Assessment of In Situ Biodegradation Potential of Benzene Using Stable Isotope

Probing (SIP)J.L. Busch-Harris, K.L. Sublette, Eleanor Jennings

and Ken Roberts Center for Applied Biogeosciences

University of Tulsa D.C. White and Aaron PeacockCenter for Biomarker Analysis

University of Tennessee Greg Davis -cba

Microbial Insights, Inc. William E. Holmes

School of Natural Resources University of Michigan

Ravi Kolhatkar and Xiaomin Yang Atlantic Richfield (a BP affiliated company)

A New Tool: Bio-Sep Bio-traps

• Demonstrate biodegradation of benzene and other hydrocarbons by indigenous microbes in – Aquifers

– Surface waters

– Sediments

– Soil • Characterize the microbial ecology of

benzene biodegradation

Bio-Sep Beads

• 3-4 mm in diameter • 25 % Nomex, 75% PAC • 74% porosity • 600 m2 of surface area/g• Surrounded by ultrafiltration-like membrane

with 1-10 micron holes • Autoclavable• Cleaned of fossil biomarkers by heating to

300 oC

SEM of Bio-Sep Beads

Surface

Interior

Biofilms Form Rapidly in Bio-Sep Beads

• High surface area

• Low shear • Concentration of

nutrients by PAC • Rapid formation of pre­

conditioning films

Live-dead stain of biofilm in Bio-Sep bead

What Do Bio-Sep Beads Collect?

• Bacteria have to enter the bead and growthere to be detected

• Slow-growing, non-growing, or dormant organisms are less likely to be collected in thebeads unless the beads offer them a significant advantage

• Organisms collected in the beads are more likely to be the more active members of thesampled community

A Simple Example

Groundwater Beads

Blue = inactive

Red = active

Total Branched Fatty Acids 6

5

4

3

2

1

0

Large O2 Infusion

Groundwater

Bio-Sep Bio-trapsPerc

ent o

f Tot

al P

LFA

0 20 40 60 80 100 120 140 160 180 200 220 240 260 Time Since Initiation of Sulfate Injection

Applications Bio-Sep Bead Samplers• Drinking water systems

– Measurable biofilms in 24 hrs – Detecting leaks in distribution

lines– Pathogen tracking– Trouble shooting

• 70-m deep storage tank on offshore platform – Microbial ecology with depth – SRB and sulfide-oxidizers

• Aqueous phase of a solvent extraction system (pH < 2) – Cause of biological fouling

• Contaminated aquifers – PCE – Hydrocarbons – MTBE

• Stream monitoring – Source tracking for coliforms

Hydrocarbon Biodegradation Assessment Using In Situ Microcosms

Bio-Sep Bio-trap

1. Load with hydrocarbon (13C-labeled and/or non-labeled)

2. Expose under in situ conditions for 4-5 weeks

3. Analyze biomass, search biomarkers for the 13C-labeling

Vapor Phase Loading of Hydrocarbon

Dry Bio-Sep beads

vacuum

valve

vacuum

p ~ 60mbar valve closed

pump

open

pump

hydrocarbon

Does Benzene Leach From the Beads?

Leaching experiment:

• 25-mL VOA vials with 50 Bio-Sep beads in each, set up in triplicate

• 10 mM sodium azide to prevent microbial growth

• Solution replaced after each sampling to avoid vapor space

• Samples analyzed quantitatively by GC-MS

Benzene Leaching in Sterile WaterAvg. benzene

Incubation concentration in time leachate (mg/L) Day 0 9.93E-04 Day 1 9.70E-04 Day 2 9.76E-04 Day 3 9.82E-04 Day 4 9.78E-04 Day 5 9.75E-04 Day 6 9.74E-04 Day 7 9.74E-04 Day 8 BDL Day 9 9.74E-04 Day 10 9.74E-04 Day 11 9.74E-04 Day 12 9.75E-04 Day 13 BDL Day 14 BDL Day 15 9.78E-04

Incubation Benzene extracted time from beads (mg/bead ±

std. dev) N=3 t0 1.05 ± 0.04 Day 15 0.99 ± 0.02

Day 30 0.97 ± 0.03

Benzene does not easily leach from the beads!

