SU927853A1 - Process for continuously culturing luminous bacteria photobacterium phosporeum - Google Patents

Description

The invention relates to microbiological login and can be used to test a number of biologically active substances. Constant light yield obtained bioluminescent bacteria to create a sensor nep- ⁵ reryvnogo action. The use of bacteria with a constant level of luminescence is also convenient for the usual use of luminous bacteria for testing environmental pollution, ¹⁰ poisons and drugs in medicine, etc. It can also be used in the microprobiological industry for the continuous production of biomass of bacteria with a high productivity of ¹⁵ and a high content of luciferase. The high luciferase content simplifies the purification of this enzyme. It can be used in scientific __ studies to study the relationship between luminescence and metabolic processes in bacteria.

A known method for the continuous cultivation of luminous bacteria Photobacterium belarerskii in a turbidostat, where the optical density of the suspension is maintained at a constant level til

However, during cultivation, bioluminescence changes by two orders of magnitude, i.e. luciferase activity in a cell varies 100 times. The use of such biomass for testing biologically active substances is extremely difficult, and the biomass is unsuitable for obtaining purified luciferase preparations.

Also known is a method of culturing Ph. phosphoreum (SMV 844), where the supply of a nutrient medium and you. The water of the finished product is maintained at a constant level by the combined signal, which consists of the light passing through the culture from the emitter (optical density) and the bioluminescence of bacteria. The ratio of s 9271 between the intensity of the passed through ^: light culture and the intensity of bioluminescence is chosen approximately equal to 1: 1. The nutrient medium used for cultivation at the time of its addition to the culture of luminous bacteria should inhibit bioluminescence. For this, a complex nutrient medium, Oxoid Nutrient Broth (CM1), is used, to which a glycerol of 1% v / v is added. and 2.5% w / v. NaCl [2].

The disadvantage of this method is the low stability of bioluminescence, which is + 8%. Bio- ι mass obtained with this more

4 luminescence of bacteria 2, a recorder with a positional regulator 3 "pump dispenser, feeding the nutrient medium to the cultivator 4, the reservoir with the nutrient medium 5 and the harvester 6.

Thermostatted at a temperature optimal for the growth and luminescence of bacteria, the cultivator is filled with a nutrient medium. A bacterial inoculum is introduced there. Cultivation is carried out with vigorous stirring and bubbling. The intensity of bioluminescence and the number of cells increases. After reaching a predetermined level of luminescence, it is satisfactory for obtaining luciferase, but is not suitable for use as a bioluminescent sensor of continuous action when it is necessary to record a change in luminescence of less than ± 8%. In addition, the method requires the use of sophisticated process control equipment by means of the position controller regulator, a metering pump is turned on, which feeds the nutrient medium to the cultivator. At the same time, the equivalent volume of culture fluid with luminous bacteria merges into the harvest collector. After diluting the culture and reducing the intensity of bioluminescence, inactivation.

The purpose of the invention is to increase the stability of the bioluminescence of bacteria.

This goal is achieved by the fact that the nutrient medium is added when bioluminescence exceeds a predetermined level, and the nutrient medium contains sodium chloride, disubstituted sodium phosphate, monosubstituted phosphate, ammonium phosphate disubstituted, magnesium sulfate, peptone and glycerol, when wearing components

Sodium Chloride Sodium Phosphate Disubstituted Potassium Phosphate Monosubstituted Ammonium Phosphate Disubstituted Magnesium Sulfate. Glycerol

Peptone

Water of the following, g / l:

28-30

8-10

1.0-1.1

0.50-0.55

0.08-0.12

1-2.5 5.0-5.1 Up to 1000 ml

The drawing shows a device used to implement the proposed method.

The device consists of a thermostatically controlled cultivator 1, a bio sensor, the cycles of the positional controller return to their original position and the flow of the nutrient medium is stopped. Then the process is repeated. The luminescence intensity is recorded continuously30 using a recorder. The feed rate of the nutrient medium by the metering pump is selected so that the duct (the ratio of the volume of medium pumped by the pump over a certain period of ₃₅ time to the volume of the cultivator) is greater than the maximum growth rate of bacteria.

