Analytical biosensor device for detecting the level of contamination of water with biodegradable organic compounds

Abstract

The invention relates to the field of ecology, and more particularly to analytical biosensor devices. The claimed device can be used for detecting (determining) the level of contamination of water with biodegradable organic compounds by measuring the biochemical oxygen demand of samples of water of various origins, including fresh surface water, subsurface water (groundwater), drinking water, wastewater and purified wastewater. An analytical biosensor device for detecting the level of contamination of water with biodegradable organic compounds comprises: a measuring cuvette (3) containing a buffer solution; a magnetic stirrer (4); and a biosensor system comprising an oxygen electrode (1) configured for connection to an appliance for recording the biosensor system response, and, arranged on said oxygen electrode (1), a receptor element (2) comprising an association of microorganisms immobilized on a carrier, the analytical biosensor device being characterized in that the microorganisms used are Paracoccus yeei VKM B-3302, Pseudomonas veronii VKM В-3835 and Rhodococcus fascians VKM Ас-2996 in a mass ratio of 1:1:1, which are immobilized on a nitrocellulsoe membrane and are coated on top with a chitosan solution that forms a hydrogel after drying. The technical result of the invention is that of increasing the upper limit of the range of detectable biochemical oxygen demand (BOD) values to 200 mg / dm3, enhancing the operational stability of the biosensor system response, as well as increasing the stable operational lifetime of the biosensor system.

Description

[0001] BIOSENSOR ANALYTICAL DEVICE FOR DETECTING THE LEVEL OF WATER POLLUTION BY BIODEGRADABLE ORGANIC COMPOUNDS

[0002] [1] Field of technology

[0003] [2] The invention relates to the field of ecology, specifically to biosensor analytical devices. The claimed device can be used to detect (determine) the level of water pollution by biodegradable organic compounds based on measuring the biochemical oxygen demand in water samples of various origins, including surface fresh water, underground (groundwater), drinking water, wastewater, and treated wastewater.

[0004] [3] State of the art

[0005] [4] Currently, to detect the level of water pollution by biodegradable organic compounds, an indicator such as biochemical oxygen demand (BOD) is used, that is, the amount of oxygen (in mg) required for the oxidation of those present in 1 dm 3water organic matter under aerobic conditions, in particular BOD5 is the amount of oxygen used by microorganisms to oxidize all dissolved organic matter in an isolated sample vessel over 5 days, BOD20 is up to 20 days, respectively. Thus, the existing standard method for determining BOD, widely used by manufacturing plants, sanitary services and water treatment plants, is based on tests with a minimum duration of at least 5 days [PNDF 14. 1:2:3:4. 123-97. Quantitative chemical analysis of water. Methodology for measuring the biochemical oxygen demand after n days of incubation (BODtotal) in surface fresh, underground (ground), drinking, waste and treated waste water. - M .: 1997. 25 p].At the same time, the specified duration of the standard method for determining BOD may cause environmentally hazardous situations due to the entry of contaminated water into water treatment facilities or its incomplete treatment during the regeneration process.

[0006] [5] To quickly determine the level of water pollution by biodegradable organic compounds, a method of assessing the BOD using biosensor analytical devices (BOD biosensors) is used. These devices utilize microorganisms capable of oxidizing a specific range of organic compounds as recognition elements.

[0007] [6] The closest analogue of the claimed invention is a biosensor analytical device for detecting the level of water pollution by biodegradable organic compounds, described in the work [Arlyapov VA, Alferov VA, Yudina N. Yu., Asulyan LD, Kamanina OA, Alferov SV, Shumsky AN, Machulin AV, Reshetilov AN Registration of BOD using Paracoccus yeei bacteria isolated from activated sludge. / / 3 Biotech, 2020, V 10, No. 207]. This device contains an oxygen electrode with immobilized Paracoccus yeei VKM B-3302 microorganisms. Immobilization of these microorganisms is carried out in polyvinyl alcohol modified with N-vinylpyrrolidone. This device allows you to determine BOD5 in the range from 0.05 to 5.0 mg / dm 3 (without sample dilution), while the duration of one measurement is up to 6 minutes, the stable operating time of the device is 45 days, and the operational stability of the biosensor system response is 7%.

