Green synthesis of metal nanoparticles loaded ultrasonic‐assisted Spirulina platensis using algal extract and their antimicrobial activity

Abstract

The synthesis of metal nanoparticles (NPs) loaded on the ultrasonic‐assisted Spirulina platensis (MNPs/UASP) was investigated using the green synthesis method. The S. platensis algal extract was taken as a reducing agent. The formations of metal NPs were characterised using UV–visible spectroscopy, Fourier transform infrared spectroscopy and scanning electron microscopy. The antimicrobial activity of different metal NPs demonstrated various inhibitory activities against one gram‐positive bacteria (Staphylocicus aureus), four gram‐negative bacteria (Klebsiella pneumonia, Proteus vulgaris, Pseudomonas aeruginosa and Escherichia coli) and one fungus (Aspergillus niger). Both CrNPs/UASP and ZnNPs/UASP show good antimicrobial activity when compared with other MNPs/UASP against microorganisms. This MNPs/UASP is effective in preventing and treating the microbial infection and water pollution in the environment.

1 Introduction

Nowadays, water resources are depreciating very fast due to the increase in world population and global warming. So the recovery and reuse of treated waste waters and the utilisation of fresh water are important to control the requirement of water. Contamination of water is a serious problem in the world, which causes diseases to humans by physical, chemical and microbiological agents [1]. Microorganisms such as Escherichia coli [2], Shigella spp. [3], Vibrio spp. [4], Klebsiella pneumonia [5], Pseudomonas aeruginosa [6], Staphylococcus aureus [7] etc. are responsible for the health acquired infections and water contamination. Activated carbon (AC) is an effective adsorbent for solving physical and chemical as well as biological water contamination problems because of its large surface area, proper pores and its excellent adsorption capacity. This AC is commonly produced by agricultural waste such as bamboo tree [8] and coconut husk [9]. The antimicrobial property of AC material can be improved by the impregnation of metallic nanoparticles (MNPs) on its surface to prevent the growth of microorganisms present in water [10]. In recent years, nanotechnology is one of the effective fields for research. Nanoparticles (NPs) are used for various applications owing to their unique properties such as optical, mechanical, electronic, magnetic and chemical properties. These properties can be attributed to their small size, large surface area‐to‐volume ratio and intrinsic surface reactivity [11, 12]. Traditionally, MNPs are synthesised by various methods such as chemical and physical methods. However, these conventional methods are very expensive and the use of toxic chemicals, such as reducing agents, organic solvents and stabilisers, which have been used for the prevention of unwanted agglomerate formation, is potentially hazardous to the environment [13, 14]. Currently, the biological synthesis method has been an excellent alternative to the traditional method because of its clean, non‐toxic and eco‐friendly nature. High pressure, temperature, energy and toxic chemicals are not necessary for this green synthesis method. This method also has various other advantages: (i) water is used as a solvent, (ii) for a long time, the synthesised NPs are safely preserved in a desiccator and, if required, can be dispersed in aqueous solution and (iii) there is no stabilising or capping agent used [15, 16, 17]. The MNPs are synthesised by various metal ions, such as silver, chromium, iron, copper, zinc and lead, using enzymes [18], microorganism [19] and plant extracts [20] as a reducing agents.

In the current study, the blue green Spirulina platensis alga is used as a reducing agent Since this microalga has potential natural elements such as proteins, vitamins, amino acids, essential fatty acids and other valuable biological substances, which are responsible for the production of MNPs [21, 22]. This study investigates the synthesis of MNPs (AgNPs, CrNPs, PbNPs, ZnNPs and FeNPs) loaded on ultrasonic‐assisted S. platensis (UASP) using the S. platensis alga extract itself used as a reducing agent in the green synthesis method. Further, the different metal NPs loaded UASP (MNPs/UASP) are characterised by using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and UV–visible spectroscopy analysis. The antimicrobial activity of MNPS/UASP is tested for various microorganisms such as S. aureus, E. coli, P. aeruginosa, K. pneumonia, Proteus vulgaris and Aspergillus niger.

