In this study, culture conditions, including dissolved oxygen (DO) content, presence of osmoprotectants, residual glucose concentration, and ammonium sulfate-feeding strategies, were investigated for decreasing the inhibition effects of acetic acid, ammonium, and osmotic stress on l-lysine fermentation by Escherichia coli. The results revealed that higher DO content and lower residual glucose concentration could decrease acetic acid accumulation, betaine supplementation could enhance osmotic stress tolerance, and variable speed ammonium sulfate-feeding strategy could decrease ammonium inhibition. Thus, with 25 % DO content, 0-5.0 g/L of residual glucose concentration, and 1.5 g/L of betaine supplementation, 134.9 g/L of l-lysine was obtained after 72 h of culture, with l-lysine yield and productivity of 45.4 % and 1.9 g/(L center dot h), respectively.