The types and concentrations of electron acceptor are the significant factors influencing the oxidation and biotransformation of organic matter in the process of pollutant biodegradation. Regulation of O2, SO42- and NO3- as electron acceptors on petroleum hydrocarbon biotransformation to final products was studied using the multiple methods including mesoscale biodegradation experiments, thermodynamic theoretical calculations and stoichiometric analyses. Petroleum hydrocarbon biodegradation ratio (PHBR) rose from 64.7 to 82.4% with dissolved oxygen (DO) (3-5 mg L- 1). PHBR increased from 57.4 to 66.1% in SO42--reducing biosystems and rose from 65.0 to 77.9% in NO3--reducing biosystems. Carbon balance was verified in different cultures. The shared functional microorganisms in different biosystems included Candida, Rhodococcus, Pseudomonas, Ochrobactrum, Marinobacter, Bacillus, Azoarcus, Alcanivorax, Acinetobacter. Pandoraea, Enterobacter and Burkholderia in anaerobic biosystems preferred to use NO3- and SO42- as electron acceptors for metabolism, and order of availability followed: NO3- > SO42-. Thermodynamic constraint showed that potentials of alkanes biotransformation to methane through hydrogenotrophic and acetoclastic methanogenesis in NO3--reducing biosystems were 7.27-7.73 and 7.25-7.70 times larger than those of SO42--reducing biosystems, respectively. Metabolism equations of microorganisms proved that anabolism and catabolism on alkanes were feasible. This work provides a support for studying the biochemical process of petroleum hydrocarbon biotransformation and lays a foundation for the realization of oil-containing wastewater bioremediation.