The overall aim of this project was to develop a passive system of long-term cometabolic transformation of mixtures of chemicals of concern (COC), by co-encapsulating a pure bacteria culture and slow releases compounds (SRCs) within hydrogel beads. The SRC would slowly release alcohols upon abiotic hydrolysis with water, which serve as substrates for growth and maintenance of the bacteria culture while also promoting aerobic cometabolism. The hydrogel bead would create a protective environment where the pure culture can grow as the SRC hydrolyses within the bead and the COC diffuse into the bead to be cometabolically transformed.

The five specific aims of the project were met including: 1) determining the pure bacteria culture to be co-encapsulated that has the ability to cometabolize a broad range of COC; 2) demonstrating that alcohols and the SRC that produce them, would promote long-term cometabolic transformations with the selected bacteria culture; 3) developing an effective method for co-encapsulation of the bacteria culture and the SRC within the hydrogel beads; 4) evaluating the co-encapsulated hydrogel beads in batch kinetic tests and groundwater microcosms; and 5) evaluating the hydrogel beads in continuous flow one-dimensional packed columns and finally, as a proof-of-concept experiment, in a large-scale physical aquifer model (PAM) where the beads were used to construct a cometabolic permeable reactive barrier (CPRB).

Technical Approach

The bacterium Rhodococcus rhodochrous strain ATCC 21198 (R. rhodochrous ATCC 21198) was selected for use in this study, since it was very effective in transforming a broad range of COC, including 1,4-dioxane (1,4-D), 1,1,1-trichloroethane (1,1,1-TCA), 1,1-dichloroethane, vinyl chloride (VC), cis-dichloroethene (cis-DCE), and 1,1-dichloroethene (1,1-DCE). Trichloroethene, however, was not effectively transformed.

Proteomic analyses was used to identify the monooxygenases involved in COC degradation. The ability of primary and secondary alcohols to support growth and degradation of COC mixtures by the microorganisms studied in Objective 1 was examined. 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 was evaluated. Technologies to co-encapsulate SRCs and selected isobutene-metabolizing bacteria were developed so that they could be co-delivered to the base of a low permeability zone for in situ remediation. The co-encapsulated systems were 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 was evaluated.


When grown on 1-butanol, 2- butanol, and 2-ethyl-1-butanol, R. rhodochrous ATCC 21198 was capable of cometabolically transforming the same range of COC, but at slower rates than when grown on isobutane. R. rhodochrous ATCC 21198 grown on 2-butanol had the largest transformation capacity and fastest degradation rates and was able to transform a mixture of 1,4-D, cis-DCE, and 1,1,1-TCA to greater than 99%.

Three orthosilicates were tested as SRC for R. rhodochrous ATCC 21198: tetrabutylorthosilicate (TBOS), tetra-sec-butylorthosilicate (T2BOS), and tetraisopropoxysilane (T2POS). The rates of hydrolysis in poisoned controls depended on the orthosilicate structure with TBOS, which produces 1-butanol, hydrolyzing more rapidly than T2POS and T2BOS that produce 2-butanol and 2-propanol, respectively. In batch tests with the orthosilicates, continuous rates of oxygen (O2) consumption and carbon dioxide (CO2) production confirmed alcohol metabolism by R.rhodochrous ATCC 21198 was occurring. The rates of metabolism, as indicated by O2 consumption and CO2 production in batch incubations (TBOS > T2POS > T2BOS) were consistent with the rates of alcohol release via abiotic hydrolysis. Both 1,4-D and 1,1,1-TCA were continuously transformed in their successive additions over 125 days, with the rates correlated with the rates of metabolism. Only a single addition of the orthosilicate was required. Alcohol concentrations in the biologically active reactors remained below the levels of detection, indicating they were metabolized rapidly after being produced. Much lower rates of O2consumption occurred in reactors containing T2BOS, which has benefits for in situ bioremediation.

A process was developed to co-encapsulate R.rhodochrous ATCC 21198 in gellan-gum (GG) hydrogel macrobeads with orthosilicates as SRC. Encapsulation with GG was found to be more effective than with alginate. TBOS and T2BOS, were encapsulated in GG hydrogels at mass loadings as high as 10% (w/w), along with R.rhodochrous ATCC 21198. In the GG beads, TBOS hydrolyzed 27 times faster than T2BOS and rates were ~4 times higher in suspension than when encapsulated. Batch kinetic tests supported the aerobic cometabolism of a mixture of 1,1,1-TCA, cis-DCE, and 1,4-D at aqueous concentrations ranging from 250 to 1000 µg/L. In biologically active batch reactors, the co-encapsulated R.rhodochrous ATCC 21198 effectively utilized the SRC hydrolysis products (1- and 2-butanol) and cometabolized repeated additions of the mixture of 1,1,1-TCA, cis-DCE, and 1,4-D for over 300 days. The transformation followed pseudo-first-order kinetics. VC and 1,1-DCE were also effectively transformed. Appropriately scaled first-order rates indicated very high extents of chemical transformation could be achieved with a residence time of a few days if co-encapsulated beads were densely packed in a column or CPRB. Studies in groundwater/sediment microcosms also demonstrated that long-term cometabolic treatment can be achieved under conditions that mimic the subsurface. Based on the lower rate of oxygen consumption while achieving similar long-term rates, T2BOS was found to be a more effective substrate than TBOS.