Example. Prepare 2 l of medium containing, g / l: _

NaCE 28-30

Nai ^ HPO ^ 1 2HqO 8.0-10

KHqPO ₄ 1.0-1.1 (NH ₄ ) q_HPO ₄ 0.50-0.55

MgS0 ₄ 7H <20 0.08-0.12

Glycerin 1.0-2.5

Peptone 5.0-5.1

This medium is poured into a cultivator ₅₀ with a capacity of 150 ml thermostatically controlled at 20 ± 0.1 ° C. After bacterial inoculation, the initial cell concentration is ^ζ 0.65 · 10 ¹ cells / ml, and the intensity of bioluminescence is 2.5 rel. Bubbling is carried out on the basis that the amount of air ⁵⁵ supplied in 1 min is equal to the volume of the cultivator, i.e.

150 ml / min, after reaching a predetermined level, equal to 40 rel.ed., the position controller closes and the metering pump for supplying the nutrient medium is turned on, providing a flow rate of 1.5 4 ^-1 . Maximum specific growth rate Ph. phosphoreum in this medium is 0.4 h. After the establishment of the stationary regime, the specific growth rate of bacteria is 0.3-0.35 h * ¹ , and the number of cells is 0.4 x x1 (G® g / ml.

Using the proposed method of cultivation allows you to stabilize the bioluminescence of Ph phosphoreum with an accuracy of + 0.6%. At the same time, complex equipment is not required. The entire installation is assembled from widespread instruments and components. The performance of the proposed method of cultivation is 2.5 times higher than in periodic mode.

Claims (3)

