Research on Aerobic Cometabolism of PCE

Aerobic cometabolism of PCE and other halogenated solvents by Pseudomonas sp.  has been well established for more than 20 years.  The following research articles were key in the development of this approach to bioremediation.  This academic research provided parallel support to CL Solutions’ successful application of aerobic cometabolism to bioremediation of hundreds of contaminated sites since 1999.

Vandenbergh, P. A., and Kunka, B. S., Metabolism of Volatile Chlorinated Aliphatic Hydrocarbons by Pseudomonas fluorescens, Applied and Environmental Microbiology, v. 54, no. 10, Oct. 1988. p. 2578 – 2579.

Deckard, L. A., Willis, J. C., and Rivers, D. B. , Evidence for the Aerobic Degradation of Tetrachloroethylene by a Bacterial Isolate, Biotechnology Letters, v16, no. 11, November, 1994. p 1221-1224.

Ryoo, D., Shim, H., Canada, K., Barbieri, P., and Wood, T.K., Aerobic Degradation of Tetrachloroethylene by Toluene-O-xylene Monooxygenase of Pseudomonas stutzeri OX1, Nature Biotechnology, vol 18, July, 2000. p 775 – 778.

Shim, H., Ryoo, D., Barbieri, P, and Wood, T.K., Aerobic Degradation of Mixtures of Tetrachloroethylene, Trichloroethylene, Dichloroethylenes, and Vinyl Chloride by Toluene-O-Xylene Monooxygenase of Pseudomonas stutzeri OX1, Applied Microbiol Biotechnol, v. 56, May 2001. p 265-269.



TCE Aerobic Cometabolism Using Cl-Out

CL-Out bioaugmentation was used to reduce the TCE concentrations in ground water at an industrial site in Louisville, Kentucky.  CL-Out microbes were injected into the TCE-contaminated ground water.  Dextrose was added to provide the carbon source for the microbial growth.  An oxygen supplement was necessary to maintain aerobic conditions in the aquifer.

Ground water sampling downgradient of the injection points showed that the CL-Out microbial population achieved a target population of 1 million cfu/ml and maintained an effective population for at least 60 days.  During that timeframe the TCE concentrations were reduced by 90%.

CL-Out Population Increase and Contaminant Destruction Trends

CL-Out Population Increase and Contaminant Destruction Trends


Bioremediation of 1,1,1-Trichloroethane and 1,1-Dichloroethene at a Confidential Site


CL-Out® bioaugmentation was used to remediate ground water contaminated by 1,1,1-trichloroethane (1,1,1-TCA) and 1,1-dichloroethene (1,1-DCE). The 1,1-DCE was formed as a product of the natural abiotic degradation of 1,1,1-TCA. The bioremediation was implemented as a pilot study and a subsequent interim action for source removal.

Remediation Approach and Results

CL-Out® was introduced into the contaminated ground water in the suspected source area by injection through temporary well points up gradient of the monitoring point. The initial application was in March 2007 and a subsequent application was completed in September 2007.

The ground water treatment results were monitored by analysis of ground water samples for the contaminants of concern. The 30-day monitoring results after the first application showed an immediate decrease in the contaminant concentrations. After 30 days, the 1,1,1-TCA concentration decreased from 1,000 µg/L to 190 µg/L and the 1,1-DCE concentration decreased from 160 µg/L to 27 µg/L . Long-term monitoring after the second application showed that the concentrations of both compounds were reduced to 1.4 µg/L. The following chart shows the long-term contaminant concentrations.



This project demonstrates several of the benefits of aerobic bioaugmentation. CL-Out® organisms were able to metabolize the parent chloroethane compound and the daughter product, which was a chloroethene compound. The metabolic diversity of the CL-Out® organisms facilitates enables the bioremediation of mingled plumes and multiple contaminants. Bioaugmentation provides active control of the site as effective organisms compatible with the site conditions were injected where they were needed for quick contaminant removal.

Remediation of an Industrial Dry Cleaning Site

Site Summary

A CL-Out bioaugmentation pilot study verified the applicability of CL-Out remediation of an industrial dry cleaning site. The pilot study also determined whether the availability of oxygen to support cometabolism would limit the bioremediations.

Geology and Hydrology

Although the impacted ground water was relatively shallow, the site geology was complicated and varied across even this small site. In general the glacial deposits at the site were mainly till with interbedded sandy and silty zones. The impacted ground water was contained within the granular deposits.


The main ground water contaminant was PCE with lower concentrations of TCE, cis -1,2 DCE and vinyl chloride. In the pilot study area, the PCE concentration was 44,200 μg/L and the total of the daughter products was 14,750 μg/L.

Pilot Study Design

A pilot study was completed in the source area in the fall of 2009. One unit of CL-Out was injected into the affected ground water in the source area. One unit of CL-Out is a 55-gallon slurry with a microbial concentration of 109 cfu/ml. The CL-Out microbes were injected with 50 pounds of dextrose to provide a carbon source to support the energy requirements of the population. Pre- and post -treatment samples were taken on February 19 and December 10, 2009. The post-treatment sample was taken after the monitoring well was purged of the injected volume. The following table shows the contaminant concentration trends in the treatment area.



The pilot study verified the applicability of CL-Out bioremediation to the site. The total concentration of CVOCs was reduced from 59,000 to 13,740 μg/L. The CL-Out bioremediation was most effective in the removal of PCE and TCE. There was an increase in vinyl chloride suggesting some incomplete reductive dechlorination, probably by native dehalogenating organisms. Full-scale treatment will be most effective with the addition of an oxygen supplement to limit the dehalogenation and promote the cometabolism of the daughter products. Overall the push-pull pilot study verified the effectiveness of CL-Out bioremediation and provided insights for making full-scale application more effective.

Bioremediation of Chlorobenzene Following ISCO

Compatable Techology To Finish Remediation

CL-Out® micorbes bioremediation of chlorobenzene continued treatment after ISCO remediation stalled.  The target isomers included chlorobenzene, 1,3-dichlorobenzene, 1,4-dichlorobenzene, and 1,2-dichlorobenzene. Before bioaugmentation, individual concentrations exceeded 50,000 µg/L. The CL-Out® treatment quickly reduced contaminant concentrations by as much as 82% more.

Remediation Approach and Results

Injection through temporary well points introduced CL-Out® micorbes into the contaminated ground water in November 2010. Only one drum of the CL-Out® was injected into the ground water.

The ground water monitoring included analysis of ground water samples for the contaminants of concern, microbial population, and known functional genes, including naphthalene dioxygenase, phenol hydroxylase, and toluene dioxygenase. The 30-day monitoring results show an immediate decrease in the contaminant concentrations. After 30 days, bioremediation reduced 62% to 82% of individual contaminants. The sampling results 30 days after bioaugmentation are compared to the pre-treatment concentrations on the following chart.


The total biomass increased from 1.5 million to 12.8 million cells per milliliter following the bioaugmentation. The concentration of the naphthalene dioxygenase functional gene also increased by more than ten fold, as shown on the following table.



This project demonstrates several of the benefits of bioremediation of chlorobenzene. Bioaugmentation provides active control of the site as effective organisms compatible with the site conditions were injected where they were needed. Furthermore, the CL-Out® aerobic cometabolism was compatible with the residual conditions following ISCO treatment. Treatment will continue to reduce the contaminant concentrations to acceptable levels.

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