Ramalingam, V. 2021. Biodegradation and molecular analysis of 1,4-dioxane and other organic contaminants in soil using metagenomic tools. Dissertation, Michigan State University, East Lansing MI.

Historically, 1,4-dioxane, a potential human carcinogen, was used as a stabilizer in 1,1,1-trichloroethane (1,1,1-TCA) formulations and is now frequently detected at chlorinated solvent contaminated sites. Bioremediation has emerged as an effective strategy to treat 1,4-dioxane. However, the distribution of 1,4-dioxane degrading species across various environmental samples is generally unknown. Additionally, 1,4-dioxane contamination typically occurs in groundwater under highly reducing conditions. There is a significant knowledge gap and a lack of information on the susceptibility of 1,4-dioxane to biodegradation under such reducing conditions. The success of organic contaminant bioremediation is often linked to the abundance of functional genes present in the soil that are associated with the degradation process. Although some information exists on the presence of these genes in contaminated soils, there is limited knowledge on the presence and diversity of these genes in uncontaminated soils. To address all these knowledge gaps, a series of studies were conducted.

The first study aims at identifying which 1,4-dioxane degrading functional genes are present in soil communities 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. The sample microcosms were amended with 1,4-dioxane thrice and live control microcosms were treated in the same manner, except 1,4-dioxane was not added. Biodegradation was observed and whole genome shotgun sequencing was carried out. Although some degraders previously linked to 1,4-dioxane degradation were detected, Nocardioides, Gordonia and Kribbella were found to be potentially novel degraders. 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.

The second study is focused on anaerobic biodegradation of 1,4-dioxane. The potential for 1,4-dioxane biodegradation was examined using multiple inocula and electron acceptor amendments. Compound specific isotope analysis (CSIA) was used to further investigate biodegradation in a subset of the microcosms. DNA was extracted from microcosms exhibiting 1,4-dioxane biodegradation for microbial community analysis using 16S rRNA gene amplicon high throughput sequencing. 1,4-dioxane biodegradation was most commonly observed in the nitrate amended and no electron acceptor treatments. However, it is important to note that the degradation was slow (approximately one year).

The third study examines a set of genes associated with organic contaminant degradation in four uncontaminated (agricultural) soils. The abundance and diversity of benA, bph, dbfA, dxnA, etnC, etnE, ppaH, npaH, vcrA, xenA, xenB and xplA were investigated using protein sequences from the Functional Gene Pipeline and Repository (FunGene). The phylogenetic trees created indicate many genera may potentially be associated with each gene including Pseudomonas, Rhodococcus, Mycobacterium and Nocardioides. From these, some strains are well studied and are known to be involved in the biodegradation of organic contaminants and others are potentially new genera that may be associated with the biodegradation of the targeted group of contaminants.

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