Nutrient Absorption Bychlorella Vulgaris F1068 Under Cetyltrimethyl Ammonium Bromide Induced

Nutrient absorption byChlorella vulgaris F1068 under cetyltrimethyl ammonium bromide induced hormesis

Qiongzhi Zhou1, Feng Li1, Fei Ge, Na Liu, Yangduo Kuang

Materials and methods

1. The measurement methods ofNH4+and TP

2. The measurement ofchlorophyll a

3. The measurement method for protein and polysaccharides contents

4. Effect of CTAB on chlorophyll autofluorescence

Captions of Figures

Fig. S1HPLC profiles for adenosine triphosphate (ATP) (Retention time, Rt, 5.491 min), adenosine diphosphate (ADP) (Rt, 5.491 min) and adenosine monphosphate (AMP) (Rt, 5.491 min).

Fig. S2The quantitative standard curves of ATP, ADP and AMP by HPLC analysis.

Fig.S3The biomassofC. vulgaris F1068 under CTAB induced hormesis (Initial cell density was 1×106cells/mL, and initial concentration of NH4+ and TP was 4 mg/L and 0.36 mg/L, respectively)(a) biomass of C. vulgaris under DTAB; (b) biomass of C. vulgaris under TTAB.

Fig. S4Chlorophyll autofluorescence (the excitation wavelength and emission wavelengthis 488 nm and 685 nm, respectively) by C. vulgaris F1068 under CTAB induced hormesis during a 7-d test period. Error bars indicate the standard deviations (n=3).The symbol stars (*)represent for the significant difference on each curve at p0.05 in one-way ANOVA.

Materials and methods

1. The measurement method ofNH4+and TP

The residual concentrations ofNH4+ in the supernatant were determined by Nessler’s reagent spectrophotometry(Liang et al., 2015). Briefly, 5 mL of the supernatant was diluted to 50 mL withdistilled water, and then 1.5 mL of Nessler’s reagent (HgCl2-KI-KOH mixed solution)and 1.0 mL of potassium sodium tartrate were separatelyadded, and then mixed well.After 10 min of reaction time, the absorbance of the samples was measured at 420 nm.Meanwhile, the residual concentrations of TP in the supernatant were alsomeasured by the Molybdenum-Antimony Anti-Spectrophotometric Method (Chen et al.,2015). In brief, 5 mL of the supernatant was diluted to 50 mL with distilled water.Then 1.0 mL of ascorbic acid and 2 mL of molybdate were added, respectively, andmixed well. After 15 min of reaction time, the absorbance at 700 nm was measured.The calibration curve was constructed with known concentrations of NH4+ or TP. The residual concentrations of NH4+ or TP were obtained from thecorresponding calibration curve.

2. The measurement ofchlorophyll a

For assay of the chlorophyll a contents of algal cells, 10 mL of each sample was centrifuged at 4000 rpm for 10 min and then collected algal cells under CTAB exposure during a 7-d test period.After being washed with ultrapure water three times, algal cells were added to 3.35mL of dimethyl sulfoxide and then incubated in a water bath at 65 °C for 20 h.After centrifugation (at 4 °C, 4000 rpm for 10 min) again, the supernatant (3.35 mL)was transferedto 10 mL bottle of brown prior to being extracted with6.7 mL of 80% acetone at 25 °C.The extracted liquid wasseparately at 645 nm and 663 nmwith a spectrophotometer (722S, Shanghai Precision Scientific Instrument Co. Ltd., China).The content of chlorophyll a was calculated as following formula:

Chlorophyll a =12.7×OD663-2.69×OD645.

3. The measurement methods for protein and polysaccharide content

For assay of theprotein content of algal cells, 100 mL of each sample were centrifuged at 4000 rpm for 15 min under CTAB exposure during a 7-d test period. Then the algal cells suspension washomogenized in 20 mL of phosphate-buffered saline (PBS) (pH 7.4) in a ultrasonic celldisruptor (SCIENTZ-ⅡD, Ningbo Scientz biotechnology Co. Ltd., China) at power of 60% in an ice bath for 10 min with vibration for 2s and interval for 3s. After centrifugation (12000rpm, 10 min, 4 oC), the contents of protein and polysaccharide were determined by the Coomassie Blue-dyeBinding assay as following:One mL of supernatantwas added to 5 mL of Coomassie Brilliant Blue G-250 (100 mg/L), and then stained for 2 min before determination at 595 nm with a spectrophotometer(722S, Shanghai Precision Scientific Instrument Co. Ltd., China).The phenol-sulfuric acid methodwas used to analyze the contents of polysaccharide as following: 0.5 mL ofthe supernatant, 1.5 mL ofultrapure water and 1 mL of 5% phenol solution were seperately added to screw cap tubes (13×100 mm), and vortex-stirred.Next,5 mL of sulfuric acid (98%) was added slowly to the crew cap tubes above. Then the tubes were closed, vortex-stirred for 5 s prior to being incubated for 30 min at 100 °C. All tubes were cooled down to room temperature before measuring the absorbance at 490 nm in a spectrophotometer(722S, Shanghai Precision Scientific Instrument Co. Ltd., China). The distilled water was used asthe blank control. (Chen et al. 2015).

4. Effect of CTAB on chlorophyll autofluorescence

Algal biomass from the test and control cultures was separately harvested by centrifugation (4000 rpm, 10 min) during a 7-day test period, and washed three timeswith PBS buffer (pH 7.4). The treated algae cells were re-suspended in PBS buffer (pH 7.4) with an initial cell density of 1×106 cells/mL. An aliquot of 5 mL of this algae suspension was taken for chlorophyll autofluorescence analysis. The fluorescence emission spectrum of the algae was recorded at 685 nm with an excitation wavelength of 488 nm by a fluorescencespectrophotometer (LS-55, Perkin-Elmer Co., USA) (Liang et al. 2013).

References

Chen B, Li F, Liu N, Ge F, Xiao H, Yang Y (2015)Role of extracellular polymeric substances from Chlorella vulgaris in the removal of ammonium and orthophosphate under the stress of Cadmium. Bioresour Technol 190:299-306

Liang Z, Ge F, Zeng H, Xu Y, Peng F, Wong M.(2013)Influence of cetyltrimethyl ammonium bromide on nutrient uptake and cell responses of Chlorella vulgaris. Aquat Toxicol 138:81-87

Nikolopoulos D, Korgiopoulou C, Mavropoulos K, Liakopoulos G, Karabourniotis G (2008). Leaf anatomy affects the extraction of photosynthetic pigments by DMSO.Talanta,76: 1265-1268

Fig. S1HPLC profiles for adenosine triphosphate (ATP) (Retention time, Rt, 5.491 min), adenosine diphosphate (ADP) (Rt, 5.491 min) and adenosine monphosphate (AMP) (Rt, 5.491 min).

Fig. S2The quantitative standard curves of ATP, ADP and AMP by HPLC analysis.

 Corresponding author: Tel.: +86 731 58298290; Fax.: +86 731 58298172;

E-mail: (F. Ge);