Superfund Research Program
Monitoring In Situ Bioremediation of TCE
In situ bioremediation, the use of naturally occurring microorganisms to destroy groundwater contaminants or convert them to harmless forms, is one of the most promising methods for addressing the ubiquitous issue of chlorinated solvent contamination at Superfund sites. However, in order to maximize the potential of this remediation approach, we need to improve our ability to understand, monitor, and manipulate the complex microbial communities performing in situ bioremediation.
In situ bioremediation of chlorinated solvents can occur by microbial reductive dehalogenation. For trichloroethylene (TCE), this involves the sequential reduction of TCE to dichloroethene (DCE), followed by conversion of DCE to vinyl chloride (VC), and finally VC to ethene. Under anaerobic conditions, the microorganisms that catalyze these reactions use the solvents as electron acceptors while using a range of substrates (e.g., lactate, methanol, H2) as electron donors/energy sources. The most serious challenge associated with the use of reductive dechlorination as a TCE remediation option is that the process does not always continue to ethene, resulting in the accumulation of DCE and VC. Both are hazardous compounds and VC is a known human carcinogen.
Supported by the Superfund Basic Research Program, Lisa Alvarez-Cohen of the University of California, Berkeley and Mark Conrad of E.O. Lawrence Berkeley National Laboratory are directing a research program with the goal of developing tools to evaluate the progress of in situ bioremediation of TCE. The Berkeley team is taking a two-pronged approach, as they work to develop:
- Stable carbon isotope analysis methods to monitor the microbial conversion of TCE to ethene
- Non-culture-based tools to monitor microbial population dynamics and community structure during the degradation of chlorinated solvents
Stable carbon isotope analysis A major barrier to implementation of in situ bioremediation techniques has been the lack of reliable field monitoring tools. Bioremediation processes are complex and occur in subsurface, heterogeneous environments. Consequently, it is difficult to distinguish between contaminant concentration changes resulting from biodegradation and those that occur due to physical processes such as groundwater transport or mixing. As a result, complete stoichiometric conversion of TCE to ethene can rarely be demonstrated in the field.
Drs. Alvarez-Cohen and Conrad are applying the technique of stable isotope fractionation to monitor the transformation of TCE and its degradation products. Degradation of organic compounds by biological enzymatic processes can cause significant shifts in the ratio of carbon isotopes 13C to 12C (delta-13C values) in both the reactants and products. Because of its lighter mass, 12C is more weakly bonded and reacts more readily than 13C. During dechlorination of TCE, 12C bonds are preferentially degraded, resulting in isotopic enrichment of the residual contaminant in 13C. Analysis of relative abundances of 12C and 13C yields information needed to assess rates of TCE degradation.
During a pilot study at the Idaho National Engineering and Environmental Laboratory's field site Test Area North, designed to investigate the treatment potential of using lactate to stimulate in situ biologic reductive dechlorination of TCE, the researchers conducted detailed, time-series stable carbon isotope monitoring. They demonstrated that:
- The isotope data were sensitive enough to provide evidence that all of the dissolved-phase TCE was fully dechlorinated to ethene.
- The addition of lactate to the source area greatly accelerated the natural process of reductive dechlorination that was already occurring.
- Isotope delta-13C data can be used to distinguish between concentration changes that occur due to physical transport process from those due to bioremediation.
Ultimately, the researchers plan to integrate isotope ratio data into a coupled reaction-transport model that could be used to monitor in situ reductive dechlorination of solvents and to quantify the total amount of contaminant that has been degraded. The results of this pilot study clearly demonstrate the sensitivity of carbon isotope measurements to the processes that occurred during the enhancement experiment.
Quite significantly, this study also established the value of lactate enrichment to act as a substrate for microbial reductive chlorination. The remediation technique yielded an estimated $15 million cost savings to the DOE for cleanup of the site.
Non-culture-based tools Because over 99% of subsurface microorganisms are non-culturable, traditional culture-based techniques for evaluating subsurface communities are inadequate. To characterize community structure and to evaluate the relative contributions of different physiological groups of bacteria to the degradation of chlorinate solvents, Dr. Alvarez-Cohen is applying a series of non-culture-based molecular methods:
- Clone library construction/clone sequencing
- Terminal restriction fragment length polymorphism (T-RFLP) analysis
- Fluorescent in situ hybridization (FISH) with rRNA probes
- Quantitative polymerase chain reaction (Q-PCR)
The research team initially used these techniques to study lactate-enriched microbial cultures from contaminated soil obtained from Alameda Naval Air Station. They evaluated the microbial communities for 1 year, over a large number of feeding cycles. In addition, they observed the impact of alternate electron donors and of brief exposure to oxygen on the anaerobic degradation processes. This was the first reported study where both PCR-based and FISH-based 16SrRNA methods had been applied in combination to characterize mixed communities performing reductive dechlorination.
This study demonstrated that used in combination, the suite of molecular techniques is a powerful tool for not only characterizing complex communities but also for tracking and monitoring specific microbial species in environmental systems. The use of clone libraries to provide peak identification for rapid T-RFLP analysis allowed for much more information to be collected with less effort than repeated clone libraries. The results from FISH alone overlooked important microbial species, but did confirm the importance of some species identified via the clone library results and highlighted some organisms that were underrepresented in the clone libraries. Preliminary results from the Q-PCR studies suggest that specific species, genes and expression can be quantified to very low detection limits, allowing for extremely sensitive analysis of both laboratory and field samples.
With respect to the TCE degradation in the test system, the researchers found that:
- Conversion of TCE in the test system began immediately with complete conversion to ethene.
- Dehalococcoides species are important elements in each of the enrichment communities that dechlorinate completely to ethene, but it is likely that other organisms (e.g., Desulfovibrio sp., Clostridium sp.) are involved either directly or indirectly in converting TCE to ethene.
- The organisms required to catalyze the final dechlorination step, from VC to ethene, are extremely oxygen sensitive or can easily be overtaken by facultatively aerobic competitors.
Such information on the ecology, physiology, and biochemistry of microbial communities involved in complete reductive dechlorination - and on perturbed communities capable of only partial dechlorination - can be applied in order to develop effective anaerobic treatment strategies for remediation of Superfund sites contaminated with chlorinated ethenes. Expanding our capabilities to monitor and predict in situ bioremediation rates through the use of innovative isotopic and molecular tools will greatly increase our ability to apply this cost-effective and efficient technology in the field, leading to extensive cost savings and risk reduction.
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To learn more about this research, please refer to the following sources:
- Richardson RE, Bhupathiraju VK, Song DL, Goulet TA, Alvarez-Cohen L. 2002. Phylogenetic characterization of microbial communities that reductively dechlorinate TCE based upon a combination of molecular techniques. Environ Sci Technol 36(12):2652-2662. PMID:12099461
- Song DL, Conrad ME, Sorenson KS, Alvarez-Cohen L. 2002. Stable carbon isotope fractionation during enhanced in situ bioremediation of trichloroethene. Environ Sci Technol 36(10):2262-2268. PMID:12038839
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