Mycosynthesized Silver Nanoparticles as Potent Growth Inhibitory ...

(1 of 6) 1600247 Harshal Sonar1 Dipali Nagaonkar2 Avinash P. Ingle2 Mahendra Rai2 1

Dr. D. Y. Patil Biotechnology and Bioinformatics Institute, Tathawade, Pune, Maharashtra, India

Research Article Mycosynthesized Silver Nanoparticles as Potent Growth Inhibitory Agents Against Selected Waterborne Human Pathogens

2

Nanobiotechnology Lab., Department of Biotechnology, SGB Amravati University, Amravati, Maharashtra, India

Search for innovative technologies for eradication of water contamination and thereby reducing waterborne diseases is a global need. Use of antimicrobial metal nanoparticles for water purification processes has opened up new possibilities to combat waterborne diseases. The present study demonstrates the synthesis of antibacterial silver nanoparticles by using fungal filtrates of Fusarium oxysporum and Phoma sorghina. Transmission electron microscopy confirmed the synthesis of isotropic, spherical nanoparticles with 20–25 nm as mean size. The mycosynthesized nanoparticles were evaluated for their growth inhibition activity against waterborne pathogens. Staphylococcus aureus was found to be the most susceptible organism to the silver nanoparticles synthesized from P. sorghina; whereas, high bacteriostatic activity of silver nanoparticles was observed against Brevibacillus borstelensis. The present findings indicate the potential of silver nanoparticles for use in water purification processes. Keywords: Antimicrobial metal nanoparticles; Growth inhibition; Waterborne diseases; Water contamination Received: April 1, 2016; revised: September 6, 2016; accepted: January 13, 2017 DOI: 10.1002/clen.201600247

1 Introduction Water contamination is a universal problem, which does not only affect human health but also has an impact on global economic and social costs. The supply of clean and affordable water to fulfill human needs is a great challenge of the 21st century. Globally, water supply faces challenge to keep up with the fast growing demand by population growth, global climate changes, and water quality deterioration. According to the World Water Development report 2014, presently, 64 billion m3 of fresh water is progressively being consumed per year [1]. Water contamination due to various microbes poses a major threat to public health. Furthermore, emergence of resistance in microorganisms to many antimicrobial agents has necessitated the continuous need to search for improved and updated water disinfection methods [2]. Numerous commercial and non-commercial technologies are being developed to solve this problem worldwide. With the start of the 21st century, nanotechnology has become the crucial technology by offering advances for water purification as well as treatment of wastewater [3, 4]. Nanomaterials possess enhanced physical and chemical properties

Correspondence: Dr. Mahendra Rai, Nanobiotechnology Laboratory, Department of Biotechnology, SGB Amravati University, Amravati 444 602, Maharashtra, India E-mail: [email protected]; [email protected] Abbreviations: AgNP, silver nanoparticle; CFU, colony forming unit; CNT, carbon nanotube; MBC, minimum bactericidal concentration; MIC, minimum inhibitory concentration; NTA, nanoparticle tracking analysis; PDB, potato dextrose broth; SPR, surface plasmon resonance; TEM, transmission electron microscopy. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

and thus hold great potential in water treatment methodologies by improving treatment efficiency. Nanoparticles ranging within the nanometer size (1–100 nm) have a great ability to be used in wastewater treatment for processes like chelation of heavy metals, adsorption for salts, etc. The novel properties of nanoparticles including high surface area and high catalytic potential can be explored more efficiently for removing toxic heavy metal ions, microbial pathogens, organic and inorganic solutes from water. The metal nanoparticles obtained after bioleaching of waste have demonstrated broad range of applications in medicine, food and water [5]. Diversity of nanomaterials such as metal-containing nanoparticles as well as nonmetallic nanostructures such as carbonaceous nanomaterials, zeolites, and dendrimers have proved to be efficient agents for water treatment. For example, plasmamodified ultra-long carbon nanotubes (CNTs) were developed with ultrahigh specific adsorption capacity for salt (exceeding 400% by weight), which was two-fold higher in comparison with conventional carbon-based water treatment techniques. Advanced water purification devices supported with these novel CNTs are expected to have superior desalination, disinfection, and filtration properties not only for salts but also organic and metal contaminants [6]. Several metal nanoparticles, for example, silver, copper, zinc, etc., have already proved its antimicrobial properties against a wide range of microorganisms. Since ancient times, the antimicrobial potential of silver has been well exploited and silver ions are being used as bactericide for burns, wounds, and dental work [7, 8]. Researchers have already recommended the applicability of silver ions as superior disinfectants for wastewater treatment containing infectious biomedical wastes [9].

