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To investigate nutrient limitation effect on the community metabolism of closed aquatic ecosystem and possible nutrient limiting factors in the experimental food chains, depletion of inorganic chemicals including carbon, nitrogen and phosphorous was tested. A closed aquatic ecosystem lab module consisting of Chlorella pyrenoidosa and Chlamydomonas reinhardtii, Daphnia magna and associated unidentified microbes was established. Closed ecological systems receive no carbon dioxide; therefore, we presumed carbon as a first limiting factor. The results showed that the algae population in the nutrient saturated group was statistically higher than that in the nutrient limited groups, and that the chlorophyll a content of algae in the phosphorus limited group was the highest among the limited groups. However, the nitrogen limited group supported the most Daphnia, followed by the carbon limited group, the nutrient saturated group and the phosphorus limited group. Redundancy analysis showed that the total phosphorus contents were correlated significantly with the population of algae, and that the amount of soluble carbohydrate as feedback of nutrient depletion was correlated with the number of Daphnia. Thus, these findings suggest that phosphorus is the limiting factor in the operation of closed aquatic ecosystem. The results presented herein have important indications for the future construction of long term closed ecological system. (C) 2011 COSPAR. Published by Elsevier Ltd. All rights reserved.