SU958495A1 - Method for determining intensity of respiration of microorganisms - Google Patents

Description

The invention relates to general and technical microbiology and can be used to study the kinetic parameters of the growth of microbial cultures, the control of fermentation processes, etc.

There is a method for determining and testing the respiration of microorganisms amperometrically using a Clark 1 electrode.

The disadvantage of the known method is that it gives underestimated values of the respiration rate, since in the process of taking a sample and transferring it to the polygraphic cell a significant part of the breathing substrates has time to be consumed.

The most ideal approach to the proposed technical essence and the achievement of the Oiviy effect is a method for determining the respiration rate of microorganisms, which provides for their continuous cultivation under aerobic conditions followed by calculation. According to this method, measurement of oxygen consumption by microorganisms is carried out directly in the fermenter by analyzing stationary concentrations of 0 and CO. in the composition of the gas mixture entering and leaving the fermenter with a subsequent calculation of the consumption of O or the release of CO from the difference 2.

This method is characterized by the need to maintain the air flow rate in the fermenter at a strictly defined level, the use of expensive instruments - gas analyzers, as well as low sensitivity and reliability of determination, oxygen consumption rate, so that this method allows to obtain reproducible results only when measuring respiratory rate dense and fast growing crops.

The aim of the invention is to improve the accuracy and sensitivity of the method.

20

The goal is achieved by the fact that according to the method of determining the intensity of respiration of microorganisms, providing for their continuous cultivation under aerobic conditions with

Claims (2)

