Screening PAHs Degrading Microbes And Its Remediation Associated With Plants

PAHs (Polycyclic aromatic hydrocarbon) is a kind of organic compound which is combined by two or more than two benzene rings, they are widely existed in the environment.Due to its special physical and chemical properties, it has fierce toxicity, such as carcinogenic, teratogenic and mutagenic effects, moreover it can be easily accumulated in the environment and can be enriched in the organism through transmitting along the food chain,thus cause a great harm to human health and ecological environment. PAHs contaminating soil have some special charactors such as disguising, irreversibility, long-term, easy mobility and cannot being degraded or disappeared completely, so the damage is more serious and remediation can be more difficult.Currently there are a lot of remediation technologies related to PAHs contaminated soil, including chemical remediation, physical remediation, phyto remediation and microbial remediation, and phyto-microbial remediation.Chemical remediation is complex, and the chemical reagent can easily cause secondary pollution.Physical remediation can not change the structure of the PAHs, besides the quantity is huge and the cost is high. Phyto remediation can not be used in large-scale remediation and commercial application, mostly stay in the laboratory stage.Microbial remediation may have great discrepancy with the experimental results due to competition or difficult to adapt to the environment.Combining high efficient degradation bacteria into mixed strains and applying in contaminated soil associated with plant can increase the number of microorganisms, which can greatly increase the remediation effect, so phyto-microbial remediation has great application prospect.This study attempted to capture the plant endophytes which can colonize in the root and degrade PAHs through growing leguminous plants, at the same time some strains are isolated from the sludge of chemical factory.all the isolated strains were found that it can tolerate the PAHs but can’t degrade it through the plate sublimation method. So we selected stored strains in ACCC,12 degradation strains were screened after measuring the size of degradation circle,10 strains whose circle diameter are more than 8 mm were finally got, according to the size from big to small, they are ACCC 02173, SL-1,02830,02172, RC99-3, F11, K13 (H25), F44-8, MCT5,02755.Second screening was performed by Fluid shaker method. Results show that strain SL-1, F11,02172,02173 are high efficient degradation bacteria, combining these four strains with rhizobium astragalus sinicus into different groups.All the groups are as below:(1) F11 (2) 02173+02830 (3) F11+ rhizobium astragalus sinicus (ACCC 13052) (4) F11+02173+02830 (5) F11+02173+ 02830+ACCC 13052 (6) ACCC 13052 (7) SL-1+02173+02830+ACCC13052Using plant associated with degradation strains to repair contaminated soil,10 days later, all the plants growed slowly, each treatment do not have significant differences. After 30 days, plant growed exuberantly,the dry weight of inoculation treatments are a little higher than that of without inoculation treatment,which showed that astragalus sinicus can tolerate high concentration of PAHs in contaminated soil, the difference between each inoculation treatment were not clear, the plant height of inoculation treatment is higher as compared with uninoculated treatment.50 days later, the dry weight of inoculated rhizobia treatments are nearly the same with the inoculation treatment, which shows that inoculating rhizobia does not significantly affect the plant biomass, treatment ABS, ABF, ABFOR were obviously higher than that of others, which were 2.68 g,2.58 g and 2.67 g respectively, the plant height of ABF0 and ABFR are significantly higher than other treatment, which is 26.1 cm and 27.3 cm respectively. The difference of nodule numbers between each treatment is noticeable, the inoculation treatment is much more higher as compared with uninoculated treatment, ABR and AFOR are the first two groups among these treatments, with a 58 per pot and 61 per pot respectively..After 50 days bioremediation, removing rate of PAHs in soil of all the treatments are obviously higher than that of control group, planting and inoculating astragalus sinicus rhizobia treatment was a little higher than single plant treatment. Removing rate of three-ring PAHs in all the treatment is very high that most of them is above 80% except ABR. Four-ring PAHs removing in ABO, ABS, ABF0 and ABF0R is desired which is close to 50%, ABS has the highest removal rate among them, at 61.04%. Removing rate of five-ring PAHs in ABF, ABF0, ABS, ABO, ABF0R, ABFR were 41.06%,43.40%,45.18%,42.95%,44.16%,28.32% repectively, six-rings PAHs were 42.68%, 54.22%,62.06%,35.97%,52.81%,40.35% repectively, total PAHs were 69.91%,70.52%, 73.98%,63.47%,69.91% and 60.46% repectively. Evaluating the removing rate of different rings PAHs and total PAHs in each treatment, ABF0R and ABS were the top two.Finally we detect the abundance of related degradation genes in the soil of each treatment, the results show that the gene abundance in all the inoculating degradation strains treatments are significantly higher than only inoculating rhizobium astragalus sinicus treatment, ABFOR, ABS, ABFO, ABO are much more higher than other treatments. The abundance of gene NAH in ABS and ABFOR were 6.14E+11 and 2.89E+11 respectively, C12O were 6.46E+08 and 3.39E+08 respectively.So ABFOR and ABS are the best efficient Phyto-microbial Remediation strains, which can be used for subsequent PAHs contaminated soil remediation experiment.The best degrading group was consisted of strain 02173,02830 and SL-1.Strain 02173 and 02830 were preliminarily identified as Pseudomonas alcaliphila and Pseudomonas corrugate, Sl-1 is type strain of Rhizobium petrolearium. SL-1 have a wide temperature range which is from 13-45℃,02173 and 02830 are from 21-45℃. all the strains can grow well at the pH condition of soil,in addtion,02173 and 02830 can adapt to the hypertonic environment.SL-1 can secrete IAA,02173 can also secrete IAA but its capacity is poor, 02830 can not secrete IAA.