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In space utilization, an enormous amount of freshwater for drinking, daily use, hydroponics. is used and recycled in a closed habitat. We have developed culture-independent techniques to analyze microbial cells at the single level, a cell itself, and also community level. By using these methods such as fluorescent vital staining (double staining with carboxyfluorescein diacetate and 4′,6-diamidino-2-phenyl indole), microcolony method and denaturing gradient gel electrophoresis (DGGE), bacterial abundance and their physiological activity in freshwater used in Closed Ecology Experiment Facilities (CEEF) “Mini-Earth” were determined.