This study aims to investigate whether overexpressing the trehalose biosynthetic gene, otsBA operon, in beta-carotene-producing recombinant Escherichia coil protects cells from toxic impurities in crude glycerol. The concentrations of potassium and methanol in crude glycerol were too low to inhibit cell growth. Cell growth and production in control cell culture were inhibited significantly in the presence of a small amount of crude fatty acids. Peroxides were generated in the presence of crude fatty acids during autoclaving and, thus, the inhibitory effect of crude fatty acids was caused primarily by these peroxides. Engineered cells overexpressing otsBA tolerated crude fatty acids (<= 42 wet-g/L), methanol (<= 7.5 g/L), and t-BuOOH (<= 60 mu M) in separate experiments and tolerated up to 60 g/L crude glycerol. These results demonstrate that overexpressing otsBA endowed cells with the capacity to tolerate the toxicity of crude glycerol for direct use. (C) 2013 Elsevier Ltd. All rights reserved.