RU2476598C2 - Method of quantitative determination of dehydrogenase activity of microorganisms - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method provides mixing a suspension of the microorganism with the substrate (10% solution of glucose) and a hydrogen acceptor, which is used as a methylene blue colorant followed by measuring the concentration of methylene blue during the enzymatic reaction. According to the rate of change of the concentration of methylene blue in the linear phase of the enzymatic reaction the dehydrogenase activity is determined.

EFFECT: invention enables to reduce time of determining the dehydrogenase activity of microorganisms and to improve the quality of its determination.

1 dwg

Description

The invention relates to analytical methods for determining the enzymatic activity of microorganisms and can be used to assess the dehydrogenase activity of activated sludge.

Currently, there is only one known method for the quantitative determination of the dehydrogenase activity of microorganisms [Guidelines for the determination of dehydrogenase activity during technological monitoring of the operation of aeration tanks. - Ministry of Housing and Communal Services of the RSFSR Order of the Red Banner of Labor, Academy of Public Utilities named after K.D. Pamfilova. - Moscow, 1978]. This method consists in the following: a sample of microorganisms (activated sludge) is placed in three centrifuge tubes, the pH is adjusted to 7 and centrifuged. Then the supernatant is drained from the tubes, and tap water and untreated wastewater are added instead. After stirring, a 0.5% solution of 2,3,5-triphenyltetrazole chloride (TTX) was added. The tubes are tightly closed, mixed and placed in a thermostat for 55 minutes at 37 ° C. At the same time, a fourth tube with sludge without TTX is placed in the thermostat, which serves as a control. After 55 minutes, the samples are centrifuged, the supernatant is drained and 10 ml of ethanol is added to the precipitate. The contents of the tubes are mixed, and then periodically shaken until the sludge flakes are completely discolored, which, depending on its quality, takes 15-30 minutes. After bleaching, the samples are centrifuged again. The colored alcohol solution from each sample is poured into a test tube, mixed and colorimetric on a photoelectric colorimeter (FEC) with a blue light filter No. 5 (490 nm) in a cuvette with a layer thickness of 0.5 cm. Dehydrogenase activity is expressed in milligrams of reduced formazan per 1 g of dry or ashless sludge substance (specific activity), or per 1 liter of a mixture (total activity).

This method of determining dehydrogenase activity is adopted as a prototype and is characterized by the following disadvantages:

1) duration;

2) labor input;

3) multi-stage;

4) the duration of cultivation (55 minutes). During this time, the cell can develop an additional amount of necessary enzymes. At the same time, the amount of dehydrogenases may decrease due to the action of proteases;

5) the specificity of the nutrient medium for microorganisms (activated sludge) is not taken into account, since under the condition of insufficient carbon nutrition, the rate of dehydrogenase reaction will be determined not directly by dehydrogenase activity, but by the concentration of the substrate;

6) for such a long time and in the case of nonlinearity of the process, the same amount of TTX can be restored with different amounts of enzyme;

7) does not take into account the possibility of the content in microorganisms of a significant amount of substances that can restore TTX without the presence of dehydrogenases.

The problem to which the present invention is directed, is to develop a method that allows you to quickly quantify the dehydrogenase activity of microorganisms and has good reproducibility.

This is achieved by the fact that in the quantitative determination of the dehydrogenase activity of microorganisms, providing for their contact with a substrate and a hydrogen acceptor, methylene blue is used as a hydrogen acceptor, and the activity of dehydroginases is determined by the rate of change of the concentration of methylene blue in the linear phase of the enzymatic reaction.

The degree of dehydrogenase activity of microorganisms in the proposed method is judged by the decrease in the concentration of methylene blue in the cell, which is continuously recorded optically. The studied suspension of microorganisms is introduced into a thermostatic measuring cell, including a signal recording system (LED, photosensor), anaerobic conditions are created in the cell; a substrate (for example, glucose) and a solution of methylene blue as a hydrogen acceptor are also introduced there. The duration of the experiment is 5-7 minutes.

An illustrative example of the implementation of the proposed method includes the following steps: 60 ml of distilled water, a certain amount of a carefully mixed suspension of activated sludge (1-2 ml), 1 ml of a 10% glucose solution are added to a 100 ml beaker. A lid was installed on the glass with an integrated system for recording the signal of the measuring cell. The volume of water was adjusted to the mark (83 ml), providing anaerobic conditions. After that, they turned on an LED with a photosensor, a stirrer. Place the beaker in a thermostat and set the desired temperature. After that, the thermostabilization of the cell and stabilization of the photosensor readings were expected, and 50 μl of a 0.2% methylene blue solution was introduced using a dispenser. The photosensor began to detect a decrease in the concentration of methylene blue during the enzymatic reaction.

