Advanced Tools for In Situ Remediation. Workshop

Transcription

1 Advanced Tools for In Situ Remediation Workshop June 22, 2011 Co-Sponsored by:

2 8:00 to 11:00 Registration/Check in Advanced Tools for In Situ Remediation Workshop Schedule June 22 and 23, 2011 RAM Center June 22, :00 Overview for Day Kevin Lund, MDEQ Remediation Division 11:15 12:30 The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation Mike Hyman, PhD., North Carolina State University 12:30 1:30 Lunch 1:30 2:15 Introduction to Compound Specific Isotope Analysis (CSIA) Robert Pirkle, PhD., Microseeps, Inc. 2:15 2:45 Applications of Molecular Biological Tools (MBTs) Greg Davis, Microbial Insights 2:45 3:00 Break sponsored by Adventus Americas Inc and MATECO Drilling 3:00 3:45 Applications of Bio Trap samplers and Stable Isotope Probing Dr. Kerry Sublette, University of Tulsa 3:45 4:30 Isotope Applications for In Situ Remediation Case Studies for Chlorinated Solvents, Petroleum Hydrocarbons, and Inorganic Compounds Julie K. Sueker, Ph.D, ARCADIS U.S., Inc 4:30 5:00 Panel Discussion, Question & Answer Period 5:30 Dinner Grilled New York Strip Steak, all you can eat crab legs, spinach salad, homemade dinner rolls, baked potatoes, fresh steamed vegetables & double chocolate Traverse City cherry brownies. 5:30 To? Happy Hour Sponsored By CES family of companies Orin/EOS/iSOC/Chemviron & Catskill

3 The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation Mike Hyman, PhD., North Carolina State University

4 The microbiology of chlorinated solvent and fuel oxygenate biodegradation Dr. Mike Hyman Department of Microbiology North Carolina State University It s a diverse microbial world out there! Eu(bacteria) and Archaea = prokaryotes Plant, animals, fungi, protozoa etc = eukaryotes Eubacteria, archaea and fungi = microorganisms Total number of microbial species 1 million 1 billion The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation

5 Prokaryotes: The unseen majority ~20,000 strains/species of prokaryotes in 1 g topsoil Prokaryotes in 5 g topsoil = ~total human population of Earth Total prokaryote population of Earth = ~5 x cells C in prokaryote biomass =~ C in plant biomass >90% of prokaryotes are in subsurfaces below 30 feet Ref #1: Whitman et al. (1998) Proc. Nat Acad. Sci. USA 95: Prokaryote structure and function Lipids DNA Polysaccharides: (cell wall) ~50,000/cell ~10 types Lipids: (membrane) ~25,000,000/cell, ~10 types Nucleic acids (cytoplasm) RNA ~250,000/cell, ~600 types DNA ~2 molecules/cell, 1 type Proteins: (cytoplasm +) ~25,000,000/cell, ~2,500 types Least specific/varied Most specific /varied The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation

6 Biodegradation in a nutshell Something to eat One or two bacteria Waste products Something to breathe Many bacteria Electron/Energy flow in biodegradation Electron Donor (reduced) Electron Donor (oxidized) Catabolism e e e e Electron transport Electron Acceptor (oxidized) Electron Acceptor (reduced) The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation

7 Aerobic glucose oxidizing bacteria (heterotrophs) Catabolism e e e e Electron transport C 6 H 12 O 6 + 6O 2 > 6CO 2 + 6H 2 O Aerobic methane oxidizing bacteria (Methanotrophs) Catabolism e e e e Electron transport CH 4 + 2O 2 > CO 2 + 2H 2 O The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation

8 Methane generating anaerobes (Methanogens) Catabolism e e e e Electron transport CO H 2 CH 4 + 2H 2 O Electron Acceptors The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation

9 More Electron Acceptors Aerobic (oxygen reducing) Aerobic (oxygen reducing) H 2 H + Acetate CO 2 Organic CO 2 Organic CO 2 Organic CO 2 Acetogen Methanogen SRB CO 2 Acetate CO 2 CH 4 SO 2 4 H 2 S Fe 3+ Fe 2+ NO 3 N 2 Aerobic (oxygen reducing) Aerobic (oxygen reducing) Carbon Isotopes Stable and unstable! The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation

10 Carbon flow in biodegradation Electron Donor (reduced) Electron Donor (oxidized) Catabolism e e e e Electron transport Electron Acceptor (oxidized) Electron Acceptor (reduced) 13 C and Biodegradation How Much Degradation? Who Did It? Who Did It And How? low/natural 13 C ABUNDANCE high/artificial ~1% 13 C ~10% 13 C ~100% 13 C The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation

11 Biology and Carbon Fractionation CSIA measures both 13 C & 12 C in specific compounds Biology discriminates against bonds with 13 C versus 12 C e.g. Plants assimilate more 12 CO 2 than 13 CO 2 e.g. Bacteria evolve more 12 CH 4 than 13 CH 4 Lighter: Less 13 C Heavier: More 13 C Compound specific isotope analysis Examines natural isotope ratios ( 2 H/ 1 H, 13 C/ 12 C, 15 N/ 14 N, 37 Cl/ 35 Cl) Compound specific rather than bulk isotopic measurement The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation

12 13 Chocolate Fractionation Decreasing total M & M s C C Increasing ratio M : M Lighter Heavier Applications of 13 C/ 12 C and 2 H/ 1 H CSIA Electron Donor (MTBE, 14D?) Metabolites (TBA, CO 2 ) Catabolism e e e e Electron transport Electron Acceptor (oxidized) Electron Acceptor (reduced) The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation

13 Applications of 13 C/ 12 C and 37 Cl/ 35 Cl CSIA Electron Donor (reduced) Electron Donor (oxidized) Catabolism e e e e Electron transport Electron Acceptor (PCE, ClO 4 ) Reduced acceptor (TCE, c DCE, Cl ) CSIA strengths and limitations Strengths Applicable to small volatiles and some ions Does not require detectable daughter product. Sensitive (LOD: ~1 ppb MTBE with P&T). Interpretation can be improved with 2D (e.g. 2/1 H and 13/12 C) Provides compelling evidence for degradation Limitations Historical, does not tell if biodegradation is current. Signal can be obscured by fresh undegraded compound. Abiotic degradation processes can impact signal. Needs to be supported with good site /plume characterization to account for heterogeneity. The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation

