Closure from the earth's atmosphere is a critical test of an ecosystem's ability to function. In our earlier testing of autotrophic Closed Ecological Systems (CESs), a C:N ratio of 26.4 (3.3 mM NaHCO and 0.125 mM NaNO3) supported algal and Daphnia populations for months, but developed extreme pH values (∼11 ungrazed, >10, grazed), suggesting that the systems were carbon-limited. Only approximately half the HCO (bicarbonate) would be expected to be available to green algae, the other portion becoming CO (carbonate). In an experiment described here, CESs were developed to explore a greater range of C:N ratios. To keep the medium from becoming too osmotically concentrated, NaNO was reduced to 0.0312 mM and NaHCO tested at 3.3, 13.2, and 26.4 mM, resulting in nominal C:N ratios of 105, 422, and 845. However, additional carbon was not beneficial to long-term survival of the organisms. The algal abundance was relatively insensitive to C:N ratio; greater concentrations of C were not beneficial. Daphnia populations were sensitive to C:N ratio and persisted longer at the lowest C:N ratio of 105. All of the C:N ratios tested in these CESs are outside of the expected range suggested from ecological studies, which is based on the Redfield Ratio of 6.625 C:N, the expected chemical composition of algae. Two potential explanations for the apparent high C demand in our CESs are suggested by the literature. The first is production of fatty algal cells, e.g., one of the algal species, Scenedesmus obliquus, is reported to produce high-lipid cells that could have a higher C:N ratio than the Redfield Ratio. The second is "carbon overconsumption," which has been suggested for N-limited marine phytoplankton communities dominated by diatoms or nutrient deficient algal communities dominated by small cells that are under-represented by chlorophyll a measurements. The unexpected C dynamics found in our CES tests could be relevant to the design of biological life support systems that must be provisioned with adequate elements for long-term ecosystem functionality. If the actual demand for C is underestimated, its storage may be inadequate.