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

放射性同位素在科学研究中得到了广泛的应用，但在放射性废物的安全使用和处理方面一直存在严重的问题。在这个项目中，我们的目标是通过光合作用生物的培养来生产~&lt；13&gt；C-标记化合物，并建立用~&lt；13&gt；C-化合物代替^&lt；14&gt；C-标记化合物用于研究的灵敏和选择性分析方法的替代方法。以纤细裸藻(Euglena Gracilis)为光合菌，初步探讨了~(13)&gt；C-CO_2的培养条件。在光照和CO2浓度控制下，对异养和自养生长进行了监测。通过研制的CO2控制器实现CO2循环：在自养条件下，裸藻首先通过开放系统中的~(12)C-CO_2通道生长。在生长的早期对数阶段，C-CO_2转变为^&lt；13&gt；C-CO_2，并继续培养，直到达到稳定期。在C-CO_2的排放过程中，为了回收利用废气，建立了闭路气路系统，但在没有CO_2监测仪的情况下，很难控制罐内的CO_2浓度。最后采用CO_2开路，将~&lt；13&gt；C-CO_2的产物一次回收到碱性捕捉器中储存使用。通过测量细胞颗粒中葡萄糖的核磁共振标记效率。细胞生长后，收获细胞，匀浆，离心法分离，收集重颗粒的副尼龙。颗粒酸解后，消化后中和并应用于^&lt；13&gt；C-NMR。目前掺入效率和标记的选择性不是很好，似乎需要进行一些修改，如^&lt；13&gt；C-CO_2的暴露时间较长或培养过程中暴露的时机。

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.