14 Opening the Black Box! What can we measure inside a bacterium or bacterial population? Carbon flow in biodegradation Electron Donor (reduced) Electron Donor (oxidized) Catabolism e e e e Electron transport Electron Acceptor (oxidized) Electron Acceptor (reduced) The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation

15 Carbon flow in biodegradation Electron Donor ( 13 C MTBE) Metabolite ( 13 CO 2 ) Catabolism 13 C e e e e Electron transport Electron Acceptor (oxidized) Electron Acceptor (reduced) Stable Isotope probing! Electron Donor ( 13 C MTBE) Metabolites ( 13 CO 2 ) Catabolism 13 C Biomolecules e e e e Electron transport Electron Acceptor (oxidized) Electron Acceptor (reduced) The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation

16 13 C-Stable Isotope Probing (SIP) DNA SIP PLFA SIP How PLFA-SIP works & what it tells you Extraction & esterification PLFA ME GC/MS GC/IRMS Microbial growth on 13 C-labeled substrate will lead to 13 C- label incorporation into newly synthesized phospholipids. Specific 13 C-PLFAs indicate role for groups of organisms in biodegradation process. 13 C-labeled metabolites ( 13 CO 2, 13 CH 4 ) verify biodegradation The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation

17 PLFA-SIP strengths and limitations Strengths: Can utilize natural abundance 13 C (~1%). Strong signal with low enrichment (~20% 13 C). Can be applied in field (e.g. Bio-Traps). Can provide estimate of degrader abundance Provides compelling evidence for BIOdegradation. Limitations: Identification of active organisms is limited 13 C cross feeding can label other trophic levels GC-IRMS analysis is not widely available. How DNA-SIP works and what it tells you DNA DNA Non metabolizers (inactive) Molecular Analysis Metabolizers (active) Microbial growth on 13 C-labeled substrate leads to 13 C incorporation into newly synthesized DNA Centrifugation separates heavy 13 C- and light 12 C-DNA. Further molecular analysis of 13 C-DNA can identify individual species of organisms, genes and pathways of degradation The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation

18 DNA-SIP strengths and limitations Strengths: Provides compelling evidence for BIOdegradation. Can identify active organisms at genus & species level. Further analysis of 13 C-metagenomic DNA. Can make use of selectively labeled substrates. Limitations: Requires highly enriched 13 C substrates ($$$). Has been applied at field scale, but not frequently. Molecular analysis can be time consuming 13 C cross feeding can label community, not just 1 o degraders. Stable Isotopes in Summary Physiological Groups Individual Species CSIA How much, how fast and how Microbial Community Inactive 12 C PLFA Active 13 C PLFA PLFA-SIP Inactive 12 C DNA Active 13 C DNA DNA-SIP Does biodegradation occur, and who does it. The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation

19 Chlorinated solvents PCE and TCE Overview of PCE and TCE PCE: (perchloroethylene, perc, tetrachloroethene) High volume chemical used as an intermediate, degreasing and dry cleaning solvent (CA phase out). TCE: (trichloroethylene, trike, 1,1,2 trichloroethene) Historically widely used as a solvent. Currently used as an intermediate in HFC refrigerant production. Both common ground water pollutants and both are persistent under aerobic conditions No organisms know to use either compound as electron donors. PCE: undergoes limited abiotic hydrolysis; PCE and TCE undergo abiotic reduction The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation

20 Dehalogenation mechanisms Many and varied. Some biotic, some abiotic, some both! Oxygenolytic: Biodegradation process that requires O 2 for monoor dioxygenases. Reductive hydrogenolysis (aka reductive dehalogenation): Most common anaerobic biodegradation mechanism for PCE and TCE. Also occurs abiotically. Dehydrochloroelimination: Common abiotic process that converts haloakanes to haloalkenes Abiotic and Biotic Transformations Aerobic Cometabolism Aerobic Metabolism Reductive Dehalogenation Abiotic Transformations The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation

21 Methane generating anaerobes (Methanogens) Catabolism e e e e Electron transport CO H 2 CH 4 + 2H 2 O Cometabolic reductive dehalogenation of PCE by methanogens Catabolism X e e e e X Electron transport CO H 2 CH 4 + 2H 2 O The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation

22 Dehalorespiration Catabolism e e e e Electron transport Pathway(s) of respiratory PCE reduction VC Respiratory process is orders of magnitude faster than anaerobic cometabolism Each reaction catalyzed by terminal reductase enzyme (e.g. tcea). Main flux is through c DCE (t DCE and 1,1 DCE are minor/unusual products). Rates typically decrease as acceptors become more oxidized. Process can often stall at c DCE in the field. While ethylene is innocuous, VC (and to less extent DCE) are more toxic than PCE and TCE The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation

23 Dehalococcoides ethenogens 195 Isolated from PCE-reducing enrichment culture Difficult organism to grow in pure culture Originally an N 2 -fixing autotroph? Fully reduces PCE to ethene and reduces other chlorinated compounds Small genome (~1.5 Mbp) Genome analysis shows presence of 17 separate reductase genes Organism Strain Phylogeny PCE- TCE TCEcDCE TCEtDCE cdce- VC Clostridium bifermentans DPH-1 Low G+C G+ x x x Dehalobacter restrictus PER-K23, TEA Low G+C G+ x x x Desulfitobacterium frappieri TCE1 Low G+C G+ x x x Desulfitobacterium metallireducens A(T) Low G+C G+ x x x Desulfitobacterium sp. Y51 Low G+C G+ x x x Desulfitobacterium sp. PCE1 Low G+C G+ x x x x Desulfitobacterium sp. PCE-S Low G+C G+ x x x Desulfitobacterium sp. KBC1 Low G+C G+ x x x x Enterobacter sp. MS1 γ-proteobacteria () () x x x Desulfomonile tiedjei DCB-1 δ-proteobacteria x x x Desulfuromonas michiganenis BB1 & BRS2 δ-proteobacteria x x x Desulfuromonas chlorethenica TT4B δ-proteobacteria x x x Geobacter lovleyi SZ δ-proteobacteria x x x Sulfurospirillum multivorans ε-proteobacteria x x x Sulfurospirillum halorespirans PCE-M2 ε -proteobacteria x x x Dehalococcoides ethenogenes 195 Green non-sulfur x () Dehalococcoides sp. FL2 Green non-sulfur () () Dehalococcoides sp. CBDB1 Green non-sulfur x x x Dehalococcoides sp. GT Green non-sulfur x x Dehalococcoides sp. BAV1 Green non-sulfur x x x Dehalococcoides (enrichment) VS Green non-sulfur x x x VCethene The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation