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Until now, a few researchers have evaluated efficacy of silver nanoparticles against waterborne pathogens causing infections. Silver and zinc nanoparticles synthesized using the aqueous extract of Calotropis procera fruit and leaves demonstrated their efficacy against Vibrio cholerae and enterotoxic Escherichia coli including biofilm inhibition [10]. Mycosynthesized silver nanoparticles were evaluated for their growth inhibition activity against Salmonella typhi, Bacillus subtilis, Staphylococcus aureus, and E. coli [11]. Similarly, in another study, silver nanoparticles successfully demonstrated activity against waterborne B. subtilis and Pseudomonas aeruginosa [12]. Casein and citrate stabilized silver nanoparticles (AgNPs) were found to be potent growth inhibitor agents against E. coli, Cronobacter sakazakii, P. aeruginosa, Enterobacter cloaceae, Pantoea agglomerans, Serratia marcescens, Aeromonas hydrophila, and Klebsiella pneumoniae [13]. Myconanotechnology is now an established branch of nanotechnology, which offers better means to fabricate nanoparticles with enhanced bioactivities as well as with prolonged stability. Among the biological systems, fungi have been considered as most efficient biosystems because of their heavy metal tolerance, simple biomass handling and cost efficiency [8, 14]. For example, silver nanoparticles synthesized by using filtrates of Aspergillus flavus, Aspergillus terreus, and Penicillium janthinellum isolated from leaves of Andrographis paniculata and Carica papaya were found to possess high growth inhibitory activity against E. coli, K. pneumoniae, Streptococcus pyogens, S. aureus, and P. aeruginosa [15]. Similarly, Fusarium and Phoma spp. were also utilized for the synthesis of silver nanoparticles with high antibacterial activity [14, 16, 17]. In the present study, fungal filtrates of Fusarium oxysporum and Phoma sorghina were used as bioreducing agents for production of silver nanoparticles. Both Fusarium and Phoma are ubiquitous fungi, which occur in a wide variety of substrates including plants, air, soil, and water. Some Phoma spp. are known to cause crop diseases such as seedling blight, leaf-spots, etc. Similarly, F. oxysporum, the common soil inhabitant exists as saprophytes and is known to be pathogenic for agricultural crops. Taking into consideration, the urgent need for the development of novel techniques to deal with waterborne pathogens, the present study was designed to synthesize silver nanoparticles and to evaluate their growth inhibitory effect on waterborne human pathogens.

2 Materials and methods 2.1 Materials Silver nitrate (AgNO3) (purity 99%) was received from HiMedia, Mumbai, India and used as received.

mycelia were separated through muslin cloth. The filtrate was collected and filtered through Whatman filter paper No.1, followed by centrifugation for 30 min at 4000 rpm. About 10 mL of fungal filtrates were treated with 90 mL of 1 mM AgNO3 and kept at room temperature for complete reduction.

2.2.2 Characterization of the synthesized silver nanoparticles 2.2.2.1 UV-vis spectrophotometric analysis The formation of silver nanoparticles was confirmed by subjecting the nanoparticle suspension to UV-vis spectrophotometry (Shimadzu-UV 1700, Japan) at the resolution of 1 nm and scanning at wavelength in the range of 200–800 nm.

2.2.2.2 Zetasizer analysis Zetasizer analysis was performed to determine zeta potential and polydispersity index of biosynthesized nanoparticles. About 30 mL of each nanoparticles solution diluted with 2 mL distilled water were added to zeta dip cells and measured by Malvern Zetasizer 90 (ZS 90, USA).

