Study On Isolation, Identification And Degradation Characteristics Of A Novel PAHs And PCBs-degrading Janibacter Sp. Strain JY11

As the representative substances of persistent organic pollutants(POPs),study on the analysis method,environmental impact and pollution repairing of polycyclic aromatic hydrocarbons(PAHs) and polychlorinated biphenyls(PCBs) is a research emphasis in all over the world.Especially microbiodegradation is considered to be a major way to wipe off PAHs and PCBs in the environment.Based on the review of related study in the worldwide,a highly effective PAHs and PCBs-degrading bacterium was isolated.The isolate was identified as Janibacter sp.with respect to its 16S rDNA sequence and fatty acid profiles,as well as various physiological characteristics,and named JY11.Further study has been done systematically on degradation ability and relevant factors that affect the degradation performance. Co-degradation of PAHs and PCBs with JY11 and P chrysosporium JY16 was also researched.The probable pathway of PAHs and PCBs degraded by JY11 were proposed with detecting the metabolites by GC-MS.The main content and experimental results are as follow:1.Extraction and analysis of PAHs from polluted soil and PCBs from transformer oil(1) Soxhlet extraction,supereritical CO₂ extraction and ultrasonic extraction were selected to extract PAHs from polluted soil gathered from Jinan Oil Refinery Factory and Shengli Oil Field.And the extracts were analyzed by GC-FID.The results showed that:16 kinds of PAHs in Jinan Oil Refinery Factory were detected with a concentration of 5.0～593.2μg/kg;12 kinds of PAHs in Shengli Oil Field were detected with a concentration of 227.7～1107.3μg/kg.The spiked recovery of each method was in the range of 88.7～102.6%and the relative standard deviation(RSD%) was in the range of 1.5～4.4%,meeting the US EPA standard of recoveries(70～140%) and RSD of repeated samples(＜20%).(2) Liquid-liquid extraction,ultrasonic extraction and solid phase extraction were selected to extract PCBs from Chinese transformer oil and the extracts were analyzed by GC-ECD.Based on the former study results on PCBs analysis in our laboratory,10 kinds of dioxin-like PCBs(PCB77,81,105,114,118,123,126,156, 167 and 189) and four common exist atropisomeric-PCBs were detected in the Chinese transformer oil.Other PCBs components were also analyzed with Arcolor 1242 standard.The recovery of internal standard of PCB209 with different extraction method was in the range of 82.3%～91.6%and the RSD%less than 5%,meeting the US EPA standard of recoveries and RSD of repeated samples.2.Isolation and identification of PAHs and PCBs-degrading bacteria16 PAHs-degrading strains were newly isolated from the polluted soil in Jinan Oil Refinery Factory,Shengli Oil Field and former Jinan Power Transformer Factory, Shandong Province of China.Two bacteria,JY11 and JY3A,were investigated. Strain JY11 was identified as Janibacter sp.with respect to its morphology,16S rDNA sequence,phylogenetic analysis,and fatty acid profiles,as well as various physiological and biochemical characteristics.The strain was Gram-positive, non-motile,non-spore-forming,short rods in young culture,0.8-1.0μm in diameter and 1.3-1.6μm long,and coccoid cells in the stationary phase of growth that are 1.0-1.2μm in diameter and 1.3-1.5μm long,occurred in pairs and sometimes in chains or in group,aerobic,oxidase-week positive,catalase-positive.On the basis of 16S rDNA sequence similarity studies,strain JY11 was shown to be most closely related to Janibacter anophelis(99.93%),J.terrae(98.48%),J.marinus(98.38%),J. limosus(98.34%),J.melonis(98.20%) and J.corallicola(97.79%). Chemotaxonomic data(menaquinone,GC content and major fatty acids) supported the allocation of the strain to the genus Janibacter.Strain JY3A was characterized as motile,rod-shaped,Gram-positive,endospore forming,single or in pairs and sometimes in chains,aerobic,oxidase-positive, catalase-positive.On the basis of 16S rDNA sequence similarity studies,strain JY3A was shown to be most closely related to B.vallismortis(99.69%),B.subtile (99.65%),B.amyloliquefaciens(99.60%),B.mojavensis(99.57%) and B.atrophaeus (99.42%).Chemotaxonomic data supported the proposal to assign the strain to the B. subtilis group of the genus Bacillus. 3.Study of degradation ability and related factors that affect the degradation performance of PAHs and PCBs by Janibacter sp.JY11(1) Degradation of PAHs by Janibacter sp.JY11PAHs with initial concentration of 100 mg/l were degraded with JY11 in liquid culture.The result showed that not only the low molecular PAHs,such as phenanthrene,fluorene and acenaphthene,but also pyrene,fluoranthene and benzo(a)anthracene have a good degradation result.Also the high molecular PAHs degraded in different degree.JY11 presented a wide degradation spectrum and high effective degradation ability.