24 Phylogeny of PCE Dehalorespirers Dehalococcoides (PCE ET) Clostridium Dehalobacter Desulfitobacterium (PCE cdce) Sulfurospirillum (PCE cdce) Geobacter Desulfuromonas (PCE cdce) Enterobacter (PCE cdce) Fuel Oxygenates MTBE and TBA The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation

25 Overview of MTBE and TBA MTBE: (methyl tertiary butyl ether) High volume chemical used in US as octane enhancer and fuel oxygenate from 1980s to mid 2000 s. Limited current use in US. Widespread current use outside US. TBA: (tertiary butyl alcohol) Limited early use as fuel oxygenate. Major biodegradation metabolite of MTBE Both common ground water pollutants, mainly from gasoline spills. Biodegrades under both aerobic and anaerobic conditions but no organisms known that utilize either compound as an electron acceptor. MTBE hydrolyzes to TBA at low ph. An analytical issue rather than environmental issue Microcosms and oxygenate biodegradation summary Electron Acceptor CO 2 CO 2 SO 2-4 Fe 3+ Mn 4+ NO - 3 O 2 CH 4 HAc H 2 S Fe 2+ Mn 2+ N 2 H 2 O degradation rate, energy yield, biomass generation frequency of conditions in gasoline impacted sites MTBE MTBE MTBE MTBE MTBE MTBE MTBE TBA TBA TBA TBA TBA The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation

26 MTBE biodegradation mechanisms Anaerobic: No pure cultures available Observed in microcosms and field work. Possible role of acetogenic bacteria. TBA metabolite is longer lived Aerobic: Cometabolic process are frequently encountered, especially with alkaneoxidizing bacteria. Cometabolic oxidation can stop at TBA or can slowly oxidize other products. MTBE and TBA metabolism occurs in a number of closely related bacteria. Initial oxidation by monooxygenases Key enzymes in TBA oxidation are phthalate dioxygenase and isobutyryl CoA mutase. C 1 metabolism is important Anaerobic MTBE biodegradation? MTBE (H 3 C) 3 C O CH 3 H 2 /CO 2 H 2 /CO 2 (H 3 C) 3 C OH TBA Acetate CH 3 COOH Methane CH 4 acetogenesis methanogenesis The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation

27 Cometabolic MTBE degradation by aerobic alkane oxidizing bacteria Catabolism X e e e e X Electron transport Commensal MTBE biodegradation The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation

28 Aerobic MTBE Metabolism Catabolism e e e e Electron transport Methylbium petroleiphilum PM1: The DE 195 of the MTBE world! Isolated from a peat moss biofilter. Grows on MTBE, TBA, and several BTEX components. Other MTBE-utilizing isolates are closely related (Aquincola, Hydrogenophaga) and have very similar physiologies. Some strains can t oxidize MTBE but can grow on TBA. Large genome (~5 Mbp) with 600 Kbp plasmid MTBE/TBA-relevant genes are on plasmid The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation

29 Phylogeny of Aerobic MTBE Oxidizers Mycobacterium* Arthrobacter* (MTBE TBA + CO 2 ) Methylibium Hydrogenophaga Polaromonas Aquincola (MTBE/TBA CO 2 ) *Cometabolism supported by gasoline n alkanes Pseudomonas* (MTBE TBA) 2D-CSIA: Aerobic or anaerobic MTBE biodegradation? Aerobic MTBE CO 2 MTBE Anaerobic TBA O 2 H 2 O CO 2 HAc monooxygenase demethylase The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation

30 Questions? The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation

31 Introduction to Compound Specific Isotope Analysis (CSIA) Robert Pirkle, PhD., Microseeps, Inc.

32 Compound Specific Isotope Analysis Robert J. Pirkle, PhD Microseeps, Inc. US EPA Guide for CSIA John Wilson USEPA Daniel Hunkeler U of Neuchatel, Switzerland Rainer Meckenstock Institute of Groundwater Ecology, Germany Torsten Schmidt U of Duisburg-Essen, Germany Barbara Sherwood-Lollar U of Toronto, Canada Compound Specific Isotope Analysis

33 US EPA Guide Chapters 1. Introduction 2. Data Quality Issues 3. Collection, Preservation and Storage of Samples 4.Interpretation of Stable Isotope Data from Field Sites 5. Strategies for Field Investigations 6. Use of Stable Isotopes for Source Identification 7. Derivation of Equations to Describe isotope Fractionation 8. Stable Isotope Enrichment Factors 9. Recommendations for the Application of CSIA 10. References Stable Isotopes in In-Situ Degradation Focus on carbon and hydrogen isotopes Can be determined in continuous flow mode Applicable to environmentally interesting concentrations Compound Specific Isotope Analysis

34 Stable Isotopes in In-Situ Degradation Compounds with Light isotopes degraded more rapidly than compounds with Heavy isotopes Product remaining becomes isotopically heavier Process of isotopic change is called fractionation Stable Isotopes in In-Situ Degradation Significant Fractionation Occurs in: Biological Oxidation Biological Reduction Abiotic Degradation In-Situ Chemical Oxidation In-Situ Chemical Reduction Compound Specific Isotope Analysis

35 Stable Isotopes in In-Situ Degradation Little or No Fractionation Occurs in: Dilution Volatilization Sorption Anaerobic Degradation of Toluene under Sulfate Reducing Conditions with Strain TRM1, Meckenstock, et al., Toluene [nm] Toluene (µm) Sulfide δ 13 C δ 13 C [ 0 /00] Sulfide [mm] Time [days] Compound Specific Isotope Analysis

36 Stable Isotopes in In-Situ Degradation Fractionation is unequivocal proof of in-situ degradation Related to the mechanism of degradation Related to the fraction of component degraded Related to the rate of degradation Used in groundwater modeling The Stable Isotope Parameters Ratio R = ([heavy] / [light]) = ([ 13 C] / [ 12 C]) x The Del Function δ x = {(R x R std ) / R std } x 1000 The Raleigh Function δ r,t = є ln f + δ r,0 Compound Specific Isotope Analysis