2.2.2.3 Nanoparticles tracking and analysis (NTA) The size of nanoparticles was determined by nanoparticle tracking analyzer (LM-20 Malvern, USA). About 5 mL of nanoparticle samples was diluted with 2 mL distilled water and injected into the nanoparticle tracking analyzer. The size of the nanoparticles was calculated by tracking the Brownian motion of nanoparticles through charge-coupled device camera connected to the instrument.

2.2.2.4 Transmission electron microscopy (TEM) The size and morphology of the mycosynthesized silver nanoparticles were analyzed by TEM (Philips, CM 200). For TEM analysis, samples were prepared by loading one drop of Ag nanoparticles suspension on conventional carbon coated copper grids and observed through TEM.

2.2.3 Antibacterial assay of AgNPs The antibacterial activities of both batches of AgNPs against waterborne pathogenic bacteria including S. aureus (ATCC-33591), E. coli (ATCC-14948), Chromobaterium violaceum (MCC-2290), Brevibacillus borstelensis (MCC-2403) were assayed by the disc diffusion method and evaluated for minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC).

2.2.3.1 Disc diffusion assay for AgNPs

2.2 Methods 2.2.1 Preparation of fungal filtrate and synthesis of silver nanoparticles The synthesis of silver nanoparticles was carried out by using fungal filtrates of F. oxysporum and P. sorghina. Both fungi were inoculated in 250 mL Erlenmeyer flasks, each of them containing 100 mL of potato dextrose broth (PDB) and later incubated at 26 2°C for 7 days. Mycelia thus grown after 7 days were harvested and resuspended in 100 mL sterilized distilled water and further incubated at room temperature for 24 h. After completion of the incubation period, © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

The standard ampicillin-loaded antibiotic discs were purchased from HiMedia, Mumbai, India. The disc diffusion assay was performed against S. aureus, C. violaceum, B. borstelensis, and E. coli on nutrient agar plates. A loopfull (10 mL) of individual test organism was allowed to grow overnight in potato dextrose broth (PDB) at 37°C. On completion of incubation period, each bacterial sp. was streaked onto the agar plates. Standard discs loaded with AgNPs as well as standard antibiotic discs loaded with individual combinations of AgNPs were placed onto the agar surface. The standard disc loaded with 1 mM AgNO3 was maintained as a control. The plates were incubated at 37°C for 24 h, and the zones of inhibition were measured. The experiment was performed in triplicate.



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Figure 1. Color change of fungal filtrate from colorless to reddish-brown for the AgNPs synthesized by (A) P. sorghina and (B) F. oxysporum.

2.2.3.2 Determination of MIC and MBC of silver nanoparticles Broth dilution method was used to determine MIC of AgNPs against bacteria. Inocula of all tested spp. containing 1 106 colony forming units (CFU) mL1 were taken into microtiter plate wells. To these wells, AgNPs with concentrations of 4, 6, 8, 10, 20, 40, 50, and 60 mg mL1 against all the bacteria were added. The microtiter plates were incubated at 37°C for 24 h. On completion of incubation period, 40 mL tetrazolium salt (prepared in 0.2 mg mL1 distilled water) was added to each microwell and observed for the final color change from pale yellow to pink. The persistence of initial color indicates bacterial growth inhibitory activity of nanoparticles. The minimum concentration required to kill 99.9% of the bacterial population (MBC) was analyzed. Samples from the wells showing no growth were streaked onto nutrient agar medium and incubated overnight. The number of viable colonies was counted and the concentration at which least number of viable colonies was observed was considered as MBC.

3 Results and discussion In the present study, F. oxysporum and P. sorghina used for the biological synthesis of AgNPs clearly showed the ability for the reduction of Ag ions into AgNPs. Further, the mycosynthesized

AgNPs were used for the assessment of their antibacterial potential against waterborne bacteria namely, S. aureus, C. violaceum, B. borstelensis, and E. coli. In this experiment, the fungal filtrates of F. oxysporum and P. sorghina after treatment with AgNO3 (1 mM) demonstrated rapid color change from colorless to dark-brown within 5–7 min in sun light. The development of dark-brown color was a clear indication of the synthesis of AgNPs in the reaction mixture (Fig. 1). The appearance of the brown color could be attributed to the surface plasmon resonance (SPR) properties of AgNPs due to the excitation of surface plasmon vibrations in the metal nanoparticles [18].

