For the successful scale-up of microbial fuel cell (MFC) systems, enrichment strategies are required that not only maximise reactor performance but also allow anodic biofilms to be robust to environmental change. Cluster analysis of Denaturing Gradient Gel Electrophoresis community fingerprints showed that anodic biofilms were enriched according to substrate type and temperature. Acetate produced the highest power density of 7.2 W m−3 and butyrate the lowest at 0.29 W m−3, but it was also found that the trophic conditions used to acclimate the electrogenic biofilms also determined the MFC response to different substrate types, with both acetate and butyrate substrates recording power densities of 1.07 and 1.0 W m−3 respectively in a sucrose enriched reactor. When temperature perturbations were introduced to investigate the stability of the different substrate acclimated electrogenic biofilms, the 20 °C acclimated acetate reactor was unaffected by 10 °C operation but all reactors acclimated at 35 °C were adversely affected. When the operating temperature was raised back to 35 °C both the acetate and butyrate reactors recovered electrogenic activity but the sucrose reactor did not. It is thought that this was due to the more complex syntropic interactions that are required to occur when metabolising more complex substrate types.