BACKGROUND: Microbial transformation of steroids has attracted widespread attention, especially the transformation of those steroids synthesized with difficulty by chemical methods. In this study, microbial transformation of androst-4-ene-3, 17-dione (AD) by Bordetella sp. B4 was investigated, and the effect of temperature on transformation was studied. RESULTS: Three metabolites were purified by preparative TLC and HPLC, and identified as androsta-1,4-diene-3,17-dione (ADD), 9 alpha-hydroxyandrost-4-ene-3,17-dione (9 alpha-OH-AD), and 3-hydroxy-9, 10-secoandrost-1, 3, 5-triene-9,17-dione (3-OH-SATD) by nuclear magnetic resonance imaging (NMR), Fourier transform infrared spectroscopy (FTIR) and mass spectroscopy (MS). It was first reported that the genus of Bordetella has the capability of AD degradation. Microbial transformation of AD was performed at 30 degrees C, 37 degrees C, 40 degrees C and 45 degrees C. The 9 alpha-OH-AD yield reached a maximum within 16 h when the strain was cultivated in media with AD as sole carbon at 37 degrees C. Surprisingly, ADD was produced by the strain cultivated at 40 degrees C but not at 37 degrees C, which was different from previous reports. It was deduced that the alcohol dehydrogenase that catalyzed the transformation of AD to ADD may be temperature sensitive. CONCLUSION: Androst-4-ene-3,17-dione was converted into 9 alpha-hydroxyandrost-4-ene-3, 17-dione and other metabolites rapidly by Bordetella sp. B4. It is anticipated that the strain Bordetella sp. 84 CGMCC 2229 can be used in the steroids industry. (C) 2009 Society of Chemical Industry