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On the basis of single factor tests, the effect of trace elements—Fe2+, Co2+and Ni2+ions—on biological methane production from heavy oil was investigated by the response surface method. A three-level Box-Behnken design was em-ployed to study the relationship between the independent variables and the dependent variable by applying initial Fe2+, Co2+and Ni2+concentration as the independent variables (factors) and using the methane production after 270 days of cultivation as the dependent variable (response). A prediction model of quadramatic polynomial regression equation was obtained. The results showed that the methane production could be as high as 240.69 μmol after optimization compared with 235.74 μmol obtained under un-optimized condition. Furthermore, the microbial communities before and after biodegradation were ana-lyzed by PCR-DGGE method. The dominant bands were recovered and sequenced. Three strains were obtained;the strain T1 has 97%similarity with Bacillus thermoamylovorans, the strain H3 has 97%similarity with Bacillus thermoamylovorans and the strain H4 has 99%similarity with Bacillus vietnamensis.