06-12-2018 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

Rødekro. Investigation 06-12-2018 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 2006-7 NA? - and 2014 effect in plume? Change in risk? Tools: Specific degradation products, isotope fractionation and dual isotope Redox, specific degraders and activity, sequencing

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

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

Flowline and plume NA 2006

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

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

Effect on redox conditions O2 nd in 2010 mix Increased NVOC Local reduction of redox conditions In 1050-1450 m change in redox Pyrite oxidation lacking More reduced Methane indicate reduction High NVOC in 2010 larger area

Activity of bacteria: Messenger RNA 06-12-2018 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

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 0-350 m and 1050 m

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

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

Effect on degradation. Isotopic fractionation Few data Few data PCE: Little left. Degradation unchanged. Very little TCE. DCE: Significant change. 0-350 m Degradation occurs. DCE: 1050 m Degradation has increased (400 m before). VC: Few detect. Only 1 with documentation for degradation.

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

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

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; 3.4-3.8 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

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 1000-1400 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

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)

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

Acknowledgements The Region of Southern Denmark for funding of sampling, analysis and reporting The European Commission, Marie Curie Actions Project No. 265063 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, www.sara.env.dtu.dk Badin et al. 2015 in prep.