Advanced Tools for In Situ Remediation Workshop June 22, 2011 Co-Sponsored by:
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, 2011 11:00 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
The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation Mike Hyman, PhD., North Carolina State University
The microbiology of chlorinated solvent and fuel oxygenate biodegradation Dr. Mike Hyman Department of Microbiology North Carolina State University michael_hyman@ncsu.edu 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
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 10 30 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: 6578 6583 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
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
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
Methane generating anaerobes (Methanogens) Catabolism e e e e Electron transport CO 2 + 4 H 2 CH 4 + 2H 2 O Electron Acceptors The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation
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
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
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
13 Chocolate Fractionation Decreasing total M & M s 12 13 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
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
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
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
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
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
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
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
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
Methane generating anaerobes (Methanogens) Catabolism e e e e Electron transport CO 2 + 4 H 2 CH 4 + 2H 2 O Cometabolic reductive dehalogenation of PCE by methanogens Catabolism X e e e e X Electron transport CO 2 + 4 H 2 CH 4 + 2H 2 O The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation
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
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 853-15A(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
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
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
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
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
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
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
Questions? The Microbiology of Chlorinated Solvent and Fuel Oxygenate Biodegradation
Introduction to Compound Specific Isotope Analysis (CSIA) Robert Pirkle, PhD., Microseeps, Inc.
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 http://www.microseeps.com/html/technicalarticles.html Compound Specific Isotope Analysis
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
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
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., 1999. Toluene [nm] 350 300 250 200 150 100 50 Toluene (µm) Sulfide δ 13 C -22-24 -26-28 δ 13 C [ 0 /00]0 2.0 1.5 1.0 0.5 Sulfide [mm] 0 1 4 8 9 10 11 Time [days] -30 0.0 Compound Specific Isotope Analysis
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
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 0.000001 Kuder (2005) Attenuation or Biodegradatio calculated from concentration data 0.00001 0.0001 0.001 0.01 0.1 1 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 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
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
A Gasoline Spill in Dana Point, CA MW - 11 318 MW - 7 106 MW - 10 < 0.5 MW - 16 18 MW - 8 25 MW - 3 164 MW - 6 490 TPHg > 1,000 mg/kg TPHg > 100 mg/kg MW - 15 1 MW - 14 26,000 MW - 12 3.6 10 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, 2003 0.058 9.6 0.30 10.9 MW-3 August, 2004 0.05 9.6 0.31 11.5 MW-8 May, 2003 0.023 11.7 0.32 11.9 MW-8 MW-7 MW-11 August, 2004 0.0043 11.7 0.46 17.1 August, 2004 0.994 23.0 0.00025 0.0093 August, 2004 1.0 44.1 0 0 Compound Specific Isotope Analysis
Application to Recalcitrant Compounds 1,000,000 NE SW -25.5 100,000-26 TBA (ug/l) & SO4 (mg/l) 10,000 1,000 100 10-26.5-27 -27.5-28 - delta 13C permil 1-28.5 0-29 0 200 400 600 800 1000 1200 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
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
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-27 0 50 100 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
} O rganic Acid C onc. (m M ) Pre-Lactate Tracer Test } 80 60 40 20 Application to Chlorinated Solvents Lactate Injection Started Lactate Injection Stopped 0 0 100 200 300 400 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) 10 5 0 0 100 200 300 400 Time (days) DCE TCE c-dce t-dce VC ethene b ) δ 13 C ( 0 /00 ) 0-10 - 20-30 - 40-50 - 60 0 100 200 300 400 Time (days) VC Ethene Concentration and Isotopic Data vs Time at TAN-25. Compound Specific Isotope Analysis
Application to Chlorinated Solvents a ) Pre-Lactate Tracer Test } Lactate Injection Started Lactate Injection Stopped Lactate Injection Resumed 35 30 25 Concentration (µm) 20 15 10 5 0 0 100 200 300 400 Time (days) TCE c-dce t-dce VC ethene b ) 10 0 δ 13 C ( 0 /00 ) - 10-20 - 30-40 - 50-60 0 100 200 300 400 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
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
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
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
13 δ C VPDB ( ) - 25.00-26.00-27.00-28.00-29.00-30.00 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 95-31.00 TCE PPG 95 TCE ICI 93 TCE DOW 92-32.00-4.00-3.00-2.00-1.00 0.00 1.00 2.00 3.00 4.00 5.00 37 δ Cl SMOC( ) Forensic Isotope Analysis of TCE Compound Specific Isotope Analysis
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 2006. 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, 2006. Compound Specific Isotope Analysis
German Industrial Site Blessing, et. al., ES&T 43, 2701 2707, 2009 Blessing, et. al., ES&T 43, 2701 2707, 2009 Compound Specific Isotope Analysis
Blessing, et. al., ES&T 43, 2701 2707, 2009 Blessing, et. al., ES&T 43, 2701 2707, 2009 Compound Specific Isotope Analysis
Blessing, et. al., ES&T 43, 2701 2707, 2009 www.microseeps.com Compound Specific Isotope Analysis
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
Applications of Molecular Biological Tools (MBTs) Greg Davis, Microbial Insights
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
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.
