Perchlorate Degradation in Acetate-fed Bioreactors · Perchlorate Degradation in Acetate-fed Bioreactors Yanguang Song Kijung Kim, Bruce Logan Department of Civil & Environ. Eng.

Perchlorate Degradation in Acetate-fed Bioreactors

Yanguang SongKijung Kim, Bruce Logan

Department of Civil & Environ. Eng.Penn State University

Perchlorate respiringculture (optional)

Supplemental carbon source with(optional) N, P addition

Backwash withchlorate, N, P,acetate

Contaminatedwater source

Perchloratefree water

Sandfilter

ATTRIBUTES:Continuous perchlorate removalClogging avoided by intermittent backwashBiofilm regeneration by side process

0.001

0.01

0.1

1

10

100

0 10 20 30 40 50 60 70

Time (day)

Perc

hlor

ate

(mg/

L)

0

20

40

6080

100

120

140EB

CT

(min

)Effluent

Influent

EBCT

Sand media

PERCHLORATE FIXED-BED REACTOR[Penn State Patent/Invention Disclosure No. 1887]

Perchlorate respiringculture (optional)

Supplemental carbon source with(optional) N, P addition

Backwash withchlorate, N, P,acetate

Contaminatedwater source

Perchloratefree water

Sandfilter

ATTRIBUTES:Continuous perchlorate removalClogging avoided by intermittent backwashBiofilm regeneration by side process

Sand media

Perchlorate Degradation: Topics

• Bacterial isolate used in tests: KJ• Bench-scale acetate-fed reactors

– Mixed culture– Pure culture (KJ)

• Effect of O2 exposure on perchlorate reduction

Perchlorate Reducing Pathway

ClO3-

ClO4-

Acetate

CO2, H2O, Biomass

Acetate

CO2, H2O, Biomass

ClO2-

Cl- + O2

Acetate

CO2, H2O, Biomass

kC

kP

kD

Chlorite and chlorate do not accumulate in water:

kD>> kC > kP

Are perchlorate-respiring bacteria abundant in nature?

One milliliter (1 mL) of water(about one teaspoon) contains:

~ 106 bacteria

~1 perchlorate-degrading bacterium

PRM Abundance: observations

• PRMs present in natural waters at ~1000/109

• PRMs in “pristine” soils, that have no evidence of perchlorate contamination: ~1/109

• At sites where perchlorate is present, have a more abundant perchlorate reducing population, ~100/109

• Dechlorosoma sp. KJ: isolated from a perchlorate-degrading packed bed bioreactor;

• Dechlorosoma sp. PDX: isolated from primary digester wastewater;

• Dechloromonas sp. JM: isolated from a hydrogen-oxidizing consortium in a autotrophic packed-bed biofilm reactor;

• Dechloromonas sp. HZ: isolated from a gas-phase anaerobic packed-bed biofilm reactor.

Several Perchlorate Respiring Bacteria Isolated in Our Laboratory

Phylogentic analysis of KJ(courtesy of Dr. Ian Head, Univ. of New Castle)

