Field Application of a Genetically Engineered Microorganism for Polycyclic Aromatic Hydrocarbon...
-
Upload
camilla-dickerson -
Category
Documents
-
view
218 -
download
0
Transcript of Field Application of a Genetically Engineered Microorganism for Polycyclic Aromatic Hydrocarbon...
![Page 1: Field Application of a Genetically Engineered Microorganism for Polycyclic Aromatic Hydrocarbon Bioremediation Process Monitoring and Control.](https://reader031.fdocuments.us/reader031/viewer/2022032016/56649edb5503460f94bea60b/html5/thumbnails/1.jpg)
Field Application of a Genetically Engineered Microorganism for
Polycyclic Aromatic Hydrocarbon Bioremediation Process Monitoring and Control
![Page 2: Field Application of a Genetically Engineered Microorganism for Polycyclic Aromatic Hydrocarbon Bioremediation Process Monitoring and Control.](https://reader031.fdocuments.us/reader031/viewer/2022032016/56649edb5503460f94bea60b/html5/thumbnails/2.jpg)
Genetically engineered microorganisms (GEMs)
Pest control
Pollution abatement
Frost protection
![Page 3: Field Application of a Genetically Engineered Microorganism for Polycyclic Aromatic Hydrocarbon Bioremediation Process Monitoring and Control.](https://reader031.fdocuments.us/reader031/viewer/2022032016/56649edb5503460f94bea60b/html5/thumbnails/3.jpg)
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
![Page 4: Field Application of a Genetically Engineered Microorganism for Polycyclic Aromatic Hydrocarbon Bioremediation Process Monitoring and Control.](https://reader031.fdocuments.us/reader031/viewer/2022032016/56649edb5503460f94bea60b/html5/thumbnails/4.jpg)
The benefit and risk related to the release of GEMs depend on their establishment in the environment
![Page 5: Field Application of a Genetically Engineered Microorganism for Polycyclic Aromatic Hydrocarbon Bioremediation Process Monitoring and Control.](https://reader031.fdocuments.us/reader031/viewer/2022032016/56649edb5503460f94bea60b/html5/thumbnails/5.jpg)
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
![Page 6: Field Application of a Genetically Engineered Microorganism for Polycyclic Aromatic Hydrocarbon Bioremediation Process Monitoring and Control.](https://reader031.fdocuments.us/reader031/viewer/2022032016/56649edb5503460f94bea60b/html5/thumbnails/6.jpg)
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.
![Page 7: Field Application of a Genetically Engineered Microorganism for Polycyclic Aromatic Hydrocarbon Bioremediation Process Monitoring and Control.](https://reader031.fdocuments.us/reader031/viewer/2022032016/56649edb5503460f94bea60b/html5/thumbnails/7.jpg)
The strain under evaluation: Pseudomonas fluorescens strain HK44
The parent strain was isolated originally from PAH contaminated soil
![Page 8: Field Application of a Genetically Engineered Microorganism for Polycyclic Aromatic Hydrocarbon Bioremediation Process Monitoring and Control.](https://reader031.fdocuments.us/reader031/viewer/2022032016/56649edb5503460f94bea60b/html5/thumbnails/8.jpg)
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.
![Page 9: Field Application of a Genetically Engineered Microorganism for Polycyclic Aromatic Hydrocarbon Bioremediation Process Monitoring and Control.](https://reader031.fdocuments.us/reader031/viewer/2022032016/56649edb5503460f94bea60b/html5/thumbnails/9.jpg)
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.
![Page 10: Field Application of a Genetically Engineered Microorganism for Polycyclic Aromatic Hydrocarbon Bioremediation Process Monitoring and Control.](https://reader031.fdocuments.us/reader031/viewer/2022032016/56649edb5503460f94bea60b/html5/thumbnails/10.jpg)
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.
