The present study has attempted to get insight into ultrasound induced enhancement in biohydrogen production from glycerol fermentation using metabolic flux analysis (MFA). A pseudo steady state metabolic flux network model was constructed and analyzed using experimentally measured glycerol uptake rate and fluxes of four metabolites, viz. acetate, butyrate, succinate and 1,3-PDO. Glycerol consumption increased by similar to 50% under sonication. Biohydrogen yield showed marked rise of similar to 40% with application of ultrasound. Butyrate and 1,3-PDO were the major products of glycerol metabolism. Sonication had major influence on carbon fluxes at the acetyl-CoA node. MFA results revealed enhanced flux towards butyrate under sonication, which was manifested in higher butyrate to acetate (B/A) ratio in products and greater H-2 generation. Biohydrogen production was also a microbial growth associated process. Finally, two theoretical alternatives for further enhancement of biohydrogen production were assessed with MFA, viz. enhancement of glycerol uptake and blocking of butyrate pathway. (C) 2017 Elsevier Ltd. All rights reserved.