Molecular Biological Tools Provide Insights at Danish MNA Site

Molecular Biological Tools Provide Insights at Danish MNA Site

Rødekro is a small town in southern Denmark with historical PCE contamination in groundwater from a central dry-cleaning facility. The dense non-aqueous phase liquid (DNAPL) tetrachloroethene (PCE) that entered the sand aquifer beneath the facility produced a 2 km long plume of PCE and degradation products. In 2006, thermal remediation with steam removed ~2 tons of DNAPL, while also mobilizing dissolved organic carbon (DOC). Released DOC benefitted natural attenuation (NA) degradation processes with contaminant concentrations decreasing up to ~1 km downgradient. Studies (Badin et al. 2016) with molecular biological tools (MBTs) commenced in 20141,2 to better understand NA processes and long-term risks associated with the plume. MBTs included: quantitative PCR (e.g., Gene-Trac® Dhc/FGA), next generation sequencing (NGS) for microbial community characterization (Gene-Trac® NGS), RNA quantification to assess gene expression and compound specific isotope analysis (CSIA) to identify degradation pathways and mechanisms.

Figure 1: cDCE Plume at Rødekro extended for more than 2 km downgradient of PCE source area From: Broholm et al., 2017.

“Analysis for microbial composition and specific degraders and their activity as well as dual stable isotopes has revealed high complexity in degradation processes and played an important role to substantiate the natural attenuation of the plume”

Role of Dehalococcoides and Dehalogenimonas:

Dehalococcoides (Dhc) and its relative Dehalogenimonas (Dhgm) are the only known microorganisms capable of reducing chlorinated ethenes to non-toxic ethene3,4. Molecular analysis indicated that Dhc/ Dhgm were most abundant, and Dhc most active, nearest the source zone at a combined 106 Dhc + Dhgm/liter of groundwater. Importantly, substantive remediation is often associated with Dhc abundance in this range. Nevertheless, Dhc VC-reductase genes (vcrA/bvcA), and associated messenger ribonucleic acid (mRNA) were not substantively

Figure 2: Aerobic/nitrate reducing conditions predominated in shallow groundwater reducing conditions in deeper groundwater. Variable redox across the Site facilitates both oxidative and reductive degradation processes. From: Broholm et al., 2017

Lack of VC-reductase suggests PCE and trichloroethene (TCE) are incompletely dechlorinated and that cis-1,2-dichloroethene (cDCE) reduction is the rate-limiting step. This is consistent with low/non-detectable (ND) concentrations of vinyl chloride (VC) and ethene in groundwater.

NGS indicated Dhgm were more abundant than Dhc at Rødekro, suggesting Dhgm are significant to biodegradation processes.

Mounting evidence suggests that Dhgm are important in chlorinated ethene remediation with reports of Dhgm dechlorinating PCE, trans- 1,2-DCE and VC and to ethene3,4,5. At Rødekro, Dhgm were identified in all NGS samples, and based on (semi-quantitative) NGS data, were generally at higher concentrations than Dhc, suggesting a significant role for Dhgm in reductive dechlorination, although this role is currently unclear.

What About Other Dechlorinators?

In addition to Dhc and Dhgm, NGS identified over 5,000 microorganisms in the groundwater with 13 potential dechlorinators, including partial dechlorinators Dehalobacter (Dhb) and Geobacter, that possibly contributed to PCE and TCE degradation to cDCE. Aerobic degraders of cDCE and VC including Polaromonas and Nocardioides were also detected and may be active in shallow aerobic groundwater. Several organisms implicated in cometabolic degradation of TCE were also identified.

NGS identified 13 potential dechlorinators out of 5,000 identified taxa attesting to the complexity of
bioremediation processes in the Rødekro groundwater.

The identification of a wide variety of dechlorinators at Rødekro indicates the biotic dechlorination processes at the site are complex and likely robust.

What About Abiotic Processes?

Dual Isotope CSIA can determine if degradation pathways are biotic or abiotic and suggested the primary cDCE degradation pathway at Rødekro was abiotic1,2. NGS data demonstrated that bacteria associated with pyrite oxidation were predominant (i.e., >20%) at the core to front of the plume. Pyrite (FeS2) and other reduced iron compounds can mediate abiotic ß-elimination of chlorinated ethenes through acetylene to ethene. Pyrite was identified as likely significant to abiotic degradation of cDCE at Rødekro.

The Overall Picture:

Figure 3 summarizes the degradation pathways identified and proposed at Rødekro. DOC released from thermal treatment spurred reductive dechlorination of PCE and TCE, primarily to cDCE, which is degraded abiotically by iron compounds (e.g., pyrite) through acetylene and possibly ethene.

The observed lack of ethene could be attributed to microbes identified in groundwater that degrade ethene to CO2 6,7 including sulfate reducing bacteria (SRB) or ethenotrophs, (e.g., Nocardiodes). Aerobic degradation of cDCE and VC – currently identified as playing a minor role – may become more important to the long-term NA; as DOC is exhausted, reductive pathways are rendered inactive. Biostimulation with an exogenous electron donor has also been identified as a viable option, based on recent (2017) detections of VC-reductases2. The overall conclusions of the studies are that concentrations have declined due to NA, and the low concentrations of VC reduces risk, it is not eliminated as the plume is still modestly extending downgradient1,2

Figure 3: Major degradation pathways at Rødekro include reductive dechlorination of PCE and TCE to cDCE, followed by abiotic ß-elimination of cDCE. Minor pathways include cDCE and VC reductive dechlorination to ethene by Dhc/Dhgm and aerobic metabolism of cDCE and VC to CO2