1.
Field application of stable isotopes and microbial techniques to an organic contaminant plume
Integrated characterization of the natural attenuation (NA) of a PCE plume after thermal source zone remediation
Mette M. Broholm1, Alice Badin2, Carsten S. Jacobsen3, Jordi Palau2, Phil Dennis4, Niels Just4, and Daniel Hunkeler2
1DTU Environment, 2University of Neuchatel, 3AArhus University, 4SiREM, 5Region of Southern Denmark

2. Rødekro. Investigation
Rødekro. Investigation
Former central dry cleaning facility ”Clip Rens” in Rødekro, Denmark
PCE DNAPL in source area, plume in sand aquifer
Degradation products TCE, DCE and a tiny bit of VC
Thermal remediation of source area in 2006 (8 years)
Studies in NA? - and 2014 effect in plume? Change in risk?
Tools:
Specific degradation products, isotope fractionation and dual isotope
Redox, specific degraders and activity, sequencing

3. Reductive dechlorination of PCE
Halorespiring bacteria: Dehalobacter (Dhb) Dehalospirillum Desulfitobacterium Desulfuromonas Dehalococcoides (Dhc)
Some types of Dehalococcoides (Dhc) Dehalogenimonas (Dhg) (tDCE)
Dhc with vinylchloride reductase gene (vcrA, bvcA), Dhg
Anaerobic conditions and hydrogen/organic donor
Specific degraders
Risk of cDCE and/or VC accumulation, if Dhc or Dhc with vcrA/bvcA are not present

4. Stable isotope fractionation for sequential reductive dechlorination
School example:
δ13C increase, as compound is degraded
End product δ13C as δ13C0 of mother compound
Intermediates:
initial formation δ13C < δ13C0
Degradation δ13C > δ13C0
Documentation δ13C > δ13C0
Isotope balance
Hunkeler et al. 1999

5. Flowline and plume NA 2006

6. Natural attenuation. Line of evidence 2007
Degradation products:
PCE and TCE degraded
Isotopic fractionation:
VC and cDCE as well as PCE and TCE is degraded
Complete dechlorination
cDCE degradation is the rate-limiting step
Specific degraders and Redox:
Redox not optimal
Low Dhc, no vcr detected
Dhc active?
Slow RD? of cDCE
VC degradation pathway?
Dhc detected, not quantifiable

7. Effect of source remediation on concentrations
PCE in source area is 2 orders of magnitude lower
In the upper part of the aquifer a significant decrease in concentrations is observed to >750 m
Centrally in the plume (1050 m) DCE and VC has decreased, no ethene
DCE continues to spread in downgradient direction

9. Effect on redox conditions
O2 nd in 2010
mix
Increased NVOC
Local reduction of redox conditions
In m change in redox
Pyrite oxidation lacking
More reduced
Methane indicate reduction
High NVOC in 2010 larger area

10. Activity of bacteria: Messenger RNA
Activity of bacteria: Messenger RNA
mRNA is a transcript of DNA
mRNA carries genetic information from DNA to the ribosome
Proteins are synthetized in the ribosome
mRNA is used multiple times
The protein will be synthetized until mRNA has been degraded by Rnase
Dhc, vcr analysis: DNA extraction and real time PCR
Activity: mRNA extraction, reverse transcriptase, real time PCR
Curtesy of Jacob Bælum, GEUS 10

11. Effect on specific degraders and activity (mRNA/DNA)
Dhc: factor 100 difference on methods
2007
Dhc more widespread in 2014, generally low level (F4-3 exception)
VcrA and bvcA (VC reductase genes) not detected
Dhc activity m and 1050 m

12. Sequencing. Bacterial composition
>5496 OTU (operational taxonomic unit)
kingdom_phylum_class _order_family_genus _species
Reported:
30 largest OTU – heat map
Comparability tree
Search for specific degraders - genus

