Formatted Title
Novel Group-6 Propane Monooxygenases Responsible for 1,4-Dioxane Biodegradation in Psychrophilic Propanotrophic Consortia
Background/Objectives
In situ bioaugmentation for 1,4-dioxane (dioxane) remediation using laboratory isolates is restricted by the low temperatures (4~14 ⁰C) at impacted aquifers. Available dioxane degraders are largely mesophilic as they were isolated at room temperature or above, yielding lower growth and dioxane biodegradation activity at aquifer-relevant temperatures. In this present study, two propanotrophic consortia were enriched and characterized given their ability to cometabolize dioxane at 14 ⁰C in the presence of propane. The diversity of soluble di-iron monooxygenases (SDIMOs) are profiled using amplicon-based sequencing to reveal the key enzymes that participate in dioxane cometabolism.
Approach/Activities
Enrichments were prepared with activated sludge samples collected from a local wastewater treatment plant. After washing with PBS, sludge samples were incubated at 14 ⁰C in two different culture media, ammonia mineral salts (AMS) and nitrogen mineral salts (NMS). Both consortia were supplemented with 3.3% of propane in the headspace. Propane consumption and dioxane removal were monitored by GC-FID. Amplicon-based sequencing was used to target the 16S rRNA gene and the SDIMO genes. Heterologous expression was used to validate the catalytic function of selected Group-6 SDIMOs as recovered from the microecological tools.
Results/Lessons Learned
After successive enrichment, steady dioxane degradation and propane consumption were observed in both consortia. 16S rRNA amplicon sequencing revealed several genera in both consortia that may contribute to dioxane degradation and cold tolerance. Genera belonging to Actinomycetes, a class harboring several genera associated with dioxane removal, Tetrasphaera, Nocardoides, and Mycobacterium in the AMS consortia, and Aquinhabitans, Nocardia, and Rhodococcus in the NMS consortia. Furthermore, genera associated with the psychrophilic character of the consortia were identified, Poloromonas, Mesorhizobium, Simplicispira, and Solitalea in the AMS consortia; while Chryseobacterium, Dyadobacter, Runella, and Chlostridium were observed in the NMS consortia. SDIMO amplicon-based sequencing detected three SDIMO groups, 3, 5, and 6. The enrichment of these three SDIMO groups could be attributed to their ability to initiate propane oxidation for growth. Particularly, four group-6 SDIMOs are present at high abundances of 72.4% and 70.6% in the AMS and NMS consortia, respectively. Their dominance in combination with the expression of these genes in heterologous hosts confirms their pivotal role in dioxane breakdown in both enriched psychrophilic consortia. This work has uncovered consortia capable of tackling dioxane contamination at environmentally relevant temperatures.