported by Dahoumane et al. (2012). The EDX analysis performed confirmed the presence of silver and chloride on the copolymer beads (Figure 1(d)). The intensity of the peak of silver indicates the formation of AgNPs. The signal of Cl is also observed which may be due to the adherence of seaweed extract used for the reduction.

Figure 1

(a) SEM image of copolymer bead without AgNPs (Blank); (b) SEM image of copolymer bead with AgNPs; (c) enlarged image of AgNPs anchored on the copolymer bead surface; (d) EDX analysis of silver nanoparticles (AgNPs) bound copolymer beads.

Figure 1

(a) SEM image of copolymer bead without AgNPs (Blank); (b) SEM image of copolymer bead with AgNPs; (c) enlarged image of AgNPs anchored on the copolymer bead surface; (d) EDX analysis of silver nanoparticles (AgNPs) bound copolymer beads.

High-resolution transmission electron microscopy

The morphology, size and the distribution of AgNPs were analyzed by HRTEM. The average size of the silver nanoparticles was observed to be 4.93–6.70 nm. The smaller particle size of AgNPs bound onto the copolymer beads render its close interaction with the microbial membranes, proving them to be good biocidal material. The HRTEM image containing AgNPs is shown in Figure 2 and in supplementary information Figure S5 (available with the online version of this paper).

Figure 2

High-resolution transmission electron microscopy image of the AgNPs formed using U. flexuosa extract along with their size measurement.

Figure 2

High-resolution transmission electron microscopy image of the AgNPs formed using U. flexuosa extract along with their size measurement.

Fourier transform infrared

The FTIR spectrum of the seaweed extract as well as the copolymer beads without AgNPs (blank) and seaweed mediated AgNPs were obtained. The IR spectrum of the seaweed extract (Figure 3(a)) showed major peaks in the lower frequency regions at 621, 764, 962, 1,120, 1,390, 1,459, 1,543 and 1,652 cm−1 which were assigned for C–Cl alkyl halide, alkenes, C–N stretch, C=C stretch and N=O bend, respectively. The IR spectrum of the macro algal extract showed that the major peaks were observed in the lower frequency regions at 1,543 and 1,652 cm−1 which were assigned for carbonyl stretching vibrations of amide I and amide II linkages. This clearly indicates the presence of protein/peptide group that serves as a stabilizing as well as reducing agent for the formation of silver nanoparticles (Gopinath et al. 2013). The peak at 1,390 cm−1 implies the role of C=C in the reduction of silver ion (Stalin Dhas et al. 2014). The infrared band detected at 2,358 cm−1 may be assigned to combination bands, namely strongly hydrogen bonded water molecules and/or hydroxyls (Frost et al. 2013). It may also correspond to the –PH in phosphine stretch (Afsheen et al. 2018) or represent a characteristic peak for CO2 (Khaleel & Nawaz 2015). The role of the asymmetric –CH bending represented by the peak at 2,927 cm−1 designates its role in the reduction of silver ions (Stalin Dhas et al. 2014). The absorbance at 3,417 cm−1 corresponded to N–H stretch as well as to O–H stretch which is characteristic of the hydroxyl functional group in alcohol and phenolic compounds. Figure 3(b) indicates the IR spectra of the blank. The FTIR spectrum of AgNPs (Figure 3(c)) showed distinct peaks at 514, 1,192, 1,378, 1,539, 1,646, 2,358, 2,936 and 3,427 cm−1. The comparison of resultant spectrum between the green macro algal extract and the copolymer bound AgNPs showed notable shift changes in the peak positions as well as the absorption bands except at 2,358 cm−1. From these results, it was concluded that halide, alkenes, amine and nitro groups which were present in the seaweed extract possessed a strong affinity to bind to the silver ions, thereby attributing their reduction to AgNPs.

Figure 3

FTIR spectra. (a) Seaweed extract only; (b) copolymer beads without AgNPs (Blank); (c) hybrid bead AgNPs synthesized using seaweed extract.

Figure 3

FTIR spectra. (a) Seaweed extract only; (b) copolymer beads without AgNPs (Blank); (c) hybrid bead AgNPs synthesized using seaweed extract.