Under conditions of excess substrate and hydrogen acceptor, the course of the reaction is linear for approximately 300 seconds, after which the measurement process is stopped. The readings of the photosensor were recorded once a second, therefore, the readings of the device were approximated by time in a linear manner (Fig. 1).

Further, based on the slope of the specified linear relationship corresponding to the linear phase of the enzymatic reaction, dehydroginase activity was calculated, which component in this case

Figure 2 shows the relationship between the readings of the device (photosensor) and the amount of methylene blue introduced into the cell, characterized by the equation y = -0.02512x + 1137.2263. That is, it is determined that in the concentration range used, an increase in the readings of the device by 1 corresponds to a decrease in the content of methylene blue in the cell by 0.02512 nmol. Microorganisms introduced into the cell, as a rule, significantly scatter and absorb light, increasing the coefficient of 1137.2263, however, the value of 0.02512 remains unchanged.

In general, the above calculations explain that the reaction rate is W = 10.68 · 0.02512 nmol / s, and taking into account that 2 ml of a suspension of microorganisms were taken for the reaction, the enzyme activity A = 10.68 · 0.02512 · 60 / 2 = 8.05 nmol / (min · ml), where 60 is the conversion per minute.

Thus, based on the slope of the indicated linear relationship corresponding to the linear phase of the enzymatic reaction, dehydroginase activity was calculated, which in this case was 8.05 nmol / (min · ml).

In general, the formula for calculating dehydrogenase activity, based on the slope of the dependence (Fig. 1) and the concentration of methylene blue, can be represented as follows:

where A is the activity of the enzyme, mol · min ^-1 · ml ^-1 ;

C is the concentration of methylene blue, mol · ml ^-1 ;

V _p is the volume of the reaction cell (in the example, 83 ml), ml;

V is the sample volume of microorganisms, ml;

t is the duration of the enzymatic reaction, min;

D is the optical density of the reaction mixture;

To ₁ is the coefficient of proportionality between C and D, equal to the product of the extinction coefficient and the path of light in solution (Bouguer-Lambert-Bera law (D = EL C)), ml · mol ^-1 ;

P - digital readings of the photosensor;

K ₂ is the coefficient of proportionality between P and D;

V _p K ₂ / K ₁ - the coefficient of proportionality between C and P, taking into account the volume of the reaction cell, mol; determined experimentally (figure 2). In our case, it is -0.02512.

dP / dt is the rate of change of the instrument readings in time, s ^-1 .

The coefficient of 10.68 in the equation y = 10.68x + 37205 is the slope of the straight line when studying the enzymatic reaction (figure 1).

The final formula can be represented:

A means (instrument) for measuring optical density, suitable for determination, is known to a person skilled in the art. In particular, a spectrophotometer (colorimeter or analogs) can be used, having an interface with a computer, having a thermostatically controlled cell, providing mixing of the solution and determining the optical density or other value associated with it. A preliminary calibration must be performed between this value and the amount of methylene blue in the cell (which is obvious to a person skilled in the art).

The experimental results are reproducible, since they are based not on one or two measurements, but on several hundred. Moreover, the error is less than 5%.

The essential features of the proposed method in comparison with the prototype are the following:

1) methylene blue is used as a hydrogen acceptor;

2) the consumption of methylene blue is determined by continuously recording the concentration of methylene blue by the photometric method;

3) registration of changes in the concentration of methylene blue is carried out within 5 minutes;

4) quantitatively, the dehydrogenase activity of microorganisms is expressed by the number of nanomoles of methylene blue per unit volume of sludge (dry matter mass of microorganisms) per unit time.

The advantages of the proposed method compared to the prototype is the following:

1) speed (the method allows you to perform analysis in 5-7 minutes);

2) significant simplification (fewer operations, automatic recording of readings, temperature control);

3) the exclusion of changes in the concentration of dehydrogenases, which may occur due to processes occurring in a living cell;

4) elimination of the influence of the color of the medium, since the measurement is not the absolute value of the optical density, but the kinetics of its change, which is especially important in the case of colored wastewater liquids.

Claims (1)

A method for the quantitative determination of the dehydrogenase activity of microorganisms, providing for their contact with a substrate and a hydrogen acceptor, characterized in that methylene blue is used as a hydrogen acceptor, and the dehydrogenase activity is determined by the rate of change of the concentration of methylene blue in the linear phase of the enzymatic reaction.

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