Biodegradable organic carbon is often added to aquifers to stimulate microbial reduction of oxidized contaminants. This carbon also stimulates fermenters, which generate important metabolites that can fuel contaminant reduction and may enhance dissolution of hydrophobic compounds. Therefore, understanding how different methods of carbon addition affect the fermentative community will enable design of more effective remediation strategies. Our research objective was to evaluate the microbial communities that developed in model aquifer columns in response to pulsed or continuous molasses input. Results indicated that the continuously fed column produced relatively low concentrations of metabolic intermediates and had a greater proportion of Bacteria and methanogens, as measured by quantitative polymerase chain reaction, near the column inlet. In contrast, the pulsed-fed column generated periodic high concentrations of metabolic intermediates, with Bacteria and methanogens distributed throughout the length of the column. The community structures of Bacteria and Archaea, measured via automated ribosomal intergenic spacer analysis, in the pulsed-fed column were significantly different from those in the control column (not fed). The microbial community composition of the continuously fed column, however, became increasingly similar to the control column along the column length. These results demonstrate that a strategy of pulsed carbon addition leads to activity that is associated with functional instability, in terms of the production of periodic pulses of fermentation products and changing carbon concentration, and may be advantageous for remediation by producing large quantities of beneficial intermediates and resulting in more homogenously distributed biomass.