2.6. Biosensor measurements
Electrochemical measurements were carried out using an Ekspert-001-4.0.1 pH meter–ionometer–BOD thermal oximeter (Ekonics-expert Ltd., Russia) coupled with a computer operated by EXP2PR specialized software (Ekonics-expert Ltd., Russia). The software enables registration and processing of biosensor signals. The maximal rate of oxygen concentration change at the addition of substrates (mg/dm3·s) was the measured parameter (biosensor response). Clark-type oxygen electrodes containing immobilized microbial cells were used as transducers. Measurements were done in a 5-cm3 cuvette. A sodium–potassium phosphate buffer solution (pH 6.8) was used; the total concentration of the salts, 20 mM. The solution was mixed by a magnetic mixer (200 rpm). A mixture of glucose and glutamic acid (GGA) at a mass ratio of 1:1, applied as the BOD5 determination standard in the Russian Federation and in international practice [2], was used as a model mixture. In accordance with the regulatory documents, a value of BOD5 equal to 205 mg/dm3 was taken to correspond to a solution containing 150 mg/dm3 glucose and 150 mg/dm3 glutamic acid (BOD5 = 0.68 × СGGA).
2.7. Immobilization of microorganisms by adsorption
To form a receptor element, microbial biomass was diluted with phosphate buffer to a concentration of 200 mg/cm3. Solutions containing pure microbial cells were mixed at a ratio of 1:1 (1:1:1 for a co-culture consisting of 3 microorganisms); 5 μl of the produced suspension was applied onto a 3 × 3 mm2 fragment of a Whatman GF/A glass fibre filter. The receptor element was dried for 10–15 min on air and fixed by means of a nylon mesh on the surface of an oxygen electrode.