Building The Community Of The Microorganisms Of Methanogenic Crude Oil Biodegradation

In the study, we got the samples from Dagang oil reservoir which was a high-temperature oil reservoir,then we isolated fermented bacterias and methanogenic archaeas using two different mediums.We Simulated the process of hydrocarbon methanogenic degradation through two different methods of cultivation and detected the amount of the VFA and methane every 40 days. After eight months ,we detected the communities of bacterias and archaeas using the PCR-DGGE technique.Finally we isolated several fermented bacteria represented by HL-3 and three methanogenic archaea represented by DL-7 and got dozens of bacterial DNA sequences and several archaea DNA sequences.Growth temperature of the strain HL-3 was at range of 40℃to 75℃(optimum temperature at 60℃) and pH range at 5.0 to 8.0 (optimum pH value at 6.5). The isolation could grow in the presence of NaCl content between 0% and 3.2% ( with optimum NaCl content at 0.25% ). Many of carbohydrates resources as glucose, ribose, mannose, xylose, cellobiose etc. might be metabolized. Metabolites of glucose were ethanol, acetate, CO2 and trace amount of propionate and butanol. (G+C)mol% content of strain HL-3 was as 33.9%, comparing to 16S rRNA sequencing similarity of T. uzonensis DSM 18761T(EF530067), the similarity level reached by 98.8%. With comparison of T. sulfurigignens DSM 17917T(AF234164), the similarity level was next reached by 98.1%. Strain HL-3 was tolerate of highe sulfite (0.1mol/L) ions and extremely highe concentration of thiosulfate (0.8 mol/L). When the concentration of thiosulfate was higher than 0.075 mol/L, The body cell would generate S element granular. The presence of H2S gas was detected inside of space at the top of serum bottle.H2/CO2 was the only substrate of strain DL-7 while formate,methanol,trimethylamine ,acetate and other secondary alcohol cannot be utilized. The optimum growthing conditions of the strain DL-7 are with the temperature at 60℃, the pH value at 7.0 to 7.5 and with optimum NaCl content at 0.25%. Without the Yeast in the medium ,strain DL-7 cannot grow. Comparing to 16S rRNA sequencing similarity of M.marburgensis DSM2133T (X15364), the similarity level reached by 99.7%.The results of the sequences of the 16SrRNA V3 region showed that there were many kinds of Thermophilic anaerobic bacteria and H2-utilizing Methanogens and that syntrophic bacterias could not be present in the process of hydrocarbon methanogenic degradation.