3.1 UV-vis spectrophotometry The reduction of silver ions into nanoparticles was confirmed by UVvis spectrophotometry in which silver nanoparticles owing to their SPR are known to possess absorption peaks at 420–450 nm [14, 19]. The fungal filtrate of F. oxysporum after treatment with aqueous silver ions was subjected to optical analysis using UV-Vis spectrophotometry and showed a sharp peak at 438 nm, which is specific for AgNPs (Fig. 2A). The AgNPs synthesized from P. sorghina showed a peak at 424 nm (Fig. 2B). It is a well-known fact that there is a very close relationship between the pattern of UV-Vis absorbance spectrum and size and shape of AgNPs [18]. Single, sharp, and

Figure 2. UV-vis spectrophotometric analysis of (A) AgNPs synthesized by F. oxysporum and (B) AgNPs synthesized by P. sorghina. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim



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Figure 3. Nanoparticle tracking analysis of AgNPs synthesized by F. oxysporum. (A) Nanoparticles size distribution histogram showing the average size and (B) NTA 3D plot of particle size distribution.

symmetrical absorbance peak of both of the AgNPs indicate the formation of quite spherical and homogenous population of nanoparticles [18]. These findings show similarity with the results reported in the past [20]. Fusarium oxysporum and P. sorghina-mediated AgNPs with narrow peak (monodisperse) indicated nanoparticles of small size, which was further confirmed by TEM analysis.

3.2 Nanoparticle tracking analysis (NTA) Nanoparticle tracking analysis (NTA) was carried out to determine the size of nanoparticles by tracking Brownian motion of nanoparticles in liquid state. The size of minimum 1000 NPs was taken into consideration to calculate average size of nanoparticle population. NTA analysis revealed an average size of 45 24 and 41 21 nm for the AgNPs synthesized from F. oxysporum and P. sorghina, respectively (Figs. 3 and 4).

3.3 Zetasizer analysis Zetasizer analysis of both batches of the silver nanoparticles was performed to determine the zeta potential. The zeta potential is the parameter used to determine the charges attained by the AgNPs present in liquid medium. Nanoparticles with high zeta potential show the repulsive force, as a result they are stable without forming aggregation [16]. In this experiment, the zeta potential of both nanoparticles was negative, which might be due to the protein capping on nanoparticles. The AgNPs formed by F. oxysporum showed lower negative zeta potential, 6.11 mV (Fig. 5A), than the nanoparticles synthesized from P. sorghina with zeta potential of 12.5 mV (Fig. 5B). Regardless of lower values of zeta potential,

both of the nanoparticles were found to be highly stable for more than 180 days, which can be attributed to capping layer derived from metabolites present in the fungal filtrate [21].

3.4 Transmission electron microscopy (TEM) Transmission electron microscopic studies of AgNPs confirmed the formation of quite spherical as well as irregular shaped nanoparticles (Fig. 6). The spherical morphology of the nanoparticles revealed that the mycosynthesized silver nanoparticles were isotropic in nature. The mean size of nanoparticles was found to be 25 and 20 nm for the AgNPs synthesized by F. oxysporum and P. sorghina, respectively.

3.5 Evaluation of antibacterial activity of nanoparticles Antibacterial activity of AgNPs was assessed against various waterborne pathogens by using the disk diffusion test and the MIC and MBC of synthesized silver nanoparticles required for growth inhibition of the pathogens were determined. The values of the inhibition zone achieved by NPs against tested organisms are given in Tabs. 1 and 2. The growth inhibitory effect of AgNPs synthesized from P. sorghina and F. oxysporum against all the tested organisms was found to be more or less similar. Encouragingly, a significant increase in the inhibition zones was observed for both batches of AgNPs in combination with standard antibiotics. AgNPs synthesized from P. sorghina showed maximum activity against S. aureus, whereas, E. coli was found to be less susceptible to AgNPs. The activity of P. sorghina-mediated synthesized AgNPs against C.

