The Study Of Hydrocarbon Degrading Bacteria Mutated By Low Energy Ions Implantation

While the development of petroleum industry, the soil has been widely and seriously polluted by petroleum hydrocarbons, which not only destroys the local soil ecological environment balance, but also causes harm to life and health of the residents and even to sustainable development of the local economy. Microbiological remediation technology has been taken as an important and effictive way to remediate petroleum-contaminated soil for its low-cost, high-efficiency, safty and free additional pollution.This paper is to conduct study in laboratory for the task of repairing petroleum contaminated soil by microbiolgical remediation technique. This research focuses on secreening and breeding microbe seeds which are both high-yield biosurfactant and hydrocarbons-degrading to enlarge the efficient microbe resource for study and applying, enhancing degradation rate and effect, investigation the characteristics and capability of the strain.By this study we want to lay foudation and provide information of the strain to this task and explorit a efficient, green and sustainable methods towards bioremediation on petroleum-contaminated soil. The results are as follow:1. Stains which are capable of producing biosurfactants and petroleum hydrocarbons degrading could be isolated by a series of step including enrichment culture hemolytic activity assay on blood agar plates and hydrolyzing oil activity assay on oil agar plates.4petroleum hydrocarbons-degrading strains, were finally isolated from crude oil-contaminated soil in Daqing oil field, named8-2,8-8,8-11,8-18.Then the fermentation supernatant of first-isolated strains was secondly screened by a drop-collapsing method, oil displacement activity and surface tension method.8-2and8-11were isolated with higher surface activity and high hydrocarbons-degrading.8-2’s ability was tested on the result of measuring:petroleum biodegradation rate was51%, oil-discharge diameter was3.8cm, and the surface tension of the medium was39.5mN m-1.8-11’s ability was tested on the result of measuring:petroleum biodegradation rate was64%, oil-discharge diameter was6cm, and the surface tension of the medium was32.7mN m-1. Different strains have a different oil degradation rate, range of30%to70%.Based on its morphological, physiological characteristics and by the comparision of its whole sequence of16S rDNA with the type strains in GenBank, showed that16S rDNA of8-2has more than99%of homogenous with ATCC19374(NR024708.1) in GenBank, therefore8-2was identified as Pseudomonas qeniculata; showed that16S rDNA of8-11has more than99%of homogenous with ATCC7061(NR043242.1) in GenBank, so8-11was identified as bacillus pumilus.2. the bacteria8-11was implanted with90×2.6×1013ions cm-2of20keV N+ions, the bacteria of mutant23which could efficiently degraded oil have been isolated from the implantation experiment. Petroleum hydrocarbon degradation was researched in this thesis, the experiments of degrading crude oil indicated that the bacteria of mutant23had a powerful ability and the biodegradation rate of Total Petroleum Hydrocarbon (TPH) came to74%, oil-discharge diameter was expand by1to2cm, the oil after biodegradation was studied by Gas Chromatography, which showed the n-alkane of oil composition was degraded completely. The study reported that the bacteria after being N+ions implanted improved its oil degradation ability and could decrease surface tension effectively; it would offer large potential application.3. The biosurfactants produced by the bacteria of mutant23made the surface tension of the medium reduce from56.1mN m-1(CK) to29.3mN m-1,showed a good surface activity, may plays an important role to promote the biodegradation rate of Petroleum Hydrocarbon. The biosurfactant produced by mutant strain23was extracted, purified and studied by various of methods, such as methylene blue-chloroform experiments, thin layer chromatography and FT-IR spectrum, results showed that the purified biosurfactant was yellow powery crystal. It was identified as glycolipid, its CMC is40mg L"1, and it can keep its surface activity in the temperature of20～100℃, salinity of0-14%solution and in a wide pH2-14. Moreover, the stability of its emulsifing activity performs well. Mutant strain23shows great stability against temperature, pH and mineralization.Oil degradation rate could increase effectively by adding biosurfactant and adding biosurfactant in advance was favorable.