Microbial Insights

Detect and quantify methanotrophs and other bacteria capable of aerobic cometabolism of chlorinated solvents

Characterization of sites impacted by chlorinated solvents such as tetrachloroethene (PCE) and trichloroethene (TCE) initially emphasizes determination of contaminants of concern (COCs) and evaluation of site geologic and hydrogeologic conditions. Following this first phase, additional site characterization often focuses on evaluation of attenuation mechanisms including biodegradation to ultimately lead to an effective corrective action. Chemical and geochemical data including the concentrations of contaminants, daughter products, and terminal electron acceptors (dissolved oxygen, nitrate, iron, sulfate, etc.) obtained during site characterization provide the first two lines of evidence to evaluate the feasibility of bioremediation as a remedial alternative. While providing valuable information, both are somewhat indirect approaches to assess biological activity. The most direct avenue to evaluate biodegradation as a treatment mechanism is to directly quantify the microorganisms or biological processes responsible for biodegradation of the contaminants of concern.

Under aerobic conditions, several different types of bacteria including methane-oxidizing bacteria (methanotrophs), ammonia-oxidizing bacteria, and some toluene/phenol-utilizing bacteria can cometabolize or co-oxidize trichloroethene (TCE), dichloroethene, and vinyl chloride (VC). In general, cometabolism of chlorinated ethenes is mediated by monooxygenase enzymes with “relaxed” specificity that oxidize a primary (growth supporting) substrate and co-oxidize the chlorinated compound. In the presence of methane, for example, methanotrophs produce methane monooxygenases which oxidize methane to methanol and can also co-oxidize TCE.

For aerobic cometabolism to be a primary treatment mechanism, three key factors must be present or supplied:

• A primary (growth supporting) substrate
• Oxygen
• Bacteria capable of producing non-specific monooxygenases

The following sections describe individual CENSUS assays, their importance in evaluating aerobic cometabolism, and provide guidelines for integrating CENSUS results into routine groundwater monitoring for common corrective actions.

CENSUS Targets for Cometabolism

Methanotrophs (qMOB): Although a variety of primary substrates including toluene, phenol, propane, and ammonia can foster production of different monooxygenases capable of cometabolic oxidation of TCE, methane is frequently the most readily available primary substrate. The qMOB CENSUS assay quantifies both types of methanotrophs and indicates the potential for cometabolic oxidation of TCE. 

Soluble Methane Monooxygenase (qsMMO): Most methanotrophs are only capable of producing particulate methane monooxygenase (pMMO) which is capable of aerobic cometabolism but often at lower rates. The qsMMO CENSUS assay targets the gene encoding soluble methane monooxygenases generally believed to support faster cometabolism of TCE.

Methanogens (qMGN): Under monitored natural attenuation (MNA) conditions, methanogens produce the methane consumed by methanotrophs as the growth supporting substrate.

Propane Monooxygenase (qPPO): In engineered remediation approaches, propane is sometimes injected as a primary substrate to promote cometabolism of TCE by propane oxidizing bacteria. The qPPO assay targets the gene encoding propane monooxygenase.

Toluene Dioxygenase (qTOD): Toluene dioxygenase catalyzes the initial oxidation of toluene and benzene but is also capable of co-oxidation of TCE if the enzyme is expressed. At mixed waste sites where TCE is a co-contaminant with petroleum hydrocarbons, expression of toluene dioxygenase under aerobic conditions could promote cometabolism of TCE.  The qTOD assay may not be appropriate for sites in which chlorinated ethenes are the only contaminants of concern and BTEX compounds are not present.

Ring-Hydroxylating Toluene Monooxygenase (qRMO): Like toluene dioxygenase, ring-hydroxylating toluene monooxygenases catalyze the initial oxidation of BTEX compounds but are also capable of co-oxidation of TCE if the enzyme is expressed. At mixed waste sites where TCE is a co-contaminant with petroleum hydrocarbons, expression of toluene monoxygenases under aerobic conditions could result in cometabolism of TCE.

Integrating CENSUS Results

Monitored Natural Attenuation (MNA):

MNA is often viewed as a “do nothing” approach that relies more on physical processes such as dilution than on biodegradation as a treatment mechanism.  Depending on site conditions, however, aerobic cometabolism can be an important component of MNA. Evaluating MNA as a corrective action and aerobic cometabolism as a treatment mechanism often depends upon integrating chemical, geochemical, and microbiological data to answer the following questions:

• Is a primary substrate present under MNA conditions?

Except at mixed waste sites, methane is likely to be the most readily available primary substrate under MNA conditions so samples should be analyzed for dissolved methane. It should be noted that the anaerobic conditions necessary for methanogenesis would not be conducive to aerobic cometabolism of TCE. However, methane and oxygen to support aerobic cometabolism of TCE may both be present at downgradient locations in the dissolved plume.

• Is oxygen available?

As mentioned previously, cometabolism of TCE by these cometabolic pathways is an aerobic process.  If the aquifer is not aerobic under MNA conditions, oxygen will have to be supplied. A lack of available oxygen may be more frequent at mixed waste sites where aerobic biodegradation of BTEX consumes available oxygen.

• Are organisms that can produce non-specific oxygenases present at the site?

The CENSUS assays qMOB and qsMMO provide direct quantification of total methanotrophs and those capable of producing soluble methane monooxygenase enzymes capable of faster cometabolism of TCE. At mixed waste sites where BTEX compounds are also present, CENSUS analysis of toluene dioxygenase and ring-hydroxylating toluene monooxygenase genes may also be appropriate.

Enhanced Bioremediation (Biostimulation):

While monitored MNA can be an effective remedial approach, aerobic cometabolism can be hindered by both a lack of primary substrate and a lack of oxygen. Therefore, biostimulation by injection of a gas phase primary substrate (methane or propane) and air is often performed to support cometabolism.  While the choice of CENSUS assay for characterization of the microbial community will depend on the choice of primary substrate, evaluating the effectiveness of biostimulation follows much the same rationale as described above for MNA. Biostimulation should result in an increase in organisms capable of cometabolism of TCE bacteria as evidenced by CENUS results, the presence of the primary substrate, and maintenance of aerobic condtions.

References

Field, J.A. and R. Sierra-Alvarez.  2004. “Biodegradability of chlorinated solvents and related chlorinated aliphatic compounds.” Reviews in Environmental Science & Bio/Technology 3: 185-254.

Oldenhuis, R. R.L. Vink, D.B. Janssen, and B. Witholt. 1989. “Degradation of chlorinated aliphatic hydrocarbons by Methylosinus trichosprium OB3b expressing soluble methane monooxygenase.”  Applied and Environmental Microbiology 55: 2819-2826.

Wackett, L.P. and D.T. Gibson. 1988. “Degradation of trichloroethylene by toluene dioxygenase in whole cells studies with Pseudomonas putida F1.” Applied and Environmental Microbiology 54: 1703-1708.