25 by the subsequent calculation, it was proposed in the process of continuous cultivation to isolate microorganisms from aerating air, to measure the consumption of oxygen dissolved in the culture medium amperometrically, to resume air supply and to register an increase in the concentration of dissolved oxygen; -c-- aL ° where C and C are, respectively, the concentration of dissolved oxygen in the feed medium (the upper limit on oxygen saturation at a given temperature) and microbial culture, μA — oxygen mass transfer coefficient, h; X - bio% 1 microorganism mass, mg / l; (- specific rate of oxygen loss, μA / h mg. The essence of the proposed method is as follows. The measurement rate of dissolved oxygen concentration (C) in the continuous chemostat culture of microorganisms is determined by the ratio of its transport from the gas phase to the liquid and microbial consumption biomass during respiration. (1) where GO and C is the concentration of dissolved oxygen in the feed medium (upper limit of oxygen saturation at a given temperature) and microbial culture, respectively, μA / l; KI, coefficient m oxygen transfer X - microorganism biomass, mg / l; q - specific rate of oxygen consumption (§ -i-μA / h-mg. The taxonomic C concentration values can be found from equation (1) provided (3c / dt O After turning off air supply cultivator mass transfer between gas and liquid stops, i.e. Ki, p (Co C) 0, and then the respiration rate of the microbial culture () is quantitatively determined by the falling speed to the concentration of dissolved oxygen et; -v Resuming the air supply to the cultivator leads to increase with speed; which can be read p by integrating equation (1) with the boundary conditions t 0, C f. - -UrtlH -h - -K f - -.t С, where the concentration of dissolved oxygen at the time of the renewal of the aeration. Equation (4) enables the graphic-analytical determination of the mass transfer coefficient K, in the coordinates-fi, t. at. Eq. (4) instead of the value C, it is legitimate to use any physical quantities linearly related, including the value of the dissolved oxygen voltage Q or the electric current in the polarographic probe used for the experimental determination of P 4, therefore the calibration of the polarographic probe is possible to produce directly in the course of growing the crop, compared the magnitude of the current with the value of C, calculated by equation (2). The magnitude of Cd for IT is determined from the temperature dependence of the solubility of oxygen in water, and K and the respiration rate of others, X, is measured experimentally. FIG. Figure 1 shows schematically an installation for cultivation; in fig. 2 - Clark's electrode on fig.Z change in oxygen concentration. Yeast culture Debariomyces formicarius VKM-U-1555 is grown at 28 ° C on a synthetic medium of the following composition, g / l; glucose 0,2; (. 0,4;. 0,1; СаС1г 2Н ,, 0 0,02; 0,8; EDTA 0,005; Biotin 0,00001; a mixture of trace elements. The cultivation is carried out at a dilution rate of O-0.069 h in the set., Not 1. The microbial culture is grown in a 600 ml glass fermentation vessel 1 with a jacket for a thermostatic liquid. The culture is mixed using a magnetic stirrer, the medium is fed by a peristaltic pump. The humidified sterile air enters the culture through the bottom of the fermentation vessel The durability of the culture volume is supported by by removing an excess of microbial suspension with an air stream through a vertical drain tube 2 mounted into a cork made of non-toxic rubber 3 cut at an angle to the horizontal plane. The dissolved oxygen sensor 4 mounted to the probe 5, a sampler 5, also suitable for the inoculum and a thick-walled capillary for the introduction of a sterile nutrient medium 6. The design of the fermentation vessel described allows you to quickly (1-2 seconds) isolate the culture from the atmospheric air by stopping its supply (closing the entrance a 7) The bubbling air escapes from the fermentation vessel through a hole in the rubber stopper, followed by establishing the level of the liquid phase within the drain tube 2 (indicated by a dotted line). To measure the concentration (voltage) of the dissolved oxygen, use the low-inertia and superior sterilization by autoclaving the modified Clarke electrode, which consists (Fig. 2) of two glass parts — the housing 8 and the rod 9 carrying the platinum cathode 10 at the end, and the silver anode 11 in the form of a plate or a spiral wound around a rod. The rod 9 is ground to the lower part of the sensor body, which allows Qa to fix the cathode in a strictly defined position. A channel 12 is left between ground surfaces (due to the fact that the rod has the shape of a circle truncated along the chord in cross section), the presence of which is necessary for continuous operation of the sensor. The lower opening of the housing 8 is covered with a fluoroplastic membrane 13 (F-4, thickness 12 µm), fixed by a ring of silicone rubber 14 and epoxy glue 15. Then an electrolyte (1N NaCl) and a rod 9 with electrodes are inserted into the case the latter is in the upper part of the body with a rubber gasket 1 and a paraphid. The final assembly of the sensor (insertion of electrofusion and installation of electrodes) is performed after autoclaving (0.5 atm, 30 min) of the housing 8 with membrane 13 as part of a fully assembled installation for the continuous cultivation of microorganisms. After the culture is stabilized (3 s after inoculation), the respiration rate is measured: air is fed to the fermenter and dissolved oxygen concentrations are recorded (Fig. 3), then air supply is restored and the increase in dissolved oxygen concentration is recorded. The value of KI is calculated. According to equation (3) -13 h; The value C according to equation (2) is equal to 459 μA O, j / and the culture respiration rate (according to equation 3) μA 3 –hour ° 2. The proposed method allows to increase the reliability and sensitivity of determining the respiration intensity of microorganisms by more than 20100 times, compared to with a known method, gives well reproducible results with a relative error of determination not exceeding .2%. DETAILED DESCRIPTION OF THE INVENTION A method for determining the intensity of respirations of microorganisms, providing for their continuous cultivation under aerobic conditions, followed by calculation, characterized in that, in order to increase the accuracy and sensitivity of the method, in the process of continuous cultivation, microorganisms are isolated from aerating air, the consumption of dissolved in the culture the oxygen medium is amperometric, the air supply is resumed, and an increase in the concentration of dissolved oxygen, e. when calculating the respiration rate, the calibration of the dissolved oxygen sensor was corrected according to the formula where GO and C are, respectively, the concentration of dissolved oxygen in the feed medium (upper limit of oxygen at a given temperature) and microbial culture, μL / L; K. — mass transfer coefficient for oxygen s / h; X is the biomass of microorganisms, q, is the specific velocity. children of oxygen, µA / h- mg. Sources of information taken into account in the examination 1.Sholts KF, Ostrovsky D.N. Cell for amperometric determination of oxygen. In Sat .. Methods of modern biochemistry, 1975, M., Science, p. 52-58. 2.Olijve W. Kok I.I. Analysis of growth of gluconobacter oxydans in glucose containing media. Arch. Microbiol 1979, 121.3, p. 283-290. TO i -9 Nf -tf 15 -13 C, nHAf / i yu no t tea