The continuous aerobic cometabolic treatment of a mixture of 1,1,1-TCA, cis-DCE and 1,4-D, each at a concentration of 250 µg/L, was studied in one-dimensional laboratory columns packed with the GG hydrogel beads co-encapsulated the with R. rhodochrous ATCC 21198 and 8% (w/w) TBOS or T2BOS. Hydraulic residence times (HRTs) in the columns ranged from approximately two days to 12 hours. In two columns containing GG beads with TBOS, over 99.5% removal of cis- DCE, 1,1,1-TCA, and 1,4-D was achieved within the first 25 days operation at an HRT of approximately two days. The development of aerobic conditions throughout the columns, through hydrogen peroxide (H2O2) addition (50 to 100 mg/L), resulted in biostimulation of R. rhodochrous ATCC 21198 within the beads throughout the columns, permitting a decrease in the HRT to 12 hours, while maintaining a chemical removal efficiency of over 99.5%. With prolonged treatment, only dissolved O2 (DO) addition (15 mg/L) was required to maintain effective treatment. After ~240 pore volumes of flow (PV), cis-DCE was replaced by 1,1-DCE (100 µg/L), which imparts a high degree of transformation toxicity. 1,1-DCE was effectively transformed with over 98% removal achieved, while 1,4-D and 1,1,1-TCA continued to be effectively transformed. After ~ 300 PV, the influent concentration of 1,1-DCE was increased to 250 µg/L. Effective transformation of all three chemicals was achieved until ~450 PV, after which all began to increase to the influent concentrations. 

The third column was packed with GG beads with T2BOS. The column was fed groundwater that contained 14mg/L DO and a mixture of cis-DCE, 1,1,1-TCA and 1,4-D, each at 250 µg/L. Due to the slower rate of hydrolysis of T2BOS, H2Oaddition was not required. Over 99% removal of 1,1,1-TCA,cis-DCE and 1,4-D was achieved with an HRT of 12 hours. After 90 PV of flow, when the chemical concentrations were increased from 250µg/L to 500µ/L and the effluent concentrations of cis-DCE and 1,4-D began to immediately increase and reached the influent concentrations after 120 PV of flow, while 1,1,1-TCA only reached about 25% of the influent concentration. Upon reducing the influent concentrations to 250 µg/L, the cometabolic transformation was restored, with over 98% removal observed. cis-DCE was then replaced by 1,1-DCE (100 µg/L), while continuing to add 1,4-D and 1,1,1-TCA. A rapid increase in all three chemicals in the column effluent was observed. The results suggest that 1,1-DCE transformation toxicity caused the cessation in the cometabolic activity in the column. At the end of the column studies, the beads were sampled and assayed for the amount of TBOS and T2BOS remaining. Approximately 56% and 98.5% of the TBOS and T2BOS originally encapsulated remained, respectively. The estimated rate of hydrolysis of T2BOS in the columns was factor of 20 lower than TBOS, which was consistent with the batch incubations. The results indicate that TBOS and T2BOS limitations were not responsible for the cessation in transformation activity in the columns, and it was likely caused from the cometabolism of 1,1-DCE.

Experiments were initiated in a 100-L PAM packed with sand (20 to 30 mesh) to evaluate the creation of a Funnel-and-Gate CPRB. The Funnel-and-Gate CPRB was created by inserting a 1-L cylinder packed with GG beads with TBOS and R. rhodochrousATCC 21198 into the PAM. Barrier walls were also inserted to direct flow into the CPRB; however, flow could bypass the CPRB, as a result of clogging and a reduction in permeability. Isobutene was used a reactive surrogate for the COC, since its cometabolism produces isobutene oxide that can be easy detected by gas chromatography. The initial phase of the study showed effective transformation of isobutene within the CPRB, with a significant fraction of isobutene converted to isobutene oxide.


This project provided 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 chemicals of concern, including 1,2,4-trichloropropane and N-nitrosodimethylamine (NDMA). Overall, the initial results show the potential of creating a CPRB using the hydrogel technology that was developed in this project. (Project Completion - 2022)


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.