Current remediation approaches for contaminants of concern (COCs) including 1,4-dioxane, chlorinated aliphatic hydrocarbons (CAHs), and petroleum hydrocarbons often rely on expensive ex situ pump-and-treat methods rather than preferable in situ treatment methods. In situ remediation is also often further complicated by COCs in low permeability zones that act as long-term sources of contamination. Aerobic cometabolic processes may be a reliable and cost effective means for in situ treatment of mixtures of COCs that are released from zones of low permeability. However, the ability to apply this process is limited by developing cost effective means of delivering the cometabolic substrate and by ensuring that effective microorganisms are stimulated on the substrates that are provided.

The overall aim of the project is to develop novel aerobic cometabolic processes based on slow-release compounds (SRCs) to treat COC mixtures of interest to DoD. These studies focus on a model isobutene-utilizing strain,Rhodococcus rhodochrous21198, that has been shown to concurrently oxidize 1,4-dioxane and diverse CAHs, including mixtures of 1,1-dichloroethane (11DCE) and 1,1,1-trichloroethane (111TCA), when grown on isobutane as a primary substrate. While in situ biostimulation using isobutane is a potential remediation strategy for COC mixtures containing 1,4-dioxane, other cometabolic strategies involving isobutane-metabolizing bacteria may be more applicable for low permeability zones.

The specific objectives of this study include the evaluation of: (1) isobutane-utilizing bacteria to cometabolically degrade COC mixtures; (2) alcohols as substrates supporting degradation of COC mixtures; (3) tetraalkoxysilanes as SRCs to produce alcohols of interest; (4) SRC and bacterial co-encapsulation technology; and (5) co-encapsulation technology in a two dimensional (2-D) physical aquifer model to treat COCs being released from zones of low permeability.

R. rhodochrous21198 and a series of related microorganisms will be grown on isobutane and their abilities to degrade individual COCs and defined COC mixtures with and without 1,4-dioxane will be quantified. Proteomic analyses will be used to identify the monooxygenases involved in COC degradation. The ability of 1^o and 2^o alcohols to support growth and degradation of COC mixtures by the microorganisms studied in Objective 1 will be determined. Proteomic analyses will be used to characterize the ability of alcohols, COCs and COC mixtures to induce expression of monooxygenases in these strains. The project team has previously shown non-toxic silico-oxide-organics (tetraalkoxysilanes) can act as alcohol-releasing SRCs.

Based on the alcohol utilization studies in Objective 2, the ability of specific SRCs to support cometabolism of COCs and COC mixtures by the pure cultures studied in Objective 1 will be evaluated. Technologies to co-encapsulate SRCs and selected isobutene-metabolizing bacteria will be developed so that they could be co-delivered to the base of a low permeability zone for in situ remediation. The co-encapsulated systems will be evaluated for their ability to support degradation of COC mixtures in both microcosm and column reactor studies.

Finally, the remediation of defined COC mixtures by co-encapsulated SRCs and bacteria using a 2-D physical aquifer model that includes zones of high and low permeability will be evaluated.

This project will provide a thorough evaluation of the potential of using SRCs to remediate COCs being released from zones of low permeability. This technology can potentially provide a low cost, passive means for the in situ treatment of COCs, and might be used with other technologies, such as anaerobic treatment followed by aerobic treatment. The technology might also be applied to other emerging contaminants of interest to DOD, including 1,2,4-trichloropropane andN-nitrosodimethylamine (NDMA). (Anticipated Project Completion - 2019)

Murnane R.A,  W. Chen, M.R. Hyman, and L. Semprini. 2021. Long-Term Cometabolic Transformation of 1,1,1-Trichloroethane and 1,4-Dioxane by Rhodococcus rhodochrous ATCC 21198 Grown on Alcohols Slowly Produced by Orthosilicates. Journal of Contaminant Hydrology, 240:1-14, doi.org/10.1016/j.jconhyd.2021.103796

Rasmussen, M.T., A.M. Saito, M.R. Hyman, and L. Semprini. 2020. Co-encapsulation of Slow Release Compounds and Rhodococcus rhodochrous ATCC 21198 in Gellan Gum Beads to Promote the Long-term Aerobic Cometabolic Transformation of 1,1,1-Trichloroethane, cis-1,2-Dichloroethene and 1,4-Dioxane. Environmental Science Processes & Impacts, 22(3):771-791.