AbstractA growth medium was developed for cultivation of Clostridium thermocellum ATCC 27405 in which “background” carbon present in buffers, reducing agents, chelating agents, and growth factors was a small fraction of the carbon present in the primary growth substrate. Background carbon was 1.6% of primary substrate carbon in the low-carbon (LC) medium, whereas it accounts for at least 40% in previously reported media. Fermentation of cellulose in LC medium was quite similar to Medium for Thermophilic Clostridia (MTC), a commonly used growth medium that contains background carbon at 88% of primary substrate carbon. Of particular note, we found that the organism can readily be cultivated by eliminating some components, lowering the concentrations of others, and employing a tenfold lower concentration of reducing agent. As such, we were able to reduce the amount of background carbon 55-fold compared to MTC medium while reaching the same cell biomass concentration. The final mass ratios of the products acetate:ethanol:formate were 5:3.9:1 for MTC and 4.1:1.5:1 for LC medium. LC medium is expected to facilitate metabolic studies involving identification and quantification of extracellular metabolites. In addition, this medium is expected to be useful in studies of cellulose utilization by anaerobic enrichment cultures obtained from environmental inocula, and in particular to diminish complications arising from metabolism of carbon-containing compounds other than cellulose. Finally, LC medium provides a starting point for industrial growth media development.