[0008] [7] The disadvantage of this device is the limited upper range of detectable values ​​of the VPK, which is 5.0 mg / dm3. 3 (without sample dilution) with operational measurement stability of 7%, given that in most cases the WPC values ​​in wastewater are more than 5.0 mg / dm3 3 The device, like its closest analogue, is also insensitive to common biodegradable organic compounds such as propanol, methanal, and arginine.

[0009] [8] Thus, the technical problem that the claimed invention is aimed at solving is the insufficiently high performance characteristics of known biosensor analytical devices for detecting the level of water pollution by biodegradable organic compounds, which, among other things, do not allow the determination of the entire spectrum of the most common hazardous substances contained in water of various origins.

[0010] [9] Disclosure of the essence of the invention

[0011]

[0010] The technical result of the invention is to increase the upper limit of the range of detectable values ​​of biochemical oxygen demand (BOD) to 200 mg / dm3 3 , increasing the operational stability of the biosensor system response, increasing the time of stable operation of the biosensor system, and expanding the spectrum of detectable biodegradable organic compounds.

[0012]

[0011] The operational stability of the response of a biosensor system shall be understood as the stability of the response of a biosensor system to the same substrate concentration when performing a large number (at least 15) of successive measurements.

[0013]

[0012] The specified technical result is achieved in a biosensor analytical device for detecting the level of water pollution by biodegradable organic compounds due to the fact that it contains a measuring cuvette with a buffer solution, a magnetic stirrer and a biosensor system including an oxygen electrode made with the possibility of being connected to a device recording the response of the biosensor system, and a receptor element placed on the oxygen electrode, which includes an association of microorganisms Paracoccus yeei VKM B-3302, Pseudomonas veronii VKM B-3835 and Rhodococcus fascians VKM Ac-2996 in a mass ratio of 1: 1: 1, immobilized on a carrier in the form of a nitrocellulose membrane and treated on top with a chitosan solution.

[0014]

[0013] In addition, according to particular cases of implementation of the invention:

[0015]

[0014] - use a chitosan solution with a concentration of 8.0 - 12.0 g / l in a 1 wt.% acetic acid solution,

[0016]

[0015] - use chitosan with a molecular weight of 50 to 190 kDa,

[0017]

[0016] - a phosphate buffer solution with a pH of 6.8 is used as a buffer solution.

[0018]

[0017] The association of microorganisms Paracoccus yeei VKM B-3302, Pseudomonas veronii VKM B-3835 and Rhodococcus fascians VKM Ac-2996, used in the biosensor system, is capable of oxidizing a wide range of organic compounds of various classes (35 compounds), which may be found in water samples of various origins (industrial and domestic wastewater, surface water), which makes it possible to obtain a high degree of correlation between the readings of the claimed device and the most common standard method for determining the VPK in accordance with PNDF 14. 1:2:3:4. 123-97.

[0019]

[0018] The use of a nitrocellulose membrane as a carrier for the immobilization of the association of microorganisms Paracoccus yeei VKM B-3302, Pseudomonas veronii VKM B-3835, and Rhodococcus fascians VKM Ac-2996 ensures high adsorption of the said microorganism cells, which prevents their leaching from the receptor element during the operation of the claimed device. The nitrocellulose membrane also exhibits increased mechanical strength and a low degree of swelling, which facilitates the long-term functioning of the receptor element. Furthermore, the nitrocellulose membrane exhibits high permeability for low-molecular compounds, which also contributes to the high sensitivity of the device.

[0020]

[0019] The use of a chitosan solution to coat an association of microorganisms immobilized on a nitrocellulose membrane: Paracoccus yeei VKM B-3302, Pseudomonas veronii VKM B-3835, and Rhodococcus fascians VKM Ac-2996, which forms a hydrogel after drying, ensures easy diffusion of the starting materials and reaction products. Moreover, the chitosan hydrogel formed after drying has mechanical strength and is insoluble in water due to its cross-linked structure.

[0021]

[0020] Brief description of drawings

[0022]

[0021] The invention is explained by figures, where:

[0022] Fig. 1 shows a general schematic view of the claimed biosensor analytical device for detecting the level of water pollution by biodegradable organic compounds.

[0023]

[0023] Fig. 2 shows a diagram of the substrate specificity (sensitivity) of the biosensor system used in the claimed device.

[0024]

[0024] Fig. 3 shows an example of a typical response of the claimed biosensor analytical device, recorded during the analysis of a sample of contaminated water, which is a graph of the dependence of the oxygen content on time.

