The method of determining the concentration of insecticide metaphos and the product of hydrolysis of organophosphorus reduction of insecticides para-nitrophenol in aqueous medium

 

(57) Abstract:

The invention relates to the field of biotechnology, Microbiology, ecology, quantitative analysis of substances and can be used to determine the presence and concentration of insecticide metaphos and the product of hydrolysis of organophosphorus reduction of insecticides para-NITROPHENOL in the aquatic environment. How is that preparing bioindicator by culturing bacteria Pseudomonas putida on a solid nutrient medium, containing no analyte. Wash the resulting biomass phosphate buffer with pH was 7.45, using a strain of Pseudomonas putida S-11, and with a pH of 8.1, using a strain of Pseudomonas putida BA-11. Incubated biomass in the same buffer. In the resulting bioindicator register the oxygen concentration using an oxygen electrode at 35°C. Add to the biological indicator test substance, registered in the obtained mixture, the oxygen concentration. On the velocity difference of oxygen consumption by bacteria determine the concentration of the analyte. The proposed invention extends the range of detectable compounds with increasing sensitivity. 2 C. p. F.-ly, 8 PL.

The invention relates to the field beating the presence and concentration of insecticide metaphos and the product of hydrolysis of organophosphorus reduction of insecticides para-NITROPHENOL.

The inventive method belongs to the category of biosensor and can be used to determine the metaphos and para-NITROPHENOL, for which there are no other biotechnological methods of detection.

At the present time to determine various aromatic compounds are widely used physical and chemical methods of analytical chemistry - gas-liquid chromatography, antivirmailservice method, chromatography in water, colorimetric and other

Recently, however, widely used biosensor analysis methods based on the use of physical and chemical sensors in combination with various biological objects (enzymes, cells, tissues, etc.,) as sensing elements.

The most known way of determination of organophosphorus and urethane compounds (J-L Marty, K. Sode and I. Karube. Biosensor for detection of organophosphate and carbamate insecticides // Electroanalysis. - 1992. - #4. - P. 249-252). Describes enzyme acetylcholine sensor based on amperometric determination of H2ABOUT2using jointly immobilized acetylcholinesterase and cholinesterase. Adding phosphorus and urethane compounds in the system causes the inactivation of acetyl exeroise sensor. Thus, it is possible to measure the concentration of paraoxon about 10 mm. The sensor is stable when stored in phosphate buffer at 4oC for 2 months and dry in the course of 1 year.

There is a way to determine the toxicity of natural and waste water, comprising contacting them with the test organisms, the culture of protozoa. As test organisms use the flagellate of the genus Chlamydomonas, forming horizontal tubes in the absence of toxic chemicals or the presence of toxic concentrations of vertical stripes accumulation, and toxicity of water is judged visually by the disappearance of these bands (the Description of the invention to author's certificate SU N 1194877A, C 12 Q 1/00 announced. 24.06.82, publ. 30.11.85).

There is also known a method of indicating the contamination of the environment by chemical substances by the impact test of the test-culture of microorganisms with subsequent monitoring of changes in the culture (the Description of the invention to author's certificate SU N 1010128A, C 12 Q 1/00 announced. 20.04.81, publ. 07.04.83).

There is a way biotesting of water and soil pollution by pollutants such as heavy metals, pesticides. The evaluation is made on the number of dead cells lni (Description of invention to the patent of Russian Federation RU N 2135994C1, 6 G 01 N 33/18 announced. 25.06.97, publ. 27.08.99).

Developed enzyme test for analysis of water pollution based on the use of meteozavisimost Gnkazy. He takes to evaluate the presence in water of heavy metals, pesticides and other toxicants on the inhibition of enzyme activity (description of the invention to the patent of Russian Federation RU N 2131925C1; 6 C 12 N 9/22, C 12 Q 1/34, C 02 F 3/34 announced. 16.07.97, publ. 20.06.99).

However, with these methods it is impossible to determine the metaphos and para-NITROPHENOL. In addition, these methods do not have sufficient sensitivity, require long preparation material and economic costs.