2 Materials and methods

2.1 Preparation of AC (UASP)

S. platensis was purchased from Arwind Enterprise, Vellore, Tamilnadu, India. This algal biomass was thoroughly washed with distilled water to separate dust and other impurities and then kept for drying in sunlight for 3 days. Later, this sun‐dried biomass was ground to fine powder and then stored in a plastic container to avoid contamination. The prepared materials were called raw S. platensis (RSP). From RSP, surface modified S. platensis (SMSP) powders were prepared to improve the surface area. In this treatment, 1:2 ratios by weight percentage of treated RSP with concentrated sulphuric acid were mixed and left for 24 h. It was then washed with distilled water several times until it reaches the supernatant pH ≈ 7 to eliminate the excessive amount of acid present in the mixture. This wetted biomass was dried for about 3 h in a hot air oven at 80°C. This dehydrated material was powdered and stored in the plastic container. Finally, UASP was prepared by using SMSP powders for increasing the adsorption capacity. For this synthesis, a 3.0 g surface modified alga with 40 ml of distilled water was taken in a 50 ml beaker. Sonication was performed for 1 h at a speed of 500 rpm and the working frequency of the ultrasonicator was 24 kHz. After the sonicated mixture was filtered using Whatman filter paper, it was dried for 24 h at 40°C in a hot air oven and stored in the plastic container [23]. This UASP was also called as AC, which was used for the antimicrobial analysis.

2.2 Extraction of S. platensis

For extraction, 10 g of S. platensis powder was taken with 100 ml of distilled water in a 250 ml beaker and this mixture was boiled for 15 min at 80°C. Then this slurry was filtered using Whatman filter paper and separated supernatant. This extracted S. platensis solution was used for further study.

2.3 Green synthesis of metal NPs loaded UASP (MNPs/UASP)

In the preparation of metallic loaded NPs (MNPs/UASP), five different metal ions (such as silver, chromium, iron, lead and zinc) were used to load on the surface of the UASP (which act as an AC). Spirulina platensis extract was used as a reducing agent. For AgNPs/UASP preparation, 1.69 g of silver nitrate (Merck Specialties Private Limited, Mumbai) with 20 ml of distilled water was dissolved in a stoppered Erlenmeyer flask. One gram of UASP powder was also added to this mixture and kept in a temperature controlled incubator at 40°C for 24 h. After 24 h, this mixture was placed in an ice bath for cooling. To this mixture, freshly prepared S. platensis alga extract (reducing agent) was slowly added and left for 24 h stirring at room temperature. Then the solution was filtered using Whatman filter paper, washed with distilled water to remove excess silver ions and finally dried for 24 h at 80°C. The same procedure was used for other MNPs preparations and only change in the amount of metal ions is used. Four grams of potassium dichromate nitrate (Merck Specialties Private Limited) were used for CrNPs/UASP synthesis. In the same manner, for the preparation of PbNPs/UASP and ZnNPs/UASP, 0.59 g of lead nitrate and 2.19 g of zinc sulphate heptahydrate (Merck Specialties Private Limited) were used. 0.01 M ferric chloride (Merck Specialties Private Limited) solution was prepared for FeNPs/UASP synthesis.

2.4 Antimicrobial activity of MNPs/UASP

The Agar well diffusion method was used to study the antimicrobial activity of the synthesised different metal NPs, which was performed to evaluate the zone of inhibition activity [24]. In this study, five bacterial strains (one gram positive and four gram negative) and one fungal strain were used which includes S. aureus (MTCC 902), K. pneumonia (MTCC 4030), P. aeruginosa (MTCC 741), E. coli (MTCC 443), P. vulgaris (MTCC 742) and A. niger (MTCC 282), respectively. For making an agar plate, 20 ml of sterile molten nutrient agar (Himedia Laboratories, Mumbai) was filled in sterile petriplates. Then each bacterial and fungal inoculum was swapped onto the individual petriplates. Using a sterile cork borer, a 6 mm diameter range of wells was made on nutrient agar plates. In these wells, metal NPs were filled with a micropipette and then incubated for 24 h (bacteria) and 72 h (fungal) at 37°C. Finally, the zone of inhibition of microbes in diameter was measured and compared with control (antibiotic) to understand the antimicrobial activity of metal NPs.

2.5 Characterisation of MNPs

MNPs/UASP was analysed by using UV–vis spectroscopy, SEM and FTIR. The formations of metal NPs were confirmed by UV spectra analysis. The surface morphology of the MNP‐UASP samples was observed by SEM analysis and the functional groups present in the different MNP‐UASPs were identified by FTIR spectroscopy using Perkin Elmer FTIR C 100566.

