1. Manipulating Deinococcus radiodurans for treatment of mixed wastes

2. The Mixed Waste Problem
Poorly characterized, complex mixtures
Difficult to treat:
chemically complicated
dangerous

3. Hanford Wastes 149 tanks built between 1944 and 1964
66 leak
37 Million gallons of waste
Low level radiation + solvents
Often just buried in pits
Contaminated soils later isolated
Conditions inside the tanks
pH often near 10 to avoid corroding tank liners
Radiation dose from a few to 5,000 rad/hour (50 Gy/hour)
Organics often in the ppm concentration, or mM depending on the organic
Millions of gallons of wash water will be generated after the tanks are emptied

4. The Mixed Waste Problem
Poorly characterized, complex mixtures
Difficult to treat:
chemically complicated
dangerous
Simplification is necessary:
Remove toxic organic component
radioactive + inorganics can be isolated using conventional methods.

5. Bioremediation and Cometabolism
Bacteria have evolved to degrade naturally occurring organics for growth or protection.
Oxygenases are able to catalyze oxidation of organics at specific carbons

6. Toluene degradation TCE degradation
T2MO
T3MO
TOD
toluene-cis-
dihydrodiol
dehydrogenase
catechol-2,3
3-methylcatechol
sMMO
T2MO
TOD
TCE epoxide
glyoxylate formate
chloral hydrate
(not in whole cells)
sMMO
T2MO
(only with sMMO)
dichloroacetate
glyoxylate formate
The University of Minnesota Biocatalysis/Biodegradation Database,
Organisms isolated that contain these oxygenases would mutate or die in radioactive waste

7. Deinococcus radiodurans
High resistance to UV, ionic radiation
Survive complete desiccation
Fast, efficient DNA repair
Complete genome sequence available
No toxic organics are known to be degraded by R1

8. Proposal Engineer D. radiodurans to
solvents
non-toxic
products
metals
Engineer D. radiodurans to
Degrade solvents
Adsorb heavy metals
Develop an onsite, low-cost treatment process using the altered strain
Remove solvents
Remove heavy metals
waste
treated
effluent
engineered
D. radiodurans

9. Steps Develop genetic tools
Clone and express broad spectrum oxygenases
Manipulate polyphosphate metabolism to achieve metal binding

10. Genetic Tools Insertion vector to create stable constructs
Expression vector and promoters to express foreign genes
Mutation system
Minimal medium
Rob Meima, Lindy Gewin, Heather Rothfuss, Amy Schmid, Alex Holland

11. Clone and express broad spectrum oxygenases
Broad spectrum oxygenases degrade many toxic solvents (toluene, TCE)
Candidates:
Toluene dioxygenase
Toluene monooxygenases
Methane monooxygenase
Target:
TCE: 1-5 nmol/min/mg protein
Toluene: nmol/min/mg protein
Heather Rothfuss

12. High Toluene Degradation with Toluene Dioxygenase80 80 80
mg protein) 70 70 70 - - - 60 60 60
/min
/min 50 50 50 40 40 40 30 30 30
degradation rate ( 20 20 20 10 10 10
500
500
500
1000
1000
1000
1500
1500
1500
2000
2000
2000
nmol
toluene added
TCE degradation: 2 nmol/min/mg protein
Heather Rothfuss

13. Solvent Resistance of P. putida DOT-T1E
Characteristics of Extremely Solvent Resistant Bacteria: Model, P. putida DOT-T1E
Growth in 1% toluene (just past saturation)
Survival of 0.3% (28mM) solvent shock when pre-grown with toluene supplied in headspace

14. Solvent Resistance in D. radiodurans
Toluene survival
1.00E+10
1.00E+09
1.00E+08
0%-
1.00E+07
0.5%-
CFU/ml
1%-
1.00E+06
1.5%-
1.00E+05
2%-
3%-
1.00E+04
0%+
0.5%+
1.00E+03
1%+
1.5%+
1.00E+02
2%+
1.00E+01
3%+ 10 20 30 40 50 60
minutes

15. Future Work
Expression of alternate oxygenases, to expand the degradative repertoire
Optimization of degradation under treatment conditions

