One of the challenges for natural attenuation is the burden of proof that it is proceeding satisfactorily falls on the proponent. And as we saw in the last lecture we need to often rely on field data collected over extensive time and special scales to demonstrate this which can be a very costly and time consuming process. One emerging approach to more rapidly assess biodegradation is the use of molecular biology methods that quantify the presence of special genetic biomarkers. Such biomarkers can help us determine who is there, what can they degrade, what type of enzymes are being made, and who is doing what. Commonly, biomarkers are used to show that specific degraders are present at greater concentrations in zones undergoing bioremediation than in background pristine areas. Let me give you an example considering the anaerobic degradation of benzene which is not always observed at contaminated sites. In this example, we observe degradation of benzene in a methanogenic microcosm using a culture that had been enriched for over ten years by Elizabeth Edwards at the University of Toronto. At that time, the responsible organisms were unknown, so we set out to address one of the most elusive questions in biodegradation which is, who initiates anaerobic benzene biodegradation? And using denaturing gradient gel electrophoresis which was state of the art ten years ago, we observed enrichment of a gene band that we identify by its nucleotide sequence to belong to Desulfobacterium species. And the corresponding 16S RNA sequence was used to design a genetic probe, a genetic primer shown here which we call ORM2. Subsequently, we decided to evaluate if bioaugmentation of aquifer columns that were not degrading benzene, if we added these methanogenic culture, would it help attenuate benzene migration? And would the biomarker be useful to assess performance? So, we got some encouraging results as you can see in the right-hand side. The highest number of benzene degraders as measured by our primer probe set, which was about 2 times 10 to the 5 cells per gram of soil, corresponded with the highest benzene degradation activity right at the sampling port where the culture had been added. We also tested this biomarker in numerous field samples and mixed cultures, some of which are known to degrade benzene anaerobically to asses the selectivity of our biomarker probe. And we got no false positives or false negatives which is encouraging. Whenever the biodegradation was observed, the biomarker was present. And if no anaerobic degradation of benzene occurred, then the biomarker was absent. This corroborates that some genetic biomarkers can be used to establish that specific degraders are present to demonstrate biodegradation potential. And that they are enrich in areas being actively bioremediated relative to background samples. But can we take this one step further and assess biodegradation rates, which is very important to determine if biodegradation is proceeding faster than migration, so that you can recommend monitored natural attenuation? Or this is also useful to provide a scientific basis for early cessation of active treatment when it is no longer removing the pollutant faster than monitored natural attenuation would. So we ran some microcosm studies using samples from contaminated sites and measured both biomarker concentrations using realtime quantitative PCR. And also measure biodegradation rates and explore whether they could be correlated so that we could predict rates based on biomarker concentration measurements. In this first example, we were interested in anaerobic degradation of toluene and use a probe for the gene that calls for Benzylsuccinate Synthase. This gene is called the bssA and is the only anaerobic enzyme gene known to degrade toluene and other alkyl benzenes. It catalyzes the additional fumerate and the eventual transformation to Benzoyl-CoA which is a pivotal intermediate in the anaerobic metabolism of aromatic compounds. The interesting thing about this enzyme is that most anaerobes that are known to degrade toluene have it. Regardless of whether they are denitrifiers, iron reducers, sulfur reducers, and we also have found this gene in the methanogenic consortia. In microcosms studies here with samples from a gasoline release site in San Diego, showed a strong correlation between the concentration of anaerobic toluene degraders inferred by the bssA biomarker and biodegradation rates. But there is a caveat, that in the field, mass transfer limitations and bioavailability may affect rates and confound this correlation. The second example involves a biomarker to assess biodegradation of 1,4-dioxane, which is an emerging pollutant often found in chlorinators following plumes. When we started this project, the genes responsible for dioxane biodegradation were unknown. But using bioinformatic and transcriptomic analysis, we identified a soluble diaro monooxygenase operon in the archetype dioxane degraders pseudonocardia dioximoran cb 1190 which we call dxmA. That's the name of the gene. This gene was induced, as you can see on the right-hand side, by both Dioxane and tetrahydrofurin as shown by reverse transcriptase qPCR. And we proceeded to develop a primer probe set to target this dioxane monooxygenase gene and measure its concentration using TaqMan chemistry which is more selective. After sequence alignments, we developed this probe that targets the gene sequence coding for the active side of this dioxane monooxygenase. So to test these biomarkers, we proceeded to obtain samples from five contaminated sites in the United States and then prepared 20 microcosm sets. These microcosm sets are illustrated here by the different colors. And after three to five months of incubation, considerable dioxane biodegradation was observed in 16 out of the 20 microcosms, that's in 80% of the microcosms. Degradation was observed in all microcosms where the biomarker was detected, even when the dioxane concentration was very low. I'm talking below 50 parts per billion as indicated by the blue arrows. And just as important, no degradation was observed when the biomarker was absent, so there were no false positives. Also, dioxane consumption, dioxane disappearance was correlated to an increase in the dxmA biomarker concentration as a result of growth of the specific degraders that are metabolizing dioxane. Finally, a significant correlation was observed between the final dxmA copy numbers and dioxane degradation rates. In contrast, copy numbers using 16s rRNA, a genetic phylogenetic biomarker that is commonly used to enumerate total bacteria, were not significantly correlated to biodegradation activity, which corroborates the selectivity of our dxmA probe. The key points of this lecture are that DNA probes can provide strong evidence of the presence of specific degraders. And their enrichment as a result of contaminant metabolism infers the potential for intrinsic bioremediation. Some generic biomarkers hold significant potential for use to predict biodegradation rates based on correlation analysis, which would be very useful to assess whether degradation is proceeding faster than migration, in support of decisions to accept monitored natural attenuation.
