Purpose: Screening scale studies were performed with eight cultures for their ability to metabolize  phenobarbital, an antiepileptic, sedative, hypnotic and substrate for CYP 2C9 and 2C19. 

Methods: The transformation of phenobarbital was confirmed and characterized by fermentation techniques, high performance liquid chromatography (HPLC), mass spectrometry (MS) and metabolism inhibition studies.

Results: Among the different cultures screened, a fungus, Rhizopus stolonifer NCIM 880, transformed phenobarbital to its metabolite, the N-glucuronide of p- hydroxy phenobarbital. HPLC data show a solvent peak at 2.4 min, culture components peaks at 4.0 and 5.4 min, respectively, and phenobarbital peak at 10.3 min, for both controls and test samples, but only the sample of  Rhizopus stolonifer showed an additional peak at 3.1 min, indicating formation of a metabolite. 

Conclusion: Microbial metabolism of phenobarbital was similar to the metabolism of the drug in mammals. Therefore, Rhizopus stolonifer can be used as a suitable in vitro model to mimic CYP 2C9 metabolism and to synthesize metabolites required for further pharmacological and toxicological studies.