In this study, the immobilization with sodium alginate (SA) for cultivating microalgae in entrapped matrix gel beads was conducted for separating it from water. Batch experiments with a period of 5 days were carried out for free and immobilized Scenedesmus obliquus simultaneously under two trophic modes, to compare the removal performances of different initial ammonium (NH4+-N) concentrations. In both free and immobilized form, the positive C-dependent effect in mixotrophy and the negative N-dependent effect in heterotrophy were observed. And the performances of immobilized form were all superior to that of free form, which showed greater tolerance to high concentration, maximally representing 96.6 +/- 0.1% removal in 50 mg/L of NH4+-N in mixotrophy. Assimilation of NH4+-N was the main removal pathway resulting the protein synthesis with the dominant component including glutamic acid (Glu), cystine (Cys), arginine (Arg) and proline (Pro). The results demonstrated a systematic understanding for NH4+-N removal in microalgae-based system.