CS233600B1 - Method of photosynthetic production of carbohydrates, non-specifically labeled with a stable carbon isotope with a high degree of enrichment - Google Patents

Abstract

The invention relates to a method for the photosynthetic production of carbohydrates, non-specifically labeled with a stable carbon isotope and with a high degree of enrichment. The essence of the invention lies in the fact that daughter cells of a synchronous population of algae with a content of 2 to 4 wt. % of carbohydrates at the beginning of the cell cycle are used for cultivation, while the production cultivation in the light takes place until the beginning of the replication of deoxyribonucleic acid, i.e. for 7 to 8 hours at a temperature of 28 to 29 ° C, after which the cultivation temperature is increased to 36 to 38 ° C and cultivated for a further 7 to 8 hours. This interrupts the synthesis of nucleic acids and proteins and achieves the exclusive synthesis of carbohydrates.

Description

The invention relates to a method for the photosynthetic production of carbohydrates, non-specifically labeled with a stable carbon isotope with a high degree of enrichment.

The invention provides a method for controlled intensive synthesis of carbohydrates, occurring during photosynthesis in a population of chlorococcal algae 13 of a suitable type, with the carbon source being CO. A high yield of the desired compounds and a high degree of their enrichment with a stable carbon isotope are achieved.

Procedures for the preparation of various organic compounds, labeled

13 radioactive / C/ or stable isotope of carbon / C/ with the help of autotrophic microorganisms, are described in a number of publications /Beán et al. 1953, Beán et Hassid 1955, Outtom et Dagliesh 1956, Ellner 1959, Liebster et al· 1961, Tovey et al. 1964, Karpov et al· 1969, Lehman et Wbber 1977, Kollman et al. 1978, 1979/· Some procedures are patented /e.g. US patent 2602047, British patent 1342098, French patent 7208482, German patent 2212092, Czechoslovak author's certificates 197918 and 199446/ _O

The vast majority of the processes described are characterized by low yields of the products produced or low specific activity (for C compounds) or low enrichment levels (for C compounds). Most of the described production methods are demanding in terms of production operations and production costs.

These shortcomings are largely eliminated by the method of photosynthetic production of carbohydrates, non-specifically labeled with a stable carbon isotope with a high degree of enrichment according to the invention 2 233 600 zu, the essence of which consists in using daughter cells of a synchronous algae population containing 2 to 4% by weight of carbohydrates at the beginning of the cell cycle for cultivation, while the production cultivation takes place in the light for 7 to 8 hours of growth at a temperature of 29 ° C, after which the cultivation temperature is increased to 36 to 38 ° C and cultivated for a further 7 to 8 hours. Production cultivation in the light takes place under conditions of maximum specific growth rate until the beginning of the replication of deoxyribonucleic acid after the 7th to 8th hour of the light period of the cell cycle, when the increase in the cultivation temperature to 36 to 38 ° C interrupts the synthesis of nucleic acids and proteins and achieves exclusive synthesis of carbohydrates. The synthesis takes place in an environment where the sole source of carbon is C.

A condition for the successful course of production cultivation is that the algae culture with which production begins has the desired properties.

Pre-cultivation in a CO2 atmosphere and the actual production cultivation are carried out in the cultivation device described in the 1979 patent application18.

An important condition for successful cultivation is the use of a synchronous population, i.e. a population in which all algal cells are at the same stage of the life cycle. Synchronous culture allows, on the one hand, to reduce the amount of carbohydrates to a minimum before the actual production cultivation, which is one of the conditions for achieving a high enrichment of the desired compounds with the C isotope, on the other hand, to intervene in the metabolism of the growing cell population at the moment that is most favorable for achieving the highest possible yield and isotopic enrichment, and on the other hand, to achieve a significantly higher population growth rate and total biomass production during the controlled production cycle compared to asynchronous culture.

The advantages of the proposed cultivation method according to the invention also lie in:

- short cultivation time and simplicity of production operations;

- exceptionally high yields of the desired compounds, as their exclusive synthesis takes place during controlled cultivation;

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- such a representation of C atoms in the molecules of the produced compounds, corresponding to the concentration of this isotope in the raw material from which it is obtained for photosynthesis.

The production of compounds according to the invention is economically efficient, since the costs of synthesizing a unit amount of product constitute only 1 to 10% of the value of the resulting product, expressed in world prices.

Example

For production cultivation, a culture of chlorococcal algae of a suitable type was used, maintained in test tubes on agar media enriched with mineral nutrient solution at a temperature of -2

C and irradiance 10 W.m FAR.- FAR means photosynthetically effective radiation, measured with a spectrally non-selective sensor, sensitive only in the range of 400 to 720 nm. /See e.g. KUBÍN: Sources of photosynthetically effective radiation and methods for its measurement, Academia Praha 1973./

a) Culture preparation and pre-production cultivation

The culture is transferred from agar to a liquid nutrient solution of the following composition: KN0 ₃ 2g.r\ MgS0 ₄ . 7 H ₂ 0 1 g _o {, KH ₂ P0

680 mg.l\ Ca/NOj/g . 4 HgO 10 mg.<~\ sodium ferric chelate 18.4 rag.l”^ /Fe content ⁺⁺⁺ 5.6 mg _o l”^/, H^BO^ 6.18 mg.l“ and trace heavy metals: Cu 0.635 mg.l“^, Mn 0.6 mg.l~\ Mo 0.96 -1 -1 mg.l , Co 0.59 mg.l · To adjust the pH to 6.8, the necessary amount of KOH is added to the nutrient solution.

