Study On The Screening And Degrading Characteristics Of Anaerobic Hydrocarbon Degrading Bacteria

In recent years, the oil pollution casused serious impact on the environment and human activities, especially the bioremediation technology used in oil polluted soil and groundwater has become a hot research topic. Bioremediation techniques include in situ and ex-situ bioremediation, and the in situ bioremediation has a great advantage in the contaminated area which polluted dispersively or the groundwater environment. Increasing evidence indicates that biodegradation of hydrocarbons takes place also in anoxic conditions. This opens new perspectives for the in situ treatment of contaminated soil and groundwater where reducing conditions below the surface limited the usefulness of O2 as an electron acceptor which could be supplied to stimulate the degradation of petroleum hydrocarbons. Under reducing conditions, other options have to be evaluated for enhancing the in situ biodegradation of organic contaminants. In this paper, firstly, the vertical distribution of physical and chemical properties of the oil contaminated soil with 16 months in situ bioremediation was studied, to analyze the role of anaerobic degradation during the in situ bioremediation process; Secondly, the Karamay oil polluted soil samples were collected, and the appropriate electronic accepter was isolated, then the thermophilic anaerobic hydrocarbon degrading bacteria which used crude oil as carbon source were isolated, and the pure degrading strain was identified by molecular biology, the optimize culture condition of the mixed culture KLAL14-2 was obtained. In addition, the environment factors effected on the mixed culture KLA14-2 was studied, and the degradation characteristics of KLA14-2 on the two different kinds of crude oil was analyzed; Finally, the range of substrate degradation and the stability of community structure were studied, the degrading characteristics of toluene and naphthalene which presented the monocyclic aromatics hydrocarbons and polycyclic aromatic hydrocarbons were investigated, and possible the metabolic pathway was speculated through ultraviolet, infrared and GC-MS methods.The experiment results showed:(1) In-situ bioaugmentation strategy was used for bioremediation of oil contaminated soil with 16 months, the oil of each soil layer was degraded in a certain extent, the removal rates of saturate and aromatic component of the surface soil was highest; As sulfate reduction and nitrate reduction took place in subsurface soil, the pH reduced from 7.86±0.03 to 7.27±0.03, and the TN reduced from 2.53±0.13 g/kg to 0.77±0.04 g/kg; The removal rate of resin and asphaltene was related to the anaerobic bacterial count. The anaerobic bioremediation strategy should be considered, when the in-situ remediation was applied in the oil contaminated area which polluted dispersively;(2) The sulfate content of Karamy oil contaminated soil was abundant, the sulfate was suitable for anaerobic hydrocarbon degrading bacteria as electron acceptor; after primary and secondary screening, the stable and high hydrocarbon degrading rate mixed culture KLA14-2 was obtained. Crude oil degradation experiments of the four pure degrading bacteria (KLA14-2-1, KLA14-2-2, KLA14-2-3 and KLA14-2-4 which were isolated from the mixed culture KLA14-2) showed that these pure strain had a low crude oil removal ability, the single hydrocarbon degrading bacteria in crude oil degrading is not appropriate; Molecular identification results showed that KLA14-2-1 and KLA14-2-3 belong to Bacillus licheniformis and Bacillus thermoamylovorans respectively, and KLA14-2-2 and KLA14-2-4 could only be confirmed have a close genetic relationship of the Desulfotomaculum and iron bacteria respectively;(3) Lower inoculum inhibited the degrading, and 2% inoculum was more appropriate, the mixed culture KLA14-2 could maintain a high oil removal rate when the temperate was above 50℃; The optimal pH range was 6-8, and the concentration of surfactant (50mg/L) which lower than the CMC could promote the oil degradation of mixed culture KLAL14-2; Sulfate-reducing have the mainly effect on the anaerobic oil degradation process, ferric iron could promote the degradation of KLA14-2, and under the nitrate-reduction condition inhibited the degradation; KLA14-2 had vary degrading characteristics with different kinds of oil, could degraded the resin component of heavy oil; (4) Substrate utilization ability experiments showed that the mixed culture were better than the pure bacteria, the KLA14-2 was ability to use 8 kinds of low concentration hydrocarbon substrates as carbon source; The PCR-ARDRA analysis showed that the KLA14-2 had a complex population structure, the culturable bacteria could be only to 21.1%, and the population structure of KLA14-2 used crude oil as carbon source was different with the original structure, the type of dominant bacteria were changed and the content increased from 63.75% to 73.75%;(5) The tolerance of mixed culture KLAL14-2 on naphthalene was worse than toluene, the anaerobic degradation of naphthalene and toluene was fitted to a non-competitive inhibition model, the toluene or naphthalene degradation ability of KLA14-2 on mixed electron acceptor conditions was better than each single electron acceptor conditions, the addition of a certain amount bicarbonate could accelerate the process of degradation of naphthalene, but toluene had little effect;(6) The major metabolic mechanism of KLA14-2 for degrading toluene were benzylsuccinate synthesis and benzoate synthesis, the major metabolic production were benzylsuccinate and benzoic acid; and the major metabolic mechanism of KLA14-2 for degrading naphthalene were carboxylation and methylation, the major metabolic production were 2-naphthoic acid, naphthyl-2-methylsuccinate, 5,6,7,8– tetrahydro -2-naphthoic acid, decalin acid and cis-2-carboxycyclohexylacetate.