Bio-indigo was produced in 3000-L batch and 5-L continuous fermentation of a recombinant Escherichia coli DH5 alpha harboring a novel gene encoding flavin-containing monooxygenase (FMO). Batch fermentation in a 3000-L fermenter produced 911 +/- 22 mg L(-1) of indigo from 2 g L(-1) tryptophan as a substrate (yield 46.9%) under the following culture conditions as follows: culture temperature 30 degrees C, pH 7.0, agitation speed 200 rpm, and aeration 3 vvm. Sufficient oxygen (aeration rate and agitation speed) was critical for bio-indigo production. For continuous fermentation in a 5-L fermenter, the volumetric productivity was found to be 11.3 mg L(-1) h(-1) up to 110 h (final accumulated bio-indigo was 23 g) with a constant dilution rate (D) of 0.084 h(-1) (constant feeding rate of 0.167 L h(-1) with medium containing 3 g L-I tryptophan). Recombinant E. coli cells have the ability to withstand the toxicity of high concentration of accumulated indigo in batch fermentation. In continuous fermentation, the recombinant cells exhibited high plasmid stability up to 110 h, after which they lost the plasmid. (C) 2010 Elsevier Ltd. All rights reserved.