The physiology of different Escherichia coli stains was analyzed for growth with glycolate as a potentially promising sustainable sole source of carbon and energy. Different E. coli strains showed large differences regarding lag phases after provision of glycolate. Whereas E. coli W showed fast adaptation, E. coli BW25113, JM101, and BL21 (DE3) needed extensive time for adaption (up to 30 generations) until the attainable µmax was reached, which, at 30 °C, amounted to 0.20-0.25 h-1 for all strains. The overexpression of genes encoding glycolate degradation did neither overcome the need for adaptation of E. coli BL21 (DE3) nor improve growth of E. coli W. Rather, high level expression of proteins involved in uptake and initial degradation steps had an adverse effect on growth. Overall, the results show a promising capacity of E. coli strains for growth on glycolate.