95

2%

Zoogloea ramigera ATCC 19544 T

Isolate KJ

Isolate JM"Dechloromonas agitata" str. CKB

Ferribacterium limneticum str. cda-1

"Dechlorosoma" sp. str. Iso2

"Dechloromonas" sp. str. CL"Dechloromonas" sp. str. NM

"Dechloromonas" sp. str. SIUL"Dechloromonas" sp. str. MissR

Rhodocyclus purpureus DSM 168 TRhodocyclus tenuis str. 2761

Isolate PDX"Dechlorosoma" sp. str. SDGM

"Dechlorosoma suilla" str. PS2"Dechlorosoma" sp. str. Iso1

Thauera aromatica DSM 6984 TThauera selenatis ATCC 55363 T

Azoarcus sp. str. BH72Azoarcus indigens str. VB32 T

Azoarcus denitrificans str. Td -15Azoarcus denitrificans str. Td -1

73

95

100

98

100

90

90100

72

56

96

67

100

100

95

100

100

63

77 84

6476

100

100

88

89

77

100

8465

100

95

2%2%

Zoogloea ramigera ATCC 19544 T

Isolate JM"Dechloromonas agitata" str. CKB

Ferribacterium limneticum str. cda-1

"Dechlorosoma" sp. str. Iso2

"Dechloromonas" sp. str. CL"Dechloromonas" sp. str. NM

"Dechloromonas" sp. str. SIUL"Dechloromonas" sp. str. MissR

Rhodocyclus purpureus DSM 168 TRhodocyclus tenuis str. 2761

Isolate PDX"Dechlorosoma" sp. str. SDGM

"Dechlorosoma suilla" str. PS2"Dechlorosoma" sp. str. Iso1

Thauera aromatica DSM 6984 TThauera selenatis ATCC 55363 T

Azoarcus sp. str. BH72Azoarcus indigens str. VB32 T

Azoarcus denitrificans str. Td -15Azoarcus denitrificans str. Td -1

73

95

100

98

100

90

90100

72

56

96

67

100

100

95

100

100

63

77 84

6476

100

100

88

89

77

100

8465

100

Classification:

Dechlorosoma sp. KJ

From: Logan et al. (2001)

KJ Perchlorate kinetics: Ks=33 mg/L (lactate grown cell suspensions)


0

1

2

3

4 R

ates

(mg/

ug-h

r)

0 100 200 300 400 500 Perchlorate Concentration (mg/L)

KJ1

PDX

Substrate kinetics: acetate

Acetate (mg/L)0 100 200 300 400 500 600

Gro

wth

Rat

e (h

-1)

0.00

0.05

0.10

0.15

0.20

0.25

0.30

oxygen



Acetate (mg/L)0 100 200 300 400 500 600

Gro

wth

Rat

e (h

-1)

0.00

0.05

0.10

0.15

0.20

0.25

0.30

oxygenchlorate



Acetate (mg/L)0 100 200 300 400 500 600

Gro

wth

Rat

e (h

-1)

0.00

0.05

0.10

0.15

0.20

0.25

0.30

oxygenchlorateperchlorate


Electron Acceptor μm (h-1) KS (mg/L)

Oxygen 0.27 ± 0.02 14 ± 1

Chlorate 0.27 ± 0.03 60 ± 25

Perchlorate 0.14 ± 0.01 470 ± 290

Kinetic constants for growth of KJ on acetate


Cell yield- KJ

0.460.44

0.50

0.00.1

0.20.3

0.40.5

0.60.7

oxygen chlorate perchlorate

Cel

l yie

ld (g

-dry

wei

ght/g

-ace

tate

)


Overall Stoichiometry

With biosynthesis: acetate/perchlorate = 2.9:1 (mol/mol)

CH3COO- + 0.35ClO4- + 0.26NH4

+ + 0.04H+→

0.26C5H7O2N (cells) + 0.7HCO3-+ 0.35Cl- +0.78H2O

Without biosynthesis: acetate/perchlorate = 1:1 (mol/mol)CH3COO- + ClO4

- → 2HCO3- + H+ + Cl-

Epifluorescent micrograph (1000 ×) of KJ stained with acridine orange





Acetate-Fed Sand Packed-bed Column

Experiments

Sampling from the side ports of the column

Acetate-fed Bioreactors

• Columns: 14 cm or 28 cm long.

• Mixed cultures: enriched on perchlorate (activated sludge samples)

• Feed: Artificial groundwater (AGW) containing 20 mg/L perchlorate and nutrients

Mixed cultures(14 cm sand column)

Critical EBCT was 34 minutes.

1

10

100

1000

20 30 40 50 60 70

EBCT (min)

Efflu

ent P

erch

lora

te (u

g/L)

series 1series 2

Detection Limit

From: Kim and Logan (2000)

1

10

100

1000

10000

100000

0 10 20 30 40 50Time (day)

Perc

hlor

ate

(ug/

L)

0

50

100

150

200

250

EBC

T (m

in)

InfluentEffluentEBCT

Mixed cultures(28 cm sand column)

Critical EBCT was ~40 minutes.

Startup






Acetate-fed Bioreactors

• Columns: 14 cm or 28 cm long.

• Pure culture: Dechlorosoma sp. KJ

• Feed: Artificial groundwater (AGW) containing 20 mg/L perchlorate and nutrients; water from the City of Redlands

Pure culture: KJ(28 cm-sand column)

0.001

0.01

0.1

1

1 10 100 1000

Empty Bed Contact Time, EBCT (min)

Perc

hlor

ate

(ppm

)

<--Startup-->Minimum EBCT2.1 min

Critical EBCT was ~2.1 minutes.