3 Result and discussion

3.1 Fourier transform infrared spectroscopy

The FTIR spectra of the pure UASP are shown in Fig. 1 a which was already reported in the previous paper [25]. In the UASP, the intense peaks observed at 3277.71 cm⁻¹ and 2918.03 cm⁻¹ confirm the presence of the stretching vibrations of the primary and secondary amines. Simultaneously, the bending vibrations were seen at 1626.51, 1531.82 and 1452.37 cm⁻¹. The stretching vibrations of aliphatic amines were observed at peak 1053.47 cm⁻¹. The FTIR spectra of the synthesised metal NPs (Ag, Cr, Zn, Pb and Fe) loaded UASP are shown in Figs. 1 b –f. For CrNP/UASP, the FTIR spectra show the peak at 544.44 and 508.32 cm⁻¹, which is due to Cr–O (M–O) stretching and the amide group observed at the peak 1630.43 cm⁻¹ shown in Fig. 1 b. Peaks at 3286.85, 2921.76, 2851.15, 1735.71, 1518.28, 1053.56, 606.24 and 548.34 cm⁻¹ were obtained in the spectra of AgNP‐UASP shown in Fig 1 c. The FTIR spectrum (Fig 1 c) reveals that bands at 3286.85, 2921.76 and 2851.15 cm⁻¹ were observed, which corresponds to the stretching vibrations of the primary and secondary amines, respectively, while their corresponding bending vibrations were seen at 1518.18 cm⁻¹. Carboxyl or carbonyl groups are also available in this same region. The peak at 1735.71 cm⁻¹ is attributed to C=C stretching (non‐conjugated). The peak at 1053.56 cm⁻¹ corresponds to the stretching of aliphatic amines and a new peak at 606.24 and 548.34 cm⁻¹, which may be due to the Ag–O (metal oxide) bond stretching. This confirms that Ag NPs have been successfully impregnated on the UASP material. Similarly, the infrared spectra for PbNP/UASP (Fig. 1 d) show that the absorption peak at 594.65 cm⁻¹, which indicates the presence of Pb–O (metal oxide) and the peak at 1161.40 cm⁻¹, confirms the presence of lead. Two peaks observed at 2922.78 and 2853.02 cm⁻¹ could be assigned to the stretching of alkene groups. The peak at 1624.70 cm⁻¹ assigned C=O stretching of amide group, N–H bending of amide group was observed at the peak 1533.19 cm⁻¹ and C–N stretching of (alkyl) amide group. In Fig. 1 e, peaks at 2922.94 and 2853.25 cm⁻¹ for ZnNP/UASP correspond to the stretching vibrations of the primary and secondary amines, respectively, and the peak at 1061.77 cm⁻¹ represents the stretching of aliphatic amines. The peaks at 1625.13, 557.75 and 510.38 cm⁻¹ confirm the Zn–O stretching and deformation vibration, respectively. Similarly, in Fig. 1 f, the new peaks appear at 577.06 and 456.96 cm⁻¹ for FeNP/UASP, which confirms the presence of Fe–O. After the impregnation of metal NPs on the UASP, the FTIR results give less intense peaks and some new peaks as well as the disappearance of peaks such as 3277.71 and 1452.37 cm⁻¹. This FTIR analysis of different metal NPs on the UASP makes it clear that the impregnation of metal NPs on the surface of the UASP material was successfully done.

Fig. 1.

FTIR images for pure UASP and different metal nanoparticles loaded UASP

3.2 UV–visible spectroscopy

UV–visible spectroscopy is an effective technique to study the formation and stability of metal NPs. UV–visible spectra of different MNPs/UASP samples are shown in Figs. 2 a –e. It was noticed that during the synthesis of MNPs in aqueous medium, the colour of solutions is changed based on the type of metal ions (Ag, Cr, Zn, Pb and Fe) present in solution, which confirms the formation of metal NPs. For AgNPs, the solution colour was changed from yellow to brown in water; this change happened after the addition of algal extract and it may be due to surface plasmon vibration in NPs [26, 27]. Similarly, for Cr NP, the colour changes from orange to green, PbNPs change from white to blue, ZnNPs change from green to white and the colour changes from yellow to green for FeNPs. Absorption peaks obtained for different MNPs are 364.2 nm (CrNPs), 449 nm (AgNPs), 383 nm (PbNPs), 379 nm (ZnNPs) and 261.8 nm (FeNPs) as shown in Figs. 2 a –e. Generally, the shape of resonance peaks depends on the nature of NPs. Small, uniform and narrow size distribution of NPs provides sharp absorbance. Similarly, broad absorbance produced by wide size distribution of NPs. In this work, the broader peak was observed for all types of MNPs, which makes it clear that the MNPs are wide size distributed and also may be due to encapsulation of the MNPs by biological components in algal extract [28, 29]. The UV–visible spectrum analysis confirms the formation of NPs (Ag, Cr, Pb, Zn and Fe) by the peak position.