16. Manipulate polyphosphate metabolism to achieve metal binding
Phosphate release at cell surface can result in metal precipitation (uranyl phosphate)
Production then degradation of polyphosphate can supply the phosphate
PO4
+ metal --> metal-Pi ppt
polyPO4
Jay Keasling, UC-Berkeley

17. Current hypothesis for the precipitation mechanism
High local Pi
concentration
Pit transporter
bidirectional UO 2 2+
soluble
HUO PO 4
Insoluble, membrane bound
Periplasm
Periplasm
Inner
Inner
Inner
membrane
membrane
membrane
PolyP
ATP
ADP
PPK
Step 1
: required step
Polyphosphate accumulation in
cytosol
Phosphate rich environment, no metal
Cytoplasm
PolyP Pi
PPX
Step 2
Inorganic phosphate excretion
Phosphate free environment, metal laden
: proposed steps
Goal: Manipulate polyphosphate synthesis and degradation

18. Polyphosphate Manipulation
Hypothesis: overexpress PPX gene, get more polyP
Identified the PPK and PPX genes
Generated mutants, which are viable
Studied promoter activity: PPK promoter is active under phosphate-limitation
Cloned and overexpressed the genes singly and together: no change in polyP
Heather Rothfuss, Alex Holland

19. Polyphosphate Manipulation
Developed a protocol to fill cell with polyP:
Starve for phosphate for several hours, then add high phosphate
Developed a protocol to release phosphate:
Starve cells for phosphate
Add metals: get almost complete removal in 2 hours! Pi
Pi + metal --> metal-Pi ppt
Alex Holland

20. Summary
D. radiodurans strains have been constructed that can degrade a variety of toxic solvents
A protocol has been developed to precipitate heavy metals
Combining the two should allow the development of a process to remove toxic solvents and heavy metals in the presence of radioactivity

21. Assess Stress Response
Treatment conditions will involve many stresses
Heat shock --solvent
pH --starvation
Ionic
Goal is to minimize waste alteration for treatment
Understanding stress response and regulatory mechanisms important for creation of an efficient bioremediation strain.
Many known stress regulators are not recognizable in the annotated genome.

22. D. radiodurans R1 is extremely resistant to environmental stress
B. subtilis 168*
9%--100X loss in 4h
0.2M—550X loss in 3h
pH4.3—1000X loss in 2h
D.radiodurans R1
EtOH % --10X loss in 8h
Salt M—10X loss in 4h
Acid pH4.2—10X loss in 8h
Amy Schmid
* Völker et al J. Bact. 181(13):

23. Heat Shock Regulators Identified: sig1 and sig2 mutants survive poorly under heat shock1 2 3 4 5 6 7 8 9
time (h)
CFU/mL WT
sig1
sig2
Amy Schmid

24. Heat shock induction of a PgroESL::lacZ fusion is decreased in sig1 and sig2 mutants
200
400
600
800
1000
1200
1400
1600 20 40 60 80
100
120
140
Time post-shift (min)
Miller Units
WT 30
sig1 30
sig2 30
WT 40
sig1 40
sig2 40
Amy Schmid

25. 2D Gel Proteomics
Comparison of 30C to 48C shows over 60 proteins induced by heat shock
Comparison of WT to mutants shows 20 of these are not induced in the mutants
Analysis of these spots by MS to determine identity ongoing (with R. Smith group, PNNL)

26. 2D Protein Gels Reveal Over 60 Heat-shock Inducible Proteins: 20 are not induced in the mutants
30
sig1 WT
sig2
48
Amy Schmid

27. Future Work
Assess global changes in response to stress using microarrays (with J. Battista and TIGR) and whole cell proteomics (with R. Smith lab, PNNL)
Assess response of D. radiodurans to simultaneous stresses, similar to those to be encountered in mixed wastes

28. Summary Solvent degradation at the target rate has been achieved
Polyphosphate manipulation is ongoing
High stress resistance is advantageous
Stress response systems being characterized
Suggests simple process design with minimal manipulation of the waste

29. Potential Batch Process
Grow cells offsite in fermentation facility, dry
Inoculate treatment system with dried cells and waste (possibly diluted)
Run system (resting cells) until solvents are removed
Remove effluent, filter cells, dry
Dispose of cells/metals