BIOGEOCHEMICAL MEASUREMENTS TO CHARACTERIZE DUAL ANAEROBIC AND

AEROBIC BIODEGRADATION PROCESSES FOR REMEDIATION OF CHLORINATED

BENZENES IN A WETLAND

Michelle M. Lorah and

Jessica Teunis, USGS, Baltimore, MD

Ethane

112TCA

12DCA

CA

1122- TeCA

TCE

12DCE

VC

Ethene

PCE

CT

CF

MeCl

Methane

CO2

124TCB,

12DCB,

Chlorobenzene *

123TCB

13DCB, 14DCB

Benzene *

CO2, CH4

??

Biodegradation Pathways

Trichlorobenzenes *

Dichlorobenzenes *

Aerobic to CO2

* Parent contaminant at site

Anaerobic reductive dechlorination • rate decreases

with decreasing number of chlorines

Oxidation reaction pathways • typically aerobic • rate decreases

with increasing number of chlorines

Conceptual model for chlorinated solvent contamination in wetland (modified from Lorah et al., 2005)

dissolved plumes

DNAPL

Aerobic micro-zones around roots

Fe2+ S2-

NH3

CH4

Stottmeister et al.(2003) Biotech.Advances

O2

Changing Paradigm

Previous paradigm for chlorinated VOCs: • Aerobic oxidation

requires measurable oxygen

• Anaerobic oxidation responsible for losses of lower VOCs at anaerobic plume fringes

Approach to evaluate biodegradation In situ microcosms with Bio-

Traps (Microbial Insights) Stable isotope probing (13C-

labeled 14DCB, CB, B) Analysis of species and

functional genes for aerobic and anaerobic biodegradation (QuantArray)

Evaluate biodegradation processes in biofilms Upflow fixed film bioreactors,

static bed Robust- can change redox

conditions and other parameters more reliably than in batch microcosms

Enrichment of aerobic degrading bacteria from anaerobic water

Bioreactor polypropylene support matrix for biofilms

GEO anions, VFAs

COC VOCs, redox

MICRO- Bio-sep beads

Sample microbes Seed with culture Load with 13C-VOCs

Amendments donor

Red Lion Creek

Standard Chlorine of Delaware Wetland Site

0.0

1.0

2.0

3.0

MNA Lactate WBC-2

Conc

entr

atio

n (m

g/L)

Fe2+

Sulfide

Methane

Sulfate

8.6

Site 107 Redox Three treatment types:

• MNA, monitored natural attenuation (no amendments)

• Lactate, biostimulated with lactate +chitin

• WBC-2, bioaugmented

ISM

Results:

0.00

0.20

0.40

0.60

0.80

1.00

1.20

MNA Lactate-Chitin WBC-2

Conc

entr

atio

n (µ

mol

/L) Site DP6 14DCB

13DCB12DCBCBBenzene

•Complete degradation of DCBs evident in WBC-2 treatment in standard ISMs

•13C-labeled ISMs showed complete degradation of monochloro-benzene in MNA and WBC-2

0

0.2

0.4

0.6

0.8

Pre-Deployment WBC-2 13C MNA 13C

Conc

entr

atio

n (m

g/bd

)

13C Loss (Site LP6)

66 % 69 %

WBC-2 13C MNA 13C k (per day) 0.019 0.021

half life (days) 37.1 33.5

2010 Bio-Traps: 13C-labeled Chloro-benzene

-500

0

500

1,000

1,500

2,000

2,500

3,000

Background MNA Lactate/Chitin WBC-2

DIC

Del

(‰)

13C Utilized for CO2, 13C Chlorobenzene

PDB-01

PDB-04

PDB-07

PDB-10 (2009)

-40

-20

0

20

40

60

80

Background MNA Lactate/Chitin WBC-2

PLF

A D

el (‰

)

13C Utilized for Biomass, 13C Chlorobenzene PDB-01PDB-04PDB-07PDB-10 (2009)

8 (NW) 104 (NW) 107 (NE) 6 (NE)

8 (NW) 104 (NW) 107 (NE) 6 (NE)

QuantArray Microbial Analysis- Anaerobic

Reductive dechlorination: DHC , Dehalococcoides spp. TCE, tceA reductase VCR, vinyl chloride reductase BV1 , vinyl chloride reductase DHBt, Dehalobacter spp. DHG, Dehalogenimonas spp.