[0025]

[0025] Fig. 4 shows the calibration dependence of the claimed biosensor analytical device.

[0026]

[0026] The elements are designated in the figures by the following positions:

[0027] 1 - oxygen electrode

[0028] 2 - bioreceptor element,

[0029] 3 - measuring cuvette,

[0030] 4 - magnetic stirrer.

[0031]

[0027] Implementation of the invention

[0032]

[0028] The claimed biosensor analytical device includes a biosensor system consisting of a converter in the form of an oxygen electrode (1), made with the possibility of connection to a device recording the response of the biosensor system, a receptor element (2) placed on the oxygen electrode (1), including an association of the bacteria Paracoccus yeei VKM B-3302, Pseudomonas veronii VKM B-3835 and Rhodococcus fascians VKM Ac-2996 in a mass ratio of 1:1:1, placed on a carrier in the form of a nitrocellulose membrane and treated on top with a chitosan solution.

[0033]

[0029] The bacteria Paracoccus yeei VKM B-3302, Pseudomonas veronii VKM B-3835 and Rhodococcus fascians VKM Ac-2996, used in the receptor element (2) of the claimed device, were cultivated in a rich mineral medium (liquid glucose-peptone nutrient medium). Composition of the liquid medium: glucose - 10 g / dm 3 , peptone - 5 g / dm 3 , yeast extract - 0.5 g / dm3 3The cell culture medium was sterilized by autoclaving at atmospheric pressure for 45 minutes. The cells were cultured aerobically for 18-20 hours in 750 cm3 shaking flasks. 3 at 29°C. The resulting biomass was then centrifuged at room temperature at 10,000 rpm for 10 minutes using a TG16WS centrifuge (Polikom LTD, Russia). The resulting centrifugate was then washed with a 20 mM phosphate buffer solution with a pH of 6.8. The precipitated cells were washed with the buffer solution, distributed into Eppendorf microtubes, and centrifuged in an Eppendorf centrifuge for 5 minutes at 10,000 rpm. The washed biomass was stored at -25°C in microtubes.

[0034]

[0030] To form a bioreceptor element (2) on a carrier in the form of a nitrocellulose membrane with an area of, for example, 10 mm 2An immobilized association of microorganisms Paracoccus yeei VKM B-3302, Pseudomonas veronii VKM B-3835 and Rhodococcus fascians VKM Ac-2996 in a phosphate buffer solution with pH 6.8 in a volume of 5 ml with a titer of 150 g / dm3 is applied using a micropipette 3 After natural drying for 1-ro hour, a chitosan solution with a concentration of 8.0 - 12.0 g / l in a 1 wt.% acetic acid solution is applied on top, using low molecular weight chitosan with an average molecular weight of 50-190 kDa (for example, manufactured by Sigma-Aldrich, Germany). Preferably, a chitosan solution with a mass concentration of 10 g / l is used, for this purpose a 1 gram sample of chitosan is transferred into 100 ml of the prepared acetic acid solution and stirred for 1-ro hour at a temperature of 35 ° C. The resulting receptor element (2) is dried in air for 1-ro hour, and after drying of the chitosan solution, a chitosan hydrogel is formed. After the above steps, the receptor element (2) is applied to the oxygen electrode (1).

[0035]

[0031] The oxygen electrode (1) (Clark electrode) is an electrochemical sensor on whose surface oxygen is reduced to form the hydroxyl ion OH'. Conditions are selected such that the current in the circuit is proportional to the amount of oxygen in the solution, i.e., to its partial pressure (P0r). If there are no reactions involving oxygen, its concentration remains constant. However, if a chemical reaction occurs in the solution that consumes oxygen, its partial pressure increases, which is detected by the device.

[0036]

[0032] The analysis of a water sample using the claimed device is carried out by placing an oxygen electrode (1) with a bioreceptor element (2) in a measuring cuvette (3) containing a buffer solution that maintains a constant pH level. The solution in the measuring cuvette (3) is then stirred using a magnetic stirrer (4). To record the response (signal) of the biosensor system, an appropriate recording device is used that determines the dependence of the dissolved oxygen content on time, for example, the Expert-009 dissolved oxygen analyzer manufactured by Econiks-Expert LLC, connected to the oxygen electrode (1) and an electronic device, for example, a personal computer, displaying the measurement results to the user.

