Grown in MSM with n-hexadecane, 5% CO2 and 100 mM NaHCO3, the bacterium lowered the surface tension of the culture supernatant from 72 to 30 mN m−1, 33 mN m−1 and 38 mN m−1 respectively within 48 h of cultivation. The ODA and the drop-collapsing test also showed obvious surface activity in all three substrate conditions (data not shown). While hydrophobicity of Bacillus sp. grown on n-hexadecane was slightly higher (75 ± 2.3%) than when grown on 5% CO2 (65 ± 3.2%) and 100 mM NaHCO3 (51 ± 2.4%) as the carbon source, there was no observable change in the values during growth. The Bacillus sp. was found to be having very good emulsifying activity (E24) at 2% hexadecane (61%), followed by 5% CO2 (53%) and 100 mM NaHCO3 (41%) respectively. This suggests that biosurfactant production does not contribute in decreasing or increasing cell surface hydrophobicity. The exact reason why some microorganisms produce surfactants is unclear. But, this strain represents a valuable source of new compounds with surface-active properties, and potential application for CO2 sequestration and bioremediation. Thus these all screening method proves that substrate like gaseous CO2 and NaHCO3 are inducer of biosurfactant production as traditionally accepted organic substrate 2% n-hexadecane (Among these three substrates, hexadecane is reported as best substrate for biosurfactant production but not involve in sequestration process).