Chemicals
All chemicals were of analytical grade and were purchased from Sigma-Aldrich (Sigma, St. Louis, MO, USA) unless otherwise indicated.
Synthesis of AgNPs-G
AgNPs-G were obtained by adding 2 mL of a 10-2 M aqueous solution of AgNO3 to 100 mL of 0.3 M β-D-glucose water solution. The mixture was boiled for 30 min under vigorous stirring. The deep yellow colour of the solutions indicated the formation of AgNPs-G. Deionised ultra-filtered 18.2 MΩ water prepared with a Milli-Q Integral Water Purification System (Merck Millipore Headquarters, Billerica, MA, USA) was used for all preparations. All glassware was washed in an ultrasonic bath of deionised water and not ionic detergent, followed by thorough rinsing with Milli-Q water and ethanol (Carlo Erba, Milan, Italy) to completely remove not ionic detergent contaminants. Finally, glassware was dried prior to use.
AgNPs-G characterization
Transmission Electron Microscopy (TEM) and UltraViolet–Visible (UV–Vis) were used to evaluate
the average and distribution size and morphology of the NPs.
TEM analysis was performed by a Hitachi 7700, at 100 kV (Hitachi High Technologies America Inc., Dallas, TX, USA). A drop of AgNPs-G solution diluted in BBM was placed onto standard carbon-supported 600-mesh copper grid. Particle size distribution has been obtained using the ImageJ program (US NIH, Bethesda, USA). A histogram was created by counting 500 particles. Optical spectra were obtained by measuring the absorption of the solution in the range between 300 and 800 nm by using a T80 spectrophotometer (PG Instruments Ltd., Leicester, UK) in a quartz cuvette with a 1 cm optical path.
The stability of AgNPs-G was assayed in BBM. In particular, the dissolution of AgNPs-G, in terms of release of Ag+, up to 10 days at r.t. in BBM culture medium was determined by atomic absorption spectroscopy (AAS; Thermo Electron Corporation, M-Series) after precipitation of AgNPs-G by ultracentrifugation (24,900g; 30 min at 4 °C). The detection limit was 1 μg/L. Triplicate readings were analysed and control samples of known Ag concentration were analysed in parallel generating data with the standard deviation of three independent samples. Silver ions dissolution degree was expressed as percentage (%) of total Ag+, as AgNO3, used to reach the highest concentration of NPs solution during treatment.
Chlorella vulgaris culture
The algae were cultured in 250 mL flasks containing 100 mL of BBM and covered with loose cotton. The flasks were placed on a shaker to keep the turbulence of culture medium simulating the natural stream of water. The cultures were kept at 23 ± 1 °C under illumination of approximately 73.6 μmol m-2 s-1 with daily cycles of 12-h light and 12-h dark. The culture cell density was monitored with a spectrophotometer (Pharmacia Biotech, Stockholm, Sweden) at 684 nm every 24 h. Cells in the exponential phase were used for all experiments.
Growth-inhibition test
The evaluation was performed following the Organization for Economic Co-operation and Development (OECD) 201 algal growth inhibition test guidelines [51]. Algae were incubated for 24 h and a week with Ag ions (0.1 μg/L and 1 mg/L of silver nitrate) and with different concentrations of AgNPs-G: 0.1, 1, 10, 100 μg/L and 1 mg/L with three replicates for each concentration. The inhibitory rate of growth was obtained by using the formula (1):
Inhibitory Rate (IR)% = (1 – N/No) x 100 (1)
where N is the density of cells/mL in the samples treated with AgNPs-G, No is the density of cells/mL in the control samples. The test was performed with three independent experiments (with three technical replicates for each repeated experiment) by using the same batch of algae and AgNPs-G.
Chlorophyll content
Treated samples were centrifuged to remove culture media. Then, 90% acetone was added to tubes. Sealed tubes were shaken to ensure that microalgae cells are in the whole solvent volume and centrifuged at 5000 rpm (5236 g) for 5 min. Chlorophyll-a concentration was determined by measuring the optical density (OD) of supernatant by spectrophotometer (Pharmacia Biotech, Stockholm, Sweden). Quantitative determination was done according to Arnon et al. (1949).
Biodistribution and subcellular localisation of AgNPs: transmission electron microscope analysis
The ultrastructural analysis of C. vulgaris treated with different concentrations of Ag ions and AgNPs-G for one day and one week was performed by TEM (Hitachi HT 7700 transmission electron microscopy) analysis.
Algae were centrifuged to remove culture media and then fixed with glutaraldehyde (2.5% in sodium cacodilate buffer 0.1 M, pH 7.2) for 2 h at 4 ºC. Then, samples were washed twice for 15 min in sodium cacodilate buffer, postfixed in osmium tetraoxide (1% in sodium cacodilate buffer 0.1 M, pH 7.2) and washed twice for 30 min in deionized H2O. Samples were stained with 0,5% uranyl acetate o.n. at 4 °C. Samples were dehydrated in a graded series of ethanol, from 30% to 100%. After dehydration, samples were embedded in Spurr resin (TAAB, Berks, UK).
Ultrathin sections of 50 nm in thickness were then cut using an ultramicrotome PowerTome PT-PC (RMC, Arizona, USA). Sections were picked up in 200 mesh copper grids and examined under a Hitachi HT7700 transmission electron microscope (Tokyo, Japan) at 75 kV.
Samples were analyzed by EDX microanalysis with the TEM module of the Auriga 405 microscope (Carl Zeiss AG, Oberkochen, Germany) for the elemental analysis of the electron-dense particles inside the cells.
X-Ray diffraction (XRD) analysis
To determine the amount of Ag+ inside algal cells, XRD analysis was performed with samples of algae treated for a week with AgNPs-G. Only AgNPs-G were used as positive control and a culture of only Chlorella vulgaris as negative control. Samples were collected, dried at 60°C and then sintered at 650°C for 4h under nitrogen protection. The analysis was performed with X-ray diffractometers (Malvern Pananalytical, United Kingdom).
ICP-OES analysis
A series of AgNPs-G stocks (0.1, 1, 10, 100 μg/L and 1 mg/L) were prepared in BBM. Algal samples with different AgNPs-G exposure concentrations and times were vacuum filtered with 0.45 μm Millipore filter to separate algae from the culture medium. Samples were acidified with HNO3 and analysed by ICP-OES to determine Ag content. The absorbed Ag by algal cells was calculated by the total Ag (TAg, also determined by ICP-OES by measuring stock solutions) minus the Ag in filtrates (FAg). Therefore, the percentage of absorbed Ag was calculated as (TAg - FAg)/ TAg x 100.