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
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
Molecular Biological Toolbox MBT: CENSUS Quantitative PCR
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
Accurate Direct sample analysis State of the Art laboratory practices Reproducible Rapid Results within days not weeks
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 emailed to project contact (7 to 10 day TAT) Results are integrated with other site parameters to evaluate site management decisions
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
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.
Dehalococcoides spp. VC Functional Genes Dehalococcoides Can t be detected using traditional techniques DCE and VC accumulation likely Potential for complete dechlorination
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
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
CENSUS Biostimulation CENSUS Bioaugmentation
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
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 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 8 *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
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
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+09 3.11E+03 MW-1 PM1 Inj. 3.54E+10 2.12E+04 MW-2 pre 9.71E+08 ND MW-2 PM1 Inj. 4.83E+09 1.06E+05 MW-3 pre 2.11E+09 ND MW-3 PM1 Inj. 3.45E+09 1.35E+04
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
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
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
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
Community Composition Community Composition 20% 45% 19% 15% 1% PLFA profile provides a fingerprint of the entire population
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
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.
Response to sulfate injections 45 198 days % of Total 40 35 30 25 20 15 10 5 0 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
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
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
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
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???
Applications of Bio Trap samplers and Stable Isotope Probing Dr. Kerry Sublette, University of Tulsa
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
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
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
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
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
Sampling Strategies Scenario A Day 0 30 60 90 30 Time Scenario B 60 90 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
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
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
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
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
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
Initial Groundwater Conditions Parameter Source (RW-1) Mid-Plume (VE-6) ORP 79 194 DO 0.48 0.34 Sulfate 842 233 Methane* 140 29 PCE 20 10 TCE ND 6 cis-dce 130 13 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 79 194 DO 0.48 0.34 Sulfate 842 233 Methane* 140 29 PCE 20 10 TCE ND 6 cis-dce 130 13 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
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 6 100 ppm RW 1 GW Flow MW 24 Not Detected Applications of Bio trap Samplers and Stable Isotope Probing
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
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