TGA analysis of benzene-loaded Bio-Sep beads

TGA Analysis of Benzene-loaded Bio-Sep Beads

0.8400

0.8600

0.8800

0.9000

0.9200

0.9400

0.9600

0.9800

1.0000

1.0200

Nor

mal

ized

Wei

ght

Without Benzene With Benzene

0 50 100 150 200 250 300 350 400 450

Temperature (C)

Biomarker of Choice: PLFA

Lipids with 13C incorporated into the phospholipid bilayer indicate utilization of the 13C-labeled compound and incorporation into biomass

Two Case Studies

• BTEX impacted aquifer in Germany

• LUST site in southern California

Germany

5660800 0 100

Saf Zz 32/02

200 300 400m 750 700 [Benzene] up to 850 mg/L650 600 550 500

5660700

5660600 450 400 350 Aquifer is virtually3005660500 free of dissolved250 200 oxygen 150

5660400 100 50 0

4514300 4514400 4514500 4514600 4514700 4514800

groundwater flow

depth profile of well Saf Zz 32/02

Wall Screen

Traps with Beads (in situ microcosm)

Weight(coated or Teflon)

Nylon line suspending Trap assembly

-17 m-56 ft

Exposure of ‘In Situ Microcosms’

Analysis of Microcosm BTEX Loading After 4 Weeks In situ Exposure

13C abundance Residual Loss (%) At% hydrocarbon (mg/trap)

natural benzene 0.1 7.9 ± 0.1 82 13C6-benzene 98.0 7.9 ± 0.1 82 natural toluene 0.1 8.0 ± 0.7 85 13C1-toluene 14.0 8.9 ± 0.7 84 blank 0.1 0.23 benzene N/A

¾ isotopic composition of contaminants unchanged • no significant exchange with aquifer • no crosstalk between traps

¾ 80 % decrease in contaminant concentration(biodegradation)

Analysis of Microcosm Biomass After In Situ Exposure

rela

tive

abun

danc

e (c

ps) 1200000

1100000

1000000

900000

800000

700000

600000

500000

400000

300000

200000

100000

6.00 12.0014.0016.0018.0020.0022.0024.0026.0028.0030.00

21:0 (int. Std.)

citric acid

14:0

i-15:0

a-15:0 15:0

16:1ω7c

i-16:0

16:0 18:1ω9c

phtalate 18:1ω7c

18:0 10me 18:010me

16:0

8.00 10.00

retention time (min)

Total lipid fatty acids profile from 13C-toluene loaded microcosm or bio-

trap

Analysis of Microcosm Biomass After

rela

tive

abun

danc

e (c

ps)

In Situ Exposure

Toluene 13C label detected in

1200000

1100000

1000000

900000

800000

700000

600000

500000

400000

300000

200000

100000

6.00 12.0014.0016.0018.0020.0022.0024.0026.0028.0030.00

21:0 (int. std.)

7556 ‰

acid? 805 ‰ 14:0

60.3 ‰ i-15:0

15.5 ‰ a-15:0 7946 ‰

15:0

13355 ‰ 16:1ω7c

i-16:0

6283 ‰ 16:0

16.0 ‰ 18:1ω9c

phtalate 9684 ‰ 18:1ω7c

92.9 ‰ 18:0

10me 18:010me 16:0

total lipid fatty acids

8.00 10.00

- 29.6 ‰

citric - 24.1 ‰

retention time (min)

Stabile isotope analysis proves toluene biodegradation and growth of microbial biomass

under in situ conditions

8.51 13.83

18.91

21.34

29.92

Analysis of Microcosm Biomass After In Situ Exposure

Total lipid fatty acids profile from13C benzene loaded bio-trap

200000

400000

600000

800000

1000000

1200000

1400000

1600000

23.59

-24.6 ‰ 12:0

21:0 (int. Std.)

14:0

16:1ω9c

16:0 18:1ω9c

18:0

phtalate

rela

tive

abun

danc

e (c

ps) - 28.8 ‰

6.00 8.00 10.0012.0014.0016.0018.0020.0022.0024.0026.0028.00

retention time (min)

8.51 13.83

18.91

21.34

29.92

Analyze Microcosms Biomass After In Situ Exposure

Benzene 13C label detected in total lipid fatty acids

200000

400000

600000

800000

1000000

1200000

1400000

1600000

23.59

-24.6 ‰ 12:0

21:0 (int. std.)

