CS199446B1 - Method for the cultivation of chlorococal algae directed at intensive synthesis of radioactive saccharides having high specific activity,marked by radionuclide c up 14 - Google Patents

Description

The invention solves a method of cultivation of chlorococal algae of the appropriate type, aimed at intensive synthesis of radioactive carbohydrates ó high specific ac.

in 14 tivite, labeled with radionuclide C.

The preparation of ¹⁴ C-labeled radioactive compounds using autotrophic organisms that use radioactive carbon dioxide as a carbon source is currently used primarily in the production of amino acids, proteins and nucleic acids worldwide. Some procedures are the subject of patents (see, e.g., U.S. Patent Nos. 100738, 121808, Authentic Certificates No. 1511, 1512, French Patent 7208482), others are secret. Also, the procedures used by ¹⁴ C-labeled carbohydrate manufacturers are not disclosed. It is believed that here also autotrophic organisms are used to produce the desired compounds.

The present method of cultivation, used by the Czechoslovak monopoly producer of radioactive compounds, the Institute for the research, production and utilization of radioisotopes in the production of radioactive carbohydrates labeled with ¹⁴ C radionuclide, is characterized by a low yield of about 1% of total incorporated radioactivity.

SUMMARY OF THE INVENTION The present invention provides a method of cultivating chlorococal ras of the appropriate type, which is directed to the intensive synthesis of high specific activity radioactive saccharides labeled with a radionuclide of < ⁴ > C.

199446-2 that the cultivation is carried out at a temperature of 35-38 ° C. For cultivation, a population of daughter cells of synchronous culture is used, which population is passing through _; before the start of the production cycle by an extended dark cycle period. In this experimental design, nucleic acid synthesis is inhibited and carbohydrate synthesis is stimulated. During a one-hour cultivation of only a few hours, which is a prerequisite for the survival of an organism growing in the presence of radioactive carbon dioxide as the sole carbon source, a significant amount of starch and sucrose is synthesized and thus high specific activity of the products.

The inhibitor of nucleic acid and protein synthesis is high for a given strain sublethal temperature. The photosynthetic activity and growth rate are the same as under optimal culture conditions.

The condition of a successful cultivation of the cultivation is that the algae seed, which starts the production, has the required characteristics. The preparation of the batch, including the apparatus in which it is carried out, is therefore part of the present invention.

Because the response of a cultured cell population to interfering with its metabolism varies at different stages of the cell's life, it is necessary to use synchronous populations, ie a population in which all the cells are at the same stage of the life cycle. Synchronous culture also makes it possible to regulate the amount of carbohydrates, especially starch, in the cells prior to the start of cultivation to ^2 COg. This starting amount of carbohydrates, which must be as low as possible in order to achieve high specific activities, is regulated both by the length of the dark period of the synchronous cycle and the temperature at which the mother cells divide into the parent cells. enzymes that accelerate the digestion of carbohydrates.

^^ preculture in an atmosphere of C0 ₂ and own production culture was performed in the culture device of the same type. It consists of a double-walled glass culture cylinder (coolant flowing through the space to ensure optimum temperature of the cultured suspension) with a working volume of 750 ml. In the culture space, the algae suspension is exposed to photosynthetically effective radiation while being vigorously stirred and saturated with carbon dioxide bubbling through the suspension in a COg / air mixture.

Production cultivator accessories consist of:

l) a U-shaped irradiation body fitted with two Nitrafot lamps of a total power of 1,000 W. The inner sides of the housing are covered by mirror surfaces, and the reflective rear wall is covered by a mirror. The application of the illumination U-body to the rear mirror wall allows complete closing of the roll-out roll in the space of the irradiation body. The intensity of the photosynthetically effective radiation (PAR) incident on the front face of the cultivator is measured by a phytoactinometer, ie a sensor, non-selectively sensitive in the photosynthetically effective radiation range of 400 to 700 nm and is continuously adjustable by an input voltage regulator.

- 3 199446

2) C02 Yyvíječe ^14> connected to the circuit of circulation mixtures C02 / air. ¹⁴ CO 2 is released in the generator from the raw material, which is Ba ¹⁴ CO 3, by dosing the acid. The circulation circuit includes an indicator of the concentration of ¹⁴ CO2 in the gas mixture, the circulation of the mixture is ensured by the pump;

3) ¹⁴ CO ₂ absorption absorber that connects to the atmosphere circuit through the circulating apparatus after the cultivation is complete or whenever it is necessary to discontinue the cultivation;

4) vacuum pumps connected through the ¹⁴ CO ₂ absorber to the gas space of the apparatus. It is used to create a vacuum that indicates the tightness of the apparatus and prevents leakage of the gaseous mixture into the external space;

5) a cooling device consisting of an ultra-thermostat connected via a contact thermometer, in a closed circuit with the cultivator.

