Mediator BOD Biosensor Based on Cells of Microorganisms Isolated from Activated Sludge

Three bacterial strains (SPB1, SPB2, and SPB3) isolated from the activated sludge of treatment facilities most efficiently oxidized (as compared to other strains) a model solution based on glucose and glutamic acid in the presence of a ferrocene mediator. The possible use of isolated strains as a basis for a mediator-type amperometric BOD biosensor was studied. Analysis of 16S rRNA genes demonstrated that the SPB1, SPB2, and SPB3 strains are 99.79–100% similar to the Paracoccus yeei BAA-599T, Pseudomonas veronii DSM 11331T, and Bacillus proteolyticus TD42T strains, respectively. It was established that the bioreceptor element based on the P. yeei SPB1 strain possessed the best characteristics. It was stable in storage at 4°С for 22 days, and the lower limit of detectable concentrations was 1.3 mg О2/dm3. In order to search for the most efficient electron transfer mediator for this strain, nine compounds, including ferrocene, thionine, methylene blue, potassium hexacyanoferrate(III), and 2,6-dichlorophenolindophenol, were screened. The mediator electron transfer signals were obtained for each of them. Ferrocene was the most efficient in terms of sensitivity and the amount of substrate oxidation that can be registered in the presence of the mediator. An analysis of ten samples of wastewater (before and after purification) and the rivers of Tula oblast demonstrated that the use of ferrocene and P. yeei SPB1 strain cells made it possible to obtain data with a high correlation (R = 0.9934) with the results of the standard method.