| Methylated phenanthrene(M-Phe)which are most ubiquitously distribute in the various environmental matrices,are major among alkylated polycyclic aromatic hydrocarbon(A-PAHs)in polycyclic aromatic hydrocarbon(PAHs)contaminated soil.Some M-Phe are more toxic and more persistent accumulation than that of phenanthrene(Phe),which result in the harmfulness to the ecological environment and human health.Besides,because M-Phe are mixed with parent PAHs(P-PAHs)in real environment,their biological effects and environmental behavior in multi-component PAHs pollution sites are also worth noticing.Aerobic biodegradation is one of the important ways to remove PAHs from contaminated environment.The PAHs biodegradation processes,metabolic pathways and mechanisms mastered is the key to successful application of bioremediation.In addition,the genotoxicity and carcinogenicity of PAHs-contaminated soil were significantly increased after microremediation due to the accumulation of PAHs metabolites that were highly polar and toxic than P-PAHs.Based on above,it is necessary to investigate the metabolic levels of PAHs to metabolites during PAHs biodegradation processes.At present,the analytical methods of PAHs and their metabolites mostly use chromatography including HPLC,GC-MS and LC-MS,which has large measuring flux and high sensitivity.Unfortunately,sample pretreatment procedures for those methods are complex,expensive and time-consuming together with heavy use of organic solvents,but beyond all else,inevitably destroy the original existence states of target analysts.Additionally,the metabolites produced in PAHs biodegradation processes were low instantaneous concentration,complexity and easy transformation,which greatly increased the difficulty of determination for PAHs metabolites.In this paper,1-methylphenanthrene(1-MP)and 3-methylphenanthrene(3-MP)are the representatives of M-Phe,Phe,pyrene(Pyr)and fluoranthene(Fla)are the representatives of low and high molecular weight PAHs,respectively.1-hydroxy-2-naphthoic acid(1H2NA)and salicylic acid(SA)are the representatives of the key metabolites of upstream and downstream pathway,respectively.On the basis of the previous derivative synchronous fluorescence spectrometry(DS-FS)with double scans,the determination conditions of DS-FS with double scans were optimized to realize the simultaneous determination of PAHs and their metabolites such as 1H2NA and SA during the PAHs biodegradation.Combined with the transformation ratios of Phe,1-MP and 3-MP to metabolites,the differences in the same metabolic pathways among Phe,1-MP and 3-MP investigated,although they were only different in the molecular configuration.In order to get closer to the real PAHs-rich environment,the synchronous fluorescence spectrometry with three scans was established to in situ detect dissolved 3-MP(Phe,1-MP),Pyr and Fla in the degradation processes of PAHs.The biodegradation characteristics of Phe,1-MP and 3-MP by Novosphingobium pentaromativorans US6-1 when the presence of Pyr and Fla.The main research results are as follows:1.The optimal DS-FS with double scans was successfully applied for in situ determination of dissolved 1-MP(Phe,3-MP),1H2NA and SA during 1-MP biodegradation.The intensities of the first derivative synchronous fluorescence peaks varied linearly with the concentrations of Phe,1-MP,3-MP,1H2NA and SA in the range of 0.20×10-7～44.89×10-7,0.20×10-7～13.04×10-7,0.20×10-7～13.04×10-7,0.22×10-7～76.30×10-7 and 0.11×10-7～52.00×10-7 mol/L,respectively.The limit of detection(LOD)of the analytical method were 0.11×10-9,0.30×10-9,0.27×10-9,0.33×10-9 and 2.90×10-9 mol/L for Phe,1-MP,3-MP,1H2NA and SA,respectively,with the percent of relatively standard deviation(RSD)less than 2%.The blank standard addition recovery of dissolved Phe,1-MP,3-MP,1H2NA and SA in mixtures were between 95.09%and 108.59%,with the percent of RSD less than 4%.Compared with fluorescence spectrometry previously reported,the sensitivity for measuring Phe,1-MP,3-MP,1H2NA and SA were increased by 4.7,1.2,2.7,1.1 and 2.2 times,respectively.2.The optimal DS-FS with double scans was used to investigate the metabolic levels of Phe,1-MP and 3-MP to 1H2NA and SA in order to demonstrate that there were different in the same biodegradation pathways of Phe,1-MP and 3-MP,although the molecular configurations of Phe,1-MP and 3-MP only had different in one methyl group and its substituted positions.With the increase of initial concentrations of Phe,1-MP and 3-MP,the instantaneous concentrations of 1H2NA and SA in the same degrading time increased continuously until PAHs were degraded completely,yet the transformation ratios of PAHs to 1H2NA and SA decreased.When the initial concentration of PAHs was 5.71×10-7 mol/L,the maximum degradation rates of Phe,1-MP and 3-MP were 7.24×10-8,3.15×10-8 and 3.67×10-8 mol/L/h,respectively.The transformation ratios of PAHs to 1H2NA were 34.9%,36.0%and 22.8%,respectively.The transformation ratios of PAHs to SA were 9.3%,17.2%and 7.2%,respectively.The metabolic levels of Phe and 1-MP to 1H2NA in the upper metabolic pathway were significantly different,1H2NA was produced through the di-oxygenation pathway at 3,4-carbon positions of Phe and mono-oxygenation pathway of methyl side chain of 1-MP,respectively.The metabolic levels of 1-MP to 1H2NA and SA were more than that of 3-MP in the mono-oxygenation pathway of methyl side chain and SA pathway.To sum up,the degradation pathways of Phe,1-MP and 3-MP were significantly different by Novosphingobium pentaromativorans US6-1.3.The synchronous fluorescence spectrometry(SFS)with three scans was successfully applied for in situ determination of dissolved 1-MP(Phe,3-MP),Pyr and Fla during 1-MP biodegradation.The intensities of the synchronous fluorescence peaks varied linearly with the concentrations of Phe,1-MP,3-MP,Pyr and Fla in the range of 0.20×10-7～13.04×10-7,0.20×10-7～13.04×10-7,0.66×10-7～6.52×10-7 and 0.66×10-7～6.52×10-7 mol/L,respectively.The LOD of the analytical method were 0.36×10-9,0.13×10-9,0.14×10-9,0.11×10-9 and 0.08×10-9 mol/L for Phe,1-MP,3-MP,Pyr and Fla,respectively,with the percent of RSD less than 3%.The blank standard addition recovery of dissolved Phe,1-MP,3-MP,Pyr and Fla in mixtures were between 80.30%and 119.48%,with the percent of RSD less than 3%.Compared with fluorescence spectrometry previously reported,the sensitivity for measuring Phe,1-MP,3-MP and Pyr were improved.4.The SFS with three scans was used to investigate the effect of Pyr,Fla,Pyr and Fla on the biodegradation of Phe,1-MP and 3-MP by Novosphingobium pentaromativorans US6-1,respectively.And the biodegradation differences of Phe,1-MP and 3-MP were also investigated when the presence of Pyr and Fla.When the degrading system containing Phe and Fla,the instantaneous degradation rate of Phe in the same degrading time was the most,that of Phe in the degrading system containing Phe,Pyr and Fla was the smallest.And the experimental results of 1-MP and 3-MP were consistent with that of Phe.In the different degrading systems,the instantaneous degradation rates of Phe were the most among Phe,1-MP and 3-MP after the incubation period of 1 h,yet that of Phe were the smallest in the incubation period of 1h. |
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