Perspectives of genetically engineered microbes for groundwater bioremediation.

Biodegradation is the main process for the removal of organic compounds from the environment, but proceeds slowly for many synthetic chemicals of environmental concern. Research on microbial biodegradation pathways revealed that recalcitrance is - among other factors - caused by biochemical blockages resulting in dysfunctional catabolic routes. This has raised interest in the possibility to construct microorganisms with improved catabolic activities by genetic engineering. Although this goal has been pursued for decades, no full-scale applications have emerged. This perspective explores the lagging implementation of genetically engineered microorganisms in practical bioremediation. The major technical and scientific issues are illustrated by comparing two examples, that of 1,2-dichloroethane where successful full-scale application of pump-and-treat biotreatment processes has been achieved, and 1,2,3-trichloropropane, for which protein and genetic engineering yielded effective bacterial cultures that still await application.

Re-evaluation of correction factors of a primary standard graphite calorimeter in 60Co gamma ray beams as a basis for the appointment of the BEV absorbed dose rate to water reference value.

The graphite calorimeter of the Federal Office of Metrology and Surveying (BEV-Bundesamt für Eich- und Vermessungswesen) was established in the 1980s as the primary standard for the absorbed dose to water for (60)Co gamma ray beams. To maintain the primary standard at an international level the graphite calorimeter and its corresponding components had to undergo a refurbishment and modernisation process. The correction factors of the graphite calorimeter were re-evaluated with Monte Carlo and experimental methods to obtain improved values. These are the correction for the effect of the gaps (1.0061), the scaling correction (0.9998), the correction for the difference in air attenuation (0.9971) and the corrections for the effective measurement depths in the graphite phantom for the graphite calorimeter (0.9886) and the CC01-105 ionisation chamber (0.9913). Consequently, it was necessary to change the reference value for the absorbed dose rate to water of the (60)Co teletherapy unit used for the calibration of secondary standard dosemeters.

Biodegradation of 1,2,3-trichloropropane through directed evolution and heterologous expression of a haloalkane dehalogenase gene.

Using a combined strategy of random mutagenesis of haloalkane dehalogenase and genetic engineering of a chloropropanol-utilizing bacterium, we constructed an organism that is capable of growth on 1,2,3-trichloropropane (TCP). This highly toxic and recalcitrant compound is a waste product generated from the manufacture of the industrial chemical epichlorohydrin. Attempts to select and enrich bacterial cultures that can degrade TCP from environmental samples have repeatedly been unsuccessful, prohibiting the development of a biological process for groundwater treatment. The critical step in the aerobic degradation of TCP is the initial dehalogenation to 2,3-dichloro-1-propanol. We used random mutagenesis and screening on eosin-methylene blue agar plates to improve the activity on TCP of the haloalkane dehalogenase from Rhodococcus sp. m15-3 (DhaA). A second-generation mutant containing two amino acid substitutions, Cys176Tyr and Tyr273Phe, was nearly eight times more efficient in dehalogenating TCP than wild-type dehalogenase. Molecular modeling of the mutant dehalogenase indicated that the Cys176Tyr mutation has a global effect on the active-site structure, allowing a more productive binding of TCP within the active site, which was further fine tuned by Tyr273Phe. The evolved haloalkane dehalogenase was expressed under control of a constitutive promoter in the 2,3-dichloro-1-propanol-utilizing bacterium Agrobacterium radiobacter AD1, and the resulting strain was able to utilize TCP as the sole carbon and energy source. These results demonstrated that directed evolution of a key catabolic enzyme and its subsequent recruitment by a suitable host organism can be used for the construction of bacteria for the degradation of a toxic and environmentally recalcitrant chemical.