The culture is grown in the above solution for 10 hours at an irradiance of 130 W.m“^, a temperature of 29 °C and a concentration of 12

C0 ₂ in a mixture with air, which corresponds to 2 X dissolved C0 ₂ in the algae suspension. Initial cultivation density of the algae suspension ^A 75q ^s 0.050. = optical density, measured at a wavelength of 750 nm in a 5 mm cuvette on a modified photometer /SPECOL/. If we multiply the optical density value by a coefficient of 0.50 /in the daughter cells of a synchronous population/ or by a value of 0.35 /in the mother cells/, we obtain an approximate value of the dry mass of the algae.

After a 10-hour irradiation period, the culture is darkened for 18 hours. In subsequent cycles, the irradiation periods are lengthened and the dark periods are shortened so that after 4 to 5 preparatory synchro233 600

- 4 nization cycles reach final values of 14 hours of irradiation and 14 hours of darkness. In order to keep the average irradiance of the population during the light period of the cycle unchanged, cultivation in the light is carried out from the second preparatory cycle in a flow chemostatic mode at a dilution rate of 0 - 0.14 h. In a stable, fully synchronous population, the optical density of the pre-production culture ranges from *750 ⁼ 0.880 to 1.150.

The mean population irradiance /E Z is determined from the irradiance c of the surface of the culture vessel ZE^Z and from the amount of FAR that passes through the cultured layer of suspension /£*/:

E with /e. - E.Z! InZE.ZE.Z· ZDOUCHA: Continuous Cultures Cit 1 t of Algae, Algal Assays in Eutrophication Monitoring,

Stuttgart 1978/,

The dilution rate 0 is equal to the specific growth rate, ^u, for a steady-state flow culture. It is most often determined from the growth curve of a single-culture based on the population doubling time, t^: /U = In 2/t^ /see e.g. OOUCHA: Candidate's dissertation, Μβδ ČSAV Třeboň 1979/«

The following data are monitored during pre-cultivation and production cultivation:

a· temperature of the suspension b· pH of the suspension c· optical density of the suspension d· concentration of CO?, dissolved in the suspension and in the gas circuit of the apparatus

e. irradiance of the surface of the culture vessel and mean irradiance of the population

f. degree of population synchrony b| Production cultivation

One hour before the end of the dark period of the preparatory synchronous cycle, the production culture cylinder is filled with 850 ml of inorganic nutrient solution, which has previously been freed from all physically dissolved CO. The air is displaced from the gas space of the apparatus and replaced with nitrogen, after which this enclosed space is partially evacuated to about 0.07 MPa/. At this point, the nutrient solution in the cylinder is saturated with CO, which is formed in the developer. 233 600

- 5 13 by blowing IN HCIO^ into a suspension of Ba ^c0 3« Equilibrium state in a nutrient solution of dissolved CO? with an atmosphere containing 2 X CO? occurs after about 30 minutes of intensive bubbling.

After saturation, such an amount of inoculum of daughter algal cells is introduced into the culture cylinder that the initial optical density of the suspension, Αγ^θ, is 0.350«. Then, growth takes place for 8 hours under autotrophic conditions at a temperature of 29 °C, when _z u ^s 0.18 h · The initial irradiance of the culture surface is

120 W.m, after the fourth hour of cultivation the irradiance increases to

-2 -2 150 Wm and after eight hours to 180 Wm. After eight hours, when the optical density A? ₅ θ reaches 1.600, the synthesis of nucleic acids and proteins is interrupted by increasing the cultivation temperature to 38 °C. At this temperature, at which the exclusive synthesis of carbohydrates takes place, the culture is grown for 8 hours.

After completion of cultivation, the algae suspension is cooled to 2 °C and centrifuged at this temperature. The obtained algal biomass and supernatant are the starting material for further processing.

In the following Table, the amounts of starch and sucrose 13 per unit volume of algal suspension and the values of C carbon enrichment during the production culture cycle are given. The carbon source for biosynthesis is either 90 X ^^C0?|or 99 X ^C0?.

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Table

Amount of carbohydrates Isotopic enrichment of product X

Hours per unit volume, culti- _; ug . ml' ¹ 90 X ¹³ C02 99 X ¹³ C02 va ce —™—··———————™·.™.

starch sucrose starch sucrose starch sucrose 0 2.9 3.4 0 0 0 0 2 29.2 5.9 81.6 35.0 88.6 40.2 4 61.3 9.2 86.0 56.2 94.8 63.0 6 104.1 11.5 87.5 62.8 96.3 69.7 8 156.8 15.3 88.5 69.5 97.2 76.5 10 258.8 90.5 89.0 86.5 98.1 95.2 12 370.1 136.1 89.3 87.6 98.2 96.3 14 473.5 156.5 89.4 87.8 98.3 96.8 16 551.2 162.4 89.5 87.9 98.4 96.9 The course of starch synthesis and sucrose during synchronous cycle production cultivation, expressed quantity of these carbohydrates in one- notes volume is shown in the figure . 1.

Claims (1)

Process for the photosynthetic production of carbohydrates not specifically labeled with a stable high-grade carbon isotope, characterized in that the cells of a synchronous algal population containing 2 to 4% by weight of carbohydrate at the beginning of the cell cycle are cultured using light production for 7 days. The growth temperature is raised to 36-38 ° C and cultured for a further 7 to 8 hours.