Biodegradation Of Phenanthrene By Massilia Spp. And Its Omics Analysis

Polycyclic aromatic hydrocarbons(PAHs)are the first known component of mutagenic,carcinogenic and teratoeenesis.Due to their characteristic of degradation-resistant,long half-life,and easy accumulated in soil,sediment,and water body,they pose huge environmental hazard to ecosystem and human health via food chain.Microbial degradation is the primary pathway to remove PAHs from soil,and bacteria are the class of microbe actively involved in the PAHs biodegradation.Based on the isolated strains of Massilia sp.WG5 and WX5 that can efficiently degrade phenanthrene,this study focused on identifying the degradation pathways and molecular mechanisms in bioremediation of phenanthrene(PHE),which was selected as the model substrate of ultimate carcinogen PAHs.The primary results and conclusions are summarized as following:(1)Our PCR results showed that the strains WG5 and WX5 had the complete identical 16S rRNAgene sequence and were identified to be Massilia sp.in Proteobacteria phylum.The difference in physiology and biochemistry between the two strains was only that the cultivation of strain WG5 was muddy in liquid LB media and the strain WX5 was flocculent.The whole genome sequencing of these two strains showed a high similarity(99.37%)and low conservation distance(0.003).The main difference between the two genomes was an insert fragment in the genome of strain WG5,which not was included in the genome of strain WX5.In this insert fragment,there was a predicted MspWG5? DNA methylation transferase with the amino acid length of 883 aa.Its recognition sequence was CAAGAC with 100.0%detection in WG5 genome.We speculated that this methylation transferase determined the different phenotype of strain WG5 from strain WX5 in liquid LB media.(2)The strains WG5 and WX5 both had the ability of efficient PHE-degradation,which could degrade 100 mg L-1 PHE totally in 48 hr.Comparing between these two strains,strain WX5 had a slightly stronger PHE-degradation ability than strain WG5 did.Strain WX5 could degrade 400 mg L-1 PHE in 5 days,while strain WG5 only degrade 80%of it.It was observed that there was a rose red component in the metabolite.This component could not be dissolved in H2O,HCl,ethyl acetate,methyl alcohol,dimethyl sulfoxide,n-hexane,acetone,and chloroform.Our analyses by UV-visible spectrum,HPLC-UV/FLD,and FTIR could not indentify the structure of this component.Further analysis by methylation derivatization and GC-MS detection showed the existence of trans-o-hydroxybenzylidenepyruvate and phthalic acid detected and identified.Based on the genome information,there were 35 related PHE-degradation functional gene analyzed.Combining the metabolites and genome information,we believed that there were three PHE-degradation pathways.One pathway was via phthalic,one was via salicylic acid,and the third was via 1-hydroxy-2-naphthoic acid to cis-2-carboxybenzalpyruvate.(3)The transcriptome RNA-Seq of strain WG5 was detected and analyzed during PHE-degradation.Base on the annotations of GO,COG,and KEGG databases,the related PHE-degradation functional gene could not be found in the differential expression genes.Matching the differential expression genes to the speculated PHE-degradation pathways found that there was no differential expression in the downstream genes of the pathway after phthalic acid.This result showed the possibility of PHE-degradation of via phthalic acid pathway,while the phthalic acid was the end product.The other pathway via cis-2-carboxybenzalpyruvate had been detected with the differential expression genes,which might be the important PHE-degradation pathway in strain WG5.Furthermore,there was a cluster of differential expression genes that cannot be matched to the speculated PHE-degradation pathways and databases.After blasting the related amino acid sequence of the gene,it was found that the similarity to the relative sequences in database was very low.Therefore,we speculated that there might be a novel pathway constructed by this cluster of genes(4)The strain WG5 showed a strong PHE-degradation ability in soil(50 mg L-1 PHE).In the soil treated with strain WG5,the concentration of PHE in the soil declined rapidly(only 9.06 mg L-1 remained after 3-days incubation).Combining the results of qPCR and high-throughput sequencing,the iHAAQ method was established and verified by an internal standard strain EDL933.The method was applied to detect the absolute abundance of bacteria communities during the biodegradation of PHE in soil.It was found that the increase of the absolute abundance of Massilia genus increased in the treatment with strain WG5 addition,which proved that the rapid PHE-degradation in the soil was due to the addition of strain WG5.Meanwhile,the heatmap and PCA analysis also showed the strain WG5 affected the bacteria communities caused by the reproduction of its own when the PHE was bioavailable.With the PHE bioavailable decreased,the influence of strain WG5 decreased,and the composition of bacteria communities returned to nearly original state.We concluded that the strain WG5 was an efficient and environmentally friendly bioremediation material for PHE-degradation in soil.