Bioremediation Of Polycyclic Aromatic Hydrocarbons Contaminated Soil By The Symbiosis Of Cowpea And Rhizobium

Polycyclic aromatic hydrocarbons(PAHs) are chemical compounds made up of at least two fused aromatic rings in a linear or clustered arrangement. They are highly hydrophobic and persistent hazardous organic compounds which can be detected in soils worldwide. Soils contaminated with PAHs pose potential risks to human health and ecological stability. They were found to have a variety of teratogenic, mutagenic and carcinogenic effects on animals, humans and plants. So their elimination is considered a high priority in many countries.Bioremediation is a relatively new in-situ method which uses plants and microbial communities in the rhizosphere to degrade PAHs. One limiting factor for bioremediation of contaminated soils is nitrogen deficiency. Therefore, legume species became the prior choice for the bioremediation of PAHs contaminated soil. Because legume has nitrogen fixing properties, it enhances the accumulation of nitrogen and organic matter. It balances the C/N ratio and increases the quantity of rhizospheric microorganisms. The mechanism of PAHs bioremediation by symbiosis of legume and rhizobium would be helpful to control soil PAHs contamination.The PAHs dissipation mechanisms were investigated to determine if the legume-rhizobium symbiosis bioremediation, especially root mucilage, plays a role in PAHs removal. Many experimental methods and techniques were applied, such as PCR, DGGE, PLFA, HPLC, 14 C radioactive isotope, root printing, resting cell and artificial neural network(ANN). The main experiments and conclusions are as following:(1) Isolation and screening of PAHs-degrading bacteria is an important aspect of the biodegradation and bioremediation of PAHs. The high efficient PAHs-degrading bacteria is the premise of bioremediation of polluted soils. A microbial consortium was obtained from the enrichment culture of slurry samples collected from an aged contaminated site. One hundred eleven strains were isolated from the microbial consortium. Among these bacteria, 43 strains could degrade PHE, 15 could degrade PYR, 27 could degrade both simultaneously, 14 and 3 could grow on PHE and PYR plate separately but had no clearzone, 9 could grow on both PHE and PYR plate but no clearzone. The biodiversity of isolates was investigated by a genomic fingerprinting technique, ERIC-PCR. The results indicated that there were 10 different ERIC-PCR groups based on the 42 representative stains finger print.(2) Seven strains which could use PAHs as a sole carbon source were isolated from aged contaminated soil. Based on the 16 S r RNA gene sequence, USACA0001-A3 was identified as Mycobacterium vanbaalenii,USACA0002-B4 was identified as Mycobacterium gilvum,USACA0003-C9 was identified as Pseudonocardia carboxydivorans,USACA0004-D17 was identified as Delftia tsuruhatensis, USACA0005-E1 was identified as Achromobacter xylosoxidans, USACA0006-F15 was identified as Pseudomonas aeruginosa,USACA0007-G6 was identified as Bacillus cereus. The PAHs dioxygenase gene from D17(721bp) and C9(320bp) were ampilified by PCR and sequenced. To our knowledge, this is the first time to study the PAHs dioxygenase gene of Pseudonocardia carboxydivorans USACA0003-C9 and Delftia tsuruhatensis USACA0004-D17.(3) The modified sublimation method was used to compare the strains’ PHE degrading ability. The speed of clear zone forming is D17>A3>C9>B4>E1> F15> G6. The top three A3、C9、D17 were chosen for further PHE degradation rate test by HPLC. In the minimal salt medium(MSM) system, while the initial concentration of PHE is 100 mg L-1, the degradation rate of PHE after 3 days incubation by D17, A3, C9 is approximately 94.4%, 85.5%,81.4%, respectively. Further experiments were carried out to optimize D17 growth conditions and the results indicated that the optimal condition was 30℃ and p H 7.0.(4) Aged PAHs-contaminated soil and freshly PAHs-contaminated soil was used by comparing the diversity of PAHs- degrading bacteria. The following eight types of PAHs: Naphthalene, Phenanthrene, Pyrene, Acenaphthene, Anthracene, Chrysene,Benzo[α]pyrene and PAHs mix, were used to culture the bacteria which adapted to use relative PAHs as a sole carbonsource. Denaturing Gradient Gel Electrophoresis(DGGE) was adopted to investigate the bacterial composition and community dynamic changes based on 16 S r RNA genes PCR amplification during enrichments batch culture. The biosurfactant was also analyzed by surface tension test. The results showed that aged PAHs-contaminated soil have more PAHs-degrading bacteria diversity than freshly PAHs-contaminated soil. Biphenyl dioxygenases gene(BPHD) was also amplified from the DNA isolated from the treatments of HS-B[α]P, HS-Mix PAHs, NS-B[α]P and NS-Mix PAHs. The similarity was 98% compared with the(bph A1) biphenyl 2,3-dioxygenase alpha encode by Rhodococcus sp.. The lowest surface tension of the biosurfactant solution was 32.5m N m-1, which indicated that powerful biosurfactant was produced by the bacteria during the degradation process of high molecular weight PAHs, such as B[α] P.(5) Nine legumes were used as plant materials for bioremediation of PAHs and petroleum contaminated soil. Through analysis of the legumes, the survival rate, biomass and the number of nodules was determined. It was concluded that cowpea(Vigna unguiculata) was superior to the other species because of its high tolerance to the PAHs toxicity by forming nodules under PAHs pressure. Cowpea was chosen as the model plant for further research on PAHs bioremediation.