UV–Vis spectrum

Figure 4(a) shows a broad peak at 480 nm. The surface plasmon resonance (SPR) peak at λmax = 480 nm was observed due to the active biomolecules present in the seaweed extract which interacted with silver ions, thereby forming AgNPs. The spectrum in the range of 420 nm to 480 nm indicates the formation of silver nanoparticles through evolvement of characteristic surface plasmon extinction (Jes et al. 2015). The polydispersity of the nanoparticles on the copolymer beads is indicated by the broadening of peak (Ghaedi et al. 2012). The UV–Vis spectrum of the methacrylic copolymer beads without silver nanoparticles (blank) is shown in Figure 4(b).

Figure 4

UV–Vis absorption spectra. Line (a) shows the AgNPs bound copolymer beads (indicated in red online); line (b) shows copolymer beads without AgNPs/Blank (indicated in blue online). Please refer to the online version of this paper to see this figure in colour: http://dx.doi.org/10.2166/wst.2018.292.

Figure 4

UV–Vis absorption spectra. Line (a) shows the AgNPs bound copolymer beads (indicated in red online); line (b) shows copolymer beads without AgNPs/Blank (indicated in blue online). Please refer to the online version of this paper to see this figure in colour: http://dx.doi.org/10.2166/wst.2018.292.

Atomic force microscopy

In order to further understand the interaction between the selected bacterial strains and the hybrid bead AgNPs, AFM imaging experiments of the bacteria treated with blank and AgNPs coated beads were carried out (Figure 5). AFM is a convenient tool for examining the changes in cell membrane morphology and surface structure (Suresh et al. 2010) and can aid in elucidating morphological changes induced by AgNPs on bacterial cells. AFM images of blank and silver nanoparticle-treated bacterial samples showed notable differences in their outer surface morphology (Shukla et al. 2012). AFM images of all the four bacterial strains treated with blank (Figure 5(a1), 5(b1), 5(c1), 5(d1)) and with AgNPs (Figure 5(a2), 5(b2), 5(c2), 5(d2)) showed a notable change in the morphology. The bacteria showed intact structure when treated with copolymeric beads without AgNPs coating (blank). The cell wall was not damaged and the shape of the cell, as well as its integrity, were maintained. While when treated with hybrid bead AgNPs, the cell surface either developed a hole or was completely ruptured. The silver nanoparticles do appear to cause significant structural changes thereby affecting the cell integrity leading to cell death. However, the precise mode of the bacterial-nanoparticle interaction is still unclear.

Figure 5

AFM images showing the interaction of Gram-positive B. subtilis and S. aureus as well as Gram-negative organisms E. coli and P. aeruginosa with copolymer beads having AgNPs anchored onto the surface and with copolymer beads without AgNPs (Control/Blank); (a1) S. aureus culture with copolymer beads without AgNPs (Blank/Control); (a2) S. aureus culture treated with AgNPs (Test); (b1) B. subtilis culture with copolymer beads without AgNPs (Blank/Control); (b2) B. subtilis culture treated with AgNPs (Test); (c1) E. coli culture with copolymer beads without AgNPs (Blank/Control); (c2) E. coli culture treated with AgNPs (Test); (d1) P. aeruginosa culture with copolymer beads without AgNPs (Blank/Control); (d2) P. aeruginosa culture treated with AgNPs (Test).

Figure 5

AFM images showing the interaction of Gram-positive B. subtilis and S. aureus as well as Gram-negative organisms E. coli and P. aeruginosa with copolymer beads having AgNPs anchored onto the surface and with copolymer beads without AgNPs (Control/Blank); (a1) S. aureus culture with copolymer beads without AgNPs (Blank/Control); (a2) S. aureus culture treated with AgNPs (Test); (b1) B. subtilis culture with copolymer beads without AgNPs (Blank/Control); (b2) B. subtilis culture treated with AgNPs (Test); (c1) E. coli culture with copolymer beads without AgNPs (Blank/Control); (c2) E. coli culture treated with AgNPs (Test); (d1) P. aeruginosa culture with copolymer beads without AgNPs (Blank/Control); (d2) P. aeruginosa culture treated with AgNPs (Test).

