Zaytseva A.S., Grishaeva A.R., Kosareva E.I.

Mediator type BOD biosensor based on active sludge
Tula State University, Russia, Tula

The conventional method for determining the biochemical oxygen demand (BOD) is based on the tests with 5 days duration. The advantage of using BOD - biosensors is reducing the analysis time from 5 days to 20 minutes. Creation of amperometric biosensors based mediators is widely used. Mediator is a compound, which can transfer electrons from the active sites of enzymes to the electrode (mediator electron transport).

Thus, Mediator type BOD biosensor was developed. Ferricyanide potassium was used as a mediator, because the working potential is decreased from 700 mV (oxygen electrode) to 288 mV (ferricyanide potassium electrode), therefore risk of side reactions with electroactive impurities comes down.

Active sludge is an enrichment culture of micro and macro organisms that remove (or change) components considered to be pollutants, however only microorganisms of active sludge were used for sensor receptor element creation. Using this biomaterial increase the university of methods, due to their oxidize activity Thus, the aim is to develop a mediator amperometric BOD-biosensor based on active sludge and ferricyanide potassium mediator for rapid analysis.

Materials and methods. Electrochemical measurements were performed using galvanopotentsiostat «IPC-micro» («Volta», Saint Petersburg, Russia), integrated with a personal computer. Range of recorded currents 5 ηA - 20 μA. Two electrode system was used for registration of sensor responses. The working electrode was a carbon-paste electrode with immobilized active sludge, reference electrode – a silver - silver chloride electrode. A constant potential was 288 mV, as it’s a redox potential of electron transport mediator ferricyanide. Measurements were performed with phosphate buffer pH 6.8. When the substrates come into the measurement cell, microorganisms oxidize it, generating current increase in the system.

Results and discussion. Firstly, working content of active sludge in the receptor cell was defined in the maximum sensor response with a constant concentration of the mediator. It is shown that working content of active sludge is 1.00 mg with a concentration of ferricyanide in the buffer solution of 0,01 M.

Active sludge have a broad substrate specificity (fig.1) and oxidize alcohols, carbohydrates, amino acids, carboxylic acids, aldehydes and sodium dodecyl sulfate, which is promising for determining BOD.

Fig. 1. Substrate specificity of Active sludge as part of the ferricyanide mediator BOD biosensor

Using the glucose-glutamic solution (GGS), analytical and metrological characteristics of BOD biosensor were obtained. The range of linear relationship between sensor responses and the BOD using a mediator- ferricyanide was 0.004 - 60 mg/l. The reproductively of the sensor was 3.8%. Time of the sensor without replacing the receptor element is 39 days.

Analysis time using this devise does not exceed 20 minutes, and measurements are not required a highly specialized personnel. Also it was determined BOD value of water and semi-fermentation products. It was shown that the use of mediator biosensor based on active sludge and ferricyanide produces data with high correlation to the conventional method.

Conclusion. In this research, it was obtained of the functioning parameters of the mediator biosensor based on active sludge, such as ferricyanide concentration (0,01 M), mass of cells on the electrode (1 mg). Analytical and metrological characteristics of BOD biosensor were also defined: long-term stability (39 days), the range of detectable concentrations of BOD (0.004 - 60 mg/l). Designed mediator biosensor based on active sludge can be used for analysis of treatment water samples.

This work was supported by the RF Ministry of the state task (project number 1714) and the grant of the President of the Russian Federation (contract № 14.Z56.14.330-MC).