0.001

0.01

0.1

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0 10 20 30 40 50 60 70

Time (day)

Perc

hlor

ate

(mg/

L)

0

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40

6080

100

120

140

EBC

T (m

in)Effluent

Influent

EBCT

As EBCT decreased:

Perchlorate was non-detectable for 84% of samples (n=147)


80

85

90

95

100

0.001 0.01 0.1 1perchlorate (mg/L)

% o

f sam

ples

less

than

th

e co

ncen

trat

ion


Reliability: After start up, perchlorate removal was stable at:

95% of time: <23 ppb

84% of time: <4 ppb


Redlands WaterPure culture KJ, (28 cm-sand column)


1.0

10.0

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0 20 40 60 80 100 120 140 160 180Time (day)

Perc

hlor

ate

(ug/

L)

0

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30

40

50

60

70

Det

entio

n Ti

me

(min

)

Redlands Effluent Redlands Influent

Detention Time

Detention Limit (4ug/L)

Redland Water

Perchlorate is completely removed using Redlands water (containing NO3

-) for detention time >11 min.

Detention time: 9 min


1.0

10.0

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1000.0

0 20 40 60 80 100 120 140 160 180Time (day)

Per

chlo

rate

(ug

/L)

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70

Det

entio

n Ti

me

(min

)

Control Effluent Control Influent

Detention Time


Control w/o NitratePure culture KJ, (28 cm-sand column)

Perchlorate is completely removed using artifical groundwater (no NO3

-) for detention time >8 min.

Detention time: 27 min

Detention time: 7.4 min


1.0

10.0

100.0

1000.0

0 20 40 60 80 100 120 140 160 180Time (day)

Perc

hlor

ate

(ug/

L)

Redlands Effluent Control Effluent

Redlands Influent Control Influent


Redland Water

Effect of NitratePure culture KJ, (28 cm-sand column)

Perchlorate degradation is more stable for groundwater with nitrate.





Problem...

When a reactor is shut down (either for maintenanceor due to mechanical problems), perchloraterespiring bacteria can be exposed to highconcentrations of DO.

High DO concentrations inhibit perchloratereduction. Our question was:

How long can an anaerobic culture be exposed tooxygen before losing the ability to reduceperchlorate?

Oxygen Exposure

0150300450600

0 30 60 90 120 150

Time (hr)

[ClO

4-].

(pp

m)

Anaerobic Control Ana-aer Control

Oxygen Exposure

0150300450600

0 30 60 90 120 150

Time (hr)

[ClO

4-].

(pp

m)

Anaerobic Control Ana-aer Control T=12h

Oxygen Exposure

0150300450600

0 30 60 90 120 150

Time (hr)

[ClO

4-].

(pp

m)

Anaerobic Control Ana-aer Control T=1h T=2h

T=4h T=8h T=12h

Oxygen Exposure

0

150

300450

600

0 30 60 90 120 150Time (hr)

[ClO

4-].

(pp

m)

Anaerobic Control Ana-aer Control T=1h T=2hT=4h T=8h T=12h T=22hT=28h T=32h

Solution...

• There should be no problems for shortexposure times (<12 h) of the bacteriato oxygen.

• Avoid oxygen exposure for longer shutdown times Do not drain the reactor.This will keep air out of the system tomaintain the culture under anaerobiccondition.

• Stop feeding the electron donor (thiswill lower pH) and nutrients (N, P).

CONCLUSIONS• It is possible to obtain high perchlorate removal rates,

with EBCT as low as 2.1 min, in packed bed reactors inoculated with pure culture (KJ). Mixed cultures require longer times (>11 min).

• Perchlorate removal was achieved in the laboratory using groundwater from the Redlands site, containing dissolved oxygen and nitrate.

• Perchlorate reduction by strain KJ can be recovered after exposure to oxygen for less than 12 hours.

ACKNOWLEDGMENTS

Funding AWWARF: Project manager Frank Blaha

National Science Foundation

Penn State University

Thank You!Q’s and A’s

Perchlorate Degradation in Acetate-fed Bioreactors · Perchlorate Degradation in Acetate-fed Bioreactors Yanguang Song Kijung Kim, Bruce Logan Department of Civil & Environ. Eng.

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