3.1. Silver tolerance and strains selection:
Silver ion tolerance experiment was conducted to correlate the tolerance to synthesis process. The six screened strains (U9, U12, U13, U17, U19 and U30) tolerate Ag+ up to 300 mg/l, only four of them grew at 500 mg/l (Fig. 2). U13 was the most tolerant by showing a tolerance percentage of 56% at 500 mg/l Ag+. Bacteria have different strategies to combat heavy metals toxicity. Biosorption of silver ions onto Gram-positive bacterial biomass in crystalline forms has been suggested as a possible mechanism of resistance (Li et al. 2011). Gelagutashvili (2013), found the efficiency of biosorption of Cr(III), Cr(VI), Cd(II), Au(III) and Ag(I) ions depends on bacterial strain and uptake conditions.
3.2. Biosynthesis of Ag NPs:
The formation of Ag NPs was observed visually by change of solution colour from colourless to brown. Streptomyces sp. U13 showed change in solution colour (Fig. 3), while the rest strains only the biomass colour was changed to greyish brown with slight change in colour of the solution. Extracellular formation of Ag NPs was confirmed by UV-Vis scan from 300 to 700 nm. The UV–visible spectra (Fig. 4) showed increased absorbance and a peak was recorded at 434 nm only in case of Streptomyces sp. U13. Several studies proved that numerous strains of Streptomyces synthesise Ag NPs (Vidhyashree et al. 2015; Abou-Dobara et al. 2017; Al-Dhabi et al. 2019; Bakhtiari-Sardari et al. 2020).
Streptomyces strains can synthesise Ag NPs intracellularly and extracellularly. Although the biomass colour of the rest tested strains was changed indicating intracellular biosynthesis, only the extracellular Ag NPs were characterized. Extraction of the intracellular Ag NPs needs application of suitable detergents or ultrasonic treatment. Economically, extracellular synthesis of Ag NPs is preferred for large-scale production because it is cheap, simple and can be easily purified. Streptomyces mechanisms for reducing AgNO3 into Ag NPs are not fully recognized. Ag NPs could be synthesized by direct contact with mycelial mass (Tsibakhashvili et al. 2011), cell free metabolites (Vidhyashree et al. 2015; Al-Dhabi et al. 2019; Bakhtiari-Sardari et al. 2020), or partially purified metabolite (Abou-Dobara et al. 2017).
UV-vis scan results reflect differences in Ag NPs characterization as well as in quantity. Increasing Ag+ concentrations increased the quantity of the produced Ag NPs, as shown in Fig. 5a and 5c. The colour of the solutions became darker, and the absorbance increased in spectrophotometric scanning. Using 100 mg Ag+/l (equivalent to 1 mM AgNO3), the highest Ag NPs concentration was obtained as shown in scan curve height (Fig. 5a). Dada et al. (2018), reported that most appropriate and suitable concentration of AgNO3 is 1 mM as where better surface plasmon resonance was obtained. Wagi and Ahmed (2020), found that increasing in Ag NPs due to AgNO3 increase is not the case for every strain and varies from species to species.
Increasing biomass weights also affecting the synthesis process by increasing the produced Ag NPs quantity (Fig. 5b), but when the biomass elevated to 100 mg (dry weight equivalent), the produced Ag NPs became greyish and precipitated rapidly. The results showed that the best conditions for synthesis of Ag NPs were 100 mg Ag+/l and 80 mg dry weight equivalent. These conditions were selected to synthesise Ag NPs to be characterized and used in the wastewater disinfection experiments.
3.3 Ag NPs characterization:
3.3.1 UV-Vis spectroscopy
Absorption band in the visible region considers the main characteristic of silver nanoparticles. Optical absorption spectra of Ag NPs are dominated by surface plasmon band which shows red end or blue end shift depends on size, shape, and aggregation of the particles (Lufsyi et al. 2015). Figure 6a showed that successfully biosynthesized silver nanoparticles, with light brown colour, had the surface plasmon absorption band with a maximum of 434 nm in the visible region. In Fig. 6b, abroad signal in the visible range with a maximum at about 414 nm was observed for commercially available synthesised silver nanoparticles (ink nanosilver) with dark brown colour due to the combined vibration of Ag NPs in resonance with the light wave (Dubey et al. 2010). The wideness of the absorption spectrum curves is good evidence of the nanoparticle size and the two observed absorption bands of Ag NPs indicate the presence of spherical or roughly spherical shape of particles (Guzmán et al. 2008).
3.3.2 X-ray diffraction studies (XRD):
The XRD pattern of biologically and commercially available synthesised silver nanoparticles was recorded in the range between 20 and 80º (2θ), step of 0.04° and 2 sec per step. The samples for XRD analysis were prepared by using a pipette to put drops of Ag NPs solution on a glass slide to achieve uniformly thin films. The XRD pattern of the biosynthesized Ag NPs sample was illustrated in Fig. 6c. The XRD pattern showed the Bragg’s diffraction peaks of Ag NPs at 38.16°, 45.04° and 64.75°, respectively corresponding to (111), (200) and (220) planes of the face centred cubic lattice. The other peak at 32.78° may be appeared due to crystallization of bio-organic phase (Sathiya et al. 2014; Selvi et al. 2016). These results are in a good agreement with reported results on previous literature (Probin et al. 2015; Vasudeva et al. 2018). Figure 6d shows that the commercially available synthesized Ag NPs (ink nanosilver) are found to be amorphous. These results are in line with previous reports (Harajyoti et al. 2011; Sayed et al. 2018).