37 Fraction of Degradation The fraction of degradation or fraction of reactant remaining, f, can be calculated from the simplified Rayleigh equation: δ r,t = Є ln f + δ r,o which after rearrangment becomes: f = exp (δ r,t - δ r,o )/Є and we can calculate f if we know Є and δ r,o, and we measure δ r,t But f can also be calculated from measured concentration, f = [reactant] t / [reactant] o = C t / C o Fraction of Degradation Kuder (2005) Attenuation or Biodegradatio calculated from concentration data F calculated from δ 13 C f ( C ) α dilution + sorption + volatilization + degradation + f ( δ ) α degradation Thus the concentration calculated from the isotopic measurements is conservative. Compound Specific Isotope Analysis

38 Rates of Reaction Since the isotopic ratio is related to the fraction of reactant remaining, it can also be used to estimate the rate of reaction along a flow path in the plume. or f = C t / C o = exp ((δ r,t - δ r,o )/Є) C t = C o exp ((δ r,t - δ r,o )/Є) Rates of Reaction C t = C o exp ((δ r,t - δ r,o )/Є) C t = C o exp (-k t t) C d = C o exp (-k d d) Therefore we can write: -k t t = (δ r,t - δ r,o )/Є or k t = (δ r,o - δ r,t )/Єt where k t is the first order rate constant in terms of time; OR -k d d = (δ r,d - δ r,o )/Є or k d = (δ r,0 - δ r,d )/Єd where k d is the first order rate constant in terms of distance. Compound Specific Isotope Analysis

39 A Gasoline Spill in Dana Point, CA MW MW MW - 10 < 0.5 MW MW MW MW TPHg > 1,000 mg/kg TPHg > 100 mg/kg MW MW ,000 MW meters MW - 9 < 0.5 Underground Storage Tanks Dispenser Islands Wilson (2005) Rates of Reaction Wilson (2005) has calculated rate constants for degradation of MTBE along flow paths, both in terms of distance and time. These projected rates along flow paths may also be used to predict the possible extent of plumes. Rates of Natural Biodegradation of MTBE Well Date Sampled Fraction MTBE Remaining (C t / C 0 ) Distance from MW-14 (meters) Projected Rate of Biodegradation with Distance (per meter) Projected Rate of Biodegradation with Time (per year) MW-3 May, MW-3 August, MW-8 May, MW-8 MW-7 MW-11 August, August, August, Compound Specific Isotope Analysis

40 Application to Recalcitrant Compounds 1,000,000 NE SW , TBA (ug/l) & SO4 (mg/l) 10,000 1, delta 13C permil Distance (ft) TBA SO4 d13c permil Day et.al.(2003) and Gulliver (2003) have shown in a parent plume of TBA that TBA is apparently degraded under sulfate reducing conditions. CSIA and Degradation Degradation chews away at the lightest stuff, leaving behind the heavy stuff. This is called fractionation. For parent molecules (i.e. what was originally released, typically PCE or TCE) fractionation is unequivocal proof of degradation. heavier lighter Compound Specific Isotope Analysis

41 CSIA and Degradation (cont.) For daughter products (not originally released but produced through degradation) fractionation alone does not prove daughter product degradation. If daughter product does not degrade, it still gets heavier as the parent gets heavier. The undegraded daughter product can get no heavier than the original (undegraded) parent product. heavier lighter CSIA and Degradation (cont.) If daughter product degrades, its lighter portion gets removed at all times. This means its isotopic ratio can get heavier than that of the parent. In chlorinated solvent daughter products, a carbon isotopic ratio heavier than the parent is evidence that the daughter product is degrading. Compound Specific Isotope Analysis

42 Application to Chlorinated Solvents 1. Song et.al. (2002) have presented a detailed timeseries isotope study of TCE undergoing enhanced bioremediation. 2. Lactate was added to stimulate bioremediation. 3. Concentration data could not determine the extent of degradation. 4. CSIA was used to show that TCE was completely converted to ethene. Application to Chlorinated Solvents TAN-31 TSF-05 TAN-10A TAN-25 TAN-26 5,000 µg/l TAN-37 SITE PLAN TAN-28 TAN-30A TAN meters TCE Isopleths TAN-49 TAN-29 1,000 µg/l 100 µg/l Source Well is TSF-05 TAN-25 and TAN-26 used to show sequential degradation using concentration and isotope data. TAN-29 and TAN-31 are background wells. Compound Specific Isotope Analysis

43 } O rganic Acid C onc. (m M ) Pre-Lactate Tracer Test } Application to Chlorinated Solvents Lactate Injection Started Lactate Injection Stopped Time (days) Lactate Injection Resumed TAN 29 TAN 25 TAN 26 TAN 31 Pilot study began with injection of clean water into TSF-05. Lactate injection began 25 days after clean water injection ended. Lactic acid rapidly fermented to butyric, propionic and acetic acids. TAN 25 and TAN 26 had significant organic acids throughout the injection period. Application to Chlorinated Solvents a ) Pre-Lactate Tracer Test 15 Lactate Injection Started Lactate Injection Stopped Lactate Injection Resumed TCE Concentration (µm) Time (days) DCE TCE c-dce t-dce VC ethene b ) δ 13 C ( 0 /00 ) Time (days) VC Ethene Concentration and Isotopic Data vs Time at TAN-25. Compound Specific Isotope Analysis

44 Application to Chlorinated Solvents a ) Pre-Lactate Tracer Test } Lactate Injection Started Lactate Injection Stopped Lactate Injection Resumed Concentration (µm) Time (days) TCE c-dce t-dce VC ethene b ) 10 0 δ 13 C ( 0 /00 ) Time (days) Concentration and Isotopic Data vs Time at TAN 26 Case 2: cis-dce degrading? - Yes Site was predominated by cis-dce Some TCE, PCE Plume crossed a geologic fault, eliminating applicability of standard concentration vs. distance analyses. VC could not be observed Was the cis-dce degrading? Compound Specific Isotope Analysis

45 Light Case 2 (cont.) cis-dce is heavier than PCE it came from (PCE parent ) cis-dce gets heavier than parent ONLY if the cis- DCE is degrading CSIA confirmed that cis-dce is degrading and the remediation is effective. Heavy Case 3: cis-dce degrading? - No Site was predominated by TCE Some cis-dce. Site was predominantly oxic. VC could not be observed Was the cis-dce degrading? Compound Specific Isotope Analysis

46 Heavy Light Case 3 (cont.) cis-dce lighter than TCE cis-dce < -30 per mil CSIA indicates degradation, if present, is slow compared to TCE degradation. CSIA confirmed what other analyses could only suggest: MNA ineffective - alternate remediation strategy required. Case 4: ISCO: Altered Hydrogeology or Successful Degradation? Site contaminated with PCE and all daughter products. Very tight formation ISCO with fracturing was the planned remediation. Was there degradation? Compound Specific Isotope Analysis

