CS197918B1 - Method for the cultivation of chlorococal algae,directed to the intensive synthesis of radioactive saccharides, having high specific activity,labeled by radionuclide c up 14 - Google Patents

Description

SUMMARY OF THE INVENTION The present invention provides a method for culturing chlorococcal algae of the appropriate type for the intensive synthesis of high specific activity radioactive saccharides labeled with ¹⁴ C radionuclide.

The preparation of radioactive compounds labeled with radionuclide ^ ⁴ C by autotrophic organisms, which use radioactive carbon dioxide as a carbon source, is currently used mainly in the production of amino acids, proteins and nucleic acids worldwide. Some procedures are the subject of patents (see, eg, US Patent No. 100738, US Patent No. 121808, French Patent No. 72-8482), others are secret. Also, the procedures used by carbohydrate producers labeled with a < ⁴ > C radioisotope are not disclosed. It is believed that here, too, autotrophic organisms are used to produce the desired compounds.

The current culture method used by the Czechoslovak monopoly producer of radioactive compounds - the Institute for Research, Production and Utilization of Radioisotopes - in the production of radioactive carbohydrates labeled with radionuclide ^ ⁴ C is characterized by a low yield of about 1% of the total incorporated radioactivity.

SUMMARY OF THE INVENTION The present invention provides a method for culturing chlorococal algae of the appropriate type, which is directed to the intensive synthesis of high specific activity radioactive saccharides labeled with a < ⁴ > C radionuclide, which comprises culturing in the presence of a specific oycloheximide inhibitor. A population of daughter cells is used for culture

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187 911 of synchronous culture, whereby this population is undergoing a prolonged dark cycle period prior to the start of the production loop. During a one-hour, only a few-hour culture, which is a prerequisite for the survival of an organism growing in the presence of radioactive carbon dioxide as the sole carbon source, significant amounts of starch and sucrose are synthesized and thus high speo-toxic product activity.

The inhibitor of nucleic acid and protein synthesis is cycloheximide. The photosynthetic activity and growth rate are the same as under optimal culture conditions.

The condition of a successful production culture is that the algae seed, which begins 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 cultured cell populations to interfering with its metaboli® varies at different stages of cell life, it is necessary to use a syhronous population (ie a population in which all cells are present) to achieve high yields and obtain a product of high specific activity. 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 the culture to about 40% CO 2. This starting amount of carbohydrates, which must be the lowest in order to achieve high specific activities, is regulated both by the length of the dark period of the synchronous loop and the temperature at which the mother cell divides into the parasitic cells. enzymes that accelerate the cleavage of glyoids.

The pre-cultures in the atmosphere of the production and cultures are carried out in a culture apparatus of the same type. It consists of a glass culture flask with a double jacket (coolant flows through the space to ensure optimum temperature of the cultivated suspension) with a working volume of 750 ml. In the culture space, the algae suspension is exposed to photosynthetically effective radiation, where it is vigorously stirred and carbonated by bubbling the suspension in a CO 2 / air mixture.

The production cultivator accessories consist of

- irradiation body U-shaped, fitted with two Nitrafot lamps with a total wattage of 1000 W. The inside of the body is covered with mirror surfaces, · the reflective rear wall is also covered by a mirror. The application of the illuminator TJ to the rear mirror wall allows complete closure of the culture vessel in the space of the radiator. The intensity of photosynthetics of the effective radiation (PAR) incident on the front wall of the cultivator is measured by a phytoactinometer (ie a sensor, non-selectively sensitive in the region of photosynthetically effective radiation in the range of 400 to 700 nm).

the CO ₂ generator connected to the CO ₂ / air circulation circuit is released in the generator from the feedstock, which is Ba 2 CO 3, by acid addition. The circulation circuit is equipped with an indicator of the concentration of CO 2 in the gas mixture, the actual circulation of the mixture is ensured by a pump,

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- an absorbent vessel for absorbing 40 Og which is connected to the atmosphere circuit by the circulating apparatus after the cultivation is complete or whenever the cultivation is to be interrupted,

- a vacuum pump connected via a 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 gas mixture into the external space,

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

Exemplary embodiment

The chlorococcal Ohlorella vulgaris BEIJE3INCK strain OM - 12/1975 is used for production cultivation, the stem culture of which 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 W.m

FEW.

