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Genetically engineered microorganisms (GEMs ) Pest control Pollution abatement Frost protection

Field Application of a Genetically Engineered Microorganism for Polycyclic Aromatic Hydrocarbon Bioremediation Process Monitoring and Control. Genetically engineered microorganisms (GEMs ) Pest control Pollution abatement Frost protection.

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Genetically engineered microorganisms (GEMs ) Pest control Pollution abatement Frost protection

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  1. Field Application of a Genetically Engineered Microorganism for Polycyclic Aromatic Hydrocarbon Bioremediation Process Monitoring and Control

  2. Genetically engineered microorganisms (GEMs) Pest control Pollution abatement Frost protection

  3. Some concerns about releasing the genetically engineered microorganisms into environment • Alter balance of fundamental processes(such as energy, carbon, and nutrient cycling) • Exchange DNA with the indigenous microorganisms

  4. The benefit and risk related to the release of GEMs depend on their establishment in the environment

  5. First release of GEMs for use in bioremediation • Oct. 30, 1996 • This study was initiated by investigators at • the Univ. of Tennessee Center for Environmental Biotechnology in collaboration with • the Environmental Science Division of Oak Ridge National Laboratory

  6. Objectives • Testing the hypothesis that a GEM can be successfully introduced and maintained in a bioremediation process • Testing the concept of using, at the field scale, reporter organisms for direct bioremediation process monitoring and control, and • Acquiring data that can be used in risk assessment decision making and protocol development for future field release applications of GEMs.

  7. The strain under evaluation: Pseudomonas fluorescens strain HK44 • The parent strain was isolated originally from PAH contaminated soil

  8. Pseudomonas fluorescens strain HK44 was created by the incorporation of • a plasmid containing a salicylate inducible operon and • the gene cassette for bacterial bioluminescence (lux) from Vibriofischerii.

  9. When naphthalene is metabolized to salicylate, the lux transcriptional gene fusion is induced and expressed to produce enzymes responsible for a bioluminescent signal at 490 nm.

  10. Thus, this bacterium is able • to biodegrade naphthalene as well as other substituted naphthalenes and other PAHs, and • to function as a living bioluminescent reporter for the presence of naphthalene contamination, its bioavailability, and the functional process of biodegradation.

  11. A unique component used: large subsurface soil lysimeters

  12. The lysimeters • permitted a semi-contained, controllable field test environment. • allowed accurate field replication and provision of control tests. • Allowed extensive sampling and numerous assays to be performed. • In situ sensors monitored temperature, bioluminescence, vapor phase volatile hydrocarbon contamination, oxygen concentration, soil moisture content, and carbon dioxide concentration.

  13. Furthermore, the lysimeters are exposed to environmental conditions over a two year period, thus providing information on in situ survivability of GEMs.

  14. 10 Soil 1 8 6 W.T Recovered Rhizobium Population (Log CFU g-1 soil) NapD NapE Soil 2 W.T 4 NapD Soil 3 NapE W.T 2 NapD NapE 0 0 10 20 30 40 50 60 70 Incubation Time (Days)

  15. The treatment zones of three lysimeters received contaminated soil inoculated with HK44; two received uncontaminated soil inoculated with HK44; and one received uninoculated, contaminated soil.

  16. Preparation of inoculum • HK44 was grown in a 500 L fermenter • after 22 h, the culture was harvested by a continuous centrifuge and the resulting cell paste resuspended in 60 gallons of saline. • This suspension was transported to the site in two 30 gallon drums.

  17. A moderate aging and weathering period was desired to better simulate soils from contaminated sites. • Unfortunately, a delay in receiving the numerous approval required for releasing the GEMs resulted in a total of 180 days of contaminant soil aging. • Consequently, approx. 95% of the naphthalene was lost through volatilization and natural biodegradation processes.

  18. Lysimeter 1--Contaminated soil inoculated with HK44 (open circle, Total viable counts and solid circle, HK44 counts) (Solid arrow: oil addition, dashed arrows: minimal medium addition)

  19. Lysimeter 5--Uncontaminated soil inoculated with HK44 (open circle, Total viable counts and solid circle, HK44 counts) (Solid arrow: oil addition, dashed arrows: minimal medium addition)

  20. Lysimeter 6--Contaminated soil without HK44 (open circle, Total viable counts and open box, indigenous tetra+ CFU) (Solid arrow: oil addition, dashed arrows: minimal medium addition)

  21. Conclusions • GEMs can be introduced and maintained in environmental soils for at least 18 months • The population of introduced strain can be affected by nutrient augmentation • Bioluminescence from GEMs can be used as a tool for monitoring and controlling the bioremediation process, AND • Report conditions are favorable for bioremediation

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