Combined Omics Approach Reveals Key Differences between Aerobic and Microaerobic Xylene-Degrading Enrichment Bacterial Communities: Rhodoferax?A Hitherto Unknown Player Emerges from the Microbial Dark Matter

Among monoaromatic hydrocarbons, xylenes, especially the ortho and para isomers, are the least biodegradable compounds in oxygen-limited subsurface environments. Although much knowledge has been gained regarding the anaerobic degradation of xylene isomers in the past 2 decades, the diversity of those bacteria which are able to degrade them under microaerobic conditions is still unknown. To overcome this limitation, aerobic and microaerobic xylene-degrading enrichment cultures were established using groundwater taken from a xylene-contaminated site, and the associated bacterial communities were investigated using a polyphasic approach. Our results show that the xylene-degrading bacterial communities were distinctly different between aerobic and microaerobic enrichment conditions. Although members of the genus Pseudomonas were the most dominant in both types of enrichments, the Rhodoferax and Azovibrio lineages were only abundant under microaerobic conditions, while Sphingobium entirely replaced them under aerobic conditions. Analysis of a metagenome-assembled genome of a Rhodoferax-related bacterium revealed aromatic hydrocarbon-degrading ability by identifying two catechol 2,3-dioxygenases in the genome. Moreover, phylogenetic analysis indicated that both enzymes belonged to a newly defined subfamily of type I.2 extradiol dioxygenases (EDOs). Aerobic and microaerobic xylene-degradation experiments were conducted on strains Sphingobium sp. AS12 and Pseudomonas sp. MAP12, isolated from the aerobic and microaerobic enrichments, respectively. The obtained results, together with the whole-genome sequence data of the strains, confirmed the observation that members of the genus Sphingobium are excellent aromatic hydrocarbon degraders but effective only under clear aerobic conditions. Overall, it was concluded that the observed differences between the bacterial communities of aerobic and microaerobic xylene-degrading enrichments were driven primarily by (i) the method of aromatic ring activation (monooxygenation vs dioxygenation), (ii) the type of EDO enzymes, and (iii) the ability of degraders to respire utilizing nitrate.

Automated summary

Xylenes, particularly the ortho and para isomers, are known to be the least biodegradable among monoaromatic hydrocarbons in oxygen-limited subsurface environments. However, little is known about the diversity of bacteria capable of degrading xylene isomers under microaerobic conditions. To address this, aerobic and microaerobic xylene-degrading enrichment cultures were established, and the associated bacterial communities were analyzed. The results showed significant differences in the composition of the bacterial communities between aerobic and microaerobic conditions, with the genus Pseudomonas being dominant in both, but Rhodoferax and Azovibrio lineages only abundant under microaerobic conditions, while Sphingobium completely replaced them under aerobic conditions. The study also identified specific enzymes and their types that play a role in xylene degradation under different oxygen conditions, revealing the importance of aromatic ring activation, types of dioxygenases, and the ability to respire nitrate for xylene degraders.