(6) A pot study was conducted to examine the effects of inoculation with rhizobium on bioremediation by cowpea grown for 35 days in aged PAHs-contaminated soil and freshly PAHs-contaminated soil. The morphology and interior structure was observed by Scanning electron micrograph(SEM). PAHs attenuation rate of cowpea roots with nodule and cowpea roots without nodule was tested by HPLC. Denaturing Gradient Gel Electrophoresis(DGGE) was conducted to analyze the diversity of PHE degrading bacteria from four microzones of cowpea rhizosphere(root surface, bulk soil, nodule surface, nodule inside). The results indicated that the addition of PHE significantly restrains cowpea growth. In freshly PAHs contaminated soil, the morphology of root nodule is pink and healthy. However, the root nodule in aged contaminated soil turn to black, and the interior structure under SEM was different from the healthy root nodule.In flask cultures, cowpea plants with nodulated roots exhibited a higher potential for PAHs attenuation in the surrounding plant solution than plants with nodule-free roots. The sublimation method indicated that PAHs utilized bacteria exist both inside and outside of the root nodule if the treatments were spiked with PHE. Denaturation gradient gel electrophoresis(DGGE) of polymerase chain reaction amplified 16 S r RNA coding genes revealed that legume nodules also contained endophytic bacteria which can degrade PAHs.Inoculation with Rhizobium significantly increased the diversity of culturable PAHs-degrading bacteria. Besides PAHs-degrading bacteria, there were many Plant growth-promoting rhizobacteria(PGPR) and biosurfactant-producing bacteria. The results suggest that the symbiotic association between cowpea and Rhizobium can stimulate the rhizosphere microflora to degrade PAHs and its application may be a promising bioremediation strategy for aged PAHs- contaminated soils.(7) The experiment of eco-toxicological effects of PHE on the inhibition of seed germination and elongation was conducted by three cowpea(Vigna unguiculata) cultivas: C1(Line UCR 232): cowpea with high mucilage; C2(Line: CB27): cowpea with low mucilage; C3(Line: 63-33(1)): cowpea with no mucilage. Five concentration gradient(0 mg L-1,50 mg L-1,100 mg L-1,300 mg L-1,500 mg L-1) was used. The results indicated that there were no inhibiting effects on cowpea seed germination. The germination rates of all three species were 100%. During the 15 days incubation period the inhibition of root and stem elongation of three cowpea species were different. The higher the PHE concentration was, the more severe the inhibition impact. But in lower PHE concentrations, the elongation of root and stem can be promoted, such as cowpea C1, which showed higher PAHs-resistant ability, the root mucilage produced by C1 may plays a role by protecting roots from the toxicity of PAHs.(8) Little is known about the effect of root mucilage on polycyclic aromatic hydrocarbons(PAHs)-polluted soil remediation. Phenanthrane(PHE) was used in this study. The objective of this study was to evaluate the PHE degradation rate with the help of three cowpea(Vigna unguiculata) cultivas(C1(Line UCR 232): cowpea with high mucilage; C2(Line: CB27): cowpea with low mucilage; C3(Line: 63-33(1)): cowpea with no mucilage.). High performance liquid chromatography(HPLC) analysis and 14C-PHE radioactive mineralization were used to monitor the PAHs degradation rate in the soil and the effect of mucilage on PAHs mineralization. Our results showed cowpea C1 with rhizobium treatment had the highest PHE degradation with 91.6% compared to the control 48.4% and 51.2% in 5 weeks. The root printing test results indicated that by adding purified mucilage, the bacteria degraded the PHE at a faster rate according to the size of clear zone. The 14C-PHE mineralization proved that root mucilage helps aged contaminated soil(HS) degrade more PHE. This is the first study to evaluate the mucilage effect on PAHs bioremediation.(9) The effect of cowpea’s root mucilage on the soil microbial communities during the PAH phytoremediation was evaluated through the analysis of denaturing gradient gel electrophoresis(DGGE) and phospholipid fatty acids(PLFA). Polymerase chain reaction denaturing gradient gel electrophoresis(PCR-DGGE) was conducted to analyze the dynamics of microbe communities during the process of plant treatments. The PCR-DGGE results showed that mucilage treatment increased the diversity of PAHs degrading bacteria of soil microbe communities, which may explain why the PHE degraded at a faster rate with C1 than other treatments. Relative changes in microbial populations of different treatments were investigated by following the dynamics of PLFA signatures. The data was analyzed by using Principal component analysis(PCA) and Artificial Neural networks(ANNs) method. The results of this investigation indicated that fungal to bacterial PLFA ratios were significantly influenced by root mucilage produced by cowpea C1, and Gram-positive to Gram-negative bacteria ratios were significantly correlated with PHE by the Kohonen self-organizing maps(KSOM) map. The microbial community composition and biomass structure associated with the rhizospheric soil and bulk soil were compared. We concluded that root mucilage could contribute to the changes in the structure of the total microbial community in the rhizosphere, which may benefit the PAHs bioremediation.