Isolation of Pseudomonas aromaticivorans sp. nov from a hydrocarbon-contaminated groundwater capable of degrading benzene-, toluene-, m- and p-xylene under microaerobic conditions

Abstract

Members of the genus Pseudomonas are known to be widespread in hydrocarbon contaminated environments because of their remarkable ability to degrade a variety of petroleum hydrocarbons, including BTEX (benzene, toluene, ethylbenzene and xylene) compounds. During an enrichment investigation which aimed to study microaerobic xylene degradation in a legacy petroleum hydrocarbon-contaminated groundwater, a novel Gram-stain-negative, aerobic, motile and rod-shaped bacterial strain, designated as MAP12^T was isolated. It was capable of degrading benzene, toluene, meta- and para- xylene effectively under both aerobic and microaerobic conditions. The 16S rRNA gene sequence analysis revealed that strain MAP12^T belongs to the genus Pseudomonas, with the highest 16S rRNA gene similarity to Pseudomonas linyingensis LYBRD3-7 ^T (98.42%), followed by Pseudomonas sagittaria JCM 18195 ^T (98.29%) and Pseudomonas alcaliphila JCM 10630 ^T (98.08%). Phylogenomic tree constructed using a concatenated alignment of 92 core genes indicated that strain MAP12^T is distinct from any known Pseudomonas species. The draft genome sequence of strain MAP12^T is 4.36 Mb long, and the G+C content of MAP12^T genome is 65.8%. Orthologous average nucleotide identity (OrthoANI) and digital DNA-DNA hybridization (dDDH) analyses confirmed that strain MAP12^T is distinctly separated from its closest neighbors (OrthoANI < 89 %; dDDH < 36%). Though several members of the genus Pseudomonas are well known for their aerobic BTEX degradation capability, this is the first report of a novel Pseudomonas species capable of degrading xylene under microaerobic conditions. By applying genome-resolved metagenomics, we were able to partially reconstruct the genome of strain MAP12 ^T from metagenomics sequence data and showed that strain MAP12 ^T was an abundant member of the xylene-degrading bacterial community under microaerobic conditions. Strain MAP12^T contains ubiquinone 9 (Q9) as the major respiratory quinone and diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine as major polar lipids. The major cellular fatty acids of strain MAP12^T are summed feature 3 (C_16:1ω6c and/or C_16:1ω7c), C_16:0 and summed feature 8 (C_18:1ω6c and/or C_18:1ω7c). The results of this polyphasic study support that strain MAP12^T represents a novel species of the genus Pseudomonas, hence the name of Pseudomonas aromaticivorans sp. nov. is proposed for this strain considering its aromatic hydrocarbon degradation capability. The type strain is MAP12^T (=LMG 32466, =NCAIM B.02668).

Keywords: BTEX; Pseudomona; biodegradation; groundwater; xylene.

FIGURE 1

Genus-level bacterial community structure of microaerobic xylene-degrading enrichment as revealed by Illumina paired-end 16S rRNA gene amplicon sequencing. Only taxa contributing more than 1% abundance were depicted.

FIGURE 2

Maximum-likelihood tree based on 16S rRNA gene sequences showing the phylogenetic relationships between strain MAP12^T and related taxa. Bootstrap values are shown as percentages of 1000 replicates. Branches signed with an asterisk occurred with every tree-making algorithm used in the study. Cellvibrio polysaccharolyticus Ka43^T was used to root the tree. Bar, 0.01 substitution per nucleotide position.

FIGURE 3

Heatmap generated with OrthoANI values between strain MAP12^T and other closely related type strains of the genus Pseudomonas.

FIGURE 4

Phylogenomic tree constructed using UBCGs (concatenated alignment of 92 core genes). For inferring the tree the FastTree algorithm was used. Bar, 0.05 substitution per nucleotide position.

FIGURE 5

Transmission electron microscopic photograph showing cell morphology and presence of flagella in strain MAP12^T. Bar = 2 μm.

FIGURE 6

Microaerobic degradation of BTEX compounds [(A): benzene, (B): toluene, (C): m-xylene, (D): p-xylene] by strain MAP12^T. Concentrations were determined by GC–MS analysis as described in the main text. The averages of triplicate experiments ± standard errors of the means, indicated by error bars, are shown.