(a) Preparation of culture

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

KNOj 1 g. I ^-1 , MgS04.7 HgO 1 gl ¹ KHgPO4 680 mg.l ¹ , Ca (NO ₃ > _2. 4 H ₂ 0 10 mg.l ¹ , ferric sodium olatlatonate 18.4 mg.l ^-1 (Pe content) ⁺⁺⁺ 5.6 mg.l ¹ ), H 2 BO 4 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. 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 flask under irradiation of 50 Wm ² , a temperature of 27 ° C and a concentration of COg in a mixture with air corresponding to 2% dissolved COg in suspension until the start density of the stationary phase of the growth curve. usually 5 days). The culture thus prepared, transferred to a cultivation cylinder of the production type, is synchronized. The synchronization is carried out by a method of alternating light and dark periods (see, for example, DOUCHA: Synohronous Cultures Algal Assays in Eutrophication Monitoring, Stuttgart 1978). Synchronous culture, diluted after each loop, is grown in an optical density range of ⁺ 0.150 (light period start) to 1,200 (after separation of maternal cells into vegetative cells) under FAR irradiation of 150 Wm ² , 27 ° C and 2% dissolved CO ₂ in suspension. . Under these conditions, it takes light period synchronous oyklu 14 hours, dark period of 8 hours and the average specific growth rate of culture / N 0.22. ^ ^1. After 3 to 4 times, the culture is ready for production cultivation.

The following data are continuously monitored and corrected during both pre-culture and production culture:

suspension temperature, suspension pH, concentration of COg dissolved in the suspension, optical density of the suspension, and PAR intensity incident on the surface of the culture cell.

The optical density is measured in a 5 mm cell at a wavelength of 750 nm on a photometer

SPECOL. If we multiply the value of the optical density by 0.65, we get (within the range of used population densities) an approximate value of dry matter of algae.

with

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(b) Production cultivation

The dark period of the last pre-production synchronous cycle runs for 13 hours. One hour before the end of the dark period of the loop, 750 ml of a new nutrient solution is prepared in the production culture flask, free of any physically dissolved non-radioactive carbon dioxide. Oxylohezlmid is added to this solution in an amount of 5 mg.l · ¹ · ¹ ·. The solution is then saturated with radioactive carbon dioxide (equilibrium in a suspension of dissolved O 2 O with an atmosphere containing 2% CO 2, occurs after about 30 minutes of vigorous bubbling). After saturation, a sufficient amount of washed racial inoculum is introduced into the vial so that the starting density of the suspension is

400 mg dry matter.ml ^-1 and the actual six-hour production cycle takes place in the light. Starting at 2 W, the 2 W irradiance of 130 Wm PAR is increased to 180 W.ra PAR after four hours. After cultivation, the algae suspension is cooled to 2 ° C and centrifuged at this temperature. The obtained algal sediment is the starting radioactive product for the actual processing.

The course of starch and saoharose synthesis in culture, expressed in µg / ml suspension and% algae dry matter, is shown in Figures 1 and 2. The specific activity of saoharose and glucose (after starch hydrolysis) during the culture is shown in Figure 3.

Claims (1)

A method for culturing chlorococcal algae directed to the intensive synthesis of radionuclide-labeled radioactive saccharides of high specific activity, characterized in that the cultures are carried out in the presence of a specific cycloheximide protein synthesis inhibitor and cultured using a synchronous culture daughter cell population. undergoes a prolonged dark period of the loop before the production cycle begins.