manipulating deinococcus radiodurans for treatment of mixed wastes
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The Mixed Waste Problem
• Poorly characterized, complex mixtures• Difficult to treat:
• chemically complicated• dangerous
Radioactive Waste Hazardous Waste(solvents, heavy metals)
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 1-100
ppm concentration, or 10-500 M depending on the organic
Millions of gallons of wash water will be generated after the tanks are emptied
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.
Radioactive Waste Hazardous Waste(solvents, heavy metals)
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
The University of Minnesota Biocatalysis/Biodegradation Database, http://umbbd.ahc.umn.edu/
Toluene degradation TCE degradation
T2MOT2MO
T2MOT2MO
T3MOT3MO
T3MOT3MO
TODTOD
toluene-cis-dihydrodiol
dehydrogenase
toluene-cis-dihydrodiol
dehydrogenase
catechol-2,3dehydrogenase
catechol-2,3dehydrogenase
3-methylcatechol
dichloroacetate glyoxylate formate
glyoxylate formateTCE epoxidechloral hydrate
(not in whole cells)
sMMOsMMO
sMMOsMMO
T2MOT2MO TODTOD
T2MOT2MO
(only with sMMO)
•Organisms isolated that contain these oxygenases would mutate or die in radioactive waste
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
Proposal
• Engineer D. radiodurans to – Degrade solvents– Adsorb heavy metals
solvents
non-toxicproducts
metals
waste
treatedeffluent
engineeredD. radiodurans
•Develop an onsite, low-cost treatment process using the altered strain
–Remove solvents–Remove heavy metals
Steps
• Develop genetic tools
• Clone and express broad spectrum oxygenases
• Manipulate polyphosphate metabolism to achieve metal binding
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
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: 10-50 nmol/min/mg protein
Heather Rothfuss
High Toluene Degradation with Toluene Dioxygenase
0
10
20
30
40
50
60
70
80
0 500 1000 1500 2000nmol toluene added
0
10
20
30
40
50
60
70
80
0 500 1000 1500 20000
10
20
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0 500 1000 1500 2000
de
gra
da
tio
n r
ate
(/m
in-m
g p
rote
in)
/min-
-
Heather Rothfuss
TCE degradation: 2 nmol/min/mg protein
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
Solvent Resistance in D. radioduransToluene survival
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
1.00E+08
1.00E+09
1.00E+10
0 10 20 30 40 50 60minutes
CF
U/m
l
0%-
0.5%-
1%-
1.5%-
2%-
3%-
0%+
0.5%+
1%+
1.5%+
2%+
3%+
Future Work
• Expression of alternate oxygenases, to expand the degradative repertoire
• Optimization of degradation under treatment conditions
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
Jay Keasling, UC-Berkeley
polyPO4
PO4
+ metal --> metal-Pi ppt
Current hypothesis for the precipitation mechanism
Innermembrane
Periplasm
Cytoplasm
Innermembrane
PolyP
ATP
ADP
PPK
Step 1: required stepPolyphosphate accumulation in cytosolPhosphate rich environment, no metal
PolyP
ATP
ADP
PPK
Step 1: required stepPolyphosphate accumulation in cytosolPhosphate rich environment, no metal
PolyP
ATP
ADP
PPK
Step 1: required stepPolyphosphate accumulation in cytosolPhosphate rich environment, no metal
Innermembrane
PolyP
Pi PPX
Step 2Inorganic phosphate excretion
Phosphate free environment, metal laden
PolyP
Pi PPX
Step 2Inorganic phosphate excretion
Phosphate free environment, metal laden
PolyP
Pi PPX
PolyP
Pi PPX
Step 2 : proposed stepsInorganic phosphate excretion
Phosphate free environment, metal laden
Pi
High local Pi concentration
Pit transporterbidirectional
UO22+
soluble
HUO2PO4Insoluble, membrane bound
Pi
High local Pi concentration
Pit transporterbidirectional
UO22+
soluble
HUO2PO4Insoluble, membrane bound
Pi
High local Pi concentration
Pit transporterbidirectional
UO22+
soluble
HUO2PO4Insoluble, membrane bound
Periplasm
Goal: Manipulate polyphosphate synthesis and degradation
Polyphosphate ManipulationHypothesis: 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
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!
Alex Holland
Pi
Pi + metal --> metal-Pi ppt
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
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.
D. radiodurans R1 is extremely resistant to environmental stress
D.radiodurans R1
EtOH 20% --10X loss in 8h
Salt 1.2M—10X loss in 4h
Acid pH4.2—10X loss in 8h
B. subtilis 168*
9%--100X loss in 4h
0.2M—550X loss in 3h
pH4.3—1000X loss in
2h
* Völker et al. 1999. J. Bact. 181(13):3942-3948
Amy Schmid
Heat Shock Regulators Identified: sig1 and sig2 mutants survive poorly under heat shock
1.E+03
1.E+04
1.E+05
1.E+06
1.E+07
1.E+08
0 1 2 3 4 5 6 7 8 9
time (h)
CF
U/m
L
WTsig1
sig2
Amy Schmid
Heat shock induction of a PgroESL::lacZ fusion is decreased in sig1 and sig2
mutants
0
200
400
600
800
1000
1200
1400
1600
0 20 40 60 80 100 120 140Time post-shift (min)
Mil
ler
Un
its
WT 30sig1 30sig2 30WT 40 sig1 40sig2 40
Amy Schmid
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)
2D Protein Gels Reveal Over 60 Heat-shock Inducible Proteins: 20 are not
induced in the mutants
30
48
WT sig1 sig2
Amy Schmid
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
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
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