Microbiological results

A soil as well as water contaminant, P. aeruginosa (ATCC 9027) (Prochnow et al. 2015), E. coli (ATCC 8739) as a faecal contamination indicator (How et al. 2013), S. aureus (ATCC 25923) (Basso et al. 2014) and B. subtilis (ATCC 6051) (Dodd & Grace 1989) as human influence indicators of water were selected for the study. The bacterial cultures were grown aerobically in LB broth at 37 °C for 24 h and maintained as stock cultures on LB agar slants at 4 °C.

Spread plate test

A preliminary experiment was conducted with AgNPs-bound copolymer beads. The zones of inhibition were measured for all the four bacterial cultures tested and represented in supplementary information (Figure S2), available online. In case of Gram-negative organisms like P. aeruginosa, the zone diameter was found to be 24 mm (Figure S2a) while for E. coli it was 15 mm (Figure S2b). The zone diameter against Gram-positive bacterial species like B. subtilis was around 18 mm (Figure S2c), while for S. aureus it was 13 mm (Figure S2d). The results indicate that AgNPs prepared using the aqueous extract of U. flexuosa exhibited good antibacterial activity against all the four selected bacterial strains.

Test tube test

The copolymer beads loaded with AgNPs reduced the bacterial count to zero with the increase in the contact time as well as the quantity of the copolymer beads. Viable cells of E. coli (Figure 6(a)) as well as P. aeruginosa, (Figure 6(b)) were reduced to zero within 6 h of contact time with 100 mg of AgNPs bound copolymer beads, while in the case of Gram-positive bacteria B. subtilis (Figure 6(c)) it reaches zero after 6 h of contact time but at a concentration of 200 mg. This may be due to the complexity of the outer cell wall of B. subtilis. In the case of S. aureus (Figure 6(d)), the count becomes zero after 6 h of contact time for 300 mg of AgNPs bound copolymer beads. This difference in bacterial reduction among the Gram-negative organisms is due to the higher permeable nature of E. coli cell wall.

Figure 6

(a) Graph showing the biocidal activity of the AgNPs against E.coli; (b) graph showing the biocidal activity of the AgNPs against P. aeruginosa; (c) graph showing the biocidal activity of the AgNPs against B. subtilis; (d) graph showing the biocidal activity of the AgNPs against S. aureus.

Figure 6

(a) Graph showing the biocidal activity of the AgNPs against E.coli; (b) graph showing the biocidal activity of the AgNPs against P. aeruginosa; (c) graph showing the biocidal activity of the AgNPs against B. subtilis; (d) graph showing the biocidal activity of the AgNPs against S. aureus.

When 200 mg of AgNPs was used, 100% reduction in the bacterial count was observed within 6 h of contact time for all the tested bacteria except S. aureus. It took nearly 8 h for the complete reduction of S. aureus when treated with 200 mg of AgNPs (Figure 6(d)). Studies suggest that both gene duplication and lateral gene transfer are important in the maintenance of the resistance mechanisms to toxic drugs or heavy metal in Staphylococcus (Chan et al. 2011).

When 300 mg of copolymer beads containing AgNPs were equilibrated in bacterial suspension they exhibited higher activity compared to the 100 mg copolymer beads within 2 h as obvious from the results. The bacterial count for all the tested strains was reduced to half or more than half with the usage of 300 mg of AgNPs coated copolymeric beads within 2 h of contact time. Complete reduction of both the Gram-negative as well as the Gram-positive viable cells was achieved within 6 h of contact time with 300 mg of AgNPs bound copolymer beads (Figure 6). This indicated that 300 mg copolymer beads with 6 h of contact time could be considered as the minimum inhibitory concentration necessary to render complete disinfection. The plate results of the test tube test are given in supplementary Figure S3 (available online).

