Mutant strain of Bacillus megaterium ACBT03 is capable of demethylation at third carbon position of colchicine and their derivatives. A wild strain of B. megaterium ACBT03 collected from field was grown at shake flask level and the most suitable sources of carbon and nitrogen were studied in order to increase the biotransformation process. Glucose in combination with glycerol having 2:1 ratio, and yeast extract plus peptone 1:3 ratio, at pH 7.0 and 28 degrees C incubation temperature were noticed to be the most suitable conditions for maximum biotransformation. The potential of demethylation of the wild strain was noticed to be very poor (20-25% of substrate supplied) when 0.1 g/l colchicine or thiocolchicine was used. In order to increase the potential for demethylation, B. megaterium ACBT03 was subjected to enrichment culture with higher concentration of colchicine. The bacterial cells were grown for 8-10 generations, in higher concentration of colchicine. The newly mutant developed through colchicine enrichment culture was named as B. megaterium ACBT03-M3. About 55% of colchicine and 60% of thiocolchicine were converted to their respective demethylation form, when this mutant was grown in 7 g/l colchicine and thiocolchicine, both at laboratory-scale (5-1-jar fermenter) and pilot-scale level (70-1 fermenter). Under optimum culture conditions the key monitoring factors to scale-up the process of demethylation were dissolved oxygen (DO) level (2.5 vvm) of culture broth and impeller tip velocity (4710 cm/min). (C) 2008 Elsevier Ltd. All rights reserved.