Supporting Information:

Toxicity of bare and surfaced functionalized iron oxide nanoparticles towards microalgae

Pey Yi Toh1, Wan Yii Tai1, Abdul Latif Ahmad1, JitKang Lim1,2, Derek Juinn Chieh Chan1*

1School of Chemical Engineering, Universiti Sains Malaysia, Nibong Tebal, Penang 14300, Malaysia.

2Department of Physics, Carnegie Mellon University, Pittsburgh, PA 15213, USA.

Preparation of working reagent for BCA protein assay

The working reagent was prepared by mixing the BCA Reagent A with BCA Reagent B in volume ration of 50:1, where:

BCA Reagent A: / Containing sodium carbonate, sodium bicarbonate, bicinchoninic acid and sodium tartrate in 0.1 M sodium hydroxide
BCA Reagent B: / Containing 4% cupric sulfate

The reagents were supplied by the Thermo Scientific.

Table S1. Statistical analysis for Figure 1 on each day of microalgae cultivation when in different concentration of magnetic nanoparticles. Statistically significant was evaluated based on one-way analysis of variance (ANOVA) followed by LSD all-pairwise comparison test at p < 0.05.

(a)

Concentration of bare-IONPs (mg/L) / Day 1 / Day 2 / Day 3 / Day 4 / Day 5 / Day 6 / Day 7
0 (Control) / A / A / A / A / A / A / A
0.15 / AB / A / A / A / A / A / A
1 / AB / A / AB / A / A / A / A
5 / AB / A / AB / A / A / A / A
10 / AB / A / AB / A / A / A / A
20 / B / A / B / A / A / A / A

(b)

Concentration of SF-IONPs (Chitosan) (mg/L) / Day 1 / Day 2 / Day 3 / Day 4 / Day 5 / Day 6 / Day 7
0 (Control) / A / A / A / A / A / A / A
0.15 / AB / A / A / A / A / A / A
1 / AB / A / A / A / A / A / A
5 / ABC / A / A / A / A / A / A
10 / BC / A / A / A / A / A / A
20 / C / A / A / A / A / A / A

(c)

Concentration of SF-IONPs (PDDA) (mg/L) / Day 1 / Day 2 / Day 3 / Day 4 / Day 5 / Day 6 / Day 7
0 (Control) / A / A / A / A / A / A / A
0.15 / A / AB / AB / AB / A / AB / AB
1 / A / AB / AB / AB / A / AB / AB
5 / A / AB / AB / AB / A / AB / AB
10 / A / AB / AB / BC / A / AB / AB
20 / A / B / B / B / A / B / B

Figure S1. TEM image of bare-IONPs, Fe3O4 (98+% purity, 20 – 30 nm) that was purchased from Nanostructured & Amorphous Materials, Inc.

Figure S2. Pre-cultivation of the Chlorella sp. cell culture in 2 L BBM medium under continuous light illumination and air bubbling.

Figure S3. Schematic diagram shows the simple setup used for microalgae cultivation under continuous light illumination and air bubbling by using 500 mL conical flask that containing 250 mL of cell culture.

Figure S4. Calibration curve of protein concentration in function of absorbance. The Albumin Standard (supplied by Thermo Scientific) used was containing bovine serum albumin (BSA) at 2 g L-1 in 0.9 % saline and 0.05 % sodium azide. The absorbance was set to zero on a cuvette filled with distilled water. The absorbance of the protein was obtained after the absorbance (562 nm) of blank standard subtracted from the absorbance (562 nm) of standard replicates.

Figure S5. Calibration curve of carbohydrate concentration in function of absorbance. The carbohydrate was quantified as sucrose equivalent. The absorbance of the carbohydrate (sucrose) was measured spectrophotometrically by UVmini-1240 Shimadzu at specific wavelength of 490 nm.

Figure S6. Images showed the greenish cell culture became brownish in colour after 20 mg L-1 of nanoparticles was added into the medium.

Figure S7. The concentration of Fe in culture medium after 20 mg/L of bare-IONPs and SF-IONPs are incubated in the medium for 7 days. The control sample is the pure culture medium without adding in nanoparticles. Fe concentration is measured by the atomic absorption spectroscopy (AAS).

Figure S8. Transmission electron microscopy (TEM) image shows the SF-IONPs (PDDA) attached on the surface of Chlorella sp. cell.

Figure S9. Microscopic imaging of Chlorella sp. at day 7 after incubated with (a) 0 mg L-1 (control group), (b) 0.15 mg L-1, (c) 1 mg L-1, (d) 5 mg L-1, (e) 10 mg L-1 and (f) 20 mg L-1 of SF-IONPs (PDDA). These micrographs were captured by an Olympus CX41RF microscope equipped with Image Pro Express 4.0.1 imaging software at 100x magnification. Scale bars: 10 μm.

Figure S10. Microscopic imaging of samples introduced with 20 mg L-1 of SF-IONPs (Chitosan) from day 0 to day 7. The SF-IONPs (Chitosan) loss its attachment on cell surface on day 6th and onward. These micrographs were captured by an Olympus CX41RF microscope equipped with Image Pro Express 4.0.1 imaging software at 100x magnification. Scale bars: 10 μm.

Figure S11. TEM micrographs show the images of the cross-sectional view of Chlorella sp. cell where the SF-IONPs (PDDA) has internalize the cells.