(2) Effects of different initial concentrations,surfactant and salicylic acid on the degradation efficiency of PAHs were studied taking phenanthrene,pyrene and benzo(a)pyrene as representative.The results showed that the degradation efficiency of pyrene and benzo(a)pyrene decreased with the increase of initial concentration, while phenanthrene can be degraded completely with different initial concentrations. Tween 80 is helpful for the degradation of PAHs.Through adding salicylic acid in the medium,the degradation efficiency of benzo(a)pyrene increased 28%compared with phenanthrene as co-metabolized substance.(3) Degradation of PCBs by Janibacter sp.JY11The degradation efficiency of PCBs with a concentration of 10 mg/l and 100 mg/l was studied separately.The result showed that the degradation efficiency decreased in some degree with the increase of initial concentration of PCBs.But JY11 presented a good performance as a whole.For PCBs with an initial concentration of 10 mg/l,the degradation efficiencies of the di and triclorine-CBs were 83%～100%;the degradation efficiencies of the tetraclorine-CB were 78%～94%; and the degradation efficiencies of penta and hexaclorine-CB were 50～65%.The degradation efficiencies of PCBs decreased along with the increase of the number of chlorine atoms.(4) In the inducing experiment by natural plant constituent,essential oil of Cordyceps kyushuensis and carvone can enhance the degradation efficiency notably; also,cymene is helpful in some degree.While,essential oil from Dracocephalum tanguticum and biphenyl nearly have no effect on the degradation efficiency.But essential oil from Amomum tsao-ko crevost et lemaire is harmful for degradation of PCBs by JY11.4.Co-degradation of PAHs and PCBs by Janibacter sp.JY11 and P. chrysosporium JY16Through adding white rot fungi,P.chrysosporium JY16,to the medium of JY11, the degradation threshold of PAHs and PCBs decreased and the degradation efficiency increased according to their co-degradation effect.In liquid culture,the degradation efficiency of the mixed bacterium and fungi is much better than JY11 and JY16.Especially the degradation efficiency of four ring-pyrene and five ring-benzo(a)anthracene.Benzo(k)fluoranthene increased at least 20%compared with JY11.For the modified polluted soil sample,73%of pyrene and 60%of benzo(a)pyrene was degraded with mixed bacterium and fungi after thirty days cultivation.The degradation efficiency increase a lot compared with the sole bacterium or fungi.Similar result was got with PCBs.The degradation result with mixed culture is better than any sole bacterium or fungi.Especially for dioxin like PCB156,167 and 189,which could not be utilized by sole JY11 or JY16, decreased by 10 percent with co-culture.PCBs in the transformer oil and modified polluted soil were degraded by the mixed bacterium and fungi.The result showed that the degradation efficiencies of PCBs in transformer oil were 8.7%～83.3%.And the degradation efficiencies of PCBs in the modified polluted soil were 18.4%～72.6%.While PCB74,dl-PCB105,118 and atropisomeric-PCB132,149 showed no degradation.5.Analysis of metabolites and proposed pathway of PAHs and PCBs degraded by Janibacter sp.JY11Phenanthrene,pyrene and benzo(a)pyrene were selected as the research targets of PAHs degradation pathway.PCB 18 and PCB77 were selected as the targets of PCBs degradation pathway study.The proposed pathway of PAHs and PCBs was concluded by detecting the metabolites by GC-MS. (1) Phenanthrene was transformed to 2,3-dihydroxy-2,3-dihydrophenanthrene firstly with dioxygenase of JY11.2,3-dihydroxy-2,3-dihydrophenanthrene was metabolized to 1-hydroxy-2-naphthoic acid,which transformed to 1-naphthol. 1-naphthol was cleaved to salicylic acid at last.(2) Pyrene was transformed to 4,5-dihydroxy-4,5-dihydropyrene firstly with dioxygenase of JY11.Then 4,5-dihydroxy-4,5-dihydropyrene was metabolized to phenanthrene 4,5-dicarboxylic acid,which transformed to 4-hydroxyphenanthrene. 4-hydroxyphenanthrene was cleaved to phthalic acid at last.(3) Benzo(a)pyrene was transformed to benzo(a)pyrene-7,8-dihydrodiol firstly with dioxygenase of JY11.And then benzo(a)pyrene-7,8-dihydrodiol was metabolized to benzo(a)pyrene-7,8-dihydroxy,which transformed to pyrene-7-hydroxy-8-carboxylic acid at last.(4) PCB18 and PCB77 have a similar probable pathway,that is JY11 attacked the 2,3 position of PCBs and which metabolized to 2,3-dihydroxy-2,3-dihydro-PCB, then transformed to 2,3-dihydroxy-PCB,and then yielded to a yellow color product, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid,which was transformed to ehlorbenzoic acid under a hydrolase.