Isolation Of Bacteria With High Efficient Biodegradation Of Polycyclic Aromatic Hydrocarbons And Their Biodegradation Ability

Biodegradation of PAHs (polycyclic aromatic hydrocarbons), particularly microbial degradation, is the main way removed from the environment. The current study of microbial degradation was mainly focused on anaerobic degradation and aerobic degradation. Studies had shown that, hypoxic environment (dissolved oxygen in water ranged from0mg/L to4.5mg/L) was widely found in swamps, lakes, soil. However, the degradation of PAHs in hypoxia condition has been seldom studied. Based on the comprehensive review of relevant research in domestic and foreign, we had collected some sediment samples contaminated with PAHs in Zhushan Bay, Taihu Lake. The content of PAHs in sediments was detected. In8%oxygen conditions, six phenanthrene-degrading bacteria and eight pyrene-degrading bacteria were initially screened. As a basis, bacterial degradation of phenanthrene and pyrene had systematic studied. Eventually, two strains of highly efficient degradation of phenanthrene and pyrene was got.Based on16S rDNA sequence analysis, phylogenetic tree construction and other means, we have identified the genus of two strains. With gas chromatography-mass spectrometry, degradation intermediates had qualitative analyzed. The metabolic pathway of the strains of phenanthrene and pyrene had preliminary studied. The main research contents and results are as follows:(1) In November,2012, sediment samples were investigated and collected from Zhushan Bay, Lake Taihu. The concentrations of dissolved oxygen in the sediments were between1.71～5.66mg/L, which was belonged to hypoxic environment. The concentrations of electron acceptors (NO3-、SO42-、Fe3+) in sediments were1.12～3.20mg/kg,55～725mg/kg,7.92～93.40mg/kg. Total16kinds of US EPA priority polycyclic aromatic hydrocarbons (PAHs) concentrations were analyzed by GC-MS. The results showed that PAHs content varied from61.2to2032.3ng/g, the mean value was1131.5ng/g. The source of PAHs in littoral zone sediments are mainly attributed to high-temperature combustion of coal, oil and other fossil fuels, but that in center of the lake was mainly attributed to oil spill. No significant PAH ecological risk was found in littoral zone of Lake Taihu according to ecological risk assessment. However, some PAH concentrations have exceeded the effective range of low (ERL) in some sampling sites. Potential harmful effects on organisms might exist, and ecological risk prevention should be in plan. TEQBaP concentrations in11sites ranged from0.284to358.29ng/g by carcinogenic risk analysis, the cancer risk was moderate level. Seven kinds of high-ring PAHs (BaA, Chry, BbF, BkF, BaP, IP, DBA, and BghiP) are a major contributor to TEQBaP value, DBA, and BaP are the biggest contributors. The TEQBap value of positions in the littoral zone had exceed the standard TEQBap target reference value (33.0μg/kg), showed that PAHs concentration in the study area had potential carcinogenicity.(2) Use phenanthrene and pyrene as sole carbon source, applied enrichment culture, regular quantitative transfer and domestication cultivation, six strains of phenanthrene-degrading and eight strains of pyrene-degrading bacteria were initially separated from sediments from Zhushan Bay under8%oxygen conditions. Then by phenanthrene/pyrene degradation experiments, the degradation efficiency of these strains had detected. Further, a strain of effectively hypoxia phenanthrene-degrading bacteria was screened, which was named as ZS1. Phenanthrene was completely degraded in10days by ZS1. Similarly, a strain of effectively hypoxia pyrene-degrading bacteria was also screened, which was named as ZS2. The degradation rate of pyrene by ZS2was nearly50%in50days. The study found that due to the different water solubility and the number of rings, hypoxia pyrene degradation efficiency is much lower than the phenanthrene. And compared with other PAHs aerobic and anaerobic degradation studies, the degradation rate of PAHs under hypoxia is between them.(3) Through bacterial morphology observation, combined with16S rDNA sequence analysis and phylogenetic tree constructed, two effectively hypoxia PAHs degrading bacteria (ZS1, ZS2) were identified. The identification results showed that, ZS1is belonged to Klebsiella sp.(named Klebsiella sp. ZS1), which is belonged the same genus with Zhao He-ping (Klebsiella sp. EMSL2), Yang Xiao-lei (Klebsiella sp. JU1) and other studies. The sequence have submitted to the GenBank database, accession number is KF974531. ZS2is belonged to Bacillus (named Klebsiella sp. ZS1). The result is same with Wu Feng-xia (Bacillusfirmus strain FC31), Zhang Hong-bo (Brevibacillus sp. J3), Hou Shu-yu (Pyl, Py2).The sequence is submitted to the GenBank database and the accession number is KJ004566.(4) Hexane/dichloromethane (1:1; V/V) mixture was used extract the intermediate shocks by liquid-liquid extraction method. The metabolites intermediate in the microbial degradation of phenanthrene/pyrene was analyzed by GC-MS. Hypoxia degradation pathway of phenanthrene and pyrene was preliminary analyzed. The main produces of Klebsiella sp. Bacteria ZS1degrade phenanthrene was ring opened hydrocarbons, one benzene ring hydrocarbons, and some sulfur and nitrogen compounds. According intermediates of phenanthrene degradation, phenanthrene was added hydroxyl at the action of the dioxygenase enzyme in aromatics, generated phthalic acid substances. And ring cleavage reaction occurred, organic carboxylic acids was generated. The main detected intermediates of Klebsiella sp. Bacteria ZS2degrade pyrene included coumarins, phthalates, dihydro-pyrene, pyrene monohydric straight chain hydrocarbons and etc. There may be two degraded ways. The first pathway:under the action of hydrogenation enzymes,4,5-dihydro-pyrene or3,5-dihydro-pyrene was generated, then phenyl ring was fractured which had generated two or three rings substances. The second pathway:under the action of monooxygenase or dioxygenase, coumarins were generated with ring cleavage reaction, and then small molecule organic carboxylic acids were continuing decomposed.