The invention relates to microbiology and can be used for testing a number of biologically active substances. Constant luminescence of the obtained yield of bacteria allows creating a bioluminescent sensor of continuous action. The use of bacteria with a constant level of luminescence is convenient and with the usual use of luminous bacteria for testing the contamination of the environment, poisons and drugs in medicine, etc. It can be used in the microbiological industry for the continuous production of bacterial biomass with high productivity and high luciferase content. The high content of luciferase simplifies the method of purification of this enzyme. It can be used in scientific studies to study the relationship between luminescence and metabolic processes in bacteria. A known method of continuous cultivation of luminous bacteria of Photobacterium belarerskii in a turbidostat, where the optical density of the suspension til is maintained at a constant level. However, in the process of cultivation, bioluminescence changes by two times, i.e. luciferase activity in the cell varies by 100 times. The use of such biomass for testing biologically active substances is extremely difficult, and the biomass is unsuitable for obtaining purified luciferase preparations. A method of cultivating Ph is also known. phosphoreum (SMB 8), where the supply of the nutrient medium and the output of the finished product at a constant level is supported by a combined signal, which is composed of light passing through the culture from the emitter (optical density) and bioluminescence of bacteria. The ratio between the intensity of the light passing through the culture and the intensity of the bioluminescence is chosen to be approximately 1: 1. The nutrient medium used for cultivation at the time of its addition to the culture of luminous bacteria should inhibit bioluminescence. For this, Oxoid Nutrient Broth (CM1) complex nutrient media is used, to which 1% v / v glycerol is added. and 2.5% wt./about. NaCI {21, The disadvantage of this method is the low stability of bioluminescence, which is 8. The bio mass obtained in this case is more satisfactory for obtaining lyephrase, but is not suitable for using it as a bioluminescent continuous sensor when it is necessary to fix the change in luminescence ± 8%. In addition, the method requires the use of complex apparatus for controlling the process of clotting. The purpose of the invention is to increase the stability of the bioluminescence of bacteria. The goal is to ensure that the nutrient medium is added when the bioluminescence exceeds the given level, and the nutrient medium contains sodium phosphate sodium disodium, phosphoric acid potassium monosubstituted, ammonium phosphate disubstituted, magnesium sulphate, peptone and glycerin, in the following ratio of components, g / l: chlorine total sodium Phosphate sodium disubstituted Potassium phosphate monosubstituted 1.0-1.1 Ammonium phosphate disubstituted 0.50-0.55 Sulfuric acid 0.08-0.12 magnesium Glycerin 1-2.5 5.0-5.1 Peptone To 1000 ml The drawing shows the device used to implement the proposed method. The device consists of a thermostatically controlled cultivator 1, a bio-luminescence sensor for bacteria 2, a recorder with position controller 3, a metering pump supplying the nutrient medium to the cultivator 4, a reservoir with nutrient medium 5, and a crop collector 6. Thermostatically controlled at the temperature optimal for the growth and luminescence of bacteria the cultivator is filled with a nutrient medium. An inoculum of bacteria is also administered there. Cultivation is carried out with vigorous stirring and bubbling. The intensity of bioluminescence and the number of cells increases. After the preset luminescence level has been reached by means of the position controller, the metering pump turns on, feeding the nutrient medium to the cultivator. At the same time, an equivalent volume of culture fluid with luminous bacteria is discharged into the crop collector. After diluting the culture and reducing the bioluminescence intensity, the contacts of the position controller return to their original position and the feeding of the nutrient medium is stopped. The process is then repeated. The luminescence intensity is recorded continuously using a recorder. The feed rate of the nutrient medium by the dosing pump is chosen so that the flow path (the ratio of the volume of the medium pumped by the pump over a certain period of time to the volume of the cultivator) is greater than the maximum growth rate of bacteria. Example. Prepared 2 liters of medium containing, g / l: NaCE28-30 .12H | jO 8.0-10 KN (P0.0.0-1.1 (NH4) LHP040.50-0.55 MhZod 7H (ip0.08- 0.12 Glycerin 1.0-2, 5 Pepto5.0-5.1 This medium is poured into a 150 ml cultivator which is temperature-controlled at 20t.. After bacterial inoculation, the initial cell concentration is 0.65 10 cells / ml, and the bioluminescence intensity is 2 , 5 rel. Units Bubbling is carried out on the basis that the amount of air supplied per 1 min equals the volume of the cultivator, i.e. 150 ml / min, after reaching a predetermined level of 40 rel., The position regulator closes and pitch dosing pump starts The average specific growth rate of Ph. phosphoreum on this medium is 0, h. After establishing the stationary mode, the specific growth rate of bacteria is 0.3-0.35 h, and the number of cells is 0, | xx1 (Gu g / ml. Using the proposed culture method stabilizes the bioluminescence of PK phos phoreum with an accuracy of t0.6%. At the same time, complex equipment is not required. The installation is assembled from widespread devices and assemblies. The productivity of the proposed method of cultivation is 2.5 times higher than in the periodic mode. DETAILED DESCRIPTION A method for continuously cultivating light bacteria of Photobacterium phosphoreum in a nutrient medium containing a carbon source and mineral salts, characterized in that, in order to improve the bioluminescence stability, the nutrient medium is added when the bioluminescence exceeds a predetermined level, and the nutrient medium contains sodium chloride. , sodium phosphate-sodium disubstituted, potassium phosphate monosubstituted, ammonium phosphate ammonium disubstituted, magnesium sulphate, peptone and hydrogen erin at the following ratio The following components, g / l: Sodium chloride Sodium phosphate dibasame8-tO scoop Potassium phosphate monoamer1, 0-1,1 scooped Ammonium phosphate bicase 0, 50-0.55 bichenate Sulfuric acid Mai- 0.08-0.12 Glycerin 1.0-2, 5 Peptone 5.0-5.1 Up to 1000 ml Sources of information taken into account during the examination 1.Fish AM Investigation of several sides of the metabolism of lucid bacteria in continuous culture. Aztoref. Candidate Dissertations, Krasno rsk, 1969. 2.Wardley-Smlth B. et all. The continous culture of luminous bacteria. 3. Appl. Bact., 1975, 39, p. 377-3 3 (prototype).