Yudina N., Savenkon A., Arlyapov V.

Tula State University, Russia, Tula

The BOD biosensor’s specifications based on yeast Debaryamyces hansenii encapsulated in dialysis membrane

Biochemical oxygen demand (BOD) is one of the most broadly used indices for monitoring the purity of aqueous environments. By definition, it represents the amount of oxygen required for biochemical oxidation of organic substances contained in the sample. The duration of classical BOD tests is 5 days (BOD₅) and more, which is too long for the on-line assessment of an ecological situation [1]. BOD assessment methods for express assays are being developed based on biosensor analyzers. As recognition elements, BOD biosensors use microorganisms capable of metabolizing a broad range of organic compounds.

The receptor element of sensor was formed by using yeast Debaryamyces hansenii. These microorganisms can oxidize many alcohols, carbohydrates, amino acids and other organic substances [2]. Dialysis membrane encapsulation allows immobilizing yeast in the softest conditions. Thus, it is possible to avoid binding the enzymes active centers of whole cells in the case of immobilization by chemical cross-linking, and also loosing of activity under adsorption on glass fiber filter and the inclusion in different gels.

The aim of this work is to determine the characteristics of the cuvette BOD biosensor yeast Debaryamyces hansenii, encapsulated in a dialysis membrane.

Materials and methods. 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. 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. A sodium–potassium phosphate buffer solution (pH 6.8) was used. 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 [1], 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×С_GGA). The method of encapsulation in dialysis membrane (Sigma-Aldrich, USA) of microorganisms was used for formation of the receptor element.

Results and discussion. This selectivity of biosensor analysis is determined by biomaterial substrate specificity of a sensor receptor element. Predominantly readily oxidizable organic substances were taken as substrates, since their ingress into water reservoirs leads to a significant reduction in the level of dissolved oxygen and subsequent eutrophication. Figure 1 presents the data for the substrate specificities of the developed biosensor.

Figure 1. Substrate specificities of the developed biosensor.

Cells Debaryamyces hansenii, immobilized by encapsulation in a dialysis membrane are capable to oxidize a very wide range of organic substances (Figure 1). These substances can be detected in the effluent of the various industries, which is promising from the standpoint of the possibility of microorganisms use for the evaluation of BOD. Dialysis membrane which used for encapsulation of yeast skips macromolecular substrates having a molecular weight less than 12 kDa. That’s why the sensor responses depend only on the enzymatic activity, as opposed to the immobilization of microorganisms by inclusion in gel or adsorption, where the sensor response was dependent on the diffusion through the matrix substrate.

The main characteristics of BOD biosensor based on yeast encapsulated in dialysis membrane (table 1).

Table 1. Characteristics of the yeast based BOD biosensor.

Characteristics	Values
Sensitivity coefficient, slope of the linear segment of the dependence of sensor response on the value of BOD5, s^–1∙10^–4	3,0± 0,3
Range of determined BOD5 values, mg/dm^3	1.3–40
Operational stability, relative standard deviation by 15 consecutive measurements, %	6,5
Long-time stability, time within which the value of biosensor response to the same concentration of GGA was no less than 25% of the initial value, temperature 20ºC, days	55
Unit assay time, duration of single measurement, min	3-4

Developed sensor surpasses analogues [2] on the long-term stability and unit assay time. Designed biosensor allows to analyze samples with a high correlation to the reference method (R = 0,9998) and in a wide range of BOD (from 2 to 32 000 mg/dm³).

References:

1. PNDF 14. 1: 2: 3: 4. 123-97. Quantitative chemical analysis of water. Methods of measurement of biochemical oxygen demand after the n-days of incubation (BOD) in surface freshwater, groundwater (groundwater), drinking water, sewage and effluent. - M .: 1997. 25 p.

2. V.A. Arlyapov, N. Yu. Yudina, L.D. Asulyan, S.V. Alferov, V.A. Alferov, A.N. Reshetilov. BOD biosensor based on the yeast Debaryomyces hansenii immobilized in poly(vinyl alcohol) modified by N-vinylpyrrolidone. Enzyme and Microbial Technology , № 53 (2013) pp. 257– 262.

Acknowledgements

The work was carried out within the framework of the State Assignment of the Ministry of Education and Science of the Russian Federation and the Federal Targeted Programme for Research and Development in Priority Areas of Development of the Russian Scientific and Technological Complex for 2014–2020 (Project No.14.574.21.0062).