47 Case 4 Concentration decrease coupled with isotopic shift (change in del) shows strong degradation at MW-A (and probably MW-23) ISCO effect unclear in MW-D, but CSIA shows degradation in MW-D. CSIA proved degradation but did the rebound undo any progress? Stable Isotopes in Forensic Investigations Compound Specific Isotope Analysis

48 13 δ C VPDB ( ) TCA PPG 95 TCA PPG 93 TCA Stan Chem 93 TCA ICI 93 TCE PPG 93 TCA Vulcan S-93 TCE Stan Chem 93 LEGEND: van Warnerdam el. al., 1995 Shouakar-Stash, et. al., 2003 TCE DOW TCE PPG 95 TCE ICI 93 TCE DOW δ Cl SMOC( ) Forensic Isotope Analysis of TCE Compound Specific Isotope Analysis

49 Potential Enrichment Paths for 3 Sources at Moffett Field identified by Dennis Goldman, Jon Gabry, Brian Maidrand and Wilson Doctor, Stable Isotope Study Orion Public Housing Area, Moffett Field, March Conclusions Stable isotope analysis provided the only line-ofevidence that there are three isotopic ratio signatures, thus three distinct TCE sources affecting the groundwater beneath the housing area at Moffett Field. At least two and likely three of the sources originate off-site and up-gradient of the housing area. This analysis has provided the Navy with the data necessary to defend to the regulatory agency that the contamination beneath the housing area originated off-site, potentially saving the Navy millions of dollars. Dennis Goldman, Jon Gabry, Brian Maidrand and Wilson Doctor, Compound Specific Isotope Analysis

50 German Industrial Site Blessing, et. al., ES&T 43, , 2009 Blessing, et. al., ES&T 43, , 2009 Compound Specific Isotope Analysis

51 Blessing, et. al., ES&T 43, , 2009 Blessing, et. al., ES&T 43, , 2009 Compound Specific Isotope Analysis

52 Blessing, et. al., ES&T 43, , Compound Specific Isotope Analysis

53 The End What does the future hold?? * Commercial 37 Cl CSIA *Vapor CSIA with application to Vapor Intrusion *Reduced detection levels using vacuum extraction. *Site specific CSIA using 2 H NMR Compound Specific Isotope Analysis

54 Applications of Molecular Biological Tools (MBTs) Greg Davis, Microbial Insights

55 Application of Molecular Biological Tools (MBTs) Greg Davis, Dora Ogles, Brett Baldwin, Anita Biernacki Microbial Insights, Inc. What do these have in common? Monitored Natural Attenuation Reductive Dechlorination of PCE Co metabolic Oxidation of TCE Anaerobic Oxidation of DCE Aerobic mineralization of benzene Anaerobic MTBE degradation

56 Answer All of these are examples of applications that Molecular Biological Tools have been applied successfully. What are Molecular Biological Tools? Tools that target biomarkers to provide information about organisms and processes relevant to the assessment and/or remediation of contaminants in the environment.

57 Traditional Microbiology When most of us think of Microbiology two words come to mind: Plate Count Disadvantages MICROBIOLOGY Microbiology Results are not very representative of insitu community ~ 1 to 10% of bacteria are culturable Highly selective based upon media conditions MBTs target Cellular Components PLFA Quantify total biomass & assess the entire microbial population MICROBIOLOGY

58 MBTs target Cellular Components CENSUS Rapidly detect and quantify specific microbial populations and processes Nucleic Acids Growth of MBTs Application Time Significant growth in the utilization of MBTs for site design and management decisions has occurred over the last decade

59 Molecular Biological Toolbox MBT: CENSUS Quantitative PCR

60 Real Time PCR Rapidly detect and quantify specific microbial populations Primarily known for it s ability to quantify a key dechlorinating bacteria, Dehalococcoides (DHC) Can target a variety of other organisms including: Methanogens SRBs/IRBs Methanotrophs Denitrifiers Dehalococcoides spp. Genetic Code

61 Accurate Direct sample analysis State of the Art laboratory practices Reproducible Rapid Results within days not weeks

62 Sensitive How does CENSUS work? Sample Collection DNA Extraction Amplification Groundwater, soil, or Bio Trap samplers collected and shipped overnight on ice (4c) Results DNA extracted from sample upon arrival Quantitative Real Time PCR is used to detect and quantify targets of interest (i.e. Dehalococcoides spp.) Assessment Results are ed to project contact (7 to 10 day TAT) Results are integrated with other site parameters to evaluate site management decisions

63 Applications include Directly evaluate the feasibility of monitored natural attenuation (MNA) Evaluate the efficacy of enhanced bioremediation approaches Assess the need for bioaugmentation Reductive Dechlorination

64 Reductive Dechlorination Target Code Contaminant Environmental Relevance Dehalococcoides qdhc PCE, TCE, DCE, VC Only known group of bacteria capable of complete dechlorination of PCE/TCE to ethene Absence of DHC suggest dechlorination of DCE and VC is improbable and accumulation of daughter products is likely Presence of DHC indicates potential for complete dechlorination even in low numbers. High concentrations correlate with increased rates of dechlorination Dehalococcoides spp.

65 Dehalococcoides spp. VC Functional Genes Dehalococcoides Can t be detected using traditional techniques DCE and VC accumulation likely Potential for complete dechlorination

66 Reductive Dechlorination Target Code Contaminant Environmental Relevance Dehalobacter qdhb PCA, TCA, PCE, TCE Capable of dechlorination of PCE and TCE to DCE Converts TCA, a common cocontaminant, to chloroethane Dehalobacter spp. Cells/mL Dehalococcoides Reductive Dechlorination Target Code Contaminant Environmental Relevance Desulfuromonas qdsm PCE, TCE Capable of dechlorination of PCE and TCE to cis DCE Desultiftobacterium qdsb PCE, TCE Capable of dechlorination of PCE and TCE to cis DCE

67 Reductive Dechlorination Target Code Environmental Relevance Total Biomass qebac Index of total bacterial biomass Methanogens qmgn Utilize hydrogen and carbon dioxide to produce methane Compete with dechlorinating bacteria for available hydrogen Sulfate Reducing Bacteria (SRBs) qsrb/irb Targets delta Proteobacteria Index of sulfate and iron reducing bacteria Competes for available hydrogen to produce hydrogen sulfide CENSUS Biostimulation