Biostimulation HRC S injection pilot test Biostimulation Applications of Bio trap Samplers and Stable Isotope Probing
Biostimulation Biostimulation Applications of Bio trap Samplers and Stable Isotope Probing
Biostimulation Biostimulation Applications of Bio trap Samplers and Stable Isotope Probing
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
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
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
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
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
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
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
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
Benzene Normalized concentration (mg/bead) 1.2 1 0.8 0.6 0.4 0.2 0 0 5 10 15 20 30 Incubation time (days) 30 MTBE and TBA 1.4 Normalized Concentration (mg/bead) 1.2 1 0.8 0.6 0.4 0.2 0 MTBE TBA 0 5 10 15 20 25 30 Incubation Time (days) Applications of Bio trap Samplers and Stable Isotope Probing
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 [ ] = 1 1000 13 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
δ 13 C of COCs MTBE More 13 C PCE TCE Less 13 C Benzene Crude oil PDB std Natural Gas -50.00-40.00-30.00-20.00-10.00 0.00 10.00 δ 13 C of COCs MTBE More 13 C PCE TCE Less 13 C Crude oil Benzene SIP results PDB std Natural Gas -50.00-40.00-30.00-20.00-10.00 0.00 10.00 Applications of Bio trap Samplers and Stable Isotope Probing
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
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
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
Xylene Concentration Map MW 7 10 ppm EX 1 100 ppm M1E1 1 ppm M2E2 10 ppm 100,000 ppb 10,000 ppb 1,000 ppb 100 ppb < 10 ppb Bio Trap Results Relative Rate Residual 13 C Xylene Concentration Sample Control MNA Pre (mg/bd) Post (mg/bd) % Loss M2E2 10 ppm 1.40 1.31 6 % Biostimulation Oxygen M1E1 1 ppm 1.40 0.84 40 % MW 7 10 ppm 1.40 1.12 20 % EX 1 100 ppm 1.40 1.17 16 % Applications of Bio trap Samplers and Stable Isotope Probing
Bio Trap Results Respiration Bio Trap Results Metabolism Biostimulation Oxygen Applications of Bio trap Samplers and Stable Isotope Probing
Bio Trap Results Metabolism 13 C/ 12 C of Biomarkers Biomass (cells/bead) Del Values Sample Total 13C Enriched % Average Minimum Maximum Control MNA M2E2 3.27E+05 2.15E+03 1% +48 50 +547 Biostimulation Oxygen M1E1 2.88E+07 2.14E+06 7% +6,288 +1,009 +10,764 MW 7 2.00E+07 6.24E+05 3 % +1,624 +348 +3,878 EX1 6.77E+07 2.17E+06 3% +1,739 +619 +3,521 Conclusions Is xylene being degraded under the current conditions? yes To what extent would biostimulation with oxygen effect biodegradation of xylene? Significant stimulation Total biomass increased (large increases in PLFA with O 2 ) Higher levels of 13 C enrichment observed in PLFA Strong evidence of microbial respiration Relative rates faster in the wells receiving oxygen Applications of Bio trap Samplers and Stable Isotope Probing
SIP Case Study Chlorobenzene Site Background Aquifer impacted by chlorinated solvents, primarily chlorobenzene (CB). Concern regarding whether observed decreases in CB concentrations were due to biodegradation rather than dilution. Oxygen addition is being considered to enhance aerobic biodegradation. Applications of Bio trap Samplers and Stable Isotope Probing
Site Specific Questions Is chlorobenzene biodegradation occurring under existing site conditions? Will oxygen addition stimulate chlorobenzene biodegradation? Study Design Assembly Unit Samplers Potential Lines of Evidence Control (MNA) Oxygen Amended COC MICRO (Bio Trap) GEO Oxygen Supplier VOCs: Differences in chlorobenzene concentrations SIP: 13 C chlorobenzene Loss 13 C compound 13 C incorporation into biomass 13 C incorporation into DIC Anions: Differences in redox conditions Applications of Bio trap Samplers and Stable Isotope Probing
