An on-line biosensor consisting of immobilized Thiobacillus ferrooxidans and an oxygen electrode was developed for automated monitoring of acute toxicity in water samples. T. ferrooxidans is an obligatory acidophilic, autotrophic bacterium and derives its energy by the oxidation of ferrous ion, elemental sulfur, and reduced sulfur compounds including metal sulfides. The assay is based on the monitoring of a current increase by addition of toxicoids, which is caused by the inhibition of bacterial respiration and decrease in oxygen consumption. Optimum cell number on the membrane was 5.0 × 108 cells. The steady-state current was obtained when concentration of FeSO 4 was above 3.6 mM at pH 3. The sensor response of T. ferrooxidans immobilized membrane for 5.0 μM KCN was within an error of 10% for 30 membranes. A linear relationship was obtained at KCN concentration in the range of 0.5-3.0 μM in a flow-type monitoring system. Minimum detectable concentrations of KCN, Na2S, and NaN3 were 0.5, 1.2, and 0.07 μM, respectively. The monitoring system contained two biosensors and these sensors were cleaned with sulfuric acid (pH 1.5) twice a day. This treatment could remove fouling on microbial immobilized membrane by natural water and ferrous precipitation in the flow cell. This flow-type monitoring sensor was operated continuously for 5 months. Also, T. ferrooxidans immobilized membrane can be stored for one month at 4°C when preserved with wet absorbent cotton under argon gas.
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