Montmorillonite

The clay minerals used in this study were kaolinite and montmorillonite (Macklin Inc., China). A batch of kaolinite or montmorillonite with particle sizes of approximately 2.5 μm was added to 200 mL BG11 medium in flasks at concentrations of 5, 50, and 500 mg/L. Next, Synechococcus sp. was inoculated. All samples were transferred to a shaker at 130 rpm for 2 h and left to stand for 2 h to allow the aggregates to settle. The absorbance of chlorophyll A of Synechococcus in the supernatants was measured to characterize changes in Synechococcus cell concentrations. Samples inoculating Synechococcus with no clay served as the controls. All samples were in triplicate.
The method for determining chlorophyll A concentration was as follows. The culture medium was centrifuged at 3,000 rpm for 10 min. Then, the supernatant was removed, and the residual was mixed with an equal volume of acetone and incubated at 4°C for 24 h. Next, the supernatant was centrifuged, and the absorbance was measured at 649 and 665 nm using a spectrophotometer. A 90% acetone solution (vol/vol) was used as a blank control. The concentration of chlorophyll A and the aggregation rate were calculated according to the following equations (46 (link), 47 (link)):
$$\text{chlorophyll\hspace{0.17em}A}\hspace{0.17em}=\hspace{0.17em}12.47A_{665}\hspace{0.17em}-\hspace{0.17em}3.62A_{649}$$
$$\text{aggregation\hspace{0.17em}rate\hspace{0.17em}}\hspace{0.17em}=\hspace{0.17em}\frac{{\text{chlorophyll\hspace{0.17em}A}}_{\mathit{Synechococcus}\hspace{0.17em}\text{only}}\hspace{0.17em}-\hspace{0.17em}{\text{\hspace{0.17em}chlorophyll\hspace{0.17em}A}}_{\text{\hspace{0.17em}clay\hspace{0.17em}group}}}{{\text{chlorophyll\hspace{0.17em}A}}_{\mathit{Synechococcus}\text{\hspace{0.17em}only}}}\hspace{0.17em}\times 100\%$$