Exemplary embodiment

Chlorella vulgaris is used for production cultivation

BEIJBRINCK, strain OM-12/1975, whose stem culture is stored in test tubes on sloping agar, saturated with mineral nutrient solution, at a temperature of 15 ° C and an irradiation intensity of 10 Wm ² FAR.

(a) Preparation of culture

The culture is transferred from agar to a liquid nutrient solution of the following composition:

KNO _3, lg.l ¹ MgSO4. 7 H20 1 gl ¹ KH2PO ₄ 680 mg.l ¹ ,

Ca (NO ₃ J _2. 4 H ₂ 0 10 mg. L ¹ , ferric sodium chelatonate 18,4 mg.l " ¹ (content

Fe ⁺⁺⁺ 5.6 mg.l ¹ }, H ^ BO ^ 6.18 mg.l ¹ and trace heavy metals:

Cu 0.635 mg.l ¹ , Mn 0.6 mg.l ¹ , Mo 0.96 mg.l ¹ , Co 0.59 mg.l ¹ . The necessary amount of KOH is added to the nutrient solution to adjust the pH to 6.8.

In the nutrient solution, the culture is grown in a 300 ml culture cylinder under irradiation of 50 Wm &^lt; ² &^gt;, 27 [deg.] C. and a CO2 concentration in a mixture with air corresponding to 2% dissolved CO2 in suspension until the density of the stationary phase of the growth curve. (typically 5 days). The culture thus prepared, transferred to a production-type cultivation cylinder, is synchronized. Synchronization of ee is accomplished by a method of alternating light and dark periods (see, for example, DOUCHA: Synchronous cultures; Algal Assays in Eutrophication Monitoring, Stuttgart 1978). Synchronous culture after each cycle is grown in an optical density range of 0.150 (beginning of the light period) to 1,200 (after division of the mother cells into daughter cells) with a FAR 150 Wm ² irradiation, 27 ° C and ² % dissolved CO 2 in suspension . Under these conditions, the synchronous cycle light period is 14 hours, the dark period is 8 hours and the average specific growth rate of the culture = 0.22 h ¹ . After 3-4 cycles, the culture is ready for production cultivation.

199446 - 4 The following data are continuously monitored and corrected during both pre-cultivation and production cultivation:

1) suspension temperature; ·

2) pH of the suspension

3) the concentration of CO ₂ dissolved in the suspension

4) optical density of the suspension

5) the FAR intensity incident on the surface

6) culture cells.

The optical density is measured in a 5 mm cell at 750 nm on a SPECOL photometer. If we multiply the optical density by 0.65, we get an approximate value of the dry matter of the algae in the range of cultivation densities used.

(b) Production cultivation

The dark period of the last pre-production synchronous cycle runs for 13 hours. An hour before the end of the dark cycle period, 750 ml of a new nutrient solution is prepared in the production culture cylinder, free of all physically dissolved non-radioactive carbon dioxide. The solution is then saturated with radioactive carbon dioxide (the equilibrium in the suspension of dissolved ¹ CO 2 with an atmosphere containing 2% ¹ CO 2 occurs after about 30 minutes of vigorous bubbling). After saturation, a sufficient amount of pro-racial inoculum is introduced into the cylinder so that the starting density of the suspension is 400 mg dry matter.ml ^-1 . Inhibition of nucleic acid and protein synthesis, accompanied by subsequent intensive carbohydrate synthesis, is achieved by culturing at a high, sublethal temperature for a given strain in the range of 35-38 ° C, at which time a light-cycle of six hours is run. Initial irradiance 130 Wm · ·. —2

The FAR will increase to 180 W.m FAR after four o'clock. After cultivation, the algae suspension is cooled to 2 ° C and centrifuged at this temperature. The obtained racial sediment is the starting radioactive product for the actual processing. The course of starch and sucrose synthesis, expressed in µg / ml suspension and in percent dry matter of ras, is shown in Figures 1 and 2. The specific activity of sucrose and glucose after starch hydrolysis during culture is shown in Figure 3.

Claims (1)

Object of the invention Method for the cultivation of chlorococcal algae directed to the intensive synthesis of high specific activity radioactive saccharides labeled with ¹⁴ C-radionuclide, characterized in that the cultivation is carried out at a temperature of 35-38 ° C using a population of synchronous culture daughter cells, before the start of the production cycle by an extended dark cycle period.