13 C Chlorobenzene Remaining Pre Deployment Control (MNA) Oxygen Amended Loss of 13 C chlorobenzene was observed in the MNA unit Post deployment 13 C chlorobenzene concentration in oxygen amended unit was substantially lower than in the control unit. 13 C Incorporation into Biomass Average Background Under natural conditions, the 13 C content of PLFA and dissolved inorganic carbon (DIC) is about 25. Applications of Bio trap Samplers and Stable Isotope Probing
13 C Incorporation into Biomass Average Background Control (MNA) Moderate level of 13 C incorporation into biomass was detected in the Control unit. 13 C Incorporation into DIC Pre Deployment Control (MNA) Moderate level of 13 C incorporation into dissolved inorganic carbon (DIC) in the Control unit. Applications of Bio trap Samplers and Stable Isotope Probing
Site Specific Q&A Is biodegradation of chlorobenzene occurring under existing Question site conditions? Yes The 13 C label was detected in both PLFA and dissolved inorganic carbon (DIC) conclusively demonstrating that chlorobenzene biodegradation was occurring. 13 C Incorporation into Biomass Average Background Control (MNA) Oxygen Amended Incorporation of 13 C into biomass in the oxygen amended unit was substantially greater than in the control unit. Applications of Bio trap Samplers and Stable Isotope Probing
13 C Incorporation into DIC Pre Deployment Control (MNA) Oxygen Amended Likewise, 13 C incorporation into DIC was greater in the oxygen amended unit. Site Specific Q&A Did oxygen addition stimulate chlorobenzene biodegradation? Yes Chlorobenzene relative half life (t 1/2 ) was substantially lower, 13 C incorporation into biomass and DIC were substantially greater in the oxygen amended unit Applications of Bio trap Samplers and Stable Isotope Probing
100% % Removal by 13 C-Amendment 80% % Removal 60% 40% 20% 0% MNA conditions Benzene MTBE TBA Chlorobenzene Naphthalene Methylene Chloride 13 C-Amendment 18000 Highest del 13 C Value ( 0 / 00 ) for FAME Observed 16000 14000 12000 10000 8000 6000 4000 2000 0-2000 MNA conditions Benzene MTBE TBA Chlorobenzene Naphthalene Methylene Chloride 13 C-Amendment Applications of Bio trap Samplers and Stable Isotope Probing
18000 16000 14000 del 13 C DIC Value 0 ( / 00 ) for DIC 12000 10000 8000 6000 4000 2000 0-2000 MNA conditions Benzene MTBE TBA Chlorobenzene Naphthalene Methylene Chloride 13 C-Amendment Questions about SIP? Applications of Bio trap Samplers and Stable Isotope Probing
What about compounds which are not used as a carbon source? + + = energy Electron Donor Electron Acceptor Biomass Waste Compound Specific Isotope Analysis (CSIA) can be used to show biodegradation of chlorinated solvents CSIA and Bio traps Compound Specific Isotope Analysis Applications of Bio trap Samplers and Stable Isotope Probing
CSIA vs. Stable Isotope Probing CSIA Isotopic fractionation results from the differences in the rates of cleavage of carboncarbon bonds involving 12 C and 13 C Stable isotope probing 13 C used as a tracer Collection of Contaminants of Concerns Bio Sep Bead COCs and daughter products are concentrated in the beads then extracted for CSIA Applications of Bio trap Samplers and Stable Isotope Probing
Remediation with Bio Sep Why Bio Sep for remediation? High densities of microbes Activated carbon Release of microbes into the environment Applications of Bio trap Samplers and Stable Isotope Probing
