Study On The Mechanism Of Pyrene Synergistic Degradation By Soil Bacteria

Pyrene is one of the most widely distributed and stable polycyclic aromatic hydrocarbons with high molecular weight?HMW PAHs?.Pyrene and other HMW PAHs have the characteristics of poor bioavailability and hard to be degraded.Hence,it is of great significance to obtain efficient degradation strain resources and explore degradation process and mechanism for the study and practice of microbial degradation of PAHs.In this research,two pyrene-degradation strains,Pseudomonas aeruginosa sp.PA06 and Achromobacter sp.AC15,were domesticated and screened from the mangrove soil samples.A series of study on the degradation characteristics of pyrene,the concomitant synthesis and impact of biosurfactants,the synergistic degradation of mix-cultured bacteria,co-metabolic degradation of pyrene and molecule omics including proteomics,genomics and transcriptomics were conducted.Results revealed the biodegradation mechanism and molecular mechanism of co-metabolic and synergistic degradation of mixed bacteria.The main achievements are listed as follows:?1?Two strains of bacteria were cultured with pyrene as the sole carbon source,and the growth curve,degradation rate,enzyme production characteristics and biosurfactant synthesis and function were analyzed.Both the Pseudomonas aeruginosa PA06 and the Achromobater AC15 showed strong ability to degrade pyrene.The maximum degradation rates of 300 mg/L pyrene were 78.9%and 43.1%,respectively.Results indicated AC15 could detect the activity of both catechol-2,3-dioxygenase?C23O?and catechol-1,2-dioxygenase?C12O?,while PA06only detected the activity of catechol-1,2-dioxygenase,suggesting that these two strains possess complementary effects on the benzene ring cleavage pathway.Moreover,Both Pseudomonas aeruginosa PA06 and Achromobacter AC15 were able to synthesize biosurfactants using pyrene as the sole carbon source.The solubility of pyrene in two culture media were 2.93 times and 1.84 times compared with that in the pure water as a control,indicating that the biosurfactant was beneficial to improve the bioavailability of pyrene.?2?The biosurfactants produced with Pseudomonas aeruginosa PA06 and Achromobacter AC15 were identified and characteristic analysis using thin layer chromatography?TLC?,fourier infrared spectroscopy?FT-IR?and ionization mass spectrometry?MS?.BS06,the biosurfactant produced from PA06,was a kind of di-rhamnolipid with molecular formula Rha-Rha-C12-C12 and molecular weight 705.In addition,BS15,the biosurfactant produced from AC15,was lipopeptide with molecular formula CH₃-?CH₂?₁₄-CO-O-Leu-Asp-Lys-Ser and molecular weight 700.Within the concentration range of 0?160 mg/L,the solubility of pyrene increased from 0.13 mg/L to 0.536 mg/L and 0.417 mg/L by BS06 and BS15,respectively.The critical micelle concentrations?CMC?of rhamnolipid BS06 and lipopeptide BS15 were 41mg/L and 49 mg/L,respectively.Under the critical micelle concentrations of the two biosurfactant,the cell surface hydrophobicity?CSH?of Pseudomonas aeruginosa PA06 was increased from 24.5%to 33.4%and 29.5%,respectively;and that of Achromobacter AC15 from19.6%to 25.8%and 23.8%,respectively.These results show both biosurfactants could change the hydrophobicity of the two strains,suggesting that the two strains may have a synergistic effect.?3?Degradation mechanism of mixed culture and co-metabolic substance was explored using two strains to degrade 600 mg/L pyrene together.In the process of degradation of pyrene,the metabolites promoted the dissolution of pyrene and the growth of both strains.The total number of viable bacteria,the total activity of two kinds of catechol dioxygenase,C12O and C23O,and the degradation rate of pyrene were greater than that of each single strain as control.The result indicated that the mixed bacteria had a synergistic degradation effect.Glucose,sucrose,?