Closest to the present invention is a method for determination of lactic acid (Luong, J. H. T., Mulchandani, A., Groom, C. A. The development of an amperometric microbial biosensor using Acetobacter pasteurianus for lactic acid.//J. Biotechnology. - 1989. - N 10. - P. 241 - 252) using a microbial biosensor-based cell strain Acetobacter pasteurianus and oxygen electrode. The method includes the interaction of the investigated samples with a bioindicator - microbial cells grown in complete nutrient medium with lactic acid, and the registration of change of oxygen concentration before and after the interaction. On smetana buffer solution at pH 6 at a temperature of 26oC.

However, this method is not intended to define the metaphos and para-NITROPHENOL, the method is characterized not high enough sensitivity, with his help determine lactic acid in the concentration range from 0.1 to 25 mm, and the complexity of the preparation of the experiment.

The technical object of the present invention is to expand the range defined compounds with increasing sensitivity.

The task is achieved in the method of determining the concentration of insecticide metaphos and the product of hydrolysis of organophosphorus nitro-aromatic insecticides of para-NITROPHENOL in the aquatic environment, including the preparation of bioindicator for analysis, which lies in the interaction of an aqueous solution of the analyte with the biological indicator, the registration of change of oxygen concentration in the environment before and after the interaction of the biological indicator with a water solution of the analyte using an oxygen electrode, and determining the presence and concentration of the analyte by changing the concentration of oxygen in the environment, where according to the invention as bioindicator using microbial cells of the genus Pseudomonas, capable to utilize opredelyaemaya of metaphos or para-NITROPHENOL, the preparation operation of the bioindicator is carried out in phosphate buffer solution with a pH corresponding to the used strain of cells of Pseudomonas and the registration of change of oxygen concentration in the environment is carried out at a temperature of 35oC. in Addition, the preparation operation of the biological indicator should be performed within 2 hours.

In well-known authors of scientific, technical and patent literature not found ways similar set of features. The result, due to the combination of these characteristics were not achieved in the known solutions. Unlike the prototype according to the invention as bioindicator using microbial cells of the genus Pseudomonas, able to dispose of analyte as the sole source of carbon, microbial cells are grown in complete culture medium without addition of metaphos or para-NITROPHENOL. The specificity of this method is the preparation operation of the bioindicator: carried out in phosphate buffer solution with a pH corresponding to the used strain of cells of Pseudomonas for two hours without adding to the environment of the cultivation of metaphos or para-NITROPHENOL. Registration of change of oxygen concentration in the environment is carried out at a temperature of 35o

The proposed method is as follows. Pre-produce the biomass of cells of the strain Pseudomonas putida C-11 (VKPM B-6708) or Pseudomonas putida BA-11 (VKPM B-6707), with the ability to utilize the designated compound as the sole carbon source. Biomass accumulated according to the used strain technology. Biomass cultivated in the buffer solution of the appropriate pH with intensive aeration in a pie rocking a certain period of time. Next, the biomass was separated by centrifugation from the medium, washed and result in work readiness, pre-determined concentration of cells in suspension to assess the required number of bioindicator for analysis. Prepare buffer solution to compare the settings with potentially the studied environment.

Then hold the interaction buffer solution with a bioindicator and removing the time dependence of changes in the concentration of oxygen in the reference environment and in an environment with a bioindicator. This technique required declare and other necessary technical equipment.

At the final stage, the interaction of the sample aqueous solution of the analyte and bioindicator and removing the time dependence of the concentration of oxygen before and after the above-mentioned interaction, which reflects the respiratory activity of the cells in relation to the defined connection. On the difference of the values of the activity of cells in the presence of a measured sample and without it determine the presence and concentration of the desired compounds.

Examples of specific performance

Example 1. Indication of metaphos cells of the strain Pseudomonas putida C-11

Pre-receive biomass of cells of the strain Pseudomonas putida C-11 (VKPM B-6708) as follows: culture cells were grown on 2% MPA for 24 hours at 30oC.

