Anaerobic 1,4-dioxane biodegradation and microbial community analysis in microcosms inoculated with soils or sediments and different electron acceptors.

1,4-Dioxane, a probable human carcinogen, is a co-contaminant at many chlorinated solvent-contaminated sites. Although numerous 1,4-dioxane-degrading aerobic bacteria have been isolated, almost no information exists on the microorganisms able to degrade this chemical under anaerobic conditions. Here, the potential for 1,4-dioxane biodegradation was examined using multiple inocula and electron acceptor amendments. The inocula included uncontaminated agricultural soils and river sediments as well as sediments from two 1,4-dioxane contaminated sites. Five separate experiments involved the examination of triplicate live microcosms and abiotic controls for approximately 1 year. Compound-specific isotope analysis (CSIA) was used to further investigate biodegradation in a subset of the microcosms. Also, DNA was extracted from microcosms exhibiting 1,4-dioxane biodegradation for microbial community analysis using 16S rRNA gene amplicon high-throughput sequencing. Given the long incubation periods, it is likely that electron acceptor depletion occurred and methanogenic conditions eventually dominated. The iron/EDTA/humic acid or sulfate amendments did not result in 1,4-dioxane biodegradation in the majority of cases. 1,4-dioxane biodegradation was most commonly observed in the nitrate amended and no electron acceptor treatments. Notably, both contaminated site sediments illustrated removal in the samples compared to the abiotic controls in the no electron acceptor treatment. However, it is important to note that the degradation was slow (with concentration reductions occurring over approximately 1 year). In two of the three cases examined, CSIA provided additional evidence for 1,4-dioxane biodegradation. In one case, the reduction in 1,4-dioxane in the samples comparing the controls was likely too low for the method to detect a significant 13C/12C enrichment. Further research is required to determine the value of measuring 2H/1H for generating evidence for the biodegradation of this chemical. The microbial community analysis indicated that the phylotypes unclassified Comamonadaceae and 3 genus incertae sedis were more abundant in 1,4-dioxane-degrading microcosms compared to the live controls (no 1,4-dioxane) in microcosms inoculated with contaminated and uncontaminated sediment, respectively. The relative abundance of known 1,4-dioxane degraders was also investigated at the genus level. The soil microcosms were dominated primarily by Rhodanobacter with lower relative abundance values for Pseudomonas, Mycobacterium, and Acinetobacter. The sediment communities were dominated by Pseudomonas and Rhodanobacter. Overall, the current study indicates 1,4-dioxane biodegradation under anaerobic and, likely methanogenic conditions, is feasible. Therefore, natural attenuation may be an appropriate cleanup technology at sites where time is not a limitation.

Enrichment of novel Actinomycetales and the detection of monooxygenases during aerobic 1,4-dioxane biodegradation with uncontaminated and contaminated inocula.

1,4-Dioxane, a co-contaminant at many chlorinated solvent sites, is a problematic groundwater pollutant because of risks to human health and characteristics which make remediation challenging. In situ 1,4-dioxane bioremediation has recently been shown to be an effective remediation strategy. However, the presence/abundance of 1,4-dioxane degrading species across different environmental samples is generally unknown. Here, the objectives were to identify which 1,4-dioxane degrading functional genes are present and which genera may be using 1,4-dioxane and/or metabolites to support growth across different microbial communities. For this, laboratory sample microcosms and abiotic control microcosms (containing media) were inoculated with four uncontaminated soils and sediments from two contaminated sites. Live control microcosms were treated in the same manner, except 1,4-dioxane was not added. 1,4-Dioxane decreased in live microcosms with all six inocula, but not in the abiotic controls, suggesting biodegradation occurred. A comparison of live sample microcosms and live controls (no 1,4-dioxane) indicated nineteen genera were enriched following exposure to 1,4-dioxane, suggesting a growth benefit for 1,4-dioxane biodegradation. The three most enriched were Mycobacterium, Nocardioides, and Kribbella (classifying as Actinomycetales). There was also a higher level of enrichment for Arthrobacter, Nocardia, and Gordonia (all three classifying as Actinomycetales) in one soil, Hyphomicrobium (Rhizobiales) in another soil, Clavibacter (Actinomycetales) and Bartonella (Rhizobiales) in another soil, and Chelativorans (Rhizobiales) in another soil. Although Arthrobacter, Mycobacterium, and Nocardia have previously been linked to 1,4-dioxane degradation, Nocardioides, Gordonia, and Kribbella are potentially novel degraders. The analysis of the functional genes associated with 1,4-dioxane demonstrated three genes were present at higher relative abundance values, including Rhodococcus sp. RR1 prmA, Rhodococcus jostii RHA1 prmA, and Burkholderia cepacia G4 tomA3. Overall, this study provides novel insights into the identity of the multiple genera and functional genes associated with aerobic degradation of 1,4-dioxane in mixed communities.

In silico analysis of soil, sediment and groundwater microbial communities to predict biodegradation potential.

This study examined soil, sediment and groundwater microbial communities for a set of key functional genes important for contaminant biodegradation. This involved PICRUSt2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) predictions based on 16S rRNA gene amplicon datasets from three separate studies with different inocula and incubation conditions, as follows: aerobic soils, oxygen-limited microcosms containing sediments and groundwater, as well as methanogenic microcosms with different inocula. PICRUSt2 predicts functional profiles of microbial communities based on marker gene (16S rRNA gene) data. The relative abundances of genera previously associated with the biodegradation of chlorinated solvents/metabolites and/or 1,4-dioxane were also determined. Predicted values for each functional gene varied between the three datasets. In all, values were high for propane monooxygenase and low for soluble methane monooxygenase. Common phylotypes associated with propane monooxygenase in two of the three datasets included Mycobacterium, Rhodococcus and Pseudonocardia. Toluene monooxygenase predicted values were greater in the oxygen-limited microcosms compared to the other two datasets. The methanogenic microcosms exhibited the highest predicted values for particulate methane/ammonia monooxygenase. The most common genera detected, previously reported as chlorinated solvents/metabolites and/or 1,4-dioxane degraders, included Pseudomonas, Sphingomonas, Rhodococcus and Rhodanobacter. Eighteen of the queried genera were not detected. As expected, more potential anaerobic degrading genera were found in the oxygen-limited and methanogenic microcosms compared to the aerobic soils. The results provide key insights as to which genes and genera may be important for biodegradation over a range of inocula and redox conditions.