biogeochemical measurements to characterize …...wbc-2. concentration (µ mol /l) site dp6 14dcb....
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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
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