Studies On The Petroleum Degradation Characteristics And The Related Gene Analysis Of Two Ochrobactrum Sp.

Microbial remediation refers to the process of metabolically transforming and degrading organic pollutants into inorganic substances by using natural or functional microbial populations in the soils.Separation of the high-efficiency pollutant-degrading bacteria is one of the key links in the bioremediation.Ochrobactrum is a class of salt-tolerant Gram-negative bacteria.Bacteria of Ochrobactrum have been reported to fix heavy metals,degrade chlorate,emulsify petroleum,and metabolize alkane.In this paper,two oil degrading bacterial strains,Ochrobactrum sp.FP1 and JP1 were identified by physiological and biochemical analysis.Their oil degrading characteristics,biological characteristics and genomics were also analyzed.The main results were as follows:By using 16 Sr DNA sequence analysis and physiological and biochemical identification,the isolated bacterial strains FP1 and JP1 were determined to belong to Ochrobactrum sp..Strain FP1 and JP1 could grow normally at the temperature range of 5-50?,with the optimum temperature at 25?.The optimum p H was p H7,and the optimum salinity was 1%.It was found that FP1 and JP1 grew well in the basal medium containing 100 mg/L of different alkanes and aromatics as the sole carbon source.The degradation characteristics of petroleum hydrocarbons of FP1 and JP1 were analyzed by GC-MS analysis.The strains were inoculated on the basal medium containing the mixed alkanes and mixed aromatic hydrocarbons with a concentration of 600 mg/L.It was found that FP1 and JP1 had strong abilities to degrade different alkanes and aromatic hydrocarbons.Among them,FP1 and JP1 have a degradation rate of more than 66.00% for each component of mixed alkanes,and a degradation rate of more than 57.00% for each component of mixed aromatics.The genomes of the FP1 and JP1 were determined and analyzed.The SMRT Link v5.0.1system was used to assemble the clean data.Based on the sequencing data,the genome sizes of FP1 and JP1 were 4862271 bp and 4862246 bp respectively,with the GC content of 56.36%.The Gene Mark S software(http://topaz.ga.tech.edu/)was used to predict the coding genes,and the results showed that the genomes contained 4587 and 4580 predicted genes.The total lengths of the coding genes were 4288296 bp and 4288746 bp,with the average lengths of934 bp and 936 bp respectively.Both strains contained 58 t RNA and 12 r RNA genes.The 4564 and 4561 genes have been annotated in the NCBI-NR database.The genes participated in a total of 137 KEGG pathways,of which 27 were related to compound degradation,including the degradation pathways of naphthalene degradation,ethylbenzene,xylene and other aromatic compounds.The genes involved in the regulation and metabolism of the carbohydrates were 252 and 250 respectively.The annotation results of the assembled gene database showed that seventeen genes in FP1 and JP1 were related to alkane degradation,including lipoprotein family protein gene(blc),long-chain fatty acid transporter(fad L),cytochrome P450 hydroxylase gene(CYP450),alkane hydroxylase gene(alk B).Thirty one and twenty seven genes were related to aromatics degradation,including degradation genes alcohol dehydrogenase gene(adh4),salicylate hydroxylase gene(xln D),3-hydroxyisobutyl-Co A hydrolase gene(hibch),catechol-2,3-Dioxygenase genes(cat E),aldehyde dehydrogenase gene(ald A),4-octaprenyl-4-hydroxybenzoate carboxy-lyase gene(bsd B)and detoxification genes such as glutathione dehydrogenase(flh A)and quinone oxidoreductase(adh T).FP1 was found to have four more aromatic hydrocarbon degradation genes than JP1,including3-hydroxyisobutyl-Co A hydrolase gene(hibch),alkene reductase gene(nem A),and enol-based coenzyme A hydratase/isomerase gene(fad B1),3-2-aminobenzoic acid-Co A ligase gene(bcl A).Through analysis of the KEGG pathways of FP1 and JP1,the degradation pathways of polycyclic aromatic hydrocarbons and single benzene ring aromatic hydrocarbons were found.In the degradation process of monocyclic aromatic hydrocarbon compounds,the methyl groups were sequentially oxidized to alcohols,aldehydes,and acids to form catechol,and catechol was cracked into aldehydes or esters,and finally degraded.Polycyclic aromatic hydrocarbon compounds were firstly degraded by opening the rings and then formed aldehydes and acids for catabolism.The protocatechuic acid 3,4-dioxygenase alpha and beta subunit genes(pca H,pca G),salicylate hydroxylase gene(xln D)and salicyl-coenzyme A 5gene were found in FP1 and JP1,which indicated that the 1-hydroxy-2-naphthoic acid,an intermediate product formed by metabolizing pyrene,was degraded by two downstream degradation pathways of phthalic acid-protocatechuic acid and phthalic acid-salicylic acid.