Aerobic In Situ Bioreactors UK site impacted by BTEX and acetone Applications of Bio trap Samplers and Stable Isotope Probing
Applications of Bio trap Samplers and Stable Isotope Probing
Toluene dioxygenase (TOD) Applications of Bio trap Samplers and Stable Isotope Probing
Fuel oil impacted groundwater 2 Bioreactor well 1/2 Monitoring wells Direction of groundwater flow ¼-slots 1¼-in solid Sch 40 pipe 15 cm Air in Mini-bio-trap chamber 1 m Pump (if required) 15 cm Applications of Bio trap Samplers and Stable Isotope Probing
ISCO Followed by Aerobic ISBR qpcr and 13 C Naphthalene SIP Applications of Bio trap Samplers and Stable Isotope Probing
PCE TCE cis-dce VC ETH Concentrating degraders for in situ treatment In situ treatment or colonization Inoculation Applications of Bio trap Samplers and Stable Isotope Probing
Transfer of DHC in a TCE plume qpcr of Bio-traps from the Donor Well and the Receptor Well after One Month 8 7 Log Cells/bead 6 5 4 3 2 1 0 Total Bacteria DHC Donor Well Receptor Well CVOC Concentrations in Receptor Well Before and After Treatment Concentration (ppb) 8000 7000 6000 5000 4000 3000 2000 1000 0 VC cdce TCE Receptor Well Before Treatment Receptor Well After Treatment - Bailed Receptor Well After Treatment - Purged Applications of Bio trap Samplers and Stable Isotope Probing
3000 2500 nzvi/ Emulsified oil DHC Bioreactor Installed 2000 1500 1000 500 0-900 -870-840 -810-720 -630-540 -450-360 -270-180 -90 0 30 60 90 120 150 [VOC], ug/l TCE cis-dce VC Ethene Time (days) Applications of Bio trap Samplers and Stable Isotope Probing
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
Isotope Applications for In-Situ Remediation Case Studies for Chlorinated Solvents, Petroleum Hydrocarbons, and Inorganic Compounds June 22, 2011 Julie Sueker,, Ph.D., P.H., P.E. 1 15 June 2011 2011 ARCADIS Outline Isotope Overview Case Studies #1: TCE Degradation in DNAPL Zone #2: CVOC Degradation Zonation in Large-Scale Plume #3: Monitoring In-Situ Benzene Degradation #4: Demonstrating Nitrate Reduction #5: Evaluating Sulfate Sources 2 15 June 2011 2011 ARCADIS Isotope Applications for In Situ Remediation
Isotope Overview 3 15 June 2011 2011 ARCADIS What are Isotopes? Atoms of same element with differing masses; same # of protons, different # of neutrons p e - p n e - p n n e - Hydrogen 1 H Deuterium 2 H, D Tritium 3 H, T Lighter Heavier Sum of protons and neutrons is the atomic mass 4 15 June 2011 2011 ARCADIS Isotope Applications for In Situ Remediation
Compound Specific Isotope Analysis (CSIA) Isotope ratio shifts (fractionation) for compounds of concern (COCs) Larger for biological and chemical processes Smaller for physical processes Differentiate between destructive and non-destructive processes Useful complement to COC concentration and geochemical data Additional line of evidence in multi-tiered assessments Zone 1: Active Microbial Degradation Zone 2: Transition Zone Zone 3: Physical Attenuation - Dilution Approximate Groundwater Flow Direction Zone 2 Stream Flow Zone 1 Source Area Zone 3 800 ft Shallow Well Deep Well 5 15 June 2011 2011 ARCADIS Compound Specific Isotope Analysis (CSIA) CSIA used to: Differentiate sources of COCs Demonstrate COCs degradation or reduction Evaluate COC degradation pathway e.g., aerobic vs. anaerobic Estimate extent of degradation and degradation rates Monitor effectiveness of engineered remediation systems 0 δ 2 H Benzene ( ) -40-80 -120 Anaerobic Aerobic 6 15 June 2011-160 -30-27 -24-21 -18 δ 13 C Benzene ( ) 2011 ARCADIS Isotope Applications for In Situ Remediation