68 CENSUS Biostimulation CENSUS Bioaugmentation

69 Aerobic Anaerobic BTEX BTEX Aerobic BTEX Target Code Contaminant Environmental Relevance Ring hydroxylating Toluene monooxygenases qrmo BTEX Catalyzes the initial (and sometimes second) hydroxylation of BTEX compounds Phenol hydroxlase qphe BTEX Catalyzes further oxidation of BTEX compounds O 2 O 2 RMO PHE Presence of indicates potential for aerobic BTEX biodegradation

70 Biostimulation Baseline 180 Days Post MW-14 MW-12 MW-4 MW-7 MW-14 MW-12 MW-4 MW-7 MW-15 MW-15 Cells/bead: ND *Quantified using initial catabolic functional genes (TOD, TOL, RDEG, RMO, & PHE) Biostimulation BTEX (mg/l) ORC Injection Aerobic Degraders Dentrifying SRB/IRBs Methanogens Sampling Event

71 Anaerobic BTEX Target Code Contaminant Environmental Relevance Benzylsuccinate Synthase qbss BTEX Targets gene encoding for enzyme in anaerobic biodegradation of benzene, toluene, and xylene BSS COO COO Fumarate Presence of indicates potential for anaerobic BTEX biodegradation Anaerobic BTEX (bssa) Cells/bead Cells/bead MNA Site 1 MNA Site 1 Sulfate Injection Site 2 bssa was 2 orders of magnitude higher in the source area Sulfate Injection Site 2 bssa increased over time following sulfate injections

72 Aerobic MTBE Target Code Contaminant Environmental Relevance MTBE utilizing PM1 qpm1 MTBE Targets Methylibium petroleiphilum PM1. one of the few bacteria isolated that is capable of growth on MTBE MTBE TBA Formate Presence of indicates potential for aerobic MTBE biodegradation Bioaugmentation Total Biomass Cells/bead MTBE degrading PM1 Cells/bead MW-1 pre 1.33E E+03 MW-1 PM1 Inj. 3.54E E+04 MW-2 pre 9.71E+08 ND MW-2 PM1 Inj. 4.83E E+05 MW-3 pre 2.11E+09 ND MW-3 PM1 Inj. 3.45E E+04

73 Cometabolism of Chlorinated Ethenes For aerobic cometabolism to be a primary treatment mechanism, three key factors must be present or supplied: A primary (growth supporting) substrate Oxygen Bacteria capable of producing non specific monooxygenases Cometabolism Target Code Contaminant Environmental Relevance Methanotrophs qmob TCE, DCE, VC Targets two types of methane oxidizing bacteria. Soluble Methane Monooxygenase qsmmo TCE, DCE, VC smmo are generally believed to support faster cometabolism of TCE TCE Cometabolism O 2 Methane Monooxygenase +CO CH 4 CH 3 OH

74 Cometabolism Target Code Contaminant Environmental Relevance Propane Monooxygenase Toluene Dioxygenase Ring Hydroxylating Toluene Monooxygenase qppo TCE, DCE, VC Propane can be added as a primary substrate to promote cometabolic oxidation of TCE qtod TCE, DCE, VC Applicable at mixed waste sites where BTEX and TCE are co contaminants Capable of cometabolism of TCE qrmo TCE, DCE, VC Applicable at mixed waste sites where BTEX and TCE are co contaminants Capable of cometabolism of TCE PLFA Phospholipid Fatty Acid Analysis Kerry Sublette, University of Tulsa

75 Phospholipid Fatty Acid Analysis Essentially the skin of the microbe Monitor microbial responses to their environment. No Prior Knowledge Required How does PLFA work? Sample Collection PLFA Extraction Gas Chromatography Groundwater, soil, or Bio Trap samplers collected and shipped overnight on ice (4c) Results PLFA biomarkers extracted from sample upon arrival Biomarkers are identified by gas chromatography with flame ionization detection (GC FID) Assessment Results are integrated with other site parameters to evaluate site management decisions

76 What can PLFA tell you? Viable (living) Biomass How many cells are present? Population Fingerprint What type of microbes are there? Microbial Activity What are they doing: active slow adapting PLFA Viable Biomass Substantial increases in biomass following electron donor addition

77 Community Composition Community Composition 20% 45% 19% 15% 1% PLFA profile provides a fingerprint of the entire population

78 PLFA Profile Profiles reflect proportions of bacterial groups Bacterial groups are composed of different fatty acids biomarkers Bacterial groups often behave like a seesaw PLFA Community Structure Substantial increase in Firmicutes (fermenters) Hydrogen production to support reductive dechlorination

79 PLFA Applications Monitor shifts in microbial community following biostimulation / bioaugmentation. Assess changes within viable biomass and community composition before and after of chemical oxidation treatment. Chemical Oxidation Pre-injection 1 Month Post 9 Months Post Cells/mL Upgradient Chem Ox. Injection Area No significant changes in biomass upgradient After 30 days of Chemical oxidation treatment biomass levels either remained constant or increased.

80 Response to sulfate injections days % of Total days 64 days 94 days 129 days 161 days 0 MW7 MW1 RW4 MW7 MW1 RW4 MW7 MW1 RW4 MW7 MW1 RW4 MW7 MW1 RW4 MW7 MW1 RW4 Anaerobic Biomarkers: Firmicutes/SRBs SRBs MBT Sampling Options

81 Sampling Considerations What questions are you trying to answer? Where to collect? Background MW 1 Source Area MW 4 MW 2 MW 3 MW 5 MW 6 MW-7 Primary Secondary Downgradient

82 Sample Collection Almost any type of sample can be analyzed by MBTs Majority are groundwater samples from monitoring wells Collected using two different approaches Bio Flo Sampling Microbes collected in a sealed filter cartridge

83 Bio Flo Approach Allows the collection of larger sample volume (more representative samples) Decreases shipping cost to the laboratory (large vs small cooler) Less chance of sample loss due to breakage DNA Concentration (ng/ul) Bio Trap Samplers Microbes collected over a period of time

84 Sampling Matrix: Bio Sep Beads Properties of Bio-Sep Beads 3-4 mm in diameter 25 % Nomex, 75% PAC 74% porosity 600 m 2 of surface area/g Surrounded by ultrafiltration-like membrane with 1-10 micron holes Autoclavable Cleaned of fossil biomarkers by heating to 300 o C Exterior of Bio-Sep Interior of Bio-Sep Questions???