Figure 4. Nanoparticle tracking analysis of AgNPs synthesized by P. sorghina. (A) Nanoparticles size distribution histogram showing the average size of 41 nm (B) NTA 3D plot of particle size distribution.

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Figure 5. Zeta sizer analysis of AgNPs synthesized by (A) F. oxysporum and (B) P. sorghina.

Figure 6. TEM studies showing morphologies of AgNPs synthesized by (A) F. oxysporum and (B) P. sorghina (both images at the scale of 50 nm).

Table 1. Antibacterial activity of AgNPs synthesized from P. sorghina

Inhibition zone (mm) Test bacterium S. aureus C. violaceum B. borstelenins E. coli

Fungal filtrate

AgNO3

AgNPs

Amphicillin

AgNPs þ Amphicillin

Nil Nil Nil Nil

11.0 0.5 11.3 1.1 14.0 1.7 12.0 0.5

16.3 2.3 14.0 1.5 15.0 1.7 12.7 0.5

30.0 1.7 37.0 0.2 38.3 2.0 25.0 9.5

33.3 0.5 39.7 0.5 39.7 0.5 28.3 1.0

All values have been represented as mean standard deviation.

Table 2. Antibacterial activity of AgNPs synthesized from F. oxysporum

Zone of inhibitions in mm Test bacterium S. aureus C. violaceum B. borstelenins E. coli

Fungal filtrate

AgNO3

AgNPs

Amphicillin

AgNPs þ Amphicillin

Nil Nil Nil Nil

15.6 1.1 13.0 2.6 15.3 3.5 14.0 1.7

14.3 0.5 14.3 3.2 17.0 1.0 14.0 2.3

30.3 2.0 33.3 5.1 34.7 5.5 21.0 3.6

33.0 1.0 35.7 4.0 35.3 2.0 22.3 2.5

All values have been represented as mean standard deviation. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim



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Table 3. Minimum inhibitory concentration of AgNPs against waterborne pathogens

AgNPs (F. oxysporum) Test bacterium

AgNPs (P. sorghina)

MIC (mg mL )

MBC (mg mL )

MIC (mg mL )

MBC (mg mL1)

10 10 10 8

20 10 20 10

8 10 10 10

10 10 20 20

S. aureus C. violaceum B. borstelenins E. coli

1

1

1

All values are significant at the 0.05% level of significance. violaceum was drastically increased when the NPs were combined with the standard antibiotic amphicillin. The findings suggest that the activity of AgNPs can be enhanced when treated in combination with antibiotics. In case of F. oxysporum-synthesized AgNPs, high antibacterial activity was noted against B. borstelensis followed by S. aureus, E. coli, and C. violaceum. Both fungal filtrates were evaluated for their effects on bacterial growth; the results confirmed the insignificant impact of filtrates on bacterial growth. The values of MIC and MBC of AgNPs are provided in Tab. 3. Both batches of AgNPs were able to inhibit the growth of waterborne pathogens at minimum concentration 8–20 mg mL1. Moreover, the silver nanoparticles showed prominent bactericidal activity at minimum concentration 10–20 mg mL1 for all tested pathogenic strains.

4 Concluding remarks The present study provides evidence of mycosynthesis of AgNPs which is a non-toxic, eco-friendly, and cheap method to yield nanoparticles strictly within the nanometer range. Both F. oxysporum and P. sorghina were found to be capable of AgNPs synthesis in extracellular environment, which were quite stable in solution due to capping of nanoparticles by the proteins present in the cell filtrate. The average size of crystalline and negatively surface charged AgNPs synthesized from F. oxysporum and P. sorghina was found to be 45 24 and 41 21 nm, respectively. Nanoparticles of both batches were spherical and isotropic in nature. The AgNPs synthesized in the present study showed promising antibacterial activity against waterborne pathogens namely, S. aureus, C. violaceum, B. borstelensis, and E. coli. The activity of AgNPs was enhanced when used in combination with commercially available antibiotics. Thus, AgNPs can be used as antimicrobial agent alone or in combination with antibiotics for development of nanoparticles supported nextgeneration water treatment devices. The authors have declared no conflict of interest.

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