The Use of Stable Isotope and Molecular Technologies to Monitor MNA and Enhance In-Situ Bioremediation

Transcription

1 The Use of Stable Isotope and Molecular Technologies to Monitor MNA and Enhance In-Situ Bioremediation Eleanor M. Jennings, Ph.D. URS Corporation

2 Introduction to Technologies Stable Isotope Techniques Stable Isotope Ratios Stable Isotope Probes Molecular Techniques Denaturing Gradient Gel Electrophoresis Quantitative Polymerase Chain Reaction 2

3 3 Introduction to Isotopic Carbon Carbon comes in different weights 12 C and 13 C are most common isotopes C Carbon Approximate 98.5: 1 ratio in naturally occurring compounds and by-products of naturally occurring compounds Not radioactive working with STABLE isotopes

4 4 Introduction to Isotopic Carbon Can distinguish between 12 C, 13 C Can artificially alter the ratio of 12 C : 13 C Thus, highly inflated 13 C levels act as isotopic tag

5 5 Stable Isotope Ratio (SIR) Analyses Also called Compound Specific Isotopic Analysis (CSIA) 1, 2, 3 Dimensional CSIA Refers to the number of elements analyzed Analysis involves a single GW sample shipped to lab Takes advantage of fact that bacteria preferentially metabolize lower isotopes versus higher isotopes Causes a shift in the 13 C: 12 C ratio 13 C Sample ( ( C/ C/ C) C) Standard

6 Non-Biologically Degraded Petroleum Hydrocarbons (-30 to -20) C Less 13 C Less Biological Degradation PDB Standard More 13 C More Biological Degradation

7 7 SIR Analyses Can Differentiate Between: Biodegraded compounds versus a fresh spill Slightly biodegraded compound versus significantly biodegraded compound Compounds that have been biologically degraded versus abiotically degraded Potential sources of a contaminant, using SIR fingerprinting

8 Introduction to Stable-Isotope Probes In-situ sampling method Can confirm presence of indigenous, degrading population Utilizes isotopically-labeled target compounds Can be any carbon-based compound Ex: benzene Bio-Trap, from Microbial Insights 8

9 9 SIP: Six Simple Steps Step 1: Baiting the Beads - obtain 13 C labeled benzene, sorb 13 C labeled benzene onto beads PAC + Nomex SEM of Bio-Sep bead; 600 m 2 internal surface area / g Bio-Trap Assembly Porosity Significant surface area to sorb labeled target compound Encourages bacterial residence within beads

10 10 SIP: Six Simple Steps Step 2: Construct in-situ sampler Step 3: Deploy into MWs, suspended within the groundwater Step 4: Incubate in-situ for 6 weeks Timeframe will vary for different compounds under different site conditions Reflects actual site conditions versus laboratory microcosms Step 5: Remove from MWs Step 6: Analyze biomarkers and residual, labeled benzene

11 11 Biomarker of Choice - PLFAs High concentrations of phospholipids fatty acids (PLFAs) in microbial cell membranes High concentration of carbon in PLFAs 13 C incorporated into PLFAs microbial membrane Result of incorporation of 13 C-label from benzene into biomass Analyze using GC-IR-MS Insight into indigenous microbial ecology and activity

12 12 Quantitative Data from PLFA Analysis Total population size What groups, how many Community profile Groups, not individual species Confirmation of compound biodegradation

13 13 Quantitative Data from PLFA Analysis Which community members are responsible for degradation Information about possible environmental stress Nutritional deficits or toxic compounds Information about current microbial population In-situ conditions

14 14 Quantified Analyses From Residual Isotopic Compound Degradation rate Under in-situ conditions, not laboratory conditions Compound mineralization to CO 2 What bacteria are doing with carbon, where microbial energy is being directed Isotopic shift Changes in the ratio of 12 C : 13 C Additional confirmation of biological degradation

15 Normalized Weig TGA Analysis MTBE on PAC and Bio-Sep Bead Compounds Do Not Easily Desorb Into Environment 0.1 g benzene per BioTrap at project inception Temperatures must reach 300 o C to leach 10 mg to groundwater Temperature ( o C) 10 C/min PAC Bead

16 16 Introduction to Technologies Stable Isotope Techniques Stable Isotope Ratios Stable Isotope Probes Molecular Techniques Denaturing Gradient Gel Electrophoresis Quantitative Polymerase Chain Reaction

17 Denaturing Gradient Gel Electrophoresis Image courtesy of Microbial Insights 17

18 18 DGGE Analysis Single GW, soil sample Determine composition of mixed microbial communities Most common members More refined detail than SIP Gives actual species present Can not prove actual degradation like SIP can Thus, often used in tandem

19 19 Quantitative Polymerase Chain Reaction (qpcr) Molecular technique that quantifies a target microbial population Class of microbes or particular species Target can be as broad or specific as needed Dependent on molecular probe used Involves a single grab sample Groundwater Soil Sediment Surface water

20 Site Introduction Groundwater impacted BTEX PCE VC Source Two gasoline retail service stations Former dry cleaner site California 20

21 Direction of groundwater flow 21

22 BTEX PCE VC 22

23 23 Questions to Answer Is there an indigenous benzene degrading population? Can benzene-degrading population be further stimulated to increase degradation efficiency? Which gas station holds the dominant responsibility for the benzene in the ditch? What is the likelihood for natural attenuation of the chlorinated compounds?