Which Isotopes do we use? Petroleum hydrocarbons δ 13 C, δ 2 H Light Hydrocarbon gases δ 13 C, 14 C, δ 2 H Chlorinated solvents δ 13 C, δ 37 Cl, δ 2 H Sulfate δ 34 S, δ 18 O Nitrate δ 15 N, δ 18 O Perchlorate δ 37 Cl, δ 18 O, Δ 17 O Chromate δ 53 Cr Metals Cu, Zn, Pb, Fe, Mn, Hg, Se, U Water δ 18 O, δ 2 H, 3 H, 3 He, 14 C Sediments 210 Pb, 137 Cs 7 15 June 2011 2011 ARCADIS CSIA Applied to a Field Site Monitoring Well Transect in COC Plume COC Concentration Source Groundwater flow direction Decreasing COC concentration along plume δ 13 C or δ 2 H Greater isotopic enrichment along plume Lesser isotopic enrichment across plume 8 15 June 2011 2011 ARCADIS Distance Isotope Applications for In Situ Remediation
Case Study #1: TCE Degradation in DNAPL Zone 9 15 June 2011 2011 ARCADIS TCE released to soil and groundwater Case Study #1: TCE Degradation in DNAPL Zone Relatively limited plume migration Low hydraulic gradient, tight soil Residual DNAPL at depths to 80 ft bgs Ongoing reductive dechlorination indicated Daughter compounds present Methane, ethene, and ethane detected Isotope analyses to support MNA for site with DNAPL 10 15 June 2011 2011 ARCADIS Isotope Applications for In Situ Remediation
Site Map D E East Plume B A C F 250 Feet West Plume Benzene Plume CVOC Plume, with TCE CVOC Plume, daughters only 11 15 June 2011 2011 ARCADIS CVOC Concentrations Source Area Sample Distance Source Area Sample Distance 12 15 June 2011 2011 ARCADIS Isotope Applications for In Situ Remediation
TCE Concentration and δ 13 C Increasing δ 13 C with decreasing TCE concentration 13 15 June 2011 2011 ARCADIS TCE and 1,2-cis-DCE δ 13 C Values Source Area Sample Distance Source Area Sample Distance 14 15 June 2011 2011 ARCADIS Isotope Applications for In Situ Remediation
Case Study #2: CVOC Degradation Zonation in Large-Scale Plume 15 15 June 2011 2011 ARCADIS Case Study #2: CVOC Degradation Zonation in Large-Scale Plume TCE released to shallow alluvial groundwater ~5,000 plume in shallow and deep alluvial groundwater DCE, VC, ethene observed in upgradient portion of plume Geochemistry and microbial data consistent with microbial degradation Evaluate δ 13 C of TCE, DCE, VC, ethene Demonstrate microbial degradation Estimate extent of degradation 16 15 June 2011 2011 ARCADIS Isotope Applications for In Situ Remediation
Site Map Stream Flow Approximate Groundwater Flow Direction 5 3 4 2 1 Source Area 8 7 6 800 ft 9 Shallow Well Deep Well 17 15 June 2011 2011 ARCADIS Shallow Groundwater 18 15 June 2011 2011 ARCADIS Isotope Applications for In Situ Remediation
Shallow Groundwater 19 15 June 2011 2011 ARCADIS Deep Groundwater 20 15 June 2011 2011 ARCADIS Isotope Applications for In Situ Remediation
Site Map Zone 1: Active Microbial Degradation Zone 2: Transition Zone Zone 3: Dilution 8 Approximate Groundwater Flow Direction Zone 3 7 5 6 Zone 2 Stream Flow 3 4 2 Zone 1 1 Source Area Zone 1 Extent TCE Degradation Shallow: 70 95% Deep: 60 65% 9 Zones 2 & 3 Extent TCE Degradation Shallow: 30 50% Deep: 15 40% 800 ft Shallow Well Deep Well 21 15 June 2011 2011 ARCADIS Case Study #3: Monitoring In-Situ Benzene Degradation 22 15 June 2011 2011 ARCADIS Isotope Applications for In Situ Remediation
Case Study #3: Monitoring In- Situ Benzene Degradation Historical accidental gasoline release ~2,500 plume benzene and 1,2-DCA Concentrations < 100 μg/l Depth to groundwater ~250 P&T operating, reduced cleanup time desired Groundwater: organic carbon and nutrient limited Soluble substrate injections initiated 23 15 June 2011 2011 ARCADIS Site Map MW-17R Shallow Groundwater Plume Deep Groundwater Plume MW-31D Groundwater Flow Direction RW-8 MW-50D MW-51D 0 100 200 24 15 June 2011 2011 ARCADIS Isotope Applications for In Situ Remediation
