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着严重的问题。本课题旨在通过培养光合生物在^<13>C-CO_2存在下生产^<13>C-CO_2标记的化合物，并为用^<13> c - _2代替^<14> c - _2标记的化合物的研究建立灵敏、选择性的分析方法替代途径。选择细叶绿藻作为光合微生物，对^<13>C-CO_2的培养条件进行了初步研究。在光照和CO_2浓度控制下，监测异养和自养生长情况。CO_2循环由本研究购买的CO_2控制器实现：在自养条件下，绿藻首先在开放系统中通过^<12>C-CO_2生长。在生长的早期对数阶段，^<12>C-CO_2变为^<13>C-CO_2，并继续培养直至达到平稳阶段。在^<13>C-CO_2通过过程中，为了回收和再利用^<13>C-CO_2，设计了一个封闭的气路系统，但在罐式发酵罐中没有CO_2监测器，很难控制CO_2的浓度。最后采用开路CO_2，输出的^<13>的C-CO_2在碱性捕集器中一次性回收，贮存备用。利用核磁共振（NMR）技术，通过测量酒窖颗粒（paramylon）中与葡萄糖结合的同位素来监测^<13> c标记的效率。生长后收获细胞，离心匀浆分馏，收集重颗粒paramylon。在酸水解颗粒后，消化物被中和并应用于^<13>C-NMR。目前的掺入效率和标记选择性都不太好，需要对其进行改进，如延长暴露时间或在培养过程中增加暴露时间。

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.