Manipulating Deinococcus radiodurans for treatment of

mixed wastes

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

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80

0 500 1000 1500 20000

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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

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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

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