During the last 10 to 15 years, the use of nucleic acid-based methods (DNA and RNA), most notably quantitative polymerase chain reaction (qPCR), has grown substantially in the environmental restoration industry.

Since the DNA is extracted directly from the groundwater, soil or sediment sample, qPCR assays targeting degrader bacteria avoid the biases associated with cultivation-based approaches to provide a more direct, accurate and sensitive method to quantify specific microorganisms or biological processes. While providing the most direct avenue to evaluate biodegradation potential, translating qPCR results such as 3.5 x 104 Dehalococcoides cells/mL into contaminant biodegradation is less readily apparent than with chemical or geochemical results.

Dehalococcoides spp.

Enlarge this picture Figure 1

Dehalococcoides spp. are hydrogen-consuming, obligate halorespiring bacteria, which have received much attention due to their ability to utilize chlorinated ethenes as electron acceptors. Chlorinated ethenes including tetrachloroethene (PCE) and trichloroethene (TCE) were widely used as dry cleaning fluids and industrial solvents, and are now prominent groundwater contaminants. Under anaerobic conditions, PCE and TCE can undergo sequential reductive dechlorination through the daughter products cis-dichloroethene (cis-DCE) and vinyl chloride (VC) to ethene. While a number of bacterial cultures including dehalococcoides, dehalobacter, desulfitobacterium, and desulfuromonas species capable of utilizing PCE and TCE as growth supporting electron acceptors have been isolated Dehalococcoides spp. may be the most important because they are the only bacterial group that has been isolated to date which is capable of complete reductive dechlorination of PCE to ethane. In fact, the presence of Dehalococcoides spp. has been associated with the full dechlorination to ethene at sites across North America and Europe. Thus, qPCR monitoring of the abundance of Dehalococcoides spp. allows site managers to evaluate the feasibility of complete reductive dechlorination under MNA conditions and the effectiveness of biostimulation (electron donor addition) to promote growth of key reductive dechlorinating bacteria and enhance bioremediation.

The accumulation of the daughter products cis-DCE and VC termed "DCE stall" is relatively common at PCE/TCE sites, especially under monitored natural attenuation (MNA) conditions. DCE stall is particularly problematic because VC is generally considered more carcinogenic than the parent compounds. Within the Dehalococcoides genus, the range of chlorinated ethenes metabolized and cometabolized varies by species and strain. For example, Dehalococcoides ethenogenes str. 195 metabolizes PCE, TCE, and cis-DCE and cometabolizes VC to produce ethene. Conversely, Dehalococcoides sp. CBDB1 utilizes PCE and TCE but does not cometabolize additional chloroethenes. Therefore, additional qPCR assays targeting vinyl chloride reductase genes (bvcA and vcrA) were developed to more definitively confirm the potential for biodegradation of VC.

Although most research has focused on chlorinated ethenes, some Dehalococcoides species are also capable of reductive dechlorination of chlorinated aromatic hydrocarbons. Again, biodegradation of individual compounds and isomers varies by isolate, however, qPCR enumeration of Dehalococcoides may also provide valuable insight into the feasibility of bioremediation of chlorobenzenes, chlorophenols and polychlorinated biphenyls (PCBs). For example, dehalococcoides sp. CBDB1 has shown broad metabolic activity utilizing pentachlorophenol (PCP), tetrachlorophenols, trichlorophenols, hexachlorobenzene, pentachlorobenzene and tetrachlorobenzenes as growth supporting electron acceptors. Dehalococcoides have also been detected in PCB-impacted sediment and transformation of numerous PCBs containing five or more chlorine substituents has been demonstrated.

Dehalobacter spp.

