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

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

l l 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

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

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

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

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

l l 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 Vibrio fischerii.

l l 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.

l l 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.

A unique component used: large subsurface soil lysimeters

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

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

Incubation Time (Days) W.T NapD NapE W.T NapD NapE W.T NapD NapE Soil 1 Soil 2 Soil 3 Recovered Rhizobium Population (Log CFU g -1 soil)

l l 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.

l l 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.

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

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)

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)

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)

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