Bacillus subtilis-based colorimetric bioassay for acute biotoxicity assessment of heavy metal ions

Abstract

This paper described a novel colorimetric bioassay based on Bacillus subtilis for acute biotoxicity assessment of heavy metal ions and real water samples. β-Galactosidase, which can catalyze the hydrolysis of o-nitrophenyl-β-D-galactopyranoside (ONPG) to produce galactose and o-nitrophenol (ONP), can be generated from Bacillus subtilis (CGMCC 1.1086) without the assistance of any inducers. When heavy metal ions exist, the activity of β-galactosidase is inhibited due to the reaction between the sulfhydryl groups in β-galactosidase and the heavy metal ions. Accordingly, the output of the ONP, a yellow chromogenic compound exhibiting a characteristic absorption peak at 420 nm, is also inhibited. Thus, acute biotoxicity of heavy metal ions can be evaluated by assessing the absorption intensity of ONP at 420 nm. To obtain an ideal colorimetric performance, working parameters such as Bacillus subtilis concentration, temperature and incubation time of the bioassay were studied and optimized. Under the optimized parameters, the acute biotoxicity of five heavy metal ions (Cu²⁺, Zn²⁺, Ni²⁺, Cd²⁺, Pb²⁺) and three real samples were evaluated. All the results suggest that Bacillus subtilis-based bioassay is a sensitive, economic, simple and promising alternative for acute biotoxicity assessment.