To obtain bioindicator cells of Pseudomonas putida C-11 fresh daily culture were washed from the agar with 0.1 M buffer solution (Na2HPO4/KH2PO4) pH was 7.45 and added into the conical flask 250 ml, containing 50 ml of 0.1 M phosphate buffer pH was 7.45. Incubation of cells that perform a pie rocking chair when mixing intensity 160 rpm at 30oC for 2 hours. Then the biomass was separated by centrifugation, for example, in a centrifuge To-24 (GDR) from the environment at 5000 rpm in ticinocom sediment resuspendable in a small amount of 0.1 M phosphate buffer pH was 7.45 and thus obtained bioindicator, are used to determine the metaphos. Optical density of cell suspension measured at photocolorimeter, wavelength =540 nm in a cuvette with an optical layer thickness of 0.5 cm, against phosphate buffer, determine the cell concentration. For the strain Pseudomonas putida C-11 1 unit of optical density corresponds to 0.67 g dry weight of cells in 1 l

As a reference solution using 0.1 M phosphate buffer with pH was 7.45, saturated with oxygen. It is placed in a thermostatted cell volume of 1 ml, equipped with a magnetic stirrer. In a cell, insert the oxygen electrode, which serves voltage of-0.6 V. as a potentiostat and a chart recorder is used, for example, polarograph Radelkis HE-105 (Hungary). In buffer solution, the concentration of oxygen is practically unchanged, register unchanged in time a current of 12 μa. The determination was carried out at a temperature of 35oC. After 3 min in a cell with a syringe dispenser enter bioindicator to a final cell concentration of 0.6-1,32 g/l to change the current time to determine the endogenous respiratory activity of the cells, it has a value of 1.9 µa/min

Then after 3-5 minutes using a syringe-dispenser in the cell entered the com is treblenija of oxygen by cells changing, respiratory activity takes the value of 13.4 µa/min

The difference in the rate of oxygen consumption by microbial cells in a buffer solution before and after adding the sample, normalized to the number of cell - specific respiratory activity (CPA) has a value of 11.5 µa/min mg dry weight of cells, which means that indication of the metaphos.

Example 2. Determining the concentration of metaphos in the aquatic environment by cells of the strain Pseudomonas putida C-11

Get the biomass of cells of Pseudomonas putida C-11 and prepare bioindicator, as shown in example 1.

The definition of CPA cells of Pseudomonas putida C-11 in relation to the commercial preparation of metaphos were carried out in 0.1 M Na2HPO4/KH2PO4the pH was 7.45 at a temperature of 35oC. Respiratory activity of the cells was calculated based on the concentration of metaphos determined using capillary gas-liquid chromatography (Methods for the determination of trace pesticides in food, feed and the environment. - M., 1983, S. 57-62). Quantitative determination of metaphos in solutions was carried out by the method of absolute calibration using solutions of reference metaphos in chloroform. The concentration of metaphos in the cell is (mm): 0,25; 0,5; 0,75; 1,0; Noi activity as shown in example 1. The dependences of specific respiratory activity of the cells from the solution concentration of metaphos presented in table 1.

Linear plot based respiratory activity of the cells on the concentration of the introduced metaphos is 0.5-3.0 mm, the minimum detectable concentration of 0.25 mm. As shown in example 1, the respiratory response of cells to the introduction of the sample is 11.5 µa/min mg dry weight of cells. On the obtained calibration dependence is determined by the concentration of metaphos, it is 0.5 mm.

Example 3. Indication and determination of the concentration of para-NITROPHENOL cells of the strain Pseudomonas putida C-11

Get the biomass of cells of Pseudomonas putida C-11 and prepare bioindicator, as shown in example 1. Prepare a series of standard solutions of para-NITROPHENOL concentrations (mm): 1,0; 5,0; 7,5; 10,0; 15,0; 20,0; 25,0; 30,0; 50,0. The concentration of para-NITROPHENOL in the cell is for each standard solution, respectively (in mm): 0,1; 0,5; 0,75; 1,0; 1,5; 2,0; 2,5; 3,0; 5,0. For each solution concentration at least five times carry out the determination of specific respiratory activity, as shown in example 1. The dependences of specific respiratory activity of the cells from Torno cell activity concentration made of para-NITROPHENOL is 0.5-2.5 mm, the minimum detectable concentration of 0.1 mm. Thus, the obtained data allow to draw a conclusion about the possibility of indication and quantification of para-NITROPHENOL.

