CENSUS – Cometabolism of Chlorinated Ethenes Detect and quantify methanotrophs and other bacteria capable of aerobic cometabolism CENSUS of chlorinated solvents Under aerobic conditions, several different types of bacteria including Environmental Relevance / methane-oxidizing bacteria (methanotrophs), ammonia-oxidizing bacteria, Target Code Contaminants Data Interpretation and some toluene/phenol-utilizing bacteria can cometabolize or co-oxidize Methanotrophs qMOB TCE, DCE, Targets two types of trichloroethene (TCE), dichlorethene (DCE), and vinyl chloride (VC). In VC methane oxidizing bacteria general, cometabolism of chlorinated ethenes is mediated by mono- (methanotrophs) Indicates the potential for oxygenase enzymes with “relaxed” specificity that oxidize a primary (growth cometabolic oxidation of TCE supporting) substrate and co-oxidize the chlorinated compound. Soluble Methane qsMMO TCE, DCE, Targets the soluble methane In the presence of Monooxygenase VC monooxygenase gene TCE Cometabolism methane, for Soluble methane mono- O 2 example, methano- oxygenases are generally trophs produce believed to support faster cometabolism of TCE methane mono- Methane Propane qPPO TCE, DCE, Propane can be added as a Monooxygenase + oxygenases which CO oxidize methane to Monooxygenase VC primary substrate to promote CH CH OH cometabolic oxidation of TCE 4 3 methanol and can also co-oxidize TCE. Toluene qTOD TCE, DCE, Applicable at mixed waste For aerobic cometabolism to be a primary treatment mechanism, three key Dioxygenase VC sites where BTEX and factors must be present or supplied: TCE are co-contaminants • A primary (growth supporting) substrate When expressed, toluene dioxygenase is capable • Oxygen of cometabolism of TCE • Bacteria capable of producing non-specific monooxygenases Ring-Hydroxylating qRMO TCE, DCE, Applicable at mixed waste Although a variety of primary substrates including toluene, phenol, propane, Toluene VC sites where BTEX and TCE Monooxygenase are co-contaminants and ammonia can foster production of different monooxygenases capable When expressed, toluene of cometabolic oxidation, methane is frequently the most readily available monooxygenases are capable primary substrate. of cometabolism of TCE Most methanotrophs are only capable of producing particulate methane monooxygenase (pMMO) which is capable of aerobic cometabolism but The combination of the qMOB and qsMMO provides quantification of total often at lower rates. Other methanotrophs are capable of producing both methanotrophs and those specifically capable of producing sMMO to aid in pMMO and soluble methane monooxygenase (sMMO) enzymes which in the evaluation of aerobic cometabolism as a treatment mechanism. general are believed to be capable of greater rates of aerobic As mentioned previously, a variety of substrates can support cometabolism of cometabolism. TCE. Propane addition has been shown to stimulate propane utilizing bacteria also capable of aerobic cometabolism. Likewise, the relaxed specificity of Microbial Insights has developed CENSUS targets for some aromatic oxygenases also permits cometabolism of chlorinated ethenes. quantification of: Depending on site conditions and corrective actions taken, these pathways may also be • total methanotrophs (qMOB) and involved in TCE degradation. • soluble methane monooxygenase (qsMMO) genes expressed by a subset of methanotrophs www.microbe.com CENSUS Cometabolic TCE Biodegradation Conclusions: • A pilot study using Bio-Trap® Samplers and quantification of • Within the radius of influence (ROI) of the injection system (MW1 • Loss of TCE methanotrophs was designed to evaluate the efficacy of a and MW16), remedial system designed to stimulate cometabolic corresponded to - total methanotrophs increased by 1-2 orders of magnitude, biodegradation of TCE. increases in - soluble methane monooxygenase genes increased by over 4 • The technology consisted of injecting a gaseous mixture of methanotroph orders of magnitude, and populations and soluble methane (carbon source), air, nitrous oxide, and triethyl phosphate to promote growth and activity of methanotrophs. - TCE concentrations decreased. methane monooxygenase gene • Injection operations were performed at one well for • Outside the system ROI (MW3), copy numbers. approximately 3 months. - no significant changes were noted in methanogens or methane • The remediation • Bio-Trap® Samplers were deployed in surrounding wells located monooxygenase genes and technology stimulated ~30 ft, 40 ft, and >150 ft from the injection well prior to and - TCE concentrations did not decrease appreciably. growth of methanotrophs during system operation. and promoted • Recovered Bio-Trap® Samplers were analyzed for total cometabolism of TCE. methanotrophs (qMOB) and Type II methanotrophs capable of producing soluble methane monooxygenase (qsMMO). MW1 1E+07 MOB MW17 1E+06 sMMO MW1 MOB Radius of Influence (ROI) 1E+05 Cells/bead sMMO Injection MW3 Downgradient 1E+04 System well 1E+07 Groundwater Trailer MOB 1E+03 1E+06 Pre-Startup Post-Startup MW18 MOB 1E+05 sMMO Cells/bead sMMO 1E+04 MW16 1E+03 Pre-Startup Post-Startup MW16 1E+08 1150 1E+07 MOB sMMO 1E+06 MOB 1E+05 MW3 1E+04 Cells/bead sMMO 1E+03 1E+02 Pre-Startup Post-Startup 10515 Research Drive Knoxville, TN 37932 Phone: 865.573.8188 www.microbe.com.