Release of silver into the filtrate

The amount of silver loaded onto the polymer beads was 9 mg/g. In this study, the silver concentration in the filtrate was below the detectable range up to 12 h. The silver release gradually increases with the increase in contact time and complete leaching was observed after 96 h (Table 1). The increase in release rate at the initial phase of contact may be due to the liberation of silver nanoparticles. Up to 12 h, the leaching of silver was below the permissible level, thereby proving these hybrid bead AgNP entities to be potential antibacterial agents.

Table 1

The release of silver from AgNPs bound to copolymer beads as a function of time

SampleTime interval for withdrawal of sample (hours)Volume of fraction collected (mL)Total silver concentration (mg/L)
Nd 
Nd 
12 Nd 
18 0.02 
24 0.034 
48 0.058 
72 0.066 
96 0.069 
SampleTime interval for withdrawal of sample (hours)Volume of fraction collected (mL)Total silver concentration (mg/L)
Nd 
Nd 
12 Nd 
18 0.02 
24 0.034 
48 0.058 
72 0.066 
96 0.069 

Nd represents not detected.

The emergence of protocols involving biological routes for the synthesis of nanoparticles constitutes an evolving facet of nanotechnology. The green synthesis approach of silver nanoparticles that defines the development of eco-friendly process without the usage of toxic chemicals is in vogue (Ramkumar et al. 2017; Kim et al. 2018; Pugazhendhi et al. 2018). This approach offers many advantages over the conventional chemical as well as physical methods. The physical approaches used for the nanoparticle production are quite laborious and expensive, and the chemical methods involve the usage of hazardous chemicals that may adhere to the nanoparticle surface making them unsuitable for biological applications (Chung et al. 2016). Chemical processes also involve high energy consumption, are cumbersome for large scale production and generate toxic by-products that pose a grave threat to the environment as well as humans. However, the biological methods have proven to be environmentally safe, rapid and energy-efficient. The selection of the environmentally friendly solvent as well as non-toxic reducing and capping agents marks the most crucial aspect of green chemistry involved in nanoparticle formation (Iravani et al. 2014). Silver nanoparticles synthesized through green method have been reported to have biomedical applications (Rasheed et al. 2017) as well as in controlling the pathogenic microbes (Ferreira et al. 2017). In addition, due to their potent antimicrobial activity, AgNPs have also been used in cosmetics (Kale & Jagtap 2018), food preservation (Kumar et al. 2017) and clothing (Wigger 2017). This environmentally friendly and biocompatible approach of using the marine macroalga U. flexuosa for the formation of hybrid bead AgNPs was therefore adopted for our study. In addition to this, the other advantages of using marine macroalgal species include their renewability, easy availability and the presence of a variety of primary as well as secondary metabolites that aid in silver reduction.

CONCLUSION

The present study reveals that hybrid bead bound AgNPs can be formed successfully using U. flexuosa via a facile single step green procedure onto the copolymer beads without causing any physical damage to the surface of beads. The stable immobilized AgNPs at a concentration of 300 mg demonstrated prominent antibacterial traits which resulted in 100% bacterial reduction within 6 h of contact time for all the bacterial strains. Complete inhibition was achieved with an increase in the contact time as well as with the increase in the amount of the AgNPs bound copolymer beads. This environmentally friendly procedure employing abundant and naturally available marine benthic algae can, therefore, be considered for antibacterial treatment of water. Such features, in a nutshell, prove these hybrid bead AgNPs coated copolymer beads, synthesized using macro algal extract, as a probable future candidate for water disinfection. However, further studies need to be carried out to assure a considerable performance of the AgNPs in more complex physiological environments.

ACKNOWLEDGEMENT

The author, Dhara Dixit, deeply acknowledges the support rendered by the Director, CSIR-CSMCRI, for providing the infrastructure to conduct this study as well as the Analytical Division (CSIR-CSMCRI) for carrying out the instrumental analysis. I also thank KERC (A Division of The Corbett Foundation) for providing the field assistance during sampling. I thank Dr M. G. Thakkar (Head, Department of Earth & Environmental Science, K. S. K. V. Kachchh University) for providing me the administrative support required for conducting this study.

Conflict of interest

The authors have declared no conflict of interest.

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Supplementary data