Example 4. Investigation of optimal pH environment in the analysis for the cells of the strain Pseudomonas putida C-11

Get the biomass of cells of Pseudomonas putida C-11 and prepare bioindicator, as shown in example 1. However, when determining the specific respiratory activity was used 0.1 M Na2HPO4/KH2PO4buffer solution with different pH values(5,6; 6,1; 6,5; 6,85; 7,0; 7,45; 7,7; 8,1, 8,45; 9,2). All other experimental conditions correspond to the conditions described in example 1. In the measuring cell is injected a solution of para-NITROPHENOL to the final concentration in the cell is 1.0 mm. The results of the experiments are given in table 3.

Thus, from the data obtained it follows that the optimal pH for the measurement of specific respiratory activity of the cells of this strain is the pH was 7.45.

Example 5. Investigation of optimal temperature analysis for the cells of the strain Pseudomonas putida C-11

Get the biomass of cells of Pseudomonas putida C-11 and prepare bioindicator, as shown in example 1. Od is 32; 35; 38; 40; 42oC).

All other experimental conditions correspond to the conditions described in example 1. In the measuring cell is injected a solution of para-NITROPHENOL to the final concentration in the cell is 1.0 mm. The results of the experiments are given in table 4.

Thus, from the data obtained it follows that the optimal temperature for the measurement of specific respiratory activity of this strain is the temperature of the 35oC.

Example 6. Indication of metaphos cells of the strain Pseudomonas putida BA-11

Pre-receive biomass of cells of the strain Pseudomonas putida BA-11 (VKPM B-6707) as follows: culture cells were grown on 2% MPA for 24 hours at 30oC.

To obtain bioindicator cells of Pseudomonas putida BA-11 fresh daily culture were washed from the agar with 0.1 M buffer solution (Na2HPO4/KH2PO4) pH of 8.1 and added into the conical flask 250 ml, containing 50 ml of 0.1 M phosphate buffer pH of 8.1. Incubation of cells that perform a pie rocking chair when mixing intensity 160 rpm at 30oC for 2 hours. Then the biomass was separated by centrifugation, for example, in a centrifuge To-24 (GDR) from the environment at 5000 rpm for 5 minutes. Precipitate resus is ovale in a small amount of 0.1 M phosphate buffer pH 8.1 and, thus obtained bioindicator, are used to determine the metaphos. Optical density of cell suspension measured at photocolorimeter, wavelength = 540 nm in a cuvette with an optical layer thickness of 0.5 cm, against phosphate buffer determine the cell concentration. For the strain Pseudomonas putida BA-11 1 unit of optical density corresponds 0,49 g dry weight of cells in 1 l

As a reference solution using 0.1 M phosphate buffer with a pH of 8.1, saturated with oxygen. It is placed in a thermostatted cell volume of 1 ml, equipped with a magnetic stirrer. In a cell, insert the oxygen electrode, which serves voltage of-0.6 V. as a potentiostat and a chart recorder is used, for example, polarograph Radelkis HE-105 (Hungary). In buffer solution, the concentration of oxygen is practically unchanged, register unchanged in time a current of 12 μa. The determination was carried out at a temperature of 35oC.

After 3 min in a cell with a syringe dispenser enter bioindicator to a final cell concentration of 0.4-1.2 g/l to change the current time to determine the endogenous respiratory activity of the cells, it has a value of 2.3 µa/min

Then after 3-5 minutes using a syringe-dispenser in the cell vby the rate of oxygen consumption of the cells is changed, respiratory activity takes the value of 28.8 µa/min

The difference in the rate of oxygen consumption by microbial cells in a buffer solution before and after adding the sample, normalized to the number of cell - specific respiratory activity (CPA) has a value of 26.5 µa/min mg dry weight of cells, which suggests the definition of metaphos.

Example 7. Determining the concentration of metaphos in the aquatic environment by cells of the strain Pseudomonas putida BA-11

Get the biomass of cells of Pseudomonas putida BA-11 and prepare a biological indicator as shown in example 6.

The definition of CPA cells of Pseudomonas putida BA-11 in respect of commercial drug metaphos were carried out in 0.1 M Na2HPO4/KH2PO4the pH of 8.1 at a temperature of 35oC. Respiratory activity of the cells was calculated based on the concentration of metaphos determined using capillary gas-liquid chromatography (Methods for the determination of trace pesticides in food, feed and the environment. - M., 1983, S. 57-62). Quantitative determination of metaphos in solutions was carried out by the method of absolute calibration using solutions of reference metaphos in chloroform. The concentration of metaphos in the cell is (mm): 0,25; 0,5; 0 the lab activity, as shown in example 6. The dependences of specific respiratory activity of the cells from the solution concentration of metaphos presented in table 5.