85 Applications of Bio Trap samplers and Stable Isotope Probing Dr. Kerry Sublette, University of Tulsa

86 Applications of Bio trap Samplers and Stable Isotope Probing Kerry Sublette, Greg Davis, Dora Ogles, Brett Baldwin, Anita Biernacki What Are Bio Trap Samplers Passive sampling tool for microbes Collects active microbes Integrated sample vs. snapshot Analyzed using molecular biological tools, analytical chemistry, and stable isotope analysis Applications of Bio trap Samplers and Stable Isotope Probing

87 How Do Bio Traps Work? Properties of Bio-Sep Beads 3-4 mm in diameter 25% Nomex and 75% PAC 74% porosity 600 m 2 of surface area/g Heat sterilized 270 o C Colonized by active microbes Cell division inside of Bio Sep bead within a bio trap Applications of Bio trap Samplers and Stable Isotope Probing

88 Types of Bio Trap Samplers Standard Bio Trap Basic design Replacement for collecting groundwater, soil, etc. Compatible with all MBTs, analytical chemistry, and stable isotope techniques Unit Samplers Supplier COC MICRO (Bio Trap) GEO Supplier Applications of Bio trap Samplers and Stable Isotope Probing

89 Assembly Control (MNA) Unit Samplers Supplier COC Treatment Option 1 MICRO (Bio Trap) Treatment Option 2 GEO Supplier Baits Include: Electron Donors Vegetable oil Molasses HRC EOS Lactate And more Electron Acceptors Oxygen (PermeOx, ORC) Nitrate Iron Sulfate Stable Isotope Compounds ( 13 C) Benzene Toluene p Xylene MTBE TBA Naphthalene Chlorobenzene And more Applications of Bio trap Samplers and Stable Isotope Probing

90 How Are Bio Trap Samplers Deployed? Purge monitoring well Monitoring Well Suspend from top of casing Deploy within the screened interval at depths of interest. If large fluctuations in the water level are anticipated suspended from a float. Screened Interval Where to Collect? Background MW 1 Source Area MW 4 MW 2 MW 3 MW 5 MW 6 MW-7 Primary Secondary Downgradient Applications of Bio trap Samplers and Stable Isotope Probing

91 Sampling Strategies Scenario A Day Time Scenario B Time How Are Bio Trap Samplers Analyzed: Molecular Biological Tools PLFA CENSUS (qpcr) DGGE Stable Isotope Probing (SIP) Chemical Analysis CSIA Dissolved Inorganic Carbon (DIC) Contaminant Concentrations Applications of Bio trap Samplers and Stable Isotope Probing

92 What Can I Do With a Bio Trap Sampler? Determine if known degraders of a COC are present Evaluate monitored natural attenuation versus enhanced bioremediation Compare effectiveness of amendments designed to stimulate bioremediation Prove that bioremediation of a specific compound is occurring Determine If Known Degraders Are Present CENSUS Dehalococcoides populations indicate the potential for complete reductive dechlorination of PCE to ethene Applications of Bio trap Samplers and Stable Isotope Probing

93 What can I do with a Bio Trap Sampler? Determine if known degraders of a COC are present Evaluate monitored natural attenuation versus enhanced bioremediation Compare effectiveness of amendments designed to stimulate bioremediation Prove that bioremediation of a specific compound is occurring Samplers Analysis Lines of Evidence COC MICRO (Bio Trap) GEO VOCs CENSUS SIP Anions Dissolved Gases Contaminant concentrations Daughter product formation Quantify specific microbial populations and processes Prove biodegradation Compare relative rates Compare degree of contaminant incorporation Redox conditions Ethene & Ethane production Supplier Applications of Bio trap Samplers and Stable Isotope Probing

94 Common In Situ Microcosm Studies Screening Remediation Alternatives Petroleum Hydrocarbon Site Chlorinated Solvent Site Control (MNA) Oxygen Addition Enhanced Aerobic Bioremediation Control Unit Evaluate MNA as treatment alternative Baseline for enhanced remediation options A Unit for Each Remediation Alternative Enhanced Anaerobic Bioremediation Control (MNA) BioStim (Electron Donor) Sulfate Addition Enhanced Anaerobic Bioremediation Bioaugmentation BioAug (Culture & Electron Donor) Passive Samplers Applications of Bio trap Samplers and Stable Isotope Probing

95 Evaluate monitored natural attenuation versus enhanced bioremediation Dehalococcoides were more than an order of magnitude greater when amended with electron donor Case Study: Baited Bio Trap Samplers Chlorinated Solvent Site in Upstate New York Applications of Bio trap Samplers and Stable Isotope Probing

96 Site Background Chlorinated VOCs released: COCs PCE, TCE, cdce, TCA, and MC Pump and Treat was used for ~4 yrs. Dual phase extraction removed ~9000+ lbs What s next Evaluate Monitored Natural Attenuation (MNA) or Enhanced Attenuation (EA) Site Characterization MNA or EA? No immediate risks No plans to sell Stable plume Minimal VC and ethene detected Build up of cis DCE MBTs only low levels of DHC in the source area Geochemical conditions were not reduced enough to support reductive dechlorination. Applications of Bio trap Samplers and Stable Isotope Probing

97 Initial Groundwater Conditions Parameter Source (RW-1) Mid-Plume (VE-6) ORP DO Sulfate Methane* PCE TCE ND 6 cis-dce TCA 59 8 VC ND ND Total CVOCs 215 ppm 38 ppm *All parameters reported as ppm (mg/l) except methane which was reported as ug/l Initial Groundwater Conditions Parameter Source (RW-1) Mid-Plume (VE-6) ORP DO Sulfate Methane* PCE TCE ND 6 cis-dce TCA 59 8 VC ND ND Total CVOCs 215 ppm 38 ppm *All parameters reported as ppm (mg/l) except methane which was reported as ug/l Applications of Bio trap Samplers and Stable Isotope Probing

98 Goals Improve understanding of site reductive dechlorination bio processes Are known dechlorinating bacteria present Evaluate potential impact of competing organisms Evaluate potential for more complete degradation Test application of HRC and HRC S as electron donor amendments Overview of Baited Bio Trap Study Control and baited Bio Trap samplers placed along a gradient across the plume MW 10 1 ppm 10 ppm VE ppm RW 1 GW Flow MW 24 Not Detected Applications of Bio trap Samplers and Stable Isotope Probing

