Stable Isotope Probing Demonstrates In Situ Biodegradation of 1,4-Dioxane. Dora Ogles (Microbial Insights) James Hatton (CH2MHILL)

Transcription

1 Stable Isotope Probing Demonstrates In Situ Biodegradation of 1,4-Dioxane Dora Ogles (Microbial Insights) James Hatton (CH2MHILL)

2 1,4-Dioxane Sources Stabilizer for chlorinated solvents (1,1,1-TCA) Solvent for paper, cotton, and textile processing Inadvertent byproduct in surfactant production

3 Physical/Chemical Properties Probable human carcinogen (0.35 µg/l) Miscible with water Low sorption Relatively low volatilization Department of Public Health, Washtenaw County, Michigan

4 Biodegradation Aerobic Growth Supporting Cordyceps sinensis (fungus) Mycobacterium sp. PH-06 Pseudonocardia dioxanivorans CB1190 (Mahendra & Alvarez-Cohen, IJSEM, 2005)

5 Biodegradation Aerobic Growth Supporting Aerobic Co-oxidation Ring hydroxylating toluene monoxygenases (RMO,RDEG,PHE) Soluble methane monooxygenase (smmo) Propane, alkenes, tetrahydrofuran (THF), etc. Toluene-2-monoxygenase (RDEG) O 2

6 Pathway Quantitative Polymerase Chain Reaction (qpcr) (HEAA) Mahendra et al ES&T 41(21): Stable Isotope Probing (SIP)

7 Stable Isotope Probing (SIP) Specially produced heavy compounds which are composed of 99% 13 C Similar characteristics as 12 C contaminant Similar environmental behavior Quantify the 13 C tracer in end-products of degradation (biomass and CO 2 )

8 How do Bio-Traps work? Properties of Bio-Sep Beads 25% Nomex & 75% PAC 3-4 mm in diameter 74% porosity 600 m 2 of surface area/g Heat sterilized 300 o C

9 Overview of SIP Approach 13 C labeled Dioxane Bio-Trap with 13 C- Dioxane loaded beads Beads analyzed following deployment Beads loaded with 13 C compound In-Situ deployment in monitoring well

10 Bio-Trap SIP Analysis Residual 13 C-Compound Contaminant Loss 13 C/ 12 C Dissolved Inorganic Carbon (DIC) Mineralization 13 C/ 12 C of Biomarkers PLFA DNA RNA Biomass

11 Site Management Options Monitored Natural Attenuation (MNA) Biostimulation (BioStim) Butanol addition Kelley et al. (2001) Bioaugmented phytoremediation Butanol addition stimulated growth of CB1190 MNA BioStim

12 In Situ Microcosm Study MNA Unit MNA Bio-Trap GEO BioStim

13 In Situ Microcosm BioStim Unit Supplier MNA Bio-Trap BioStim GEO Supplier

14 13 C Incorporation into Biomass 1.E+07 1.E+06 MW-9 MW-37 1.E+05 Cells/bead 1.E+04 1.E+03 1.E+02 1.E+01 1.E+00 MNA 13C Enriched Biomass (Cells/bd) MNA Total Biomass (Cells/bd)

15 Unit of measure Amount of 13 C relative to 12 C is expressed by the δ 13 C notation δ 13 C 13 C ( / ) [ ] = Sample ( 13 C/ 12 C) Standard The standard is a specific carbon-containing mineral from a specific location: Pee Dee Belimnite (PDB) 12 C Units of δ 13 C are or per mill

16 13 C Incorporation into DIC DIC δ 13 C ( ) MW-9 Ave. Background MNA MNA MW-37

17 13 C Incorporation into Biomass Average PLFA δ 13 C ( ) MW-9 Ave. Background MNA MNA MW-37

18 Co-oxidation Potential 1.00E E+05 MW-9 MW E+04 Gene copies/bead 1.00E E E E+00 MNA MNA Toluene Monooxygenase (RMO) Phenol Hydroxylase (PHE) Soluble Methane Monooxygenase (smmo)

19 Conclusions MNA Units qpcr Moderate abundances of genes encoding oxygenases capable of co-oxidation of dioxane Incorporation into Biomass Detection of 13 C enriched PLFA demonstrated that dioxane biodegradation occurred within the passive microbial sampler Incorporation into DIC Although low, 13 C enriched DIC was detected indicating dioxane mineralization had occurred

20 In Situ Microcosm BioStim Unit Supplier MNA Bio-Trap BioStim GEO Supplier

21 13 C PLFA MNA vs BioStim 1.E+07 1.E+06 MW-9 MW-37 Cells/bead 1.E+05 1.E+04 1.E+03 1.E E E E E+04 1.E+01 1.E+00 MNA BioStim MNA BioStim 13C Enriched Biomass (Cells/bd) Total Biomass (Cells/bd)

22 13 C PLFA MNA vs BioStim PLFA δ 13 C ( ) MW-9 MW-37 Ave. Background MNA BioStim MNA BioStim

23 13 C Incorporation into DIC DIC δ 13 C ( ) MW-9 MW Ave. Background MNA BioStim MNA BioStim

24 Co-oxidation Potential 1.00E E+05 MW-9 MW-37 Gene copies/bead 1.00E E E E E+00 MNA BioStim MNA BioStim Toluene Monooxygenase (RMO) Phenol Hydroxylase (PHE) Soluble Methane Monooxygenase (smmo)

25 Conclusions BioStim Units qpcr Confirmed potential for co-oxidation Stable Isotope Probing Demonstrated that 13 C dioxane biodegradation and mineralization occurred in situ BioStimulation (butanol addition) Overall results did not conclusively demonstrate enhanced biodegradation vs MNA

26 Conclusions Dioxane biodegradation occurred under existing site conditions at both locations Bacteria harboring oxygenase genes capable of dioxane co-oxidation were present Butanol addition may not have appreciably enhanced dioxane biodegradation

27 SIP Advantages Relatively conclusive evidence of contaminant biodegradation in situ No knowledge of biodegradation pathway or organisms responsible is needed. Applicable to a wide variety of common contaminants

28 SIP Limitations Greater contaminant concentrations in sampler Potential desorption (dioxane) Some 13 C compounds can be expensive to synthesize

29 SIP Limitations Generally not appropriate for compounds used as electron acceptors SIP-PLFA cannot identify degraders δ 13 C ( ) i15:0 i16:0 c16:1w7c 16:1w5c c17:1w6c cy17:0 18:1w9c 18:1w7c cy19:0 10me16:0 10Me18:0 c14:0 c16:0 c17:0 c18:0 c18:2n6c c20:4n6 c20:5n3

30 Questions???