Linear plot based respiratory activity of the cells on the concentration of the introduced metaphos is 0.5-3.0 mm, the minimum detectable concentration of 0.25 mm. As shown in example 6, the respiratory response of cells to the introduction of the sample is 26.5 µa/min mg dry weight of cells. On the obtained calibration dependence is determined by the concentration of metaphos, it is 0.75 mm.

Example 8. Indication and determination of the concentration of para-NITROPHENOL cells of the strain Pseudomonas putida BA-11

Get the biomass of cells of Pseudomonas putida BA-11 and prepare bioindicator, as shown in example 6. Prepare a series of standard solutions of para-NITROPHENOL concentrations (mm): 1,0; 5,0; 7,5; 10,0; 15,0; 20,0; 25,0; 30,0; 50,0. The concentration of para-NITROPHENOL in the cell is for each standard solution, respectively (mm): 0,1; 0,5; 0,75; 1,0; 1,5; 2,0; 2,5; 3,0; 5,0. For each solution concentration at least five times carry out the determination of specific respiratory activity, as shown in example 6.

The dependences of specific respiratory aktivnosti respiratory activity of the cells on the concentration of introduced para-NITROPHENOL is 0.5-2.5 mm, the minimum detectable concentration of 0.1 mm. Thus, the obtained data allow to draw a conclusion about the possibility of indication and quantification of para-NITROPHENOL.

Example 9. Investigation of optimal pH environment in the analysis for the cells of the strain Pseudomonas putida BA-11

Get the biomass of cells of Pseudomonas putida BA-11 and prepare bioindicator, as shown in example 6. However, when determining the specific respiratory activity was used 0.1 M Na2HPO4/KH2PO4buffer solution with different pH values(5,6; 6,1; 6,5; 6,85; 7,0; 7,45; 7,7; 8,1; 8,45; 9,2).

All other experimental conditions correspond to the conditions described in example 6. In the measuring cell is injected a solution of para-NITROPHENOL to the final concentration in the cell is 1.0 mm. The results of the experiments are shown in table 7.

Thus, from the data obtained it follows that the optimal pH for the measurement of specific respiratory activity of the cells of this strain is the pH of 8.1.

Example 10. Investigation of optimal temperature analysis for the cells of the strain Pseudomonas putida BA-11

Get the biomass of cells of Pseudomonas putida BA-11 and prepare bioindicator, as long as the temperatures (24; 28; 32; 35; 38; 40; 42oC). All other experimental conditions correspond to the conditions described in example 6. In the measuring cell is injected a solution of para-NITROPHENOL to the final concentration in the cell is 1.0 mm. The results of the experiments are shown in table 8.

From the data obtained it follows that the optimal temperature for the measurement of specific respiratory activity of this strain is the temperature of the 35oC.

Thus, the use of the proposed method to determine the concentration of metaphos and the product of hydrolysis of organophosphorus reduction of insecticides para-NITROPHENOL in aqueous media with high sensitivity to the investigated substrates.

1. The method of determining the concentration of insecticide metaphos and the product of hydrolysis of organophosphorus reduction of insecticides para-NITROPHENOL in aqueous medium, characterized in that the prepared bioindicator by culturing bacteria Pseudomonas putida capable of dispose of analyte as the sole carbon source, on a solid nutrient medium, containing no analyte, flushing obtained biomass phosphate buffer with a pH corresponding to intracel oxygen using an oxygen electrode at 35°C, then add to the biological indicator test substance, registered in the obtained mixture, the oxygen concentration and the velocity difference of oxygen consumption by bacteria determine the concentration of the analyte of interest.

2. The method according to p. 1, characterized in that the cultivated strain of Pseudomonas putida S-11, and washing and incubation is carried out in phosphate buffer pH was 7.45.

3. The method according to p. 1, characterized in that the cultivated strain of Pseudomonas putida BA-11, and washing and incubation is carried out in phosphate buffer pH 8.1.

 

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