99 Bio Trap Results: 100 ppm 10 ppm 1 ppm Background Bio Trap Results: 100 ppm 10 ppm 1 ppm Background Applications of Bio trap Samplers and Stable Isotope Probing

100 Bio Trap Results: 100 ppm 10 ppm 1 ppm Background Bio Trap Summary Observations: HRC S was most effective in stimulating DHC HRC S helped control competition with SRB/IRBs Methanogens could still be a problem Time Frame: ~75 days for the entire study ~60 days of in situ incubation of the Bio traps ~14 days for MBTs Total Cost 1/3 cost of traditional microcosm study Applications of Bio trap Samplers and Stable Isotope Probing

101 Biostimulation HRC S injection pilot test Biostimulation Applications of Bio trap Samplers and Stable Isotope Probing

102 Biostimulation Biostimulation Applications of Bio trap Samplers and Stable Isotope Probing

103 Biostimulation Biostimulation Applications of Bio trap Samplers and Stable Isotope Probing

104 Case Study: Baited Bio Trap Samplers Gasoline Impacted Aquifer Site Background Shallow aquifer impacted by gasoline. Benzene, toluene, ethylbenzene, xylenes (BTEX) are the contaminants of concern. Dissolved oxygen (DO) concentrations are typically low (<1 mg/l). Concentrations of alternative electron acceptors (nitrate, iron, sulfate) were also fairly low. Considering oxygen addition to stimulate BTEX biodegradation. Applications of Bio trap Samplers and Stable Isotope Probing

105 Site Specific Question Question Will oxygen addition promote growth of bacteria capable of aerobic biodegradation of BTEX? Study Design Assembly Unit Samplers Potential Lines of Evidence COC VOCs: Differences in benzene concentrations Control (MNA) MICRO (Bio Trap) CENSUS: Differences in populations of aerobic benzene utilizing bacteria SIP: 13 C benzene incorporation into biomass and CO 2 Oxygen Amended GEO Anions: Differences in redox conditions Oxygen Supplier Applications of Bio trap Samplers and Stable Isotope Probing

106 In Situ Microcosm CENSUS Results (PHE) Control Unit Oxygen Unit O 2 O 2 RMO PHE PHE population is almost 2 orders of magnitude greater in the oxygen amended unit In Situ Microcosm CENSUS Results (TOD) Control Unit Oxygen Unit TOD population is more than 2 orders of magnitude greater in the oxygen amended unit Applications of Bio trap Samplers and Stable Isotope Probing

107 In Situ Microcosm CENSUS Results (TOL) Control Unit Oxygen Unit TOL population detected only in the oxygen amended unit Questions so far? Applications of Bio trap Samplers and Stable Isotope Probing

108 What can I do with a Bio Trap Sampler? Determine if known degraders of a COC are present Evaluate monitored natural attenuation versus enhanced bioremediation Compare effectiveness of amendments designed to stimulate bioremediation Prove that bioremediation of a specific compound is occurring SIP Stable Isotope Probing Applications of Bio trap Samplers and Stable Isotope Probing

109 What is Stable Isotope Probing? Coupling MBTs with stable isotope compounds to link biodegradation to the responsible microbes Stable isotope compounds Specially produced heavy compounds which are composed of 99+% 13 C Natural compounds are 99% 12 C Same characteristics as original compound Behave similar to the natural compound Used as tracers to increase our understanding of contaminant fate Applications of Bio trap Samplers and Stable Isotope Probing

110 Microbes colonize beads Bio Sep Bead 13 C labeled compounds sorbed to Bio Sep beads Bio Trap colonized by indigenous microorganisms Microbes utilize target compound Bio Sep Bead Some microbes that colonized the Bio Sep bead can utilize 13 C labeled target compound. Applications of Bio trap Samplers and Stable Isotope Probing

111 13 C Incorporation into biomass and CO 2 CO 2 CO 2 CO 2 CO 2 CO 2 CO 2 CO 2 CO 2 13 C is incorporated into new cells growing in the beads and in CO 2 Overview of Bio Trap SIP Approach 13 C labeled Benzene Bio-Trap with 13 C- benzene loaded beads Beads analyzed following deployment Beads loaded with 13 C compound In-Situ deployment in monitoring well Applications of Bio trap Samplers and Stable Isotope Probing

112 Benzene Normalized concentration (mg/bead) Incubation time (days) 30 MTBE and TBA 1.4 Normalized Concentration (mg/bead) MTBE TBA Incubation Time (days) Applications of Bio trap Samplers and Stable Isotope Probing

113 Bio Trap SIP Analysis Residual 13 C Compound Local relative rate 13 C/ 12 C Dissolved Inorganic Carbon 13 C/ 12 C of Biomarkers PLFA DNA RNA Mineralization (C for energy) Metabolism (C for growth) Unit of measure Amount of 13 C relative to 12 C is expressed by the δ 13 C notation 13 δ C ( ( 13 C/ Sample [ ] = C/ 12 C) C) Standard The standard is a specific carbon containing mineral from a specific location: Pee Dee Belimnite (PDB) 12 Units of δ 13 C are o / oo or per mill Applications of Bio trap Samplers and Stable Isotope Probing

114 δ 13 C of COCs MTBE More 13 C PCE TCE Less 13 C Benzene Crude oil PDB std Natural Gas δ 13 C of COCs MTBE More 13 C PCE TCE Less 13 C Crude oil Benzene SIP results PDB std Natural Gas Applications of Bio trap Samplers and Stable Isotope Probing

115 Stable Isotope Probing (SIP) Bio Trap Study Industrial facility in New Jersey evaluating air sparging for a p xylene contaminated site Site Background Industrial Site in New Jersey Impacted by finishing products (paints and coatings) Leaking UST Contaminant of Concern p Xylene Applications of Bio trap Samplers and Stable Isotope Probing

116 Questions to Answer Is p xylene being degraded under the current conditions? To what extent would biostimulation with oxygen effect biodegradation of xylene? Is xylene being degraded following O 2 injection? Some thing to eat Some thing to breath + O 2 Electron Donor (p xylene) + = Electron Acceptor energy PLFA CO 2 Applications of Bio trap Samplers and Stable Isotope Probing

117 Phospholipid Fatty Acid Analysis Essentially the skin of the microbe Bio Trap Sampler Overview Bio Trap samplers baited with 13 C p xylene Deployed in locations where concentration varied (100, 10, and 1 ppm) Samplers were analyzed using SIP Samplers were deployed for ~30 days. Applications of Bio trap Samplers and Stable Isotope Probing