3.3.3 Fourier transform-infrared spectroscopy analysis (FTIR):
FTIR analysis was performed for biosynthesized and commercially available Ag NPs. The biosynthesized Ag NPs shows absorption peaks at 3449.06, 2092.39, 1637.27, 1460.81, 1259.29 and 1161.9 as shown in Fig. 6e. The functional groups corresponding to these absorption peaks are O-H stretching, C=O stretching, C=O stretching of amide groups of proteins, amino and amino-methyl stretching groups of protein, C–O –C stretching and C–O stretch (Probin et al. 2015; Vasudeva et al. 2018). The sharp absorption beak at 1637.27 cm-1 indicates that proteins are interacting with biosynthesized Ag NPs without affecting on their secondary structure during reaction with Ag+ ions or after binding with Ag NPs (Fayez et al. 2010). The IR spectroscopic analysis confirms that presence of proteins acting as capping agent to prevent agglomeration of nanoparticles and provide stability to the medium (Sathyavathi et al. 2010).
FTIR spectrum of the commercially available Ag NPs (Ink nanosilver) was presented in Fig. 6f. It shows that seven peaks located at 3470.28, 2096.24, 1639.2, 1436.71, 1163.83, 1114.65 and 1039.44 cm−1are assigned for O–H, C ≡ C, C=C, N–H, C–O, C–O and C–N stretch vibration mode, respectively. The same results were reported on previous literature (Preetha et al. 2013; Kumari et al. 2016).
3.3.4 High resolution transmission electron microscope (HR-TEM):
ISO 21363:2020 (Nanotechnologies – Measurements of particle size and shape distributions by transmission electron microscope) has published recently by international organization for standardization (ISO) to study the morphology of nanoparticles. The size and shape of the two samples of silver nanoparticles were obtained by using the nanometrological technique HR-TEM. The samples were prepared by dropping Ag NPs solution onto a micro copper grid. TEM images Fig. 6g and 6h revealed that the biosynthesized Ag NPs and commercial Ag NPs (ink nanosilver) well dispersed and had spherical shape. The size of biosynthesized Ag NPs ranging from 5 to 37 nm with mean diameter 22.75 ± 10.88 nm and the size of ink Ag NPs ranging from 2 to 26 nm with mean diameter 16.523 ± 7.792 nm.
3.4 Wastewater disinfection experiments:
Reduction of total coliform count after column experiment using the two tested substrates loaded with Ag NPs was shown in Fig. 7a. The results revealed that after 6 hrs the reduction percentage of total coliform count was 100% in limestone gravel column while 50% reduction was recorded in foam column. Silver discharged from limestone gravel column was less than 0.01 mg/l which is below the standards to be discharged into the aquatic environment (Standards and specifications for liquid wastes, 2015). This result may be due to that Ag NPs are stable and remains in zero-valent state, but unreduced Ag+ can loaded onto the substrate and released much easier again (Pasricha et al. 2012). It worth to note that the used substrates have different binding capabilities to Ag NPs. Previous work by Pasricha et al. (2012) reported that cotton fabrics bind to Ag NPs more than wool and nylon.
The type of substrate controls the amount of Ag NPs that loaded on it and thus control the disinfection process. Moustafa (2017) removes 98.5% of 41 × 105 CFU total coliform/100 ml after 24 h by foams that have been pre-soaked in 1118.6 mg/l Ag NPs. In this experiment, foams removed 50% of 25×105 CFU/ml after 6 hrs only, may be due to the lower loaded Ag NPs and contacting time. Limestone gravel is a high porous substrate and used in wastewater treatment processes (Fahim et al. 2019; El-Shahawy et al. 2020). Due to the higher efficiency of limestone than foam in binding and disinfection, limestone was chosen to test different disinfection conditions. As shown in Fig. 7b, the reduction percentage of total coliform count was 100% after 6 hrs of using continuous flow mode or static mode. Continuous flow mode was performed at 2 ml/min, Mthombenia et al. (2012), also found that 2 ml/min was the most efficient flow rate.
The efficiency of disinfection system depends on different parameters, one of them is the concentration of the disinfectant. The increased concentration of Ag NPs ensures the complete disinfection process (Mpenyana-Monyatsi et al. 2012; Moustafa, 2017), but on the other hand it will certainly rise the cost and increase the discharged silver. Hence using lower but efficient Ag NPs concentration to coat the substrate will reduce the cost. The results showed that after half an hour the two tested concentration (200 and 400 mg/l) eliminate about 50% of the initial total coliform count. With time passing columns loaded with 400 mg/l concentration gradually reduce the count more than 200 mg/l but both concentrations were completely eliminating the total coliform after 150 min (Fig. 8). 200 mg/l of the biosynthesized Ag NPs was considered the efficient concentration.
The efficiency of Ag NPs in certain field depends on their characteristics. The biosynthesis process produced heterogeneous Ag NPs suspension in both shape and size. The commercial Ag NPs were more homogenous in shape and size. The efficiency of both biosynthesized and commercial Ag NPs was determined. After 90 min there was no difference in reduction percentage, and both completely eliminate the total coliform after 150 min (Fig. 9).