46 ‰ 14:0

13414 ‰ 16:1ω7c

998 ‰ 16:0

191 ‰ 18:1ω9c

264 ‰ 18:0

phtalate

rela

tive

abun

danc

e (c

ps) - 28.8 ‰

6.00 8.00 10.0012.0014.0016.0018.0020.0022.0024.0026.0028.00

retention time (min)

Stabile isotope analysis proves benzene biodegradation and growth of microbial biomass

under in situ conditions

Polar Lipid Fatty Acid (PLFA) Profiles of Viable Microorganisms Enriched in Bio-traps

31.38

550000

500000

32.05

450000toluene 400000

350000baited 300000

250000

200000

150000

37.29

100000same PLFAs, 37.14 31.4950000 39.48

30.32 35.02

24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00different TIC: ZEITZ_1B.D

900000abundance 850000

800000 14:0 16:1ω7c 16:0 18:1ω9c 18:1ω7c 18:0 fatty acid750000

70000037.13

650000

600000

550000

500000

450000

400000

350000

benzene 31.37

300000

250000

baited 32.06200000

150000

100000

37.29

50000 26.08 37.94

24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00

Time >

Yucaipa, CA

• Site of gasoline LUST• Is there intrinsic bioremediation of benzene at this

site? • Will nitrate stimulate the intrinsic bioremediation of

benzene? • Bio-Sep in situ microcosms (non-baited and nitrate

baited), both with 13C-benzene, installed in triplicateinto MW20 suspended 30 cm below water table withfloat; incubated for 45 days

• Specific activity of 13C-benzene only about 8%

Geochemistry of MW20 and Unimpacted Background Well

Parameter Background well MW20

Benzene ND 6.6 µg/L

TEX ND ND

MTBE ND 1.2 µg/L

pH 7.4 7.5

Nitrate-N 9.4 mg/L 8.6 mg/L

Sulfate 23 mg/L 19 mg/L

Alkalinity 140 mg/L 130 mg/L

TDS 280 mg/L 250 mg/L

Bio-Sep® Tube-in-Tube Nitrate-baited Sampler

Nitrate-baited

beads

Standard Bio-Sep® beads

PFA tubing

Nylon spacer

SEM of Nitrate-baited Bio-Sep Beads

Surface

KNO3 crystals

Interior

Relative Abundances of Phospholipid Fatty Acids Structural Groups and Key Fatty Acids in Non-baited and Nitrate-baited Bio-traps

Phospholipid structural group or fatty acid

Non-baited Bio-traps*

Nitrate-baited Bio-traps*

Terminally branched

1.33 ± 1.12 2.00 ± 0.17

Monoenoics 72.2 ± 2.8 73.9 ± 1.1 Branched Monos 0.1 ± 0.17 0.4 ± 0 Mid-branched Sats 0.6 ± 0.17 0.6 ± 0.17 n-Sats 23.0 ± 1.89 21.5 ± 0.45 16:1ω7c 32.0 ± 3.5 35.5 ± 1.9 16:0 20.5 ± 2.1 19.7 ± 0.70 18:1ω7c 22.3 ± 4.0 24.7 ± 2.5 18:1ω9c 9.7 ± 3.1 5.9 ± 3.6 cy19:0 2.0 ± 0.55 2.0 ± 0.15

*Mean ± std.dev. (n=3)

δ13C Values of Individual Fatty Acids Derived from Phospholipids From the Non-

baited and Nitrate-baited Bio-traps.

Phospholipid fatty acid

Non-baited Bio-traps

Nitrate-baited Bio-traps

16:1ω7c +5699 ± 161* +6095 (n=2)*** 16:0 +5342 ± 240 +5762 (n=2) 18:1ω7c +3514 ± 756 +4037 ± 251 18:1ω9c +754 (n=1)** +1137 (n=1) cy19:0 +1055 (n=1)

The δ 13C of natural benzene is about -26 o/oo. Values more positive reflect increasing enrichment with 13C. * Mean ± std. dev. (n=3) **One observation *** Avg. of two observations

12 %

13C

-ben

zene

13C-benzene Enrichment in Yucaipa Bio-traps

11

10

9

8

7 Mean Mean±SE Mean±SD Outliers 6

Pre-deployment Post-incubation Nitrate Extremes Post-incubation Controls

Yucaipa Conclusions

• Bacteria indigenous to the aquifer at Yucaipa are capable of biodegradation of benzeneunder aquifer conditions.

• No evidence that nitrate stimulated benzene biodegradation or had a significant effect onthe subsurface microbial community structure.

• Incorporation of 13C-benzene into PLFA was easily detected with low specific activity inloaded beads

What’s Next?

• Deployment of in situ microcosms in sediments and soil

• 13C-fuel oxygenates

• 13C incorporation into DNA – from labeled 16S rDNA we can potentially identify degraders

Acknowledgement

This work was funded by

the Integrated Petroleum Environmental Consortium (IPEC)

and Atlantic Richfield, a BP affiliated company