![Page 11: Field Application of a Genetically Engineered Microorganism for Polycyclic Aromatic Hydrocarbon Bioremediation Process Monitoring and Control.](https://reader031.fdocuments.us/reader031/viewer/2022032016/56649edb5503460f94bea60b/html5/thumbnails/11.jpg)
A unique component used: large subsurface soil lysimeters
![Page 12: Field Application of a Genetically Engineered Microorganism for Polycyclic Aromatic Hydrocarbon Bioremediation Process Monitoring and Control.](https://reader031.fdocuments.us/reader031/viewer/2022032016/56649edb5503460f94bea60b/html5/thumbnails/12.jpg)
![Page 13: Field Application of a Genetically Engineered Microorganism for Polycyclic Aromatic Hydrocarbon Bioremediation Process Monitoring and Control.](https://reader031.fdocuments.us/reader031/viewer/2022032016/56649edb5503460f94bea60b/html5/thumbnails/13.jpg)
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.
![Page 14: Field Application of a Genetically Engineered Microorganism for Polycyclic Aromatic Hydrocarbon Bioremediation Process Monitoring and Control.](https://reader031.fdocuments.us/reader031/viewer/2022032016/56649edb5503460f94bea60b/html5/thumbnails/14.jpg)
Furthermore, the lysimeters are exposed to environmental conditions over a two year period, thus providing information on in situ survivability of GEMs.
![Page 15: Field Application of a Genetically Engineered Microorganism for Polycyclic Aromatic Hydrocarbon Bioremediation Process Monitoring and Control.](https://reader031.fdocuments.us/reader031/viewer/2022032016/56649edb5503460f94bea60b/html5/thumbnails/15.jpg)
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)
![Page 16: Field Application of a Genetically Engineered Microorganism for Polycyclic Aromatic Hydrocarbon Bioremediation Process Monitoring and Control.](https://reader031.fdocuments.us/reader031/viewer/2022032016/56649edb5503460f94bea60b/html5/thumbnails/16.jpg)
![Page 17: Field Application of a Genetically Engineered Microorganism for Polycyclic Aromatic Hydrocarbon Bioremediation Process Monitoring and Control.](https://reader031.fdocuments.us/reader031/viewer/2022032016/56649edb5503460f94bea60b/html5/thumbnails/17.jpg)
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.
![Page 18: Field Application of a Genetically Engineered Microorganism for Polycyclic Aromatic Hydrocarbon Bioremediation Process Monitoring and Control.](https://reader031.fdocuments.us/reader031/viewer/2022032016/56649edb5503460f94bea60b/html5/thumbnails/18.jpg)
![Page 19: Field Application of a Genetically Engineered Microorganism for Polycyclic Aromatic Hydrocarbon Bioremediation Process Monitoring and Control.](https://reader031.fdocuments.us/reader031/viewer/2022032016/56649edb5503460f94bea60b/html5/thumbnails/19.jpg)
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.
![Page 20: Field Application of a Genetically Engineered Microorganism for Polycyclic Aromatic Hydrocarbon Bioremediation Process Monitoring and Control.](https://reader031.fdocuments.us/reader031/viewer/2022032016/56649edb5503460f94bea60b/html5/thumbnails/20.jpg)
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.
![Page 21: Field Application of a Genetically Engineered Microorganism for Polycyclic Aromatic Hydrocarbon Bioremediation Process Monitoring and Control.](https://reader031.fdocuments.us/reader031/viewer/2022032016/56649edb5503460f94bea60b/html5/thumbnails/21.jpg)
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)
![Page 22: Field Application of a Genetically Engineered Microorganism for Polycyclic Aromatic Hydrocarbon Bioremediation Process Monitoring and Control.](https://reader031.fdocuments.us/reader031/viewer/2022032016/56649edb5503460f94bea60b/html5/thumbnails/22.jpg)
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)
![Page 23: Field Application of a Genetically Engineered Microorganism for Polycyclic Aromatic Hydrocarbon Bioremediation Process Monitoring and Control.](https://reader031.fdocuments.us/reader031/viewer/2022032016/56649edb5503460f94bea60b/html5/thumbnails/23.jpg)
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)
![Page 24: Field Application of a Genetically Engineered Microorganism for Polycyclic Aromatic Hydrocarbon Bioremediation Process Monitoring and Control.](https://reader031.fdocuments.us/reader031/viewer/2022032016/56649edb5503460f94bea60b/html5/thumbnails/24.jpg)
![Page 25: Field Application of a Genetically Engineered Microorganism for Polycyclic Aromatic Hydrocarbon Bioremediation Process Monitoring and Control.](https://reader031.fdocuments.us/reader031/viewer/2022032016/56649edb5503460f94bea60b/html5/thumbnails/25.jpg)
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