Preparation of ^<13>C-labeled compounds by using photosynthetic microbes and development of its sensitive detection system.

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Radioisotopes have been widely used in scientific researches, however, there always exist serious problems in the safety usage and the treatment of radioactive wastes. In this project we aimed to produce ^<13>C-labeled compounds by cultivating photosynthetic organisms in the presence of ^<13>C-CO_2, and to establish the alternative way of sensitive and selective analytical methods for the researches using ^<13>C-compounds instead of ^<14>C-labeled ones. Euglena gracilis was chosen as a photosynthetic microbe and the culture conditions using ^<13>C-CO_2 was preliminarily examined. Heterotrophic and autotrophic growth were both monitored, under the control of light and CO_2 concentrations. CO_2 circulation was achieved by a CO_2 controller purchased in this research : in the autotrophic condition, Euglena was first grown by the passage of ^<12>C-CO_2 in the open system. In the early logarithmic phase of growth ^<12>C-CO_2 was changed to ^<13>C-CO_2 and the culture was continued until it reached the stationary phase. During the passage of ^<13>C-CO_2, a closed system of gas circuit was deviced to recover and re-use of waste ^<13>C-CO_2, however, it found difficult to control the concentration of CO_2 without a CO_2 monitor in the jar fermentor. Finally the open circuit of CO_2 was adopted and the output of ^<13>C-CO_2 was once recovered in a alkaline trap and stored until use.Efficiency of ^<13>C-labeling was monitored by NMR by measuring the incorporated isotope into glucose in the cellar particles, paramylon. After growth the cells were harvested, homogenized and fractionated by centrifugation and the heavy particles paramylon were collected. After acid hydrolysis of the particles, the digest was neutralized and applied to the ^<13>C-NMR.At present efficiency of incorporation and selectivity of labeling were not so good that some revision seemed to be required, such as longer exposure of ^<13>C-CO_2 or timing of the exposure during cultivation.