Study On Degradation Mechanism And Metabolic Pathway Of Phenanthrene By Biodemulsifier-producing Bacterium Achromobacter Sp. LH-1

Polycyclic aromatic hydrocarbons?PAHs?,which are composed of two or more benzene rings positioned in a line or by way of clustering,have recently led to increasingly remarkable pollution of water and soil as a result of naturally occurring and human activities.As a representative of PAHs,phenanthrene?PHE?has been listed as a priority environmental pollutant in many countries.Meanwhile,it has been regarded as a model compound for studying PAHs because of its special structure.Biodegradation of PHE has garnered extensive research interest on account of no secondary pollution,saving time,high efficiency and low cost.However,the application of the method is limited due to the hydrophobicity of PHE.Biodemulsifier is a novel biosurfactant,and is considered to be of excellent surface activities.Biodemulsifier has hydrophilic head and hydrophobic tail and is capable of promoting the demulsification efficiency of water in oil?W/O?and oil in water?O/W?emulsions effectively.Besides,it is biodegradable.Both contribute to the widely application of biodemulsifier in the demulsification of kerosene and waste water in oil field,and so on.when the biodemulsifier concentrations exceed their critical micelle concentrations?CMC?,PHE will be enclosed by the thus formed micelles,greatly promoting the solubility of PHE in water.Therefore,applying biodemulsifiers produced by microorganisms to the environment contaminated by PAHs can not only remarkably degrade PAHs in the environment,but also cause no secondary pollution to the environment.Contrary to the extensive utilization of biodemulsifiers in dealing with oily waste water,few researches have been focused on PHE pollution.Therefore,further study on biodemulsifier-accelerated degradation of PHE would serve as theoretic basis for the restoration of PHE-contaminated environment,making it possible to be applied to environmental control,especially to the bioremediation of environmental pollution such as petroleum hydrocarbons.Strain LH-1 can grow using PHE as the sole carbon source to produce biodemulsifiers.Herein,utilizing PHE as the target pollutant,we investigated the growth of LH-1 and the degradation characteristics of PHE and study on the fermentation and degradation kinetics of LH-1 by nonlinear fitting using origin 9.0 software.The degradation pathways of PHE by strain LH-1 are explored by Liquid Chromatography-Mass Spectrometry?LC-MS?,and the promotion mechanism of biodemulsifiers on PHE degradation by strain LH-1 cells is experimentally supported by Gas Chromatography-Mass Spectrometry?GC-MS?,determination of membrane permeability of strain LH-1by?-galactosidase releaseetc,etc.The main findings are as follows:?1?LC-MS was used to identify degradation products of PHE degradated by strain Achromobacter sp.LH-1 at different cultivation times.The results showed that eighteen products were obtained.After analyzing these metabolites,we concluded that PHE was degraded by LH-1 through multiple pathways.In addition to the typical salicylic acid and phthalate pathways,PHE can also be degraded by two other unusual pathways.?2?Research was conducted on the fermentation kinetics of the PHE-degrading strain Achromobacter sp.LH-1.On the basis of Logistic model,Luedeking-Piret model and modified Luedeking-Piret equation,fermentation process properties undoubtedly referred to.We obtained respectively the kinetic equation of strain growth,substrate degradation and production formation.Experimental data were processed by Origin 9.0 software,and batch-fermentation model parameters were obtained.The fact that the model predicted values coincided with the experimental values.?3?Having explored the mechanism of demulsifier promoted degradation of PHE by Achromobacter sp.LH-1,it was found that when NH₄NO₃ concentration was 5 g/L,the demulsification rate of whole culture solution was reduced more than 80%after 7 d culture.And the LH-1 cells grew well,indicating that the production of biodemulsifier by strain LH-1 cells was significantly inhibited.The effects of different nitrogen source concentrations and external biodemulsifier on adsorption and degradation of PHE by strain LH-1 were investigated.The results showed that the degradation rate of PHE reached 81.46%,94.02%,96.06%,respectively,and the maximum adsorption of PHE reached40.17%,32.81%,60.01%after 7 days of culture.The maximum cell surface hydrophobicity?CSH?of the strain LH-1 when cultured at low concentration of nitrogen source?1 g/L NH₄NO₃?and low concentration of nitrogen source?1 g/L NH₄NO₃?adding biodemulsifier?40 mg/L?were 3.07 and 3.93times higher than those of culturing at the high-concentration nitrogen source?5 g/L NH₄NO₃?.The effect of biodemulsifier on cell membrane permeability of strain LH-1 was evaluated using the release amount of o-nitropheny l-?-D-galactopyranoside?ONPG?by strain LH-1 cells.The maximum release amount of o-nitropheny l-?-D-galactopyranoside?ONPG?by strain LH-1 cells when cultured at the low concentration of nitrogen source?1 g/L NH₄NO₃?and low concentration of nitrogen source?1 g/L NH₄NO₃?adding biodemulsifier?40 mg/L?were 1.75 and 2.08 times higher than those of culturing at the high-concentration nitrogen source?5 g/L NH₄NO₃?.