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The goals of this study were to predict the genes associated with the biodegradation of organic contaminants and to examine microbial community structure in samples from two contaminated sites. The approach involved a predictive bioinformatics tool (PICRUSt2) targeting genes from twelve KEGG xenobiotic biodegradation pathways (benzoate, chloroalkane and chloroalkene, chlorocyclohexane and chlorobenzene, toluene, xylene, nitrotoluene, ethylbenzene, styrene, dioxin, naphthalene, polycyclic aromatic hydrocarbons, and metabolism of xenobiotics by cytochrome P450). Further, the predicted phylotypes associated with functional genes early in each pathway were determined. Phylogenetic analysis indicated a greater diversity in the sediment compared to the groundwater samples. The most abundant genera for sediments/microcosms included Pseudomonas, Methylotenera, Rhodococcus, Stenotrophomonas, and Brevundimonas, and the most abundant for the groundwater/microcosms included Pseudomonas, Cupriavidus, Azospira, Rhodococcus, and unclassified Burkholderiaceae. Genes from all twelve of the KEGG pathways were predicted to occur. Seven pathways contained less than twenty-five genes. The predicted genes were lowest for xenobiotics metabolism by cytochrome P450 and ethylbenzene biodegradation and highest for benzoate biodegradation. Notable trends include the occurrence of the first genes for trinitrotoluene and 2,4-dinitrotoluene degradation. Also, the complete path from toluene to benzoyl-CoA was predicted. Twenty-two of the dioxin pathway genes were predicted, including genes within the first steps. The following phylotypes were associated with the greatest number of pathways: unclassified Burkholderiaceae, Burkholderia-Caballeronia-Paraburkholderia, Pseudomonas, Rhodococcus, unclassified Betaproteobacteria, and Polaromonas. This work illustrates the value of PICRUSt2 for predicting biodegradation potential and suggests that a subset of phylotypes could be important for the breakdown of organic contaminants or their metabolites. 

Co-contamination with chlorinated compounds and 1,4-dioxane has been reported at many sites. Recently, there has been an increased interest in bioremediation because of the potential to degrade multiple contaminants concurrently. Towards improving bioremediation efficacy, the current study examined laboratory microcosms (inoculated separately with two soils) to determine the phylotypes and functional genes associated with the biodegradation of two common co-contaminants (cis-dichloroethene [cDCE] and 1,4-dioxane). The impact of amending microcosms with lactate on cDCE and 1,4-dioxane biodegradation was also investigated. The presence of either lactate or cDCE did not impact 1,4-dioxane biodegradation one of the two soils. Lactate appeared to improve the initiation of the biological removal of cDCE in microcosms inoculated with either soil. Stable isotope probing (SIP) was then used to determine which phylotypes were actively involved in carbon uptake from cDCE and 1,4-dioxane in both soil communities. The most enriched phylotypes for 13C assimilation from 1,4-dioxane included Rhodopseudomonas and Rhodanobacter. Propane monooxygenase was predicted (by PICRUSt2) to be dominant in the 1,4-dioxane amended microbial communities and propane monooxygenase gene abundance values correlated with other enriched (but less abundant) phylotypes for 13C-1,4-dioxane assimilation. The dominant enriched phylotypes for 13C assimilation from cDCE included Bacteriovorax, Pseudomonas and Sphingomonas. In the cDCE amended soil microcosms, PICRUSt2 predicted the presence of DNA encoding glutathione S-transferase (a known cDCE upregulated enzyme). Overall, the work demonstrated concurrent removal of cDCE and 1,4-dioxane by indigenous soil microbial communities and the enhancement of cDCE removal by lactate. The data generated on the phylotypes responsible for carbon uptake (as determined by SIP) could be incorporated into diagnostic molecular methods for site characterization. The results suggest concurrent biodegradation of cDCE and 1,4-dioxane should be considered for chlorinated solvent site remediation.