Study On Construction Of Highly Effective Petroleum Alkane Degradation Bacteria And Crude Oil Degradation Gene Engineering

Along with the increasement of oil exploitation and usage, a large amount of petroleum pollutants was entering our environments and causing serious environmental pollutions, which were harmful to both human and other organisms. So far, the microbial remediation has been considered to be a relatively effective and safety method for dealing with petroleum pollution all over the world. The principle of this method was shown as follows:After being screened and tamed from natural environment, various petroleum contaminants degrading strains were growing in the contaminated areas and degrading the petroleum contaminants. As the oil degrading bacteria were often mixed flora, there may be a competitive inhibition during process of the bacterial growth, which caused the change of the strain ratio and non-ideal treatment effect. Therefore, in order to enhance the degradation efficiency of oil pollution and reduce the rivalry among the compound bacteria, various genetic engineering bacterias, which can degrade petroleum effectively, were constructed by using genetic engineering method. As the ideal bacteria in harmless treatment of environmental pollution, these engineering bacteria have lots of advantages. such as degrading a variety of harmful substance, high efficiency of degradation and strong adaptability, etc.In this study,10strains, which could degrade petroleum hydrocarbons were isolated from the contaminated soil in Changqing, Shengli and Kelamayi oil production factory. After screening, the bacteria with highest efficiency of degrading petroleum which named BS3was selected and identified by morphological observation, physiological and biochemical tests, antibiotic resistance tests and some molecular biological experiments which can determine its phylogenetic position. Then, the bacteria’s growth characteristic and its effect on degrading petroleum were tested, the result showed that the strain lack of aromatic hydrocarbon degradation ability. To strengthen the degradation ability, aromatic degradation effects of aromatic-degrading bacteria BNF1saved in our laboratory was confirmed, and the degradation gene was located, cloned and expressed in E.coli. Took pUT/mini-Tn5suicide plasmid vector as the recombinant expression vector, the degradable gene from plasmid of strain BNF1was insert into the chromosome of the strain BS3. The genetic engineering of crude oil degradation bacteria was constructed successfully by using triparental conjugation. The growth characteristics, degradation characteristics and stability were studied. Meanwhile, based on the genetic engineering bacteria mentioned above, both immobilization treatment and indoor bioremediation simulation experiment of oil contaminated soil were done. Taking corn seed germination conditions as an important indicator of evaluation on repairing effect, five different treatments were tested on the effects of petroleum contaminated soil bioremediation. On the basis of the laboratory simulation experiment, the in situ bioremediation experiment was performed in a drilling waste mud pool of Changqing oil production factory, which could provide feasibility of technical methods and applications on treatment of petroleum contaminated soil later.The main results were shown as follows:1) Ten strains with petroleum hydrocarbon degradation ability were isolated from contaminated soil which collected from Chanqing, Shenngli and Kelamayi oil production factory, the strain named BS3was chosen for best degradation effect on crude oil in screens. By using bacterial morphology, physiological tests, antibiotic resistance testing and16S rRNA gene sequence analysis, strain BS3was identified as Acinetobacter (Acinetobacter sp.).2) Petroleum-degrading strain BS3{Acinetobacter sp.)’s growth characteristics, environmental factors which affected petroleum hydrocarbon degradation efficiency and specific components for petroleum hydrocarbon degradation in detail were tested. We found that the optimal concentration of petroleum hydrocarbon for growth of BS3was600mg/L. The optimal pH, temperature, inoculum and number of revolutions shaker was7,28℃,1%and120～200r/min. The strain BS3was efficient at alkanes degradation on crude oil, total alkane degradation rate reached81%. While the aromatic component degradation was ineffective, the degradation rate was only21%.3) The biodegradability of the PAHs-degrading bacteria BNF1(Pseudomonas putida) was verified and dioxygenase activity was detected. By using enzymatic studies, plasmid elimination and PCR amplification methods, catechol2,3-dioxygenase gene (C23O) was located in the plasmid of Pseudomonas putida BNF1. After being amplified by PCR and sequenced, C23O gene was924bp, encoding an enzyme product of307amino acids. By using the expression vector pET-30a(+), C23O gene was expressed, the results indicated that the dioxygenase mainly existed in the interior of the strain cell as intracellular enzyme, while the extracellular concentration was minimal.4) The C23O gene was recombinated into pUT/mini-Tn5transposon vector to construct transgenic vector pUT/mini-Tn5-C230. By using triparental conjugation technology, C23O gene was inserted into the chromosome of the host strain BS3. Then, a genetically engineered bacteria Acinetobacter sp. BS3-C23O was screened. Next, the growth characteristics, the substrate specificity of the degradation enzyme activity and stability factors of genetic engineering BS3-C23O were studied. The results showed that the recombinant strain BS3-C23O displayed a broad substrate specificity, high enzyme activity, and favorable stability. The strain had good biodegradability to degrade both alkanes and aromatics, and the growth characteristics did not change significantly, these characteristics are essential for handling oil pollution in environment.5) Based on the genetically engineered bacteria with efficiency crude oil biodegradability, immobilized agents was prepared and simulation experiment of bioremediation was carried out against the petroleum contaminated soil. The results showed that there was a significant reduction of petroleum hydrocarbons in soil after sixty days’restoration experiment. However, oil degradation rate in the experimental group was higher than the control group, which indicated that the use of immobilized microbial agents, with daily dig in the soil to improve the oxygen content, had greatly enhanced the restorative effects of petroleum contaminated soil. While the petroleum hydrocarbons in soil was degradated, the soil toxicity was decreased greatly, the soil was slowly improved and was adapted to the growth of plants. The preliminary in situ restoration experiment was taken in drilling waste mud pool of Changqing oil production factory on the basis of laboratory simulation experiments. After degradation experiment, the content of water in the mud changed to50%-60%; pH reduced to7.8; the content of salt was reduced by50%; the total content of petroleum hydrocarbons were reduced beyond the level of80%; the organic matter content increased significantly, which had achieved certain effect.