| The study of biological degradation and soil remediation technology to the petroleum pollutants in typical region has become focus in the domestic and international environment, biological field. In the light of regional and importance for Longdong Loess Plateau polluted by petroleum, the study of sorption behaviors of crude oil in soil and law of natural biodegradation of petroleum contaminated soil is applied. Then oil-degradation bacterial strain are selected from local microorganisms in soil, the oil-degradation mixed bacterium and adaptal factors has been optimized and constructed; and technology conditions of aggrandizeing bioremediation, such as plowing, added sawdust, nitrogen and phosphorus-nutrient agent are studied on repairing process, the role and influence of these measures are studied. Lastly, the mixed bacteria strains from the molecular level are identificated and analyzed from homology, put forward preservation conditions for every bacteria. The main study results are as follows:(1) Static experiments for sorption behaviors of crude oil for loess soil in Longdong area are carried out, the adsorption isotherms of crude oil in loess soils are well described by the Langmuir and linear equation. The adsorption coefficients are0.013and0.012mg/1; The adsorption capacity of oil in the five media in turn is:Silica sand, Kaolin, Loessal soil, High pressure sterilized soil, Diatomite; The increase of pH, temperature and silica sand are not favorable to adsorption of crude oil in soil, increasing the salinity can increase adsorption of crude oil. Situ-biodegradation to depredate petroleum pollutants in soil under simulating natural conditions are applied,and find that the half-life of degradations of petroleum oil in no-polluted soil added0.15%,0.3%,0.5%petroleum are17.2,18.2,23.7d, the bio-degradation coefficients are0.0401,0.0388,0.0293mg/d; In different petroleum concentrations, petroleum degradation rate shows trend of decline with increase of petroleum’s addition. This performance shows that excessive concentration of petroleum can inhibit microbial natural degradation efficiency.(2) The soil samples come from Huaqing/XiFeng oil areas of Gansu province which have being polluted by oil for a long time. Using crude oil as the sole carbon source,13strains are screened and isolated with the enrichment and separation method, the strains can all grow up in the pH5-9, and in oil medium (0.5%standard oil), at the pH8, the oil degradation rates reach a maximum (more than95%). The degrading appears that after being treated with various bacteria and the mixed bacterium, firstly, the higher molecular weight n-alkanes are degraded into lower molecular weight n-alkanes, and the n-alkanes in odd-numbered carbon are degraded into n-alkanes in even-numbered carbon. Every bacterium degradating hopane needs higher energy than n-alkanes, Aromatic hydrocarbons are degradated most difficultly, the degradation rate is lowest, mainly form is demethylation effection for the side of methyl on fused ring, which is the process of natural gas. Several bacteria and mixed bacterium all have stronger demethylation effect for methyl on fused ring, among them F1, A5, D4’s performance is more outstanding. Mixed bacterium’s degradation rate is higher than that of each single bacterium for n-alkanes (except for a positive role:n-tridecane). In process of orthogonal and optimizational experiment, Pseudomonas (A6) plays an important role on maintenance of high degradation rate in mixed strains. The bacteria agent’environmental conditions of crude oil degradation are:salinity is8g/l, pH is8, at30℃,400-600mg/1oil concentration conditions, the mixed strains’degradation rate is highest, and the stability and adaptability are better than individual strains. Therefore, the mixed bacterium has stronger adaptability to different soil and water environment in the practical application.(3) Joining the mixed bacterium into soil and tillage technology, on the one hand, the soil environment is improved at bacteria, water, oxygen and other aspects; on the other hand tillage is in favor of volatilization of the small molecule metabolite by microbial decomposition, so as to greatly accelerate the speed of removal of residual oil. Under the condition of bacterium content increasing one order of magnitude by adding mixed bacterium into the soil, the half-life reduces nearly4times than the natural soil (without bacteria).When soil moisture is24.1%, the mixed bacterium is added into the soil, bacteria content reachs1.09×10, the half-life of treatment of5%soil polluted by petroleum is41days, after61d, degradation rate reachs84.64%by adding mixed bacterium into soil and tilling technology. Adding leavening agents (sawdust) can increase water retention and ventilation capacity of soil, but also has advantageous to improve dehydrogenase activity in the bioremediation process. In the samples turned every day, add leavening agents, on the sixty-second day, measuring enzyme activity has increased to about4.0, is about2times higher than not added leavening agents. Remediation ability of petroleum contaminated soil with mixed bacterium and organic fertilizer jointly is improved:after48days, under oil contents of50g/kg in the contaminated soil, the oil degradation rate of2%,4%,8%bacterium reach68.01%,80.42%,78.47%, which are clearly improved the biodegrading efficiency higher than CK degradation rate. The4%mixed bacterium’s degradation efficiency is most significant. In organic fertilizer, Nitrogen and Phosphorus content is the main factor affected oil degradation rate, only the amount needed to a certain range could have active degradation efficiency, the4%organic fertilizer can achieve the best degradation efficiency.(4) Through molecular identification, strain A6is Pseudomonas aeruginosa genus, D4is Montessori Pseudomonas, Acinetobacter lwoffii species as A5, F1is yellow class of Nocardia genus, F2is dark green streptomyces. Every bacteria Can be freezed dried powder or be made from various slant of culture medium, at37℃, after24h cultured, conserved in4℃refrigerator spare. |
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