| The oil spill occurred frequently in recent years,along with the oil pollution becoming a serious environmental problem for coastal countries.Study found that 98%of the total polycyclic aromatic hydrocarbons(PAHs)in the oil was alkylated polycyclic aromatic hydrocarbons(A-PAHs).And after spilled into the water environment,the content of PAHs in sediment was still as high as above 70%.Scientists pointed out that Phenanthrene(Phe)and alkylated Phe widely distributed on the oil spill area.And alkylated Phe was the main PAHs which resulted in development and reproductive toxicity for marine fish.Thus,research about A-PAHs gradually become the focus of environmental expert's attention.Biodegradation is one of main methods to get rid of PAHs in the environment.Some conducted studies on dissolved PAHs biodegradation mainly focus on parent PAHs,partial reaserches involving A-PAHs mainly concentrated on the alkylated naphthalene.Howerver,research about biodegradaton of dissolved A-PAHs with more than three benzene rings is rare.Based on this,firstly,this paper studied biodegradation characteristics of single Phe and methyl phenanthrene(MP):1-MP,3-MP,4-MP and 9-MP by the Novosphingobium pentaromativorans US6-1 and the trans-membrane mechanism.Secondly,the variation of cell membrane surface properties of bacterium US6-1 in the presence of Phe or MP and affinity parameters between the five kinds of PAHs and US6-1 were investigated.Thirdly,the biodegradation of dissolved multi-component Phe,3-MP or 9-MP by the bacterium US6-1 and preliminary discussion on the mechanism were studied.Finally,under the condition of laboratory simulation,the influence of LMWOAs:oxalic acid,citric acid and tartaric acid on the biodegradation process of Phe,3-MP and 9-MP were studied.The results of the study are as follows:1)Synchronous fluorescence spectrometry was used to study the biodegradation process of dissolved one-component Phe and MP by bacterium US6-1,and the mechanism of the membrane process were also examined.The result showed that for Phe and MP,the synchronous fluorescence linear range were 0.08-6.00×10-6 mol/L and 0.08-1.20×10-6 mol/L,respectively,and the linear correlation coefficient>0.9912.It meant that this method could be used for studying the biodegradation process of dissolved MP.The detection limits of Phe,1-MP,3-MP,4-MP and 9-MP were 5,17×10-10?3.74×10-10?7.17×10-10?3.12×10-10?4.23×10-10mol/L.The sensitivity,linear ranges and detection limits of the proposed method were lower than researches.Different positions of methyl substituent on Phe ring produced different space steric effect,affecting the biodegradation of MP.The degradation rate trend was:4-MP(a)<9-MP(?)<1-MP(?)<3-MP(?).It was a general trend that isomers with methyl substitution in the ?-position degrade more rapidly compared to isomers with methyl substitution in the ?-position.The actual maximumal biodegradation rate with the theory of maximumal trans-membrane flux had good linear relationship(R2 =0.8969),showed that trans-membrane was the limiting step during the biodegradation process of Phe and MP.NaN3 had little effect on the degradation of Phe,but inhibited MP degradation and the inhibition degree was related to substituent position.This showed that space steric hindrance of methyl substituents changed the way of A-PAHs acrossing the membrane,and depended on the substituent positions.2)To further understand the mechanism of difference during Phe and MP degradation process,the influence of Phe or MP on bacterium US6-1 were investigated.The ratio of 3-OH fatty acid to the total fatty acid content on the bacterium US6-1 membrane surface in the presence of MP were lower than of Phe,which were:4.22%for Phe,3.76%for 1-MP,3.03%for 3-MP,2.64%for 4-MP,2.79%for 9-MP,respectively.It meant that the content of lipopolysaccharides(LPS)decreased,the membrane surface hydrophobicity increased to a larger degree in the presence of MP than Phe.Hydrophobic experimental results were Phe(34.6%)<3-MP(40.10%)<1-MP(41.31%)<4-MP(60.73%)<9-MP(68.4%),further verifying cell surface hydrophobicity of bacterium US6-1 in the presence of MP were higher than that of Phe.The order of the five kinds of PAHs biodegradation rate was opposite with hydrophobic experimental results,which meant that the hydrophobicity was not the main factor causing differences of MP isomer degradation.Besides,the influence of MP on the cell membrane fluidity and stability of bacterium US6-1 were different from Phe,which further suggesting the membrane process of MP were different from Phe.The Ks value by the Lineweaver Burk plot equation of PAHs were:4-MP>9-MP>1-MP>3-MP>Phe.It meant that the combination of methyl substituent hindered the PAHs interaction with degradating enzyme,making the degradation rate of MP decreased.3)Synchronous fluorescence spectrometry was used to study the biodegradation process of two-component and three-component Phe,3-MP,9-MP with Fluorene(Flu),1-Methyl fluorene(1-MF)or Pyrene(Pyr).The result showed the linear range of MP were 0.08-1.20×10-6 mol/L.The detection limits of Phe,3-MP and 9-MP were 1.24×10-9?1.34×10-9?1.42×10-9 mol/L.The sensitivity,linear ranges and detection limits of the proposed method were consistent with researches.Results showed that the bacterium US6-1 gave priority to utilize Phe and MP,then Flu and 1-MF,finally Pyr.Compared to one-component system,the degradation of Phe or MP was suppressed in the multi-component systems,and the inhibition in three-component system were stronger than in two-component system.The bidegradation of Flu or 1-MF were promoted with the presence of Phe or MP.During the biodegradation process of Pyr,Phe or MP functioned as promotion but Flu and 1-MF functioned as inhibition in multi-component system.And in three-group system,the biodegradation patterns of Pyr depended on the promotion and inhibition getting balanced with respect to each other.In addition,in multi-component system,the biodegradation of MP was inhibited to larger degree than Phe,and the biodegradation of other PAHs were promoted to larger degree than Phe.In other words,the presence of methyl substituent made the PAHs existed longer in enviroment.4)Influence of LMWOAs:oxalic acid,citric acid and tartaric acid on the biodegradation of dissolved Phe,3-MP and 9-MP by bacterium Novosphingbium pentaromativorans US6-1 were individually studied using synchronous fluorescence.Results showed that 0.2 mmol/L LMWOAs promoted the biodegradation of Phe.0.2 mmol/L oxalic acid inhibitied the biodegradation of 3-MP and 9-MP,while 0.2 mmol/L tartaric acid or citric acid promoted these processes.The presence of 2.0 mmol/L oxalic acid promoted the degradation of the three PAHs,but 2.0 mmol/L tartaric acid inhibited these processes.Oxalic acid at two concentrations,i.e.0.2 mmol/L and 2.0 mmol/L improved the cell surface hydrophobicity of US6-1.For Phe and 3-MP,the results of two-factor variance analysis showed that the types,concentrations of LMWOAs,and the interaction of these two factors played main role during the biodegradation process of Phe and 3-MP(p<0.05);for 9-MP,these three factors were not the key factors influencing the biodegradation of 9-MP(p>0.05).In conclusion,the comparative study of the dissolved Phe and MP biodegradation process,which is helpful to understand the PAHs biodegradation process,and provide a reference for predicting more complex PAHs degradation process,and scientific guidance and data to support for PAHs behave in water environment. |
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