Isolation And Identification Of Degrading Bacteria In The Diesel Contaminated Soil And Their Degradation Capability Study

The rapid development in the oil industry today, the environment is also faced with serious pollution problems. Diesel is one of the most used fuel in transportation. And it causes soil contamination in the course of processing, unreasonable waste disposal as well as the accident occurred, particularly underground storage tank leakage. In this situation, the repair work carried out so many. Among them, microbiology-repaired was carried out in recent years., because of the economic, efficient and environment friendly advantages. In this paper, we took the soil sample from the Tongzhou district, Beijing. Simulated underground storage tank, preparde a length, width and height were one meter sand box device, then Man-made polluted the siol from diesel to last for one year. Take the soil sample from which top-down distribution of the points, use the bio-enrichment separation methods, the four diesel-degrading bacterial strain were taken. And then, study this bacterial strains, and the combination method of indoor shaking-flask in which five factors(temperature, pH, nitrogen, phosphorus source, salinity) were considered and the indoor Shaking- flasked experiment, and the main results are as follows:(1) The microbiology experiment was carried out to enrich, separate, screen and identify by means of molecular biology to indigenous dominant bacteria in the sand box, four diesel-degrading strains:B-1, B-2, B-3, B-4. then through morphological observation and 16S rDNA sequence analysis, the results showed these strains belong to Microbacterium sp. Brevundimonas sp., Tetrathiobacter kashmirensis, Pseudomonas sp. .This is also the first report about biodegrading diesel contaminated soil using strain B-3, and B-4 is Pseudomonas sp., a very typical of diesel-degrading bacteria. So in this paper, the study key is B-3, the B-4 as a frame of reference.(2) The indoor shaking-flask experiment showed that the two diesel-degrading bacteria B-3 and B-4 had the optimal growth and diesel degradation conditions: initial temperature is 25℃~30℃, pH is 7~7.5, optimal nitrogen source is NH4Cl, optimal phosphorus source is K2HPO4 or KH2PO4, the salinity is 0.5%~5%.(3) Under the optimum culture conditions, we studied degradation characteristics of strain B-3 and strain B-4. The degradation rate of strain B-3was obviously higher than that of strain B-4 for different initial diesel concentration. When the diesel fuel concentrations below 0.378mg/g, the degradation efficiency of strain B-3 is relatively low. When it more than 0.378mg/g, as the concentration increases, strain B-3 degradation efficiency gradually increase, up to 60.98%. Then strain B-4 optimal concentration in diesel is 0.252mg/g, the degradation rate is 48.97%. When the concentration exceeds this range, it has made them can not be tolerated. This two bacteria degradation rate decreased. It showed that bacteria themselves have the tolerance range of pollutants. Because of pollutants in high concentration, it would produced a toxic effect on the strains, inhibit the growth of bacterial.(4) The indoor simulation experiments of the degrading diesel oil contaminated soil. Especially in the soil medium, understanding the degrading performance of strain B-3 and strain B-4. The overall degradation rate of bacteria at the temperature of 30℃and 35℃, the degradation effect is good. B-3 was superior to B-4. When the low-temperature range is 20℃and 25℃, the experiment lasted for 15 days, as the temperature increased, the degradation rate of strain B-3 was positively correlated with temperature. B-4 was also the same with this. Therefore, in the actual restoration project, when it lasted for 15-20 days, to provide a micro-nutrient for bacterias. So that, with adequate nutrition, micro-organisms will play a high degradation.