Dehalobacter is another genus of anaerobic bacteria capable of reductive dechlorination of chlorinated compounds including PCE and TCE. Unlike most Dehalococcoides isolates that have been characterized, Dehalobacter pure cultures studied to date can utilize PCE and TCE but cannot metabolize or cometabolize the daughter products cis-DCE and VC. While the range of chlorinated ethenes utilized appears relatively limited, members of the genus Dehalobacter have the somewhat unique ability to utilize chlorinated ethanes including 1,1,1-trichloroethane (1,1,1-TCA) and 1,1,2-trichloroethane (1,1,2-TCA). TCAs were extensively used in industrial applications as degreasers and are therefore common co-contaminants at PCE/TCE-impacted sites. The presence of 1,1,1-TCA is especially problematic at PCE-impacted sites due to inhibition of reductive dechlorination of chlorinated ethenes particularly VC. Under aerobic conditions, TCA can be co-oxidized by methane-, ammonia-, and butane-oxidizing bacteria. Due to the presence of chlorinated ethanes as co-contaminants at PCE sites and the fact that impacted groundwater is often anoxic or anaerobic, anaerobic mechanisms for biodegradation of chlorinated ethanes are particularly pertinent.

The range of chlorinated ethanes biodegraded by Dehalobacter varies by species and strain. Dehalobacter restrictus strain TCA1 converts 1,1,1-TCA to chloroethane with the transient accumulation of 1,1-dichlorethane as an intermediate. 1,1,2-TCA did not serve as a growth-supporting electron acceptor for strain TCA1, however, Grostern and Edwards attributed the dichloroelimination of 1,1,2-TCA to vinyl chloride to a Dehalobacter species. The Grostern and Edwards study is particularly interesting because it was performed with mixed culture (Dehalococcoides sp. KB-1 and Dehalobacter sp. MS), chlorinated ethenes (TCE and daughter products), and 1,1,1-TCA. In cultures containing both organisms, degradation of cis-DCE and VC to ethene by Dehalococcoides proceeded only after dechlorination of 1,1,1-TCA by Dehalobacter thus demonstrating important roles for both genera at PCE/TCE sites co-contaminated by TCA.

Desulfitobacterium spp.

As with Dehalobacter spp., some members of the Desulfitobacterium genus can utilize PCE, TCE and 1,1,2-TCA as growth-supporting electron acceptors. In addition, some Desulfitobacterium spp. have demonstrated relatively broad specificity and may play an important role in the biodegradation of chlorinated aromatic hydrocarbons. For example, many species within the Desulfitobacterium genus are capable of ortho dechlorination of chlorinated phenols including EPA priority pollutants pentachlorophenol (PCP) and 2,4,6-trichlorophenol. One particular isolate, D. frappieri PCP-1, is also capable of meta and para dechlorination of some tri- and di-chlorophenols. Additionally, when induced by the presence of chlorophenols, strain PCP-1 can cometabolize a broad range of chlorinated aromatics including pentachloroanilines and pentrachloronitrobenzene.

Methanotrophs and aerobic cometabolism of chlorinated ethenes

Groundwater sampling with Bio-Flo filter

Under aerobic conditions, several different types of bacteria including methane-oxidizing bacteria (methanotrophs), ammonia-oxidizing bacteria, and some toluene/phenol-utilizing bacteria can cometabolize TCE, DCE and 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. Although a variety of primary substrates can foster production of different monooxygenases capable of cometabolic oxidation, methane is frequently the most readily available primary substrate.

Most methanotrophs are only capable of producing particulate methane monooxygenase (pMMO), which is capable of aerobic cometabolism but often at lower rates. Other methanotrophs are capable of producing both pMMO and soluble methane monooxygenase (sMMO) enzymes which in general are believed to capable of greater rates of aerobic cometabolism. qPCR assays targeting total methanotrophs and the sMMO gene were developed to aid in the evaluation of aerobic cometabolism as a treatment mechanism.

Summary

Bioremediation, by definition, is dependent upon the activity of subsurface microorganisms. However, site microbiology is often viewed as a "black box" where contaminants enter and daughter products form. Advances in molecular biology such as qPCR offer an unprecedented opportunity to peer into the black box to more directly evaluate bioremediation.

If 1,1,1-TCA is present as a co-contaminant, enumeration of dehalobacter could aid in determining whether TCA would be degraded or persist, potentially inhibiting cis-DCE and VC biodegradation. Similar scenarios could be envisioned for other contaminants, but the overlying premise remains the same: The judicious selection of qPCR assays and careful interpretation the results in light of chemical and geochemical data provides a more complete understanding of in-situ bioremediation that will aid in science-based site management decisions. PE