[0037]

[0033] The said device has sensitivity to a wide range of biodegradable organic compounds such as methanol, ethanol, propanol-1, butanol-1, 2-methylpropanol-1, 2-methylpropanol-2, 3-methylbutanol-1, propanetriol-1,2,3, sorbitol, xylose, glucose, galactose, mannose, fructose, sucrose, lactose, methanal, methanoic acid, ethanoic acid, ethanedioic acid, propanedioic acid, citric acid, EDTA, potassium benzoate, 4-nitrophenol, 2,4-dinitrophenol, glycine, serine, glutamine, tyrosine, aspartic acid, glutamic acid, arginine, sodium DDS, sodium DDBS.

[0038]

[0034] Below is described a specific example of determining the BOD of a water sample using the claimed device, demonstrating the principle of its operation.

[0039]

[0035] An oxygen electrode (1) with a receptor element (2) placed thereon, containing an immobilized association of the microorganisms Paracoccus yeei VKM B-3302, Pseudomonas veronii VKM B-3835 and Rhodococcus fascians VKM Ac-2996 in a mass ratio of 1:1:1, is immersed in a measuring cuvette (3) with a volume of 5 ml with a 30 mM phosphate buffer solution with a pH of 6.8 and the level of dissolved oxygen, reflecting the background oxygen content in the medium, is recorded. Then a sample containing biodegradable organic substances in a volume of 100 μl is added. Measurements are carried out with continuous stirring using a magnetic stirrer (4) at room temperature. After recording the response of the biosensor system, which is a graph of the dependence of the oxygen content on time (Fig. 3), the measuring cuvette (3) is washed with a buffer solution.Next, the value of the maximum rate of oxygen consumption is calculated and the BOD5 value is determined using a previously constructed calibration curve (Fig. 4).

[0040]

[0036] The calibration dependence is constructed using a glucose-glutamate mixture (GGM) solution prepared in accordance with the requirements of PNDF 14. 1:2:3:4. 123-97. In accordance with the specified regulatory document, it is assumed that BOD5 is equal to 205 mg / dm3 3 , corresponds to a solution containing a total of 300 mg / dm 3 glucose and glutamic acid.

[0041]

[0037] The linear range of the dependence of the response of the biosensor system on BOD5 is in the range of 0.1 - 200 mg / dm3 3 (without sample dilution). The duration of a single measurement does not exceed 6 minutes. The stable operating time of the biosensor system, during which the microorganism activity decreases by no more than 25%, is at least 60 days.

[0042]

[0038] Table 1 presents comparative characteristics of the claimed device for detecting the level of water pollution by biodegradable organic compounds and its closest analogue.

[0043] Table 1.

[0044]

[0045]

[0039] Thus, the claimed device makes it possible to significantly increase the upper limit of the range of the determined BOD value, while increasing the operational stability of the measurements and increasing the duration of stable operation of the biosensor system.

Claims

CLAUSES OF THE INVENTION 1. A biosensor analytical device for detecting the level of water pollution by biodegradable organic compounds, comprising a measuring cuvette (3) with a buffer solution, a magnetic stirrer (4) and a biosensor system including an oxygen electrode (1) configured to be connected to a device recording the response of the biosensor system, and a receptor element (2) placed on the oxygen electrode (1), which includes an association of microorganisms immobilized on a carrier, characterized in that the microorganisms used are Paracoccus yeei VKM B-3302, Pseudomonas veronii VKM B-3835 and Rhodococcus fascians VKM Ac-2996 in a weight ratio of 1:1:1, immobilized on a nitrocellulose membrane and treated from above with a chitosan solution, which forms a hydrogel after drying.

2. A biosensor analytical device for detecting the level of water pollution by biodegradable organic compounds according to claim 1, characterized in that a chitosan solution with a concentration of 8.0 - 12.0 g / l in a 1 wt.% acetic acid solution is used.

3. A biosensor analytical device for detecting the level of water pollution by biodegradable organic compounds according to paragraphs 1 and 2, characterized in that chitosan with a molecular weight of 50 to 190 kDa is used.

4. A biosensor analytical device for detecting the level of water pollution by biodegradable organic compounds according to claim 1, characterized in that a phosphate buffer solution with a pH of 6.8 is used as a buffer solution.

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