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

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

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

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

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.

The strain under evaluation: Pseudomonas fluorescens strain HK44

The parent strain was isolated originally from PAH contaminated soil

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.

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.

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 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.

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)

10

0 10 20 30 40 50 60 70

0

2

4

6

8

W.T

NapD

NapE

W.T

NapD

NapE

W.T

NapD

NapE

Soil 1

Soil 2

Soil 3

Rec

over

ed R

hiz

obiu

m P

opu

lati

on

(Log

CF

U g

-1 s

oil)

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.

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.

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.

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

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