-ketoglutaric acid and sodium citrate all can promote the degradation of pyrene,in which the most effect was from sodium citrate.In the co-metabolism system of 600 mg/L pyrene and 1.4 g/L sodium citrate,the total number of live bacteria,total enzyme activity,pyrene solubility and degradation rate were all greater than that of the control group?without addition of sodium citrate?.among which,pyrene solubility was 2.5 times that of the mixed bacteria control group,and pyrene degradation rate reached 74.6%,1.57 times that of the control group.?4?Achromobacter AC15 was cultured with sodium citrate or pyrene as the sole carbon source,respectively.The protein expression was detected using method of proteomics.A total of 359 proteins were identified and 109 were significantly differentially expressed proteins,of which 52 proteins were upregulated and 57 were downregulated.Under pyrene stress conditions,the expressions of naphthalene dioxygenase,di-hydrodiol naphthalene dehydrogenase and catechol-2,3-dioxygenase which were involved in the PAHs degradation pathway are up-regulated,indicating that the initial oxygenation of pyrene by Achromobacter AC15 may be mainly in the way of dioxygen,and the degradation of benzene ring may be mainly in meta-cleavage pathway.In addition,the up-regulated expression of some enzymes involved in energy metabolism and TCA cycle indicated these factors were important for pyrene degradation.Furthermore,amino acid metabolism-related enzymes,such as glutamate dehydrogenase,aspartic aldehyde dehydrogenase and type I glyceraldehyde3-phosphate dehydrogenase,down-regulated expression;Also,fatty acid synthesis-correlation enzymes,including enoyl hydrase,?-ketoethylacyl carrier protein synthase II,acetyl-Co A carboxylase and carboxyl transferase down-regulated expression.Results confirmed that the lipopeptide synthesis of peptide chain and aliphatic chain was reduced under the condition of pyrene as sole carbon source.Significant difference protein analysis associated with pyrene degradation and lipopeptide synthesis provided theoretical support for the synergistic degradation of mixed bacteria and the co-metabolic degradation of pyrene.?5?Pseudomonas aeruginosa PA06 was cultured with sodium citrate or pyrene as the sole carbon sources,respectively.The gene transcription of PA06 was studied by methods of genomics and transcriptomics.The genomic reads were annotated by COG to obtain 5858functionally genes.The number of known genes detected in transcriptome samples PA06-2?using sodium citrate as the carbon source?and PA06-4?using pyrene as the carbon source?were 5672 and 5476,respectively.There were 1786 genes with significant differences,of which1343 were up-regulated and 443 were down-regulated.Under pyrene stress conditions,the expressions of cytochrome P450,genes cyp107B1,pob A,hpa B,kyn A,xyl X,ant B,ant A,cat A,hmg A,phh A,xyl Z were up-regulated.The results revealed that the initial oxygenation of PA06on PHAs degradation in two ways,the mono-oxygen and the di-oxygen way.Moreover,the up-regulated expression of genes pob A,hpa B,xyl X,ant B,ant A,cat A,hmg A,phh A,xyl Z indicated that there are two ways of benzene ring opening with single oxygen and double oxygen.And there are many ways of benzene ring degradation,including ortho-cleavage pathway and urinary black acid pathway.The ortho-cleavage pathway could be concluded to be predominant from the upregulated expression genes.Some genes associated with energy metabolism,TCA cycle and glyoxylate acid metabolism were up-regulated expression,indicating that these factors have important significance in pyrene degradation.Furthermore,the down-regulated expression of genes pqs H,rhl R and las I indicated that QS signal molecular system showed negative regulation under the condition of pyrene as the sole carbon source.The biosynthesis of rhamnolipid precursor d TDP-L-rhamnolipid and?-hydroxy fatty acid weakened distinctly,and thus the biosynthesis of rhamnolipid reduced significantly.Genetic analysis of significant differences associated with pyrene degradation and rhamnolipid synthesis provided theoretical support for the synergistic degradation of mixed bacteria and the co-metabolic degradation of pyrene.