Fig. 2.

UV–visible spectra for various metal nanoparticles

(a) CrNPs, (b) AgNPs, (c) PdNPs, (d) ZnNPs, (e) FeNPs

3.3 Scanning electron microscopy

SEM analysis provides information about the size and topography of pure UASP and various MNP/UASPs as shown in Figs. 3 a –f. Owing to the ultrasonication of SMSP, rougher and irregular pores developed on the surface of UASP as shown in Fig. 3 a. The SEM images of the MNP/UASP (Ag, Cr, Pb, Zn and Fe) clearly show that the distribution of MNPs on the surface of the UASP material is shown in Figs. 3 b –f. These NPs are partially filled on the surface of the UASP material, i.e. unfilled pores are also available on the surface of UASP. So, more active sites in UASP surface are helpful to attract more number of NPs on it. Finally, the SEM analysis results confirmed that the metal NPs (Ag, Cr, Pb, Zn and Fe) were successfully impregnated on the UASP material.

Fig. 3.

Scanning electron microscope images

(a) Pure UASP, (b) CrNP‐UASP, (c) AgNP‐UASP, (d) PbNP‐UASP, (e) ZnNP‐UASP, (f) FeNP‐UASP

3.4 Antimicrobial activity of MNPs/UASP

In this study, the antimicrobial activities of the various synthesised metal NPs (MNPs/UASP) such as AgNP/UASP, CrNP/UASP, PbNP/UASP, ZnNP/UASP and FeNP/UASP are analysed using the Agar well diffusion method against six different types of microorganisms such as one gram‐positive bacteria (S. aureus), four gram negative bacteria (E. coli, P. aeruginosa, K. pneumonia and P. vulgaris) and one fungi (A. niger). The white and cloudy areas show the bacterial and fungal growth while the transparent areas around the MNPs reveal the inhibition zone in millimetres as shown in Figs. 4 a –f. The inhibition zone diameters are mainly obtained based on the type of metal ions, algal extract and microorganisms. These MNPs are having the antimicrobial activity due to the interaction of positively charged metal ions and negative charged microbial cell membrane, which causes the membrane rupture and cell lysis [30]. The measured values of zone of inhibition of MNPs are listed in Table 1, which makes it clear that chromium and zinc NPs are having greater antimicrobial activity against both bacterial and fungal strains, when compared with all other metal NPs. Thus, these synthesised metal NPs are used enormously for various applications, especially in wood healing, cancer therapy, cotton fabrics and textiles, water paint etc. [31].

Fig. 4.

Antimicrobial activity of synthesised metal nanoparticles

Table 1.

Antimicrobial activity of MNPs loaded UASP synthesised using S. platensis

Microorganisms	Zone of inhibition, mm
	AgNP/UASP	CrNP/UASP	PbNP/UASP	ZnNP/UASP	FeNP/UASP
S. aureus	8	17	6	14	1
P. aeruginosa	5	15	5	13	1
K. pneumonia	4	12	7	9	3
E. coli	9	14	6	16	2
P. vulgaris	7	12	8	11	1
A. niger	6	19	7	13	1

4 Conclusion

The green synthesis method is an effective, non‐toxic and eco‐friendly method, which was used for the preparation of metal NPs (MNPs) such as AgNPs, CrNPs, PbNPs, ZnNPs and FeNPs loaded on the UASP using the same algal extract as a reducing agent. The surface morphology and functional groups present in the MNPs/UASP were confirmed by the SEM and FTIR techniques. The maximum absorbance and their respective band gap energy of MNPs were determined by using UV–visible spectroscopy analysis, which confirmed the formation of nanoparticles. The antimicrobial activities of different MNPs/UASP were studied against six microorganisms such as one gram‐positive bacteria (S. aureus), four gram‐negative bacteria (K. pneumonia, P. vulgaris, P. aeruginosa and E. coli) and one fungus (A. niger). The result concluded that the antimicrobial activity of CrNPs and ZnNPs was greater than all MNPs against the selected microorganisms.