- Many studies have described the response of the facultative anaerobe, Escherichia coli, to anaerobic conditions, yet they all investigated free-living (planktonic) cells because attempts to cultivate anaerobic E. coli biofilm were mostly unsuccessful. We challenged these findings and cultivated E. coli strain MG1655 biofilm under both aerobic and anaerobic conditions, characterizing the mature biofilm architecture and global gene expression profile. We used RNA sequencing technology to compare stationary phase planktonic cells with mature biofilm, cultured with and without oxygen. Our results suggest that gene expression patterns significantly differ between biofilm and planktonic cultures cultivated under the same oxygenic conditions. The anaerobic E. coli biofilms were slow growing and patchy compared to aerobic biofilms, yet some features were unchanged like the production of extracellular polymeric substances. A closer inspection of the mRNA data revealed that essential cell processes were attenuated in anaerobic biofilms, including protein synthesis, information transfer, cell structure, regulation and transport. Our results suggest that lack of oxygen imposes severe stress on mature biofilms thus limiting the cells’ activity. We further propose that E. coli does not favor growing in anaerobic biofilms and when forced to do so, the cells prevail by attenuating their activity in order to survive.