24 24 Question 1 Is there an indigenous benzene degrading population? Methods used to answer: Stable Isotope Probes

25 Sampling locations for stable-isotope probes 25

26 26 Question 2 Can this population be further stimulated to increase degradation efficiency? Methods used to answer: Information from SIP, coupled to DGGE

27 Sampling locations for DGGE analyses 27

28 Question 3 Which gas station holds the dominant responsibility for the benzene in the ditch? Method to answer: Stable Isotope Ratios 28

29 Sampling locations for SIR analyses - Performed before SIP 29

30 Question 4 What is the likelihood for natural attenuation of the chlorinated compounds? Method to answer: Quantitative Polymerase Chain Reaction (qpcr) 30

31 PCE Degraders Capable of dechlorinating PCE to ethene Commonly found at Cl-solvent sites Not the only microbe that can dechlorinate Dehalococcoides sp. strain FL2 31

32 Sampling locations for qpcr analyses 32 32

33 Results 33

34 34 Stable Isotope Probe Results Presence of substantial, robust benzene-degrading population confirmed Significant incorporation of isotopic label into biomass Higher incorporation at plume periphery than in plume center Population was very active, however was showing signs of nutritional deficits both in plume center and periphery

35 35 DGGE Results Benzene Plumes Results agreed with SIP data Population in plume center Primarily sulfate-reducers Identified species associated in literature with BTEX degradation Population at plume periphery Iron-reducing bacteria Identified species associated in literature with BTEX degradation

36 36 Responsible Party? Geochem data detected BTEX from both plumes reaching ditch Question: Who was primarily responsible for impacting the ditch?

37 Sampling locations for SIR analyses 37

38 δ 13 C = -11 δ 13 C = 4 δ 13 C = 231 δ 13 C = 640 δ 13 C = 8 δ 13 C = 121 δ 13 C = 1489 δ 13 C = 187 δ 13 C =

39 δ 13 C = -11 δ 13 C = 4 δ 13 C = 231 δ 13 C = 640 δ 13 C = 8 δ 13 C = 121 δ 13 C = 1489 δ 13 C = 187 δ 13 C = 423 δ 13 C data from this service station demonstrated much higher levels of biological degradation than ditch samples 39

40 δ 13 C = -11 δ 13 C = 4 δ 13 C = 231 δ 13 C = 640 δ 13 C = 8 δ 13 C = 121 δ 13 C = 1489 δ 13 C = 187 δ 13 C = 423 δ 13 C confirmed that this service station not heavily impacting ditch 40

41 41 MNA Potential of Chlorinated Plume Needed to discern the potential to naturally attenuate the chlorinated compounds Performed qpcr to quantify Dehalococcoides population Known to fully dechlorinate PCE to ethene Coupled with DGGE analysis data

42 7.46 x 10 3 Moderate Dehalococcoides population DGGE revealed two robust dechlorinating populations Work cooperatively PCE to ethene 5.33 x

43 43 Benefits to this Project Detailed profile of microbial community members in benzene and solvent plumes SIP, DGGE, qpcr Appropriate site amendments to be developed Resulted in more rapid contaminant degradation 85% increase benzene degradation rates 56% increase PCE to ethene conversion rates Allowed site to approach site closure more rapidly Estimated years saved Substantial savings over lifespan of project

44 44 Benefits to this Project Determined which service station is primarily responsible for ditch contamination SIR Helped determine financial responsibility for ditch remediation along with site hydrogeological, geochemical data

45 45 Summary Stable isotope and molecular tools can assist with determining best remediation design Can have a significant impact on effectiveness of remediation system Can help discern most effective MNA / bioremediation strategy Determine how to most efficiently implement remediation enhancements

46 46 Summary Can determine if an MNA / bioremediation strategy is working to its full potential Monitor remediation strategy Determine new closure timeline and associated, reduced costs

47 Summary Can help demonstrate MNA / bioremediation to regulators State level: CA, MD, and other states Federal level: Multiple EPA regions Can convince regulators to sometimes allow less expensive remediation methods by proving effectiveness Can reduce lifespan of remediation project by years, reducing lifespan costs Reducing years of O&M, reporting costs $15-20K saved per year at small sites Much more at larger sites 47

48 48 Importance of Technologies Can be used on wide range of contaminants Hydrocarbons Fuel Oxygenates Chlorinated Solvents Manufacturing Wastes Chem / Pharm Materials Use is accepted by scientific community Uses backed by years of peer-reviewed research International acceptance of their use

49 Questions??? 49