J < <

1.0E+01

1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

1.0E+08

DHC tceA vcr BVC DHBt DHG BCR bssA assA

Cells

/mL

MNA LAC WBC-2

BTEX, PAHs and alkanes: BCR, Benzoyl coenzyme A reductase bssA, benzylsuccinate synthase assA, alkylsuccinate synthase

-------Reductive dechlorination-------

QuantArray Microbial Analysis- Aerobic

pMMO, particulate methane monooxygenase sMMO, soluble methane monooxygenase TCBO, trichlorobenzene dioxygenase RDEG, toluene monooxygenase 2 RMO, toluene monooxygenase

< < < 1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

1.0E+08

pMMO sMMO TCBO RDEG RMO PHE EDO PM1 ALKB

Cells

/mL

MNA LAC WBC-2

NA NA

PHE, phenol hydroxylase EDO, ethylbenzene/isopropyl- benzene dioxygenase PM1, Methylibium petroliphilum PM1 ALKB, alkane monooxygenase

Bioreactors 2012-13

Native vs. bioaugmented Three vessels (1 liter

each) in each bioreactor Native microbe seeded

bioreactor (C) Anaerobic

dechlorinating culture WBC-2 seeded in two bioreactors (D, E)

Varied from anaerobic to aerobic conditions in C

SCD Bioreactors- ORP (median residence time~ 40 hr; pH~7.0-7.5)

-500

-400

-300

-200

-100

0

100

200

3003/

12

3/22 4/

1

4/11

4/21 5/

1

5/11

5/21

5/31

6/10

6/20

6/30

7/10

7/20

7/30 8/

9

8/19

8/29 9/

8

9/18

9/28

10/8

10/1

8

10/2

8

11/7

OR

P, in

mill

ivol

ts

2012

Native Bioreactor, C CTankC2C4C6CWaste

aerobic 6/20-8/25

added buffer

aerobic 10/10-10/20

-500

-400

-300

-200

-100

0

100

200

300

3/12

3/22 4/

1

4/11

4/21 5/

1

5/11

5/21

5/31

6/10

6/20

6/30

7/10

7/20

7/30 8/

9

8/19

8/29 9/

8

9/18

9/28

10/8

10/1

8

10/2

8

11/7

OR

P, in

mill

ivol

ts

2012

WBC-2 Bioreactor, D DTankD2D4D6DWaste

stopped all donor addition,10/5/12

aerobic 10/10-10/20

0

500

1,000

1,500

2,000

2,500

3,000

3/12

3/22 4/

1

4/11

4/21 5/

1

5/11

5/21

5/31

6/10

6/20

6/30

7/10

7/20

7/30 8/

9

8/19

8/29 9/

8

9/18

9/28

10/8

10/1

8

10/2

8

11/7To

tal C

Bs

+Ben

zene

, in

µg/

L

Native (C ) Bioreactor aerobic 6/20-8/25

added buffer + nutrients 7/27/12

aerobic 10/10-10/20

Inflow Tank

Waste

0

500

1,000

1,500

2,000

2,500

3,000

3/12

3/22 4/

1

4/11

4/21 5/

1

5/11

5/21

5/31

6/10

6/20

6/30

7/10

7/20

7/30 8/

9

8/19

8/29 9/

8

9/18

9/28

10/8

0/18

0/28 11/7

Tota

l CB

s +B

enze

ne ,

in µ

g/L

WBC-2 (D) Bioreactor aerobic 10/10-10/20

Inflow Tank

Waste

SCD Bioreactors- Total CBs+Benzene

SCD Bioreactors- Aerobic vs. Anaerobic

• occurrence of aerobic and anaerobic degradation by native bacteria • aerobic degradation much faster than anaerobic biodegradation • WBC-2 able to efficiently degrade chlorinated benzenes and benzene anaerobically • accumulation of daughter products not evident

0.0

2.0

4.0

6.0

8.0

0 10 20 30 40

Conc

entr

atio

n (u

mol

/L)

Time (hrs)

C Reactor 8/13/12 aerobic

124TCB

123TCB

14DCB

13DCB

12DCB

CB

B

0.0

2.0

4.0

6.0

8.0

0 10 20 30 40Time (hrs)

D Reactor 8/10/12 anaerobic

124TCB

123TCB

14DCB

13DCB

12DCB

CB

B

Enrichment of Aerobic Native Culture

0

2,000

4,000

6,000

8,000

0 20 40 60

Conc

netr

atio

n (u

g/L)

Time (hrs)

15BN-B 124TCB

12DCB

Chlorobenzene

8 L of wetland groundwater from 15B filtered (0.2 µm)

Filtrate placed in tryptone-yeast extract broth (Difco ISP Medium 1)

Spiked with CB, 12DCB, 14DCB, and 124TCB

Incubated aerobically on shaker First order 124TCB 12DCB CB

k (per hr) 0.051 0.071 0.15 half life (hrs) 13.6 9.8 4.6

r2 .972 .999 .968

Conclusion

Wetland Sed, Sand, Chitin, dual biofilm-GAC

Aquifer (~40 ft thick)

• Both anaerobic (WBC-2 and native microbial biofilms) and aerobic (native) degradation processes are effective – Aerobic degradation

fastest but logistically difficult for in situ treatment of wetland sediments and groundwater

– Concentrations as high as 100 mg/L successfully tested

• Plan to utilize both cultures in a reactive mat with biofilms on GAC. Column tests are underway