Identification of Novel 1,4-Dioxane Degraders and Related Genes from Active Sludge by Combination of 16S rRNA and Shotgun Sequencing

The identity and function genes of the microorganisms involved 1,4-dioxane biodegradation are largely unknown due to the high phylogenetic diversity of 1,4-dioxane degraders. In this study a 1,4-dioxane degradation consortium was enriched from domestic waste water treatment plant active sludge. The dynamic changes of the bacterial community showed the importance of an unclassified genus belonging to Xanthobacteraceae which increased significantly in abundance during the enrichment process. Species with relative high abundance (> 0.3 %) were identified in relation to their metabolic activity in the biodegradation process through shotgun sequencing. The acetylene test and nested PCR strategy targeting soluble di-iron monooxygenase genes revealed a novel monooxygenase involved in the 1,4-dioxane degradation. The functional gene investigation revealed that Xanthobacter sp. 91, Xanthobacter sp. 126 and Rhizobiales bacterium presented the most likely monooxygenases for the hydroxylation of 1,4-dioxane. Moreover, the glycolate oxidase genes responsible for utilization of intermediates through TCA cycle mostly existed in Xanthobacter sp. 126, whose function can be proved by the adding of specific glycolate oxidase inhibitor a-hydroxy-2-pyridinemethanesulfonic acid. The failure to isolate any pure 1,4-dioxane degradation clone further confirmed the biodegradation of 1,4-dioxane was not ascribed to simply one strain but was a result of synergstic effect of different strains. The degradation pathway of 1,4-dioxane including identified enzymes and degraders was proposed on the basis of the three major intermediates (2-hydroxy-1,4-dioxane, ethylene glycol and oxalic acid), on community information and on functional genes. These findings on functional genes and the cooperative role of different species could potentially allow reconstruction of a bacterial consortium for remediation of sites contaminated by 1,4-dioxane.