13. Sequencing. Chlorinated ethene degraders
~5500 OTU. Search for chlorinated ethene degraders
Potential degradation
Complete RD
RD of PCE+TCE
Ox VC+DCE
Genus
Dhc
Dhg
f_Dehalococ-coidetes* (m.Dhc&Dhg)
Dhb
Dehalo-bacter Gb
Geo-bacter
Clostri-dium
Aceto-bacterium
Desulfo-vibrio
Sporo-musa
Methano-sarcina Po
Polaro-monas
Myco-bacterium
Nocar-dioides
Methylo-sinus
B23-2
5.21·104
1.66·106
3.04·106
1.30·105 Nd
7.42·105
2.60·104
2.61·104
B23-3
1.63·105
3.72·106
9.18·104
2.04·105
1.33·105
4.38·105
2.04·104
B34-2
7.05·104
8.28·105
4.72·106
2.64·105
9.70·105
4.58·105
1.76·105
8.81·104
3.88·105
2.52·104
3.52·104
B34-3
9.28·104
3.31·105
1.78·106
3.18·105
2.66·105
6.65·104
5.70·105
B34-4
3.74·105
2.50·106
1.50·104
1.95·105
B34-6
4.61·105
9.99·105
5.77·104
3.85·104
B58-2
8.34·104
5.17·105
3.33·104
1.12·106
1.76·104
B58-6
1.06·105
6.60·104
9.78·105
9.24·104
5.28·104
2.64·104
B61-1
4.40·104
5.50·103
6.05·104
1.26·105
1.65·104
7,.2·105
B61-3
5.90·103
4.66·105
1.77·104
4.60·105
5.90·104
B74-3
8.30·104
2.77·104
1.80·105
1.94·105
8.29·104
1.24·104
1.81·106
2.76·104
1.11·106
kontrol
1.24·106
5.52·104
3.36·106
2 Complete RD (Dhc, Dhg). Dehalogenimonas (Dhg), vc reductase gene: closely related to Dhc, complete dechlorination of DCE
7 RD of PCE+TCE
4 Aerobic oxidation of VC, 1 VC&cDCE. Polaromonas.
Very complex composition of bacteria in many samples suggest several different concurrent degradation processes

14. Effect on degradation. Isotopic fractionation
Few data
Few data
PCE: Little left. Degradation unchanged. Very little TCE.
DCE: Significant change m Degradation occurs.
DCE: 1050 m Degradation has increased (400 m before).
VC: Few detect. Only 1 with documentation for degradation.

15. Dual isotope example: stable C and Cl RD
Example - reductive dechlorination
C stays in degradation product
Cl is separated
Isotope balance for Cl demands
Isotope data for Cl
No other source of Cl
Lin. corelation δ13C : δ37Cl
Depend on degradation proces and –mechanism
Depend on bakteria type
Depend on other conditions?
Hunkeler et al. 2009

16. Dual isotope: Example PCE: RD biotic to TCE 2,7 RD biotic to DCE 0,7
Badin et al. 2014

17. Dual stable isotopes – C and Cl
Linear correlation – could indicate dechlorination?
Correlation coefficient indicative of degradation process
PCE (3):
Biotic RD
PCE→TCE, Sulfurospirillum: 2.7
PCE→DCE, Sulfuroospirillum: 0.7
PCE→DCE, Desulfitobacterium:2.5
TCE (2,7):
Biotic RD: 4.8;
Abiotic RD: 5.2
DCE (2,0) > 750 m:
Abiotic RD: 3.0; 3.1
Biotic RD, Dhc-vcr: 11.6
Aerobic biotic oxidation: 32.3

18. Rødekro. Summary Near treated source area: PCE – decreased 100 times
NVOC release has led to RD
cDCE degradation documented
Little VC produced
Dhc activity, no vcr
RD of PCE to DCE
Upper: cDCE/VC aerobe oxidat.
Around m:
Redox conditions changed
FeS formed, green rust?, pyrite?
cDCE degradation increased
No VC accumulation
Dhc activity, Mixed degraders
cDCE abiotic and biotic degr.
>1500 m:
Continued spreading of cDCE

19. Rødekro. Conclusion and perspective
Mass much smaller and decreasing
Reduced conditions induced by NVOC release
Degradation increased
Mixed degradation processes
Risk decreased (not eliminated)
Future evolution in conditions and degradation?
Stimulation potential revealed for:
Biotic (ERD) degradation
Biotically induced (FeS?, green rust, pyrite) abiotic degradation (ISCR)

20. NA documentation methods. Summary
Isotope fractionation
Very strong tool
Documentation of cDCE and VC degradation
Important for risk assessment
Dual isotope
New potentially strong tool
Continued research for better process understanding and pathway identification
Specific degraders and genes
Good tool
Need for expansion to more degraders – incl. Dhg
Activity of specific degraders
New strong tool
Which degraders are active?
Use with care
Sequencing
New interesting tool
Continued research will strengthen our microbial and process understanding

21. Acknowledgements
The Region of Southern Denmark for funding of sampling, analysis and reporting
The European Commission, Marie Curie Actions Project No for funding of the PhD project of Alice Badin and stable isotope and microbial analysis
University of Toronto assisted with interpretation of sequencing results
Technicians at DTU Environment, Region of Southern Denmark, and GEUS assisted with field and laboratory work
References: Broholm et al. 2015,
Badin et al in prep.