A rapid and simple respirometric biosensor with immobilized cells of Nitrosomonas europaea for detecting inhibitors of ammonia oxidation

As obligate chemolithotrophs, ammonia-oxidizing bacteria (AOB) grow very slowly and are known to be extremely sensitive to a wide variety of inhibitors. Since it is generally accepted that inhibition of ammonia oxidation by AOB results in a total failure of nitrogen removal, it is necessary to develop a method to detect inhibitors of ammonia oxidation in wastewater. Since ammonia oxidation accompanies oxygen consumption, ammonia oxidation can be easily evaluated by measuring oxygen consumption rate using a dissolved oxygen (DO) probe. In this study, a rapid and simple respirometric biosensor using the pure culture of Nitrosomonas europaea was developed. N. europaea was cultivated in a continuous fermentor operating at the dilution rate of 0.008 h−1 to obtain physiologically constant cells and was immobilized onto the dialysis membrane through filtration. DO, determined by the biosensor, started to increase 30 s later after ammonia oxidation inhibitor was fed, and a new steady-state DO was obtained in 10-30 min. For this DO profile, steady-state kinetics was applied to evaluate ammonia oxidation efficiency. The concentration of a toxic compound causing 50% decrease of oxygen-consumption activity (EC50) was determined for different chemicals. The EC50 values obtained with the biosensor (0.018 mg l−1 for allylthiourea, 0.027 mg l−1 for thioacetamide, 1.10 mg l−1 for phenol and 0.01 mg l−1 for thiourea) indicated that the developed biosensor was highly sensitive to a variety of the inhibitors. It was also shown that the biosensor is applicable for on-line real time monitoring.