Summary of the Dehalococcoides & Dehalobacter Detection Project

Chlorinated solvents are prevalent and problematic groundwater contaminants because of their tendency to form large, dissolved-phase plumes, their recalcitrant nature and the subsequent risk to human health. Remediation approaches are frequently based on bioremediation, which relies on establishing and maintaining significant populations of microorganisms capable of reductive dehalogenation. The majority of monitoring methods involve quantitative polymerase chain reaction (qPCR), which is performed in the laboratory on an expensive thermal cycler. The objective of this work was to explore other quantification methods, which could potentially be more specific, less expensive, less sensitive to inhibition, field deployable, or enable a larger selection of functional genes to be quantified.

This project developed loop mediated isothermal amplification (LAMP) to quantify microorganisms (16S rRNA gene & reductive dehalogenase genes) associated with reductive dehalogenation. LAMP assays for three reductive dehalogenase (RDase) genes (vcrA, bvcA, tceA) were compared to qPCR assays using two commercially available bioaugmentation cultures (KB-1, SDC-9). The developed LAMP assays for vcrA and tceA genes were validated by comparing quantification on the hand held platform, the Gene-Z, and a real time thermal cycler using deoxyribonucleic acid (DNA) isolated from groundwater samples from a SDC-9 bioaugmented site. Other experiments tested DNA amplification from Dehalobacter with and without crude lysis and varying concentrations of humic acid. A new technique was developed where filters were amplified directly within disposable Gene-Z chips (direct filter amplification, DFA). Another approach for quantifying RDase genes (without DNA extraction) was developed requiring only low cost laboratory equipment (a bench top centrifuge and a water bath) and less time and resources compared to qPCR. Shotgun sequencing was used for the quantification of taxonomic and functional biomarkers associated with chlorinated solvent and 1,4-dioxane bioremediation.

The comparison of LAMP primers and qPCR indicated quantification was similar over a large range of gene concentrations. Also, the quantitative increase in gene concentrations over one growth cycle of KB-1 and SDC-9 using LAMP primers was comparable to that of qPCR. The developed LAMP assays for vcrA and tceA genes were validated by comparing quantification the Gene-Z and a real time thermal cycler using DNA isolated from eight groundwater samples obtained from a SDC-9 bioaugmented site. A visual based SYBR green LAMP-Most Probable Number (MPN) approach was developed, offering a low cost and user-friendly alternative to qPCR for quantifying RDase genes in groundwater samples.

The taxonomic analysis of the shotgun sequencing data revealed numerous genera previously linked to chlorinated solvent degradation, including Dehalococcoides, Desulfitobacterium and Dehalogenimonas. Reads aligning with both aerobic and anaerobic biomarkers were observed across all sites. Aerobic solvent degradation genes, etnC or etnE, were detected in at least one sample from each site, as were pmoA and mmoX. The most abundant 1,4-dioxane biomarker detected was Methylosinus trichosporium OB3b mmoX. Reads aligning to thmA or Pseudonocardia were not found.

The visual based SYBR green LAMP-MPN approach offers three key advantages compared to existing methods: time, cost and the potential in situ application. The use of centrifuged cells, instead of DNA, reduces the time and cost required for sample preparation (no DNA extraction). Also, compared to qPCR, the LAMP assay has a shorter run time and the visualization of amplification products is immediate. The assay requires only basic laboratory equipment (benchtop centrifuge and water bath), and does not require an expensive real time thermal cycler. With additional development and validation, it is possible that the method could be applied in the field. Additionally, the dUTP-UNG (Deoxyuridine triphosphate - Uracil DNA glycosylase) system reduces the probability of false positives due to carry over contamination and increases the overall robustness of visual detection with SYBR green LAMP. The regression equations generated for SYBR green LAMP assay with MPN technique can be used to calibrate the assay to relate the data to traditional qPCR data.

The work illustrates the importance of shotgun sequencing to provide a more complete picture of the functional abilities of microbial communities. The approach is advantageous over current methods because an unlimited number of functional genes can be quantified. Additional work should focus on RDase detection limits for shotgun sequencing data and comparisons to data generated with qPCR.

In summary, LAMP would be beneficial at sites containing groundwater with higher humic acid contents, as LAMP amplification is less sensitive to inhibition, compared to qPCR. LAMP would be beneficial if funds for monitoring were limited, as the only equipment needed include an incubator and a waterbath. However, the individuals performing the assays would still need basic skills in microbiology/molecular methods. In comparison, qPCR requires an expensive thermal cycler. Further, LAMP can be performed without DNA extraction, which also reduces costs.

Dang, H., Y.H. Kanitkar, R.D. Stedtfeld, P.B. Hatzinger, S.A. Hashsham, and A.M. Cupples. 2018. Abundance of Chlorinated Solvent and 1,4-dioxane Degrading Microorganisms at Five Chlorinated Solvent Contaminated Sites Determined via Shotgun Sequencing. Environmental Science and Technology, 52(23):13914–13924.

Kanitkar, Y.H., R.D. Stedtfeld, P.B. Hatzinger, S.A. Hashsham, and A.M. Cupples. 2017. Development and Application of a Rapid, User-friendly and Inexpensive Method to Detect Dehalococcoides sp. reductive Dehalogenase Genes from Groundwater. Applied Microbiology and Biotechnology, 101:4827–4835.

Kanitkar, Y.H., R.D. Stedtfeld, P.B. Hatzinger, S.A. Hashsham, and A.M. Cupples. 2017. Most Probable Number with Visual Based LAMP for the Quantification of Reductive Dehalogenase Genes in Groundwater Samples. Journal of Microbiological Methods, 143:44-49.

Kanitkar, Y.H., R.D. Stedtfeld, R.J. Steffan, S.A. Hashsham, and A.M. Cupples. 2016. Development of Loop Mediated Isothermal Amplification (LAMP) for Rapid Detection and Quantification of Dehalococcoides spp. Biomarker Genes in Commercial Reductive Dechlorinating Cultures KB-1 and SDC-9. Applied and Environmental Microbiology, 82:1799-1806.

Stedtfeld, R.D., T. M. Stedtfeld, F. Samhan, Y.H. Kanitkar, P.B. Hatzinger, A.M. Cupples, and S.A. Hashsham. 2016. Direct Loop Mediated Isothermal Amplification on Filters for Quantification of Dehalobacter in Groundwater. Journal of Microbiological Methods, 131:61-67.

Stedtfeld, R., T. Stedtfeld, M. Kronlein, G. Seyrig, R. Steffan, A.M. Cupples, and S.A. Hashsham. 2014. DNA Extraction-free Quantification of Dehalococcoides spp. in Groundwater using a Hand-held Device. Environmental Science and Technology, 48:13855-13863.