Evaluating the aerobic xylene-degrading potential of the intrinsic microbial community of a legacy BTEX-contaminated aquifer by enrichment culturing coupled with multi-omics analysis: uncovering the role of Hydrogenophaga strains in xylene degradation

Abstract

To develop effective bioremediation strategies, it is always important to explore autochthonous microbial community diversity using substrate-specific enrichment. The primary objective of this present study was to reveal the diversity of aerobic xylene-degrading bacteria at a legacy BTEX-contaminated site where xylene is the predominant contaminant, as well as to identify potential indigenous strains that could effectively degrade xylenes, in order to better understand the underlying facts about xylene degradation using a multi-omics approach. Henceforward, parallel aerobic microcosms were set up using different xylene isomers as the sole carbon source to investigate evolved bacterial communities using both culture-dependent and independent methods. Research outcome showed that the autochthonous community of this legacy BTEX-contaminated site has the capability to remove all of the xylene isomers from the environment aerobically employing different bacterial groups for different xylene isomers. Interestingly, polyphasic analysis of the enrichments disclose that the community composition of the o-xylene-degrading enrichment community was utterly distinct from that of the m- and p-xylene-degrading enrichments. Although in each of the enrichments Pseudomonas and Acidovorax were the dominant genera, in the case of o-xylene-degrading enrichment Rhodococcus was the main player. Among the isolates, two Hydogenophaga strains, belonging to the same genomic species, were obtained from p-xylene-degrading enrichment, substantially able to degrade aromatic hydrocarbons including xylene isomers aerobically. Comparative whole-genome analysis of the strains revealed different genomic adaptations to aromatic hydrocarbon degradation, providing an explanation on their different xylene isomer-degrading abilities.

Keywords: Aromatic hydrocarbons; Biodegradation; Catechol 2,3-dioxygenase; Groundwater; Hydrogenohaga; Xylene.

Fig. 1

Degradation process of xylene isomers in the aerobic enrichments containing (A) m-xylene, (B) p-xylene, and (C) o-xylene. Xylene concentrations were measured by GC–MS analysis at the 5th week of enrichment as described in the main text. Means of duplicate enrichments are given (with standard error)

Fig. 2

Bacterial community structure of meta- (M1, M2), para- (P1, P2), and ortho-xylene-degrading (O1, O2) enrichment cultures as revealed by 16S rRNA gene-based T-RFLP

Fig. 3

(A) Cluster analysis of the 16S rRNA gene-based T-RFLP electropherograms of the duplicate enrichment cultures at the 5th week by Bray–Curtis algorithm. (B) Principal component analysis 16S rRNA gene-based T-RFLP electropherograms. M1 and M2, m-xylene degrading enrichments; P1 and P2, p-xylene-degrading enrichments; O1 and O2, o-xylene-degrading enrichments

Fig. 4

Genus-level bacterial community structure of enrichments M1, P1, and O1 as revealed by Illumina paired-end 16S rRNA gene amplicon sequencing. Only taxa contributing more than 1% abundance were depicted

Fig. 5

Aerobic degradation of (A) m-xylene, (B) p-xylene, and (C) benzene by Hydrogenophaga sp. strain D2P1. 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

Fig. 6

Aerobic degradation of (A) o-xylene, (B) toluene, and (C) benzene by Hydrogenophaga sp. strain D2P3. 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

Fig. 7

Physical map of the phenol degradation gene clusters of Hydrogenophaga sp. strains D2P1^T and D2P3