Use of a glycerol-limited, long-term chemostat for isolation of Escherichia coli mutants with improved physiological properties

The evolution of Escherichia coli MG1655 mutants was followed over 126 d in a glycerol-limited chemostat at a dilution rate of 0.05 h(-1). This corresponds to a total of 217 generations at a doubling time of 13.9 h. After this time, nearly 90% of the chemostat population consisted of evolved mutant strains as determined by their altered colony morphologies on plates. Two mutants were isolated that exhibited generally improved growth phenotypes in batch cultivations on glycerol, glucose or the gluconeogenic substrate acetate. Higher specific growth rates and increased biomass yields were found for both mutants. For one mutant, this behaviour was combined with significantly reduced secretion of overflow metabolites when either glycerol or glucose was the carbon source. Additionally, during all growth phases of a batch cultivation, this mutant exhibited increased resistance to a variety of adverse conditions including heat shock, osmotic stress and nutrient deprivation. It also displayed significantly shorter lag phases.