Arlyapov V.A., Yudina N.Yu., Ermilin I.E., Anufrieva O.M., Kozlova T.N.

Tula State University, Russia, Tula

BOD biosensor based on the association of yeast and bacterial microorganisms

Biochemical oxygen demand (BOD) is one of the most broadly used indices for controlling the purity of aqueous media; by definition, it represents the amount of oxygen required for the biochemical oxidation of organic substances contained in a sample. The traditional BOD assaying technique requires incubation of an oxygen-saturated sample for 5, 10 or 20 days (BOD₅, BOD₁₀ or BOD₂₀, respectively). An alternative are express methods of BOD detection using biosensor analyzers based on the application of microorganisms capable of oxidizing a broad range of organic compounds. A drastic distinction of this method from the standard technique is a much shorter time of assay, reduced from 5 days to 10–20 min.

To create response elements BOD sensors are used either pure cultures of micro-organisms with certain consumer characteristics (broad spectrum of oxidized substrates, resistance to adverse environmental factors), an association of micro-organisms (artificial association, activated sludge). Using associations microorganisms can significantly improve the range of oxidizable substrates and consequently the correctness of the BOD. At the same time BOD biosensors based on the association of microorganisms may have insufficient stability, which cause a change of association with time. BOD - biosensors are based on a complex microbial population such as active silt, have the best ability to detect a wide variety of substrates, but because of instability over time consortium such biosensors give less reproducible results.

The aim of the present work was to study the stability in time of association of yeast and bacterial microorganisms and creation on their basis response elements for BOD sensors. The objects for the creation of associations were pure cultures of yeast: Pichia angusta VKM Y-2518, Pichia angusta VKM Y-1397, Pichia angusta VKM Y-2559, Arxula adeninovorans VKM Y-2676, Arxula adeninovorans VKM Y-2677, Debaryamyces hansenii VKM Y-2482 , Candida baidini VKM Y-2356 and bacteria Gluconobacter oxydans sbsp. industries VKM B-1280. Cultures of micro-organisms were kindly provided by the All-Russian Collection of Microorganisms of the Institute of Biochemistry and Physiology of Microorganisms (Pushchino, Moscow region).

In the course of work on the basis of the length of the main phases of growth of pure cultures of micro-organisms and their substrate specificity, were made up 4 association as potentially possible response elements for BOD sensors. Methods of sowing on solid medium, light microscopy, a method using amperometric biosensor and method of Koch assessed the stability of associations created within 4 months. Evaluation of stability over time has shown the stability of associations: A. adeninovorans VKM Y-2677 + P. angusta VKM Y-1397 + C. baidini VKM Y-2356; A. adeninovorans VKM Y-2677 + P. angusta VKM Y-1397; G. oxydans VKM B-1280 + P. angusta VKM Y-2518.

Association of A. adeninovorans VKM Y-2676 + P. angusta VKM Y-2518 + D. hansenii VKM Y-2482 is not stable, and by the end of the study it is dominated by a strain of P. angusta. Instability of the association is likely associated with a higher growth rate and the flow of active metabolic processes of microorganisms P.angusta VKM Y-2518. Linear and exponential phase of growth data of yeast cells are just 4 hours.

Defined the basic analytical and metrological characteristics of biosensors based on three stable associations. The association of A. adeninovorans VKM Y-2677 + P. angusta VKM Y-1397 + C. baidini VKM Y-2356 has the broadest substrate specificity and can be most efficiently used when creating BOD biosensor. At the same time, the biosensor based on microorganisms P.angusta VKM Y-1397 and A.adeninovorans VKM Y-2677 has the highest sensitivity among the developed biosensors (lower limit determined by the content of 1.1 dm³ BOD₅). Thus, the two associations with the best characteristics are used for the analysis of real samples of wastewater.

Determination of BOD₅ effluent standard dilution method was carried out in accordance with current regulations of the Russian Federation (PND F 14. 1:2:3:4. 123-97). Table 1 shows the values of BOD, determined using a biosensor and BOD values determined by standard dilution.

Table 1. The results of the analysis of real samples.

The origin of the real sample	Microorganisms		Standard dilution method
	A.adeninovorans Y-2677, P.angusta Y-1397, C.baidini Y-2356	P.angusta Y-1397, A.adeninovorans Y- 2677
River water	7,3±0,7	7,7±0,8	7,6±0,8
Water from the city pond	3,7±0,4	4,2±0,4	3,4±0,4
Thawing water	5,1±0,5	4,8±0,5	4,0±0,4
The water from the treatment plant	75±3	72±4	76±3

Thus, the values of BOD₅ determined using the biosensor based on association of microorganisms coincide with the BOD₅ values obtained by the standard method with account for the confidence interval. The results indicate a possibility of using the developed biosensor analyzer as a prototype pilot model of devices for commercial production.

The work was supported by the Federal Goal-oriented Program “Scientific and Scientific-Pedagogical Cadres of Innovative Russia” for 2009–2013, agreement No 14.B37.21.0561 and a grant of the President of the Russian Federation for state support of young Russian PhD scientists, Agreement No 16.120.11.4341-MK.