Microbial Biomass 1,000,000 MW-50D RW-8 100,000 Cells/mL filtered 10,000 1,000 100 Oct-03 Dec-03 Feb-04 Apr-04 Jun-04 Jul-04 Sep-04 Nov-04 25 15 June 2011 2011 ARCADIS Benzene Concentration 26 15 June 2011 2011 ARCADIS Isotope Applications for In Situ Remediation
Benzene δ 13 C (time) 27 15 June 2011 2011 ARCADIS Benzene δ 13 C & Concentration 28 15 June 2011 2011 ARCADIS Isotope Applications for In Situ Remediation
Case Study #4: Demonstrating Nitrate Reduction 29 15 June 2011 2011 ARCADIS Case Study #4: Demonstrating Nitrate Reduction O O N + O 2/3-2/3-2/3- Former fertilizer distribution facility Elevated NO 3 and NH 3 in soil and groundwater Intermittent layers of silt, sand, and clay Perched groundwater Addition of 580,000 lbs sugar via infiltration ponds 1998-2006 by previous consultant NO 3 reduction elevated CH 4, CO 2, N 2 O in soil gas May have caused radial flow offsite Isotope investigation evaluate ongoing microbial NO 3 reduction for MNA remedy δ 15 N and δ 18 O of NO 3 Offsite NO 3 associated with onsite NO 3? Fractionation primarily via nitrate reduction 30 15 June 2011 2011 ARCADIS Isotope Applications for In Situ Remediation
Source Area Groundwater Sugar Additions 31 15 June 2011 2011 ARCADIS δ 15 N and Nitrate Concentration Increasing δ 15 N with decreasing NO 3 concentration Two potential sample groupings 32 15 June 2011 2011 ARCADIS Isotope Applications for In Situ Remediation
Nitrate δ 15 N and δ 18 O NO 3 fertilizer General Denitrification Trend Direction nitrified NH 4 in fertilizer soil N manure and septic waste 33 15 June 2011 2011 ARCADIS Case Study #5: Evaluating Sulfate Sources 34 15 June 2011 2011 ARCADIS Isotope Applications for In Situ Remediation
Case Study #5: Evaluating Sulfate Sources Cement manufacturing facility with limestone quarry Elevated concentration of sulfate in groundwater Naturally occurring sulfate in bedrock Leaching of sulfate from CKD δ 34 S and δ 18 O of sulfate to evaluate sulfate sources 35 15 June 2011 2011 ARCADIS Sulfur Isotope Signatures Medium δ 34 S ( ) Ocean sulfate +20.7 Marine evaporites +5 to +35 Reduced sulfur, sulfide minerals 50 to 0 Anthropogenic & terrestrial biogenic atmospheric sulfur +3 to +7 Oceanic biogenic atmospheric sulfur +16 to +23 Gulf of Mexico salt domes, S 0 +2 to +6 36 15 June 2011 2011 ARCADIS Sueker, BBL, Inc. 2001 BBL, Inc. Isotope Applications for In Situ Remediation
Sulfate Expected Compositions 37 15 June 2011 2011 ARCADIS Sulfate 3 Sources Identified 38 15 June 2011 2011 ARCADIS Isotope Applications for In Situ Remediation
Summary 39 15 June 2011 2011 ARCADIS On the Horizon Analytical precision continually being improved Metals: Cr, Se, Cu, U, Zn, etc. Laboratory microcosm studies define isotopic shifts for additional COCs Combining molecular biology and isotopic tools to evaluate specific degradation pathways 40 15 June 2011 2011 ARCADIS Isotope Applications for In Situ Remediation
Take Home Messages Incorporating isotope analyses can provide considerable added value for relatively small additional cost For successful investigations: Define goals for utilizing isotopic tools Understand strengths and limitations of tools employed Evaluate supporting data requirements for data interpretation Have something to compare your data to (i.e., pre-remedy implementation, source area, or time ) 41 15 June 2011 2011 ARCADIS Questions? Mother Goose and Grimm 2001 Grimmy, Inc. Dist. By Tribune Media Services. All rights reserved. 42 15 June 2011 2011 ARCADIS 42 Isotope Applications for In Situ Remediation
Imagine the result Contact Information: Julie K. Sueker 303.231.9115 julie.sueker@arcadis-us.com us